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A study of prenatal and postnatal development in the oldfield mouse, Peromyscus polionotus

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Title:
A study of prenatal and postnatal development in the oldfield mouse, Peromyscus polionotus
Creator:
Laffoday, Samuel Kanada, 1924-
Publication Date:
Language:
English
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viii, 124 leaves. : ; 28 cm

Subjects

Subjects / Keywords:
Eggs ( jstor )
Embryos ( jstor )
Female animals ( jstor )
Gestation period ( jstor )
Litter size ( jstor )
Lumens ( jstor )
Mating behavior ( jstor )
Reproduction ( jstor )
Species ( jstor )
Uterus ( jstor )
Mammals -- Embryology ( fast )
Oldfield mouse ( fast )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

Notes

Bibliography:
Includes bibliographical references (leaves 99-101).
General Note:
Manuscript copy.
General Note:
Vita.

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University of Florida
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|University of Florida
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This item is presumed in the public domain according to the terms of the Retrospective Dissertation Scanning (RDS) policy, which may be viewed at http://ufdc.ufl.edu/AA00007596/00001. The University of Florida George A. Smathers Libraries respect the intellectual property rights of others and do not claim any copyright interest in this item. Users of this work have responsibility for determining copyright status prior to reusing, publishing or reproducing this item for purposes other than what is allowed by fair use or other copyright exemptions. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder. The Smathers Libraries would like to learn more about this item and invite individuals or organizations to contact the RDS coordinator (ufdissertations@uflib.ufl.edu) with any additional information they can provide.
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ocm13434049
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A STUDY OF PRENATAL AND


POSTNATAL


DEVELOPMENT IN THE OLDFIELD MOUSE,

Peromyscus polionotus











By
SAMUEL K. LAFFODAY


A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY











UNIVERSITY OF FLORIDA
June, 1957














ACKNOWLEDGMNTS


Many individuals have contributed most generously of their

time, knowledge, and experience to this work. Dr. H. B. Sherman, Professor of Biology at the University of Florida until his retirement in 1955, suggested the problem and guided me through the initial phases of the work. To him I should like to express my sincere thanks. For their valuable criticisms suggestions, and interpretations, I should like to thank Drs. Lewis Berner, J. H. Gregg, G. K. Davis, J. D. Kilby, Pierce Brodkorb, J, W, Brookbank, and J. N. Layne. Dr. Layne helped especially with the analysis and interpretation of data on postnatal development. Several of uy fellow students contributed in various ways to the work. Robert Haubrich and Claude Adams cared for the laboratory animals from time to time; John Outterson assisted with the field work, and Robert Weigel and Joe Davis with the photography* To all of them I should like to express my appreciation. Dr. E. Ruffin Jones, Jr., as chairman of my supervisory comittee advised me on my academic program, guided me in my research, and gave me every consideration and continued inspiration. It is a pleasure to express to him my sincere appreciation. Finally, my wife, Joanne Andrews Laffoday, deserves special acknowledgment for the assistance which she has given me. She helped with the field collections, with the writing and typing of this report, and throughout has provided moral and financial support. I should like, therefore, to expresa to her my thanks for her many contributions.


ii














TABLE OF CONTENTS


ACKNOWLED(MENTS . . . . . . . . . . . . . . . . . . . . . . . iLIST OF TABLES. . . . . . . . . . . . . . . . . . . . . . . . v

LIST OF ILLUSTRATIONS ....*......... .. *. . vi

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . 1

METHODS AND KATERIALS . .... ......... .. . . . . 6

DESCRIPTION OF PRENATAL DEVELOP1iT . . . . . . . . . . . . 13

Zero Hours . . . . . . . . . . . . . . . . . . . . . 13
Fifteen Hours . . . . . . . . . . . . . .. * . . . 13
Twenty-Four Hours . . . . . . . . . . . . . . . . . . . 15
Forty-Eight Hours . . . . . . . . . . . . . . . . . . 15
Sixty Hours . . S . . . . . , . . . . . . . 16
Seventy-Two Hours . . . . . . . . . . . . . . . . . . . 16
Ninety-Six Hours . . . . . . . . 17
Five Days . . . . . . . . . . . . . . . . . . . . . . 18
Six Days .o . . . . . . 20
Seven Days . . . . . . . . . . . . . . . . . * . . . . 20
Eight Days . . . . . . . . . . . . . . . . . . 21
Nine Days . . . . . . . . . . . . . . . . . * *. . 22
Ten Days . . . . . . . . . . . . . . . . . . . . . 25
Eleven Days . . . . . . . . . . . . . . . . . . . . . . 28
Twelve Days . . . . . . . . . . . . . . . . . . . . . 28
Thirteen Days . *.. . . . * . e * . * . . . . . * 32
Fourteen Days . . . . . * . . . . * *. . . . . . . . 37
Fifteen Days . . . . . . . . . . . . . . . . . . . . . 44
Sixteen Days . . . . . . . . . . . . . . . . . . . . . . 51
Seventeen Days . . . .. . . . . . . . . .. . . . . . . 56
Eighteen Days . . . . * . . . oO. .. . # 60

PRENATAL GROWTH . . . . . . . . . .... . . . . . . . . . . 64

Measurements of Uterine Swellmngs at Implantation Sites . 64 Weights and Volumes of Embryos . a . . a . 05. . . ... . 65
Measurements of Certain Body Parts of Embryos from
Twelve Through Eighteen Days of Development . . . . . 66

PARTURITION . . . . . . ...... . C . . . . * . . 68


iii













TABLE OF CONENTS--Contired


LITTER SIZE AND SEX RATIO.......... . ..... 72

Litter Size . . . . . . . . . . . .. ... . . . . . 72
Sex Ratios . . . . . . . . . . . . . . . . . . . . . . 72

GESTATION . . . . . . . . . . . . . . . . . . . . . * . . . . 74

Gestation Length in Non-Lactating Females . . . . . . . . 74 Influence of Lactation on Gestation. .. ...... 74
Post-Partum Estrus and the Normal Estrous Cycle . . . . . 75 POSTNATAL GROWTH AND DEVELOPMENT . . . . .. . . . ...... 77

Weights at Birth .................... 77
Postnatal Growth Measurements. .0 0 *. * . . . . .. 77 Development . . . . . . . . . . . . . . . . . . . . * . 79

DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . 84

SUMMARY AND CONCLUSION . . . . . . . . ............ 96

LITERATURE CITED . . . . . . . . . . . . . . . . . . . . . . . 99

PLATES AND FIGURES . . . . . . . . . . . . . . . . . . . . . . 103

BIOGRAPHICAL SKETCH . . . . . . . . . . . . . . . . , 0 124


iv













LIST OF TABLES


Table Page


1. List of Eggs and Embryos Studied. . . .... . 10


2. Mean Measurements in Millimeters of Uterine Swellings . * . . . . . . . . . . . . . . . 64


3. Mean Weights and Volumes of Embryos 12 to 18 Days of Age . . . . . . .. . .. . .. .. 65


4. Average Measurements of Body Parts of Embryos in Millimeters . . . . . . . . .. . .. ..* 67


5. Relative Frequencies of Different Litter
Sizes in P. polionotus . . . ...... ..... 72


6, Average Daily Growth Measurements in Millimeters and Weights in Grams of
P. polionotus from 2 to 60 Days of Age . . . .... 78


V













LIST OF ILLUSTRATIONS


S..

A.

B.

C.

D.

E.

F.

G.


S.. 0 0 0 * * 0 0 0 0 * 0 4 * 0 * * * 0 0 * * * * * * Recently ovulated egg. Pronuclear stage. Two cell stage. Four cell stage. Eight cell stage. Morula stage. Blastocyst stage.


. . . . . . . . * . 0 . . . . . . . . 0 . . . . * . * ..

A. Longitudinal section of embryo five days of age. B. Longitudinal section of embryo six days of age. C. Longitudinal section of embryo seven days of age. D. Longitudinal section of embryo eight days of age. E. Longitudinal section of embryo nine days of age
showing three cavities.

F. Longitudinal section of embryo nine days of age
showing a break in the chorion allowing a direct
connection of the exocoelom with blood-filled
spaces of the ectoplacental cone.


. . . . . . . . . . . . . . . . a . 0 0 . . . 0 . .

A. Longitudinal section of embryo nine days of age
showing formation of exocoelom.

B. Sagittal section of embryo nine days of age
showing somite formation.

vi


Plate


I .


II III


Page 103


105


















107












LIST OF ILLUSTRATIONS--Continued


C. Frontal section of embryo ten days of age
showing allantois connected to chorion.


. 0 .0 0 . . * 0 0 0 0 0

Embryo ten days of age. Embryo twelve days of age. Embryo thirteen days of age. Embryo fourteen days of age. Embryo fifteen days of age.


. . . . 0 . . 0 * . . * . 0 Embryo sixteen days of age.

Embryo seventeen days of age. Embryo eighteen days of age.


0 0~ 0 * 0 0 0 0 60


VI . . .




VII...

A.

B.


VIII . . .

A.

B.


. . . * . . 0 . . 0 0 . . . . . * . . 0 . . . * .

Litter of mice two to four hours old. . . . . .. . . . . . . .. * 0 & * & 0 0 0 a 0 & * 0 &

Litter of mice four days of age. Litter of mice eleven days of age. . . # 0 0 * . & 0 * . 0 . 0 * . . . . * . . .

Dorsal view of mouse twenty-four days of age. Side view of mouse twenty-four days of age.


vii


Plates


Page


I..












V . .


a . A.

B.

C.

D.

E.


. .

A.

B.

C.


109


113 115






117












LIST OF ILLUSTRATIONS-Continued

Plates Page

.. . D v o o h d o *ge.9

A. Dorsal view of mouse thirty days of age.

B. Ventral view of mouse thirty days of age.,

C. Side view of mouse forty days of age.



Figure

1 . . . . . . . . . . . . . . . . . . . . . . . . . . . 121

Graphs showing measurements of certain body
parts of embryos from twelve through
eighteen days of gestation.

2. . . . . . . * . . . . . . . . . & . * . . . . 123

Graphs showing growth rates of selected body
dimensions and weights from birth to sixty
days of age.


viii














INTRODUCTION


The present state of knowledge concerning the embryonic development and other aspects of reproduction in mammals in markedly incomplete. Only a small fraction of the more than 3,000 species of recent mammals have been studied in any detail, and the majority of these are domestic or laboratory forms. The paucity of developmental studies on wild species in large part stems from the difficulties attendant upon maintaining many kinds under captive conditions, not to mention the problems of inducing breeding in the laboratory and of obtaining knoun-age embryological stages. Despite the fragmentary nature of the data, there has been a distinct tendency to apply the findings based on the study of the embryology of a single or of a few species to the broader divisions of classification. The chief justification for such extrapolation seems to lie in the assumption that the intrauterine environment is relatively constant in all mamals, at least in the placental forms, as are the requirements for maintenance of the embryo. As a consequence the pattern of embryonic development, freed from the selective processes of differing environmental conditions, would be expected to be relatively conservative and probably closely similar even throughout the higher taxonomic divisions. However, Simpson (1945) has pointed out that "Embryonic structure is no more free from the effects of new mutations, from adaptation, convergence, divergence, and the like than is any other phase of the life

1









2

cycle." Thus, although embryos or ontogenetic processes may be more similar than adults in closely related animals, they may also be more dissimilar. Further, it must be borne in mind that the morphological, physiological, behavioral and other characteristics exhibited by the adult and utilized in assessing relationships between species are the end result of modification of embryonic processes or chronologies through the action of differing genie constitutions. Therefore divergence in adult structure would be expected to be associated with ontogenetic differences, although the latter may be hard to detect. Evidence presently available does indicate the existence of significant differences in embryonic structure or processes among mammals at the higher levels of classification. Less is known of the kinds and extent of such differences between lower taxonomic units, and the significance of those that have been pointed out will not become clear until many more careful studies are made of the embryology of numbers of related species.

Modern systematic approach relies upon all aspects of the biology of species in its attempt to arrive at evolutionary synthesis. The period of embryonic development is an important stansa in the life cycle of any species and might be looked upon as an important source of evidences of relationships. However, since such data are not easy to obtain, it has played only a relatively minor role in the delimitation of the lower taxonomic categories. But when such information can be procured it may prove valuable in augmenting and supplementing the usually employed adult morphological taxonomic criteria.









3

Among recent mammals the order Rodentia comprises a vast array of species in which extensive adaptive radiation, convergence, and other factors often obscure phyletic relationships and clearly provide a situation -where all available data should be employed. Various aspects of reproduction have thus far proved useful in the taxonomy of various groups of rodents. Among the features that have been employed are: the developmental history of the fetal mebrahes, characteristics of the newborn young, length of gestation, nature of the estrous cycle, and the anatomy of the genital organs. The value of details of embryonic development in this connection has not been well explored.

Considerable attention has been given to the classification of rodents in the families Muridae and Cricatidae, where many of the aspects of reproduction mentioned above have been utilized in addition to "classical" taxonomic characters. The status of the two groups is at present not fully agreed upon by taxonomists. Some workers, Ellerman (1941) include both under the Muridae, whereas others, Simpson (1945) afford each full family rank.

A considerable amount of work has been done on the embryology and development of several species of these rodents extensively used as laboratory animals. The embryology of the house mouse, jKup musculus and rat, Rattus norvegicus, both murids, is comparatively well known as the result of studies by Huber (1915), Long and Burlingame (1938) and Snell (1941). A recent account of the general chronology of prenatal development in a cricetid, the hamster, Mesocricetus, is that of Boyer (1953). Differences in the chronology and details of development in









4

these forms are apparent but their importance from a taxonomic standpoint cannot be evaluated until similar data are available for species of other genera in the two groups. With this consideration in mind the present investigation was undertaken.

The oldfield mouse, Peromyscus polionotus, was selected for

study. The mice of this genus are typical cricetid types of wide distribution in North America, ranging from Atlantic to Pacific coasts and from the tree line in the north to Panama, Hamilton (1939). The genus contains more named forms than any other in North America, 56 species with numerous geographic races being listed by Miller and Kellogg (1955). Various species of Peromyscus occur in a great variety of habitats and are frequently the commonest of small mamals in an area, often playing an important role in the ecology of the region. Their diversity of form and wide geographic and ecological distribution, coupled with the ease by which they can be captured and maintained under laboratory conditions, have made these mice the subject of numerous taxonomic, ecological, physiological, behavioral, and genetic studies. Such investigations have contributed much to our understanding of differentiation and speciation in mammalian stocks.

Although postnatal development and other details of reproduction have been studied in various members of the genus, it appears that the only published reference to embryological davelop-mnt in the group is the mention by Ryder (1887) of "inversion" of the germ layers. Smith (1939) in an unpublished master's thesis reported upon the early embryology of P. polionotus from the pronuclear stage to the formation









5


of the blastocyst.

The main objective of the present study was to follow the sequence of embryological development as copletely as possible from conception to term. Since the general features of embryology in this species are similar to those of the laboratory mouse, rat, and hamster, no effort was made to describe in great detail all particulars of differentiation and organogenesis. Rather, attention was directed principally to those features that seemed to offer a basis for critical comparisons between the various genera. Since prenatal development is merely the initial phase in a continuous growth process to adulthood, observations on postnatal growth and development were considered as also falling logically within the scope of the investigation. Data pertaining to other aspects of reproduction, such as length of gestation, parturition, estrous cycle, litter size, and sex ratios of the young at birth, which were accumulated incidental to the principal objectives of the work are also presented.














METHODS AND MATERIALS


The mice used in establishing the laboratory colony were obtained from burrows in the Ocala National Forest in Marion County, twenty-three miles east of Ocala, Florida. Some animals were captured along the road bed of State Highway 20, from Central Tower east to Juniper Springs. Collections were also made on the Salt Springs road from Central Tower north for a distance of approximately six miles. This population has been referred to as Peroawsncus polionotus subgriseus by Schwarts (1954).

Burrows could be seen along the shoulders of the road with little difficulty and could be dug easily. Only burrows that were plugged a short distance from the entrance were excavated, as these were usually occupied by a pair or by a family of mice, while unplugged burrows were usually unoccupied. After determining the direction of the burrow a small minnow seine was laid on the ground several feet back from the entrance in hopes of covering the exit from the escape tunnel. In some cases, after removal of the plug from the entrance tunnel, the introduction of a piece of rubber tubing into the burrow would cause the mice to flee by way of the escape tunnel and emerge under the minnow seine. Often, however, the mice would not leave the nest until it had been completely exposed by digging. If they then ran into the escape tunnel and remained there instead of coming to the surface the introduction of the rubber tubing into the escape tunnel would promptly drive them out.

6









7

Mice were housed in the laboratory in cages measuring

9 x 15 x 9 inches in width, length, and height, respectively. The top and bottom were formed of cookie sheets and the sides of 1/4 inch hardware cloth. The tops were secured by two coil springs and were easily removed. Coarse wood shavings were placed on the floor and a one quart nesting jar containing a small amount of cotton was kept in each cage. A water bottle was attached to the outside with a delivery tube extending into the cage.

The diet consisted chiefly of Purina Laboratory Chow pellets on which they fed ad lib. Lettuce was put into the cages once or twice weekly; and while matings were being made small amounts of beef liver were offered to the animals and these were readily accepted. The diet was also supplemented from time to time with whole yellow corn.

To increase the laboratory stocks wild mice taken in the field as pairs were kept paired in the laboratory. A single pair was kept in each cage. Usually they would not reproduce for six months or more but with production of the first litter, there would often result a sustained littering period for three to five litters. It was soon discovered that the most productive method of securing litters from laboratory-raised animals was by placing as many as four females to a cage and introducing a proven male for a period of about two weeks. Successful pregnancies could be detected within fifteen days after the date of removing the male and the gravid animals were then isolated. This method proved quit* effective and is several cases when as many as twenty females were used all of them became pregnqut as a result of being with the male for the










a


two-week period.

In an effort to obtain carefully timed embryos, and to measure the length of the gestation period in non-lactating females, frequent attempts were made to observe actual coitus. However, this was soon abandoned as the presence of an observer discouraged mating. The material used for this study was obtained by determining the onset of estrus through vaginal smears. Then a proven male was introduced into the cage with the female at about 8:30 P. M. and was removed the next morning at approximately 8:30 A. M. A vaginal smear was then taken and the slide checked for the presence of sperm. If sperm were found the mouse was killed with ether after an appropriate period and the genital tract was removed and fixed in Lavdowsky's mixture, Guyer(1947). By sacrificing pregnant females at appropriate intervals a sequence of embryos was obtained representing each of the first eighteen days of the gestation period except the eleventh. The ages of embryos in this paper were all calculated from the time of finding sperm in the vaginal fluid. Obviously this method does not provide the exact age of an embryo, but it is impossible to determine the exact time of fertilization with any known method. Griffith and Farris (1942) indicated that various points of reference have been used in the timing of embryos, but the exact age of the embryos could not be determined by the time of copulation, ovulation, or fertilization.

The embryos from which sections were to be prepared were processed by the usual paraffin technique, sectioned at 10 micra, stained with Standard Alum-Hematoxylin and Triosin (pH 5.4-5.6), and mounted









9

with Permount. In material representing the first four days post copulation, both sides of the Fallopian tubes and the entire uterus were sectioned. For stages from five through twelve days sections of the uterus containing implants were excised and the embryos were sectioned in uero. In each case embryos were sectioned both transversely and parallel to the long axis of the uterine horn. In addition, embryos ten days of age and older were removed from the uterus, dissected free of all fetal membranes and sectioned transversely. A description was made of external form and the extent of internal development from ten through eighteen days of age.

A number of measurements was made to determine changes in embryo size and volume with age. Due to the small size of the specimens no volumetric or weight measurements were attempted for embryos ten days of age arid under and all measurements were made with a compound microscope and an ocular micrometer. For specimens ranging from twelve through eighteen days gestation measurements of length and width were made with the aid of a pair of fine (bolt-controlled) dividers under 5x magnification. Volumetric measurements were also made of this series of embryos. The method employed was to select micro-vials of a size appropriate for the embryo to be measured. Each of these vials was ringed with a diamond point marking device, and its volume to that line was determined. The embryo was placed on filter paper and rolled around with the aid of a camels hair brush until it appeared to be dry and then introduced into the previously standardized vial. The vial was filled up to the marked ring from a 5 ml. Burette that could be










10

read to 1/100 of a ml. The measured amount of water that it took to fill the vial containing the embryo, subtracted from the known volume of the vial gave a difference which represented the volume of the embryo, Each embryo was measured three times and an average was obtained. Volume determinations made in this manner were quite consistent, often yielding identical readings for each of the three measuremert s.

Weights were also recorded for this series of embryos after they had been blotted as previously described, placed in stoppered weighing bottles, and weighed on an analytical balance.

The description of prenatal development is based upon a study of sixteen whole specimens and of fifty-four eggs and embryos which were sectioned. The stages studied are listed in Table 1.


TABLE 1

LIST OF EGGS AND EPM YOS STUDIED Number of
Age Specimens Stage of Development
0 hours 5 Newly ovulated eggs surrounded by
corona radiata

15 hours 4 Pronuclear

24 hours 3 2-cells

48 hours 3 4-cells

60 hours 3 4-cells

72 hours 5 Morula in lower end of tube, and
blastocysts in uterus

96 hours 4 Blastocysts, morula, and 2-cell stage
in uterus









11


TABLE 1 - Continued

Number of
Age Specimens Stage of Development

5 days 2 Implanted embryonic vesicle with beginning of proamniotic cavity

6 days 3 Egg cylinder, proamniotic cavity restricted to embryonic ectoderm

7 days 3 Proamniotic cavity continuous into
extra-embryonic ectoderm; posterior amniotic fold beginning

8 days 3 Presence of 3 cavities; amniotic,
exocoelom, and ectoplacental

9 days 4 Allantois present, Somites present in
one embryo, not yet developed in others 10 days 4 Body form U-shape, allantoic stalk joins
chorion, and appearance of first aortic arch

11 days 0 - -

12 days 4 Body form tightly coiled C; limb buds
present

13 days 4 Tail distinct; anterior limb buds
fan-shaped

14 days 3 Face formed, features not yet discernible as mammalian

15 days 3 Resemble adult; vibrissae follicles an
each side of snout

16 days 4 Digits outlined but fully webbed; mammae
present; no hair follicles on body 17 days 3 Digits of forelimb. deeply notched; plantar tubercles present; hair follicles present zn body

18 days 4 Digits fully formed; eyes half closed

During the course of this investigation data were obtained an










12

parturition, litter size, sex ratios at birth, gestation, post-partum estrus, and the normal estrous cycle in P. polionotue.

Observations on postnatal growth and development were made on thirteen litters of mice totaling forty-four individuals. These were carefully observed from the time of birth up to fourteen days, with measurements and weights being taken at two-day intervals. From fourteen to thirty days measurements were made every four days, and from thirty days to fifty or sixty days, weekly measurewants were made. Weights and measurements were started on sane litters at two days of age and at three days of age on other litters, and all measurements after four and five days of age were made on anaesthetized animals. The measurements taken were those generally employed by mammalogists, that is total length, tail length, hind foot length, and length of ear from crown.














DESCRIPTION OF PRENATAL DEVELOPMENT


Zero Hours

Two recently ovulated eggs were found in the right Fallopian tube and three in the left of a specimen killed immediately upon finding motile sperm in a vaginal smear. Each egg appeared as a somewhat spherical, homogeneous mass of lightly staining cytoplasm. There were no visible nuclei in any of the sections, nor were any polar bodies present. It could not be ascertained whether or not these eggs were fertilized, although sperm were present in the Fallopian tubes at the level of the ova. Each ovum was surrounded by a corona radiata which consisted of a dense mass of deeply-staining cells, anu a wide zona pellucida was evident between the egg and the corona cells (Pl. IA). The maximum dimensions in the plane of section of these ova were, in micra: 62 x 46.5; 69 x 54.2; 62 x 54.2; 62 x 46.5; 62 x 50.


Fifteen Hours

Embryos representing the pronuclear stage were obtained from a mouse killed fifteen hours after a few non-motile sperm and sperm fragments were found in the vaginal smear. In most pregnant females which had mated many motile sperm were found in the vaginal smears. It seems apparent, therefore, that a longer period of time had elapsed between copulation and the making of the smear in this particular specimen than was usually the case. However, under the method of age determination used in this work, this must nevertheless be recorded 13









14


as a fifteen-hour stage.

The four embryos found were centrally located in the large

lumen of the ampulla of one Fallopian tube and were not in close association with the mucosal lining of the tube. A few sperm were also visible lying in the lumen of the tube. A disintegrated corona radiata appeared in the form of mucoid-like strands surrounding each embryo. The embryos were surrounded by a fairly thick sona pellucida and between this membrane and the cytoplasm of the egg was a clear perivitelline space. The sperm centrum appeared as a slight conical projection. The second polar body was located in the perivitelline space producing a slight bulge in the encompassing sona pellucida (Pl. IB). Various degrees of fusion of the pronuelei were evident in the series of embryos. Fusion was complete in one, in progress in two others, and had just commenced in another. In the latter case the pronuclei were in close juxtaposition but the nuclear membranes were still intact. The pronuclei in each case were located approximately in the center of the egg. Size differences were evident in the pronuclei, the smaller perhaps representing the male pronucleus as suggested by Huber (1915) and Smith (1939). The larger pronucleus was elliptical in shape and the smaller one appeared more round. The greatest length and width, in micra of the larger pronucleus were 18 x 14; and for the smaller pronucleus 14 x 14.4. One of the embryos measured 77.5 x 77.5 micra in greatest length and width, respectively, vhile the other three were the same sise, 77.5 x 54.2 micra.









15


Twenty-Four Hours

Three embryos obtained from a specimen killed twenty-four hours subsequent to finding sperm in her vaginal fluid had undergone the first cleavage division and were in the two cell stage. All were in the isthmus of the same Fallopian tube, while the other contained several degenerating ova. The cytoplasm of each blastomere was granular and the nucleus was round and centrally located. The blastomeres were surrounded by the sona pellucida, but there was no evidence of corona radiata cells at this stage (Pl. IC). The greatest length and width of one typical two cell embryo were 93.0 and 62.0 micra; the blastomeres were equal in size. The nucleus of each blastomere had a diameter of 18 micra.


Forty-Eight Hours

Each of three embryos obtained at this stage was composed of four cells and was still located in the Fallopian tubes. They were distributed two on one side and one on the other. The two embryos in the isthmus of the same tube were located very close together as they both appeared in several of the same sections. Smith (1939) obtained two four cell stage embryos in a female sacrificed 47+ hours after copulation.

The plane of section did not pass through all four of the cells in any of these forty-eight hour embryos, so that individual sections show no more than three cells (Pl. I,D). The sona pellucida surrounding the embryo was still distinct and of the greatest diameter











16

of the embryo at this stage was 62 micra.


S=.* Hours

Three embryos from a female sacrificed at sixty hours were

also in the four cell stage and exhibited no discernible advance over those at forty-eight hours. However, they were slightly larger, with average maximum diameters of 77.5 and 62.0 micra.


SeventyTwo Hours

Embryos representing the seventy-two hour stage were obtained from two different females. These two sets of embryos showed significant differences in development. Three embryos from one female were located in the distal end of the Fallopian tube and each was in the eight cell stage of development (Pl. I,E). The most distally located embryo was 120 micra from the tubo-uterine junction. One embryo measured 62.0 micra in length and width while another was more elongate and measured 77.5 x 46.5 micra. The third was damaged in suctioning and could not be measured. Fragments of the sona pellucida still surrounded each embryo. Whether the appearance of the zona pellucida at this stage of development was natural or due to injury in preparation could not be determined.

The two embryos of the other set were in the uterine horns

and had already attained the blastocyst stage. Implantation had been initiated, as the embryos were embedded in pits in the wmaosa on the antimesometrial side of the uterus. In one embryo the inner call mass









17

was toward the lumen while in the other it was toward the mucosa of the uterus. The trophoblast consisted of from fourteen to sixteen cells, and the inner cell mass was composed of from eight to ten cells. The average dimensions of the two blastocysts were 77.5 x 62.0 micra in length and width, respectively. The subgerminal cavity was parabolic in shape and measurements in micra of the greatest length and width of each cavity were 38.75 and 46.5.


Ninety-Six Hours

A considerable variation in degree of development was exhibited by the four embryos secured from a single female at ninety-six hours. Three embryos representing as many different stages of development were found in the left horn. The embryo which was located nearest to the ovary was in the morula stage. Distal to this was an apparently healthy two cell stage lying in the uterus, and still farther from the ovary was an embryo of twelve to sixteen cells.

The late blastocyst found at this stage of development was

more elongate than the blastocyst seen in the seventy-two hour stage. Its greatest length and width measurements were 93.0 x 46.5 micra. The segmentation cavity measured 62.0 x 31.0 micra, the greatest width of the cavity being at the level of the base of the inner cell mass. This embryo was lying free in the uterine lumen near the neck of a mucosal pit on the antimesometrial side (Pl. IG).

The morula stage was lying free in the uterus and occupied a position about equi-distant from either side of the wall (Pl. I,F). It was nearly spherical in shape and a segmentation cavity was









18


starting at one end of the embryo. It had a maximum length and width of 62.0 x 54.25 micra.

The two cell embryo was lying eccentrically in the uterine lumen toward the antimesometrial side, No discernible difference could be seen between this embryo and the twenty-four hour old embryos previously described. Each cell measured 31.0 x 31.0 micra.

The twelve to sixteen cell stage embryo was also free uterine. Only one section of this embryo was found in its proper place on the slide although several other sections which had been displaced during staining were seen, hence no detailed description could be made. The greatest width and length measurements taken from this single section were 77.5 x 62.0 micra.


Five Days

Embryos at five days of age were implanting, and the implantation sites were discernible upon gross examination as areas of slight swellings and increased vascularity along the uterine horns. In the fixed uterus, dimensions of one of these swellings were: length 4 mm., width 2.5 mm., depth 3.5 mm. The uterus in the area not directly concerned with implantation was almost uniformly round and had an outside diameter of 2.0 mm. Snell (1941) in his description of implantation in the white mouse, states that there is an appreciable swelling in the uterus at the implantation site one day after implantation (five days after mating).

Of the t hree embryos obtained at this age two were sectioned for detailed study. The embryos were deeply lodged in the uterine









19

mucosa on the antimesometrial side, and the cellular organization of the uterine mucosa In the region of the implanted embryo was changed into cell masses and cords of cells surrounded by sinusoids (Pl. II,A). The mesometrial portion of the pit formed by the embryo was filled with degenerated uterine epithelial cells.

The embryos of five days of age showed a marked increase in size over the previous stage examined. The old germinal cavity had elongated and formed the yolk cavity which was delimited by a thin layer of trophoectoderm (Reichert's membrane) with scattered distal entodermal cells applied to its surface. The inner cell mass had become differentiated into an egg cylinder extending about half way to the floor oiC the yolk cavity. The ectoplacental cone, extra-embryonic ectoderm, and embryonic ectoderm were easily recognizable. A constriction marked the line of junction of the extra-embryonic and embryonic ectoderm. The latter consisted of a single layer of cuboidal cells with large round nuclei surrounding a small cleft, the proamniotic cavity; whereas the former was composed of an irregular mass of cells with elongate nuclei. A single layer of large cuboidal embryonic entodermal cells formed the outer layer of the portion of the egg cylinder that protruded into the yolk cavity. The total length and width of the egg cylinder, excluding the ectoplacental cone, was 108.5 x 46.5 micra. The ectoplacental cone, a conspicuous conical-shaped mass of cells extending toward the lumen of the uterus, measured 46.5 micra in length.








20


Six Days

Three six-day embryos were deeply embedded in the antimesometrial wall of the uterus, the lumen of the latter being reduced to a very narrow alit toward the mesometrial side. The individual embryos did not differ signl ficantly in their development. The egg cylinder which extended almost to the ventral wall of the yolk cavity had increased in size from the condition at five days (Pl. II,B). This was due, apparently, to an increase in the number of cells rather than to the growth of individual cells* The greatest length and width measurements of one of the embryos at this age were 155.0 and 62.0 micra, while the length of the ectoplacental cone was 62.0 micra. The proamniotic cavity was present as a median slit confined to the embryonic ectoderm and parallel to the long axis of the egg cylinder.


Seven Ras

In three embryos obtained at seven days of age the elongated

egg cylinders had completely invaginated into the yolk cavity (Pl. IIC). The proamniotic cavity was continuous dorsally as far as the ectoplacental cone, and was considerably wider than the slit-like cavity of the previous stage. There was an infolding of cells at the junction of the embryonic ectoderm with the extra-embryonic ectoderm which marked the beginning of the posterior amniotic fold. Although a few mesodermal cells were present in this area, they did not yet constitute a definite layer. Dorsal to the posterior amniotic fold, another infolding of cells from one side of the extra-embryonie ectoderm into the proamniotic cavity









21

was apparent. The significance of this invagination could not be determined. Snell (1941) considered a similar structure in the white mouse embryo as being possibly due to very rapid growth occurring in the posterior wall of the egg cylinder at this stage.

The distal entodermal lining of the trophectoderm was uniform ly established and consisted of a single layer of spindle-shaped cells. The embryonic entodem had changed from the cuboidal shape previously noted to a thin layer of flattened and elongated cells. However, the entodermal cells lining the extra-embryonic ectoderm were still cuboidal and closely packed, forming a fairly thick layer. The greatest main axis and width measurements of a representative egg cylinder of this age, excluding the ectoplacental cone, was 294.0 x 93.0 micra.

Decidual cells had extended across the lumen of the uterus and had joined the mesometrial side of the uterine wall in these seven day embryos.


Eight Days

Among the significant advances of embryos obtained at eight

days of age was the presence of a distinct layer of mesoderm that had been proliferated from the region of the primitive streak (Pl. IID,). From their origin at the junction of the embryonic and extra-embryonic ectoderm, the mesodermal cells had spread in all directions. The enlarged posterior amniotic fold had almost made contact with the anterior amniotic fold. The latter consisted of a small projection of cells into the proamniotic cavity from the sone of junction of the extra-embryonic and embryonic eotoderm at the anterior end of the









22

developing embryo. The large cavity in the posterior amniotic fold

was not yet lined with mesoderm.

The fore-gut was apparent as an invagination of the embryonic entoderm on the anterior side of the embryo, at the junction of the embryonic and extra-embryonic ectoderm. The greatest length and width measurements of a representative embryo at eight days of age were 620.0 x 201.5 micra. The ectoplacental cone measured 155.0 micra.

Red blood corpuscles completely filled the spaces between the cell masses of the uterine mucosa in the region of the ectoplacental cone, and also surrounded Reichert's membrane. i addition, in some sections erythrocytes were observed between Reichert's membrane and the embryo proper and even in the proamniotic cavity.

The uterine lumen was completely obliterated at this stage of development.


Nine Days

There was marked variation in development among four embryos of the same litter after nine days of gestation. In the embryo showing the earliest stage of development, the amnion had formed thereby delimiting the amniotic cavity. The exocoelom was in the process of being formed, as four large cavities appeared in the mesoderm of the posterior and lateral amniotic folds. In sagittal section a total of six distinct cavities could be seen in place of the old proamniotic cavity (Pl. III,A)-. The most ventral of these constituted the newly formed amniotic cavity, the most dorsal the ectoplacental cavity, and the four smaller cavities marked the developing exocoelom. The









23

head process was a broad layer of cells located at the ventral extremity of the egg cylinder. It appeared to be continuous with the mesoderm layer from the primitive streak and was directed toward the anterior end of the developing embryo. The most anterior extension of the head process was reduced to a very thin layer that appeared to be continuous with the flattened embryonic entoderm. The total' length and width measurements were 775.0 x 310.0 micra respectively and the length of the ectoplacental cone was 341.0 micra. The allantois had not begun to develop in this embryo.

A second embryo of this same litter was somewhat further developed. Rather than the six cavities seen in the preceding embryo, this embryo had only three cavities: the amniotic, the ectoplacental and the exocoelom (which was fully formed). The ectoplacental cavity was much smaller than the other two cavities. The amnion consisted of a ventral layer of embryonic ectoderm and a dorsal layer of mesoderm while the chorion consisted of a ventral layer of mesoderm and a dorsal layer of extra-embryonic ectoderm. The allantois was present as a small projection of mesodermal cells from the posterior end of the embryo into the cavity of the exocoelm (Pl. II,F). The mesodermal layer limiting the allantoic projection was continuous with the mesodermal layer of the amnion. The anterior part of the wmbryonic ectoderm adjacent to the invaginating entoderm of the fore-gut was elevated as the head fold. The notochord had differentiated fram the head process and was located on the ventral side of the egg cylinder. On the ventral side of the notochord there was visible a slight









24

U-shaped depression similar to that which Snell (1941) termed the archenteron in the white mouse. The egg cylinder was more elliptical in shape than in previous stages and its broadest part was at the region crossed by the amnion. The greatest length and width of this embryo were 852.5 and 403.0 micra, respectively.

In a third embryo of this litter the allantois extended about midway into the cavity of the exocoelom. The size of the ectoplacental cavity was greatly reduced, forming a small crescent-shaped cavity. The paraxial mesoderm had differentiated as a dense layer, although segmentation into somites had not begun; a slight embryonic coelom had formed in the intermediate mesoderm. Measurements of the greatest length and width were 861.0 x 589.0 micra.

In the most advanced embryo of this litter the head fold was large and its cavity was filled with head mesenchyme cells. Somite formation had begun, and five somites could be counted. The archenteron still persisted as an entodermal invagination on the ventroposterior side of the embryo (Pl. III,B).

The three embryos of this litter which were in the presomite stage and still maintained the egg cylinder form, possessed mattrnal blood within their exocoeloms and amniotic cavities. Since this blood had the same appearance as that surrounding Reichert's membrane and filling sinusoids in the uterus, a close search was made of all sections for a possible source of entry into these cavities. In one embryo a channel was discovered which appeared to have been formed by the mesodermal layer of the chorion pushing through the ectodermal










25

layer of the chorion and extending across the ectoplacental cavity encompassing blood sinusoids in the ectoplacental cone (Pl. IIG). By this means maternal blood was channeled directly into the excooelom. No source of entry for blood into the amniotic cavity was observed.


Tan Day

There were four embryos in the litter taken after ten days of gestation, and they were all fixed in utero. Two were sectioned in utero; one was removed from the uterus and fetal membranes and sectioned, and the other was removed from the uterus and fetal membranes and kept as a reference.


External

At this stage the body is U-shaped as the original axis that was established at the formation of the primitive streak and the head fold is largely maintained. The head process is at the upper right side of the U, and the tail region is at the upper left. The dorsal side of the animal is bent inward making the concavity of the U, and the posterior part of the embryo is buried to the right. The area of the future mid-gut forms the bottom of the U (Pl. IVA).

The total body length of a single fixed specimen was approximately 2.86 mm. This measurement was taken using a binocular dissecting microscope and an ocular micrometer. Due to the shape of the embryo, it was necessary to make several short measurements along the body and to add these together for the total length. No doubt, this









26

method is subject to error, but vith fixed material it was about the only one which could be employed.

The only conspicuous external feature was the fairly large bulbous swelling representing the heart on the ventral side of the animal just beneath the head process.


Internal

A study of sectioned material showed that the mandibular arch was fully formed and possessed the first pair of aortic arches. The stomodeum appeared as a deep groove in sagittal section. The oral plate had ruptured in one of the embryos, while in another embryo of this same litter, it was still intact. Cephalic to the stomodeal opening was the triangular shaped pre-oral gut. The first pharyngeal pouches were forming and in the median venural wall of the pharynx the thyroid plate had begun to evaginate.

The fore-gut continued posteriorly to about the lower level

of the pericardial chamber where it terminated in the anterior intestinal portal. The mid-gut was an open groove thus resembling the condition described by Snell (1941) in the white mouse of eight days and eighteen hours development. At this stage, the length of the hind-gut approached that of the fore-gut. The somatopleure of the coelom was continuous with or closely applied to the amnion, while the somatopleure of the extra-embryonic coelom constituted the wall of the yolksac, The allantoic stalk was a loosely arranged mesodermal projection extending through the extra-embryonic coelom and joining the chorion (Pl. 1iIc).









27

The anterior cardinal veins could be seen in the mesoderm of the head and embryonic blood cells were visible lying in the lumen of these vessels. The first aortic arch was ccmlete.

The ventricle and sino-atrial regions of the heart were distinct and numerous embryonic blood cello were enclosed by the erdocardium of the ventricle. The omphalo-mesenteric veins and the allantoic veins were confluent just posterior to the sino-atrial region of the heart. The paired dorsal aortae extended posteriorly from the first aortic arch, which contained embryonic blood cells in its lumen. Although there were at least eight scmites at this stage the exact number could not be determined. They consisted of uniform masses of cells without any organization into dermatome, myotome, and sclerotome regions and lacked a myocoele.

The notochord was an obvious structure extending from the region of the mid-brain to the caudal end of the embryo. Even though it first differentiated from the head process several days earlier in development, the notochord appeared to be still joined to the dorsal entodermal wall of the gut. This is in line -with the observations of Snell (1941) on the white mouse. He found that: "For a considerable period notochord and gut entoderm remain joined. Eventually, however, the two halves of the gut entoderm grow across the ventral surface of the notochord and unite in the mid-ventral line, leaving the notochord as an axial, rod-like structure between ectoderm and entoderm." In the head region, the neural groove was still open and the optic vesicles had beon differentiated from the diencephalon as large lateral









28

evaginations. Closure of the neural groove began in the region of the fifth somite and continued caudally for approximately 150 micra, beyond which the right and left neural folds were closely approximated.


Eleven Days

As previously noted no embryos of this stage were obtained.


Twelve Days

There were four embryos obtained for this age and one was sectioned in utero, while the others were removed from the uterus and fetal membranes before being studied. Giant cells were quite numerous in the decidua adjacent to the embryo both laterally and ventrally. Such cells were first seen in the uterus with five-day-old embryos. It was not determined whether the giant cells persisted beyond twelve days as embryos were not sectioned in utero beyond this stage. Snell (1941) reports giant cells as a conspicuous feature lying between Reichert's membrane and the decidua in sections of the white mouse embryos of six to fourteen days.


External

At the twelve-day stage the body was tightly coiled with the right side of the head overlying the short tail (Pl. IVB). The mesencephalon protruded anteriorly as a hump on each side of the diencephalon and the optic vesicles were apparent as wide evaginations. An auditory pit was present on each side in the region of the hind brain just dorsal to the hyoid arch. Both anterior and posterior limb buds were present, the anterior being slightly larger than the posterior.









29

The heart as seen from the left side of the animal was a round, bulbous, membranous swelling lying between the cephalic tip of the telencephalon and the anterior limb buds. The tall was well defined although short and still sharply bent ventrally. Laterally, the prominent mandibular and hyoid arches were apparent. The third arch was also present, but

was quite small and was somewhat obscured by the hyoid arch. The man. dibular arch had not yet fused to form the mandible. Nasal pits were present, but the naso-lateral, maxillary, and naso-medial processes had not yet fused.


Internal

Nervous

The brain had attained the five-vesicle stage and in cross section through its anterior portion, the lateral telencephalic vesicles appeared as obvious lateral evaginations. The infundibulum in the floor of the diencephalon was in contact with the tip end of Rathke's pocket. The optic vesicles had constricted on each side of the brain to form the optic stalk. The optic cup had formed and the lens vesicle was in the optic cup, although the former had not yet separated from the superficial ectoderm. Otocysts were well formed at the level of the posterior region of the hind brain and had become completely separated from the overlying ectodem. In this area the seventh and eighth cranial nerve ganglia appeared adjacent to the ventro-lateral side of the newly formed otocyst. The large fifth ganglion was present, as were ganglia nine and ten and the roots of nerves eleven and twelve. Histologically the spinal cord was somewhat differentiated into the inner opendynal,









30

middle mantle, and outer marginal layer of cells. Numerous blood vessels were present in the mantle layer. At the anterolateral margin of the maxillary arch, the superficial ectodern had thickened and evaginated slightly to form the olfactory pit. Digestive

The oral cavity consisted of the stomodeal opening. The

tongue primordia were not recognizable as such. Rathke's pocket appeared as a hollow tube extending from the roof of the oral cavity to the posterior tip of the diencephalon. The first three visceral arches were present and the hyomandibular cleft on the left side appeared to have broken through. The third visceral arch contained the third aortic arch, and in the floor of the pharynx the thyroid diverticulum was present. Immediately distal to the bifurcation of the esophagus and trachea, the trachea evaginated laterally to form two bronchi. The left bronchus at this stage was larger than the right. The esophagus was a short structure resembling a slit-like tube and was roughly 150 micra long. In transverse sections, the stomach appeared as a long slit-like structure running dorso-ventrally in the body. A slight curvature of the stcsach produced a convexity toward the left side of the embryo. Pancreatic diverticula were present at the ventral limits of the stomach. An intestinal loop was present in the body stalk. The liver was quite unorganized at this stage, consisting of scattered cords of cells with numerous blood filled spaces between the cords. The transverse uroreetal fold had divided the cloaca into a dorsal rectum and a ventral urogenital sinus.









31


The anal plate still persisted.


Circulatory

The obvious vessels anterior to the heart were the common cardinals from which the anterior cardinals issued anteriorly and became elaborately branched in the head region. The posterior cardinals were well formed and unbranched, while the dorsal aorta was paired in the trunk region and gave off numerous segmental arteries to the body wall. The first and second aortic arches had degenerated but the third was large and still intact at this stage. The heart was a large structure and consisted of an auricle and a ventricle which were in broad communication with each other. As a result of the formation of trabeculae carneae the ventricle and conus arteriosue had become thick walled in contrast to the thin membranous wall of the auricle. The interventricular septum had started to form on the ventral floor of the ventricle but at this stage it was merely a raised ridge of fibrous tissue. The interatrial septum had also begun to form and was present as a slight conical projection of cells from the dorsal roof of the auricle. The vitelline veins at this stage were being broken up by the liver cells and appeared indistinct but could still be recognized suptying into the sinus venosus. The left umbilical vein had become large although it was still connected to the sinus venosus. Also, the vitelline arteries were still paired channels, and hence had not yet given rise to the anterior mesenteric artery.










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Exeretory

The mesonephroi were not massive at this stage of development. The primordium of the metanephric duct which was solid except for its hollow end paralleled the mesonephric duct for a very short distance. Endocrine

Rathke's pocket was in contact with the infundibulum presaging the formation of the pituitary body and the thyroid primordium was represented by a tubular mass of cells between the paired hyoid arches in the floor of the pharynx. Ienital

Yedial to the mesonephros vas a concentration of deeply staining cells which constituted the beginning of the genital ridges. Muscular and Integumentary

The somites had started to differentiate into regions, but the myocoel was indistinct.


Thirteen Days

By the thirteenth day stage the body was in the form of a C and was tightly coiled (Pl. IV,C). From the region of the posterior limb bud, the caudal part of the trunk and the tail raised up and curled around so that the tail rested against the ventrolateral side of the pharyngeal arches on the right side of the animal. The head, at this stage, constituted at least half of the entire embryo. The mesencephalon was a large dome-shaped prominence while the hindbrain









33


had a transparent membranous roof.. The olfactory pits were more prominent than in the previous stage, The naso-lateral and naso-medial processes were in contact ventrally with the maxllary process, bit were as yet incompletely fused. The eyes were quite evident and were located at the dorsal apex of the groove between the naso-lateral process and the maxillary process. The lens was distinct but there was no sign of eye lids. There was no pinna visible in the region of the auditory pit. On the ventral side of the embryo the heart bulge between the anterior limb buds was very prominent, and the extreme ventral part of the ventricular bulge was resting against the naso-medial process. The large liver protuberance appeared to be continuous caudally with the heart bulge. On the ventro-lateral side of the trunk between the anterior and posterior limb buds was a slight ridge produced by the mesonephros. The anterior limb buds were somewhat fan-shaped and appeared to be slightly longer than the posterior limb bud, whose shape was hemispherical.


Internal

Nervous

The layers of the neural tube were much thicker and the neurocoel had become somewhat smaller than in the previous stage. The marginal layer had the appearance of white matter, and surrounded the cord except on the dorsal side. Numerous mesenchymatous fibers extended through the white matter of the cord and penetrated deeply into the mantle layer, forming blood vessels. In the region of the trunk, sympathetic ganglia were evident. The olfactory pits were much deeper than









34

in the previous stage and had formed large nasal chambers separated from each other by a wide naso-medial process with naso-lateral processes on each side. Jacobsen's organ was present as an evagination and thickening of the ectoderm of the medial side of the nasal chambers. At this stage there was no trace of the first cranial nerve. The eye had advanced considerably since the previous stage as the lens had become separated from the outer ectodermal layer. It was quite spheroidal and possessed a central cavity. The optic cup had a very thick inner sensory layer and a thin outer pigmented layer. The otocyst had increased in size and the endolymphatic duct was a conspicuous structure between the otocyst and the lateral plate of the hind brain. The ectodermal wall of the otocyst was much thicker on the ventral side adjacent to the eighth cranial nerve ganglion than on the dorsal side. The upper portion of the otocyst had begun to constrict, presaging the formation of the upper vestibular pouch and the lower cochlear pouch. Ganglia of the seventh and eighth cranial nerves had separated and the seventh nerve could be traced down into the hyoid arch.


Digestive and Respiratory

The lateral extensions of the third pharyngeal pouches were

thick masses of cells constituting the primordia of parathyroid three, and thymus three. The laryngotracheal groove was present in the floor of the pharynx in the region of the fourth aortic arch. On each side of the groove, a large arytenoid swelling had pushed up toward the roof of the pharynx. The fourth pharyngeal pouch had evaginated laterally and turned ventrally. The terminal ends of the fourth pouch









35

were thick showing the primordia of the parathyroid four, thymus four, and the post-branchial bodies. Where the esophagus and trachea sepa-rated, the lumen of the esophagus was compressed dorso-ventrally while the trachea was compressed laterally. Mesenchyme surrounding the trachea was more densely arranged and stained much darker than adjacent tissue vhich suggested the beginning of cartilage formation in this area. As the esophagus and trachea extended posteriorly, their lumens became so reduced in size that they were barely detectable. The trachea remained the larger of the two tubes and increased in size again before bifurcating into right and left bronchi. The right bronchus remained several times larger than the left until it again divided. Five lung lobes were seen in sections at this stage, four on the right and one on the left side. The right medial lobe while connected with the other lobes of the right side was somewhat distinct and was enclosed in its own pleural sac. The stomach had increased still more in size, and the ventral lobe of the pancreas had become a conspicuous globose mass of tissue just ventral to the stomach. The common bile duct and gall bladder were discernible at this stage. In following the tail sections anteriorly a very small tail gut could be seen. The cloacal mebrane was still present.


Circulatory

The greatest change in the heart was an increase in thickness of the wall of the ventricle. The four chambered condition was obvious in the heart as the interatrial septum had extended deeper into the atrial cavity, and the interventricular septum had become more prominent.









36
The eaMbasre of the heat wr* Aull broadly eamoeted. The largo left mbUiliwal vela hd invaded th Liver prqpw and given rise to the dueta. venoume %hb we eanriwmt with the proximal end of the vitallino veins. In the prwvidm etag, the vit.lU.e veins hAd *ready broken up to foam the patio prtal system. The right mbilclal vain was fairly wi&a1 amn lay immaswpicnouely in ti. lateral body wafl. The peroistent distal vads of the viteflln veins drrined blood from the region of the yolk am and intostimne Into the liver. Just ventral to the vitelline veins was the superior nmenterIe artery vhieh In the previous step was the paired vitelline artery. In the region or the anterior limb bude, the subalavian artery extended from the dorwal &*ra into each lib bud. The mablevian veins were draind by the posterior ardinal veins. The eternal ilic arteries arose from large abilloal eateries an each side and extended into the posterior liab buds. The iliae veins of the posterior limb buds were now formed and were drained by the posterior cardinal. Anteriorly the vrtbral artery was located Jost ventral and closely applied to the nerral tbe. The ifterrAl esrotd arteries lay jest dorel to the Pharin and the esteawl oaraWU arteries were preesmt In the Uiemoo of the firet visceral arab. The velms of te head reiton emeAfted of the large anteree oardinals, mbich were the est onspWevome vessels sees in sectiass, and the external jugular veifs which were forais is the sandibular arch region. The third, fouwth, and elAh aortie areho were Intaet at Mi etage.









37


Excretory

The mesonephroi were much larger than in the previous stage and numerous collecting tubules were apparent in cross sections. A condensation of mesenchyme forming the nephrogenous tissue of the metanephros was visible around the expanded proximal and of the ureter. The combined mesonephrie and metanephric ducts led into the urogenital sinus near its junction with the rectum. Endocrine

The thyroid primordia were located between the paired hyoid arches, and the lateral extensions of the third and fourth pharyngeal pouches were forming parathyroid three and four, and thyms three and four. The infundibulum had fused Aith Rathke's pocket presaging the formation of the pituitary body.


Genital

The cells of the genital ridge had lost their mesenchymal appearance and were large ovoid cells. A great deal of mitotic activity was evidenced in tissue of this area.


Fourteen Days

After fourteen days of gestation the body was still C shaped and torsion was no longer obvious, and the main axis of the embryo was lying in one plane. Due to cervical flexure, the head was at right angles to the trunk, and the anterior tip of the snout rested on the heart prominence (Pl. IN, D). The greatest bend in the trunk was in









38

the region of the posterior limb buds, while between the anterior and posterior limb buds the trunk had straightened slightly from the previous stage. The tail curled to the right and its tip rested on the right side of the face just cephalad to the eye. The limb buds had not changed in appearance from the previous day, except that the posterior limb buds had become constricted so that they too were fanshaped. The mesencephalon still protruded as a hump, and the roof of the medulla of the brain was slightly more opaque than in the previous age. The mid-dorsal line of superficial ectoderm above the spinal cord all the way to the caudal end of the trunk was still quite transparent. The face was forming, although the features could not be identified as mammalian. The naso-lateral process had fused with the medial nasal process, and both had in turn fused with the maxillary process. The mandible had just barely fused in the mid-ventral line. Although the mouth was open, the tongue was not apparent in the oral cavity. The ventral body wall was transparent enough to allow the visceral organs to be seen. At this stage, the umbilical hernia was a slight twist of the intestine at the base of the umbilical stalk. A small genital tubercle was present.


Internal


Nervous

The meninges of the brain appeared as a thin membrane outside of the marginal layer of the brain. The telencephalis vesicles had continued evaginating both laterally and anteriorly, until they










39

extended far craniad of the original roof of the telencephalon, and the lamina terminalis was recessed in a deep groove between the cerebral hemispheres. The cavities of the lateral ventricles were in broad communication 4ith the third ventricle, and the infundibulum had continued its evagination from the floor of the diencephalon and had turned down over the distal tip of Rathke's pocket. The walls of the neural tube had thickened considerably since the last stage described, and had compressed the lumen laterally so that it appeared club-shaped with the narrow end toward the ventral side of the tube. Ventro-laterally the tube was much thicker than elsewhere representing primordia of the ventral horns. The neural tubes at this stage were richly supplied with blood vessels.

Several changes were evident in the eyes. There was a covering of superficial ectoderm over the lens, and between the lens and this covering was a thin band of loose mesenchyme cells. These cells diverged to each side of the lens and formed an aggregation on the floor of the optic cup between it and the lens. The cells that had already reached this position had lost their mesenchymal appearance and were multinucleated. The lens at this stage was somewhat elliptical and its hemispherical cavity was restricted to its upper half. This cavity was bounded on all sides by lens epithelium, except for the floor, which consisted of cells from the inner wall of the lens, among which fiber formation had started. A dorsal and lateral pouch were forming from the semi-circular canal portion of the otocyst. Ventral to this area, the otocyst was bent medially and continued as










40

an elongated cochlear duct which was adjacent to, and parallel with, the first pharyngeal pouch. This pouch had narrowed presaging its new role as the auditory tube, but as yet, its distal end showed no signs of enlarging into a tympanic cavity. The nasal pits had deepened and had broken through into the oral cavity, thus establishing the choanae. The vomero-nasal organ (Jacobson's organ) was now in the form of tubular sacs extending for a distance through the nasal septum. At this extreme distal end they were closed, but proximal to this point a lumen was present. Its ectodermal wall was quite thick. Fibers representing the olfactory nerve extended from the floor of the nasal pit to the brain.


Digestive

The proximal end of Rathke's pocket as it extended upward from the roof of the oral cavity had become greatly reduced in sise and existed as a small solid cord of cells. The tongue was deeply undercut, and was a wide, flat U-shaped structure projecting from the pharynx into the mouth cavity. The epithelial layer bordering the upper. and lower jaws was beginning to thicken forming the labiodental ledge. Salivary glands had started to differentiate on each side of the oral cavity just ventral to the jaw-cheek groove. The thyroglossal duct was evident as an evagination from the floor of the pharynx. The larynx was a solid epithelial plate in the floor of the pharynx. A short connection existed between the trachea and esophagus at their cephalic ends. Where they first separate, the esophagus was larger but further caudad; it was reduced to a minute










41

tube, and the trachea was somewhat larger. Both of these tubes were surrounded by a condensation of mesenchyme. Each lobe of the lung possessed branching bronchi, and cells of this area were still mesenchymatous in appearance. The stomach showed a further increase in size although no folds or rugae had yet formed. The ventral lobe of the pancreas now appeared to be larger than the dorsal lobe but both had grown somewhat since the last stage. The liver had filled the peritoneal cavity to the extent of producing ventro-lateral bulges in the body wall. Its cellular substance had become more condensed so that it more closely resembled the adult liver. The gall bladder was saccate and was closely applied to the ventral side of the liver. There was only a faint trace of the tail gut in the form of a solid mass of entodermal cells just ventral to the caudal artery. It persisted in the tail region for only a few sections.


Circulatory System

In the head the anterior cardinal vein had become the internal jugular vein with several tributaries from the brain region and the short external jugular was present. Paired internal carotid arteries were evident, also the basilar artery was seen ventral to the vmrelencephalon. More posteriorly, the basilar artery divided into two vertebral arteries. The hyaloid artery was complete to the eye. The third aortie arches were partially broken down and the remaining portion was now the proximal end of the internal carotid artery. Leading frim the ventricle of the heart mas a thick walled tube, the bulbous cordis, which divided into the ascending aortic trunk on the 3eft side, and









42

the pulmonary trunk on the right. The sixth aortic arches were confluent with the pulmonary trunk and the ductus arteriosus was present in the left side. Posterior to the aortic arches, the dorsal aorta remained paired for a short distance, then the two descending aortae fused, The coeliac artery branched off from the aorta and was seen in the omentum between the stomach and the pancreas. In the heart, the endocardial cushion of the atrioventricular canal had developed to the extent that each atrium emptied largely into the ventricle of the same side. However, since the interventricular septum had not yet fused to the endocardial cushion the ventricles were still in comunication with each other through a small passage just ventral to the cushion. The right and left atria, although separated by the septum primum, were also still in communication with each other through the interatrial foramen. The sinus venosus emptied into the rigt atrium and the left common cardinal had swung over across the mid-line and entered the sinus venosus. Caudally the sinus venosus received the old right vitelline vein which had become the inferior vena cava. The ductus venosus had become a large vessel in the liver. It received the large left umbilical vein from the body wall and the portal vein from the liver and emptied into the sinus venosus. The much smaller right umbilical vein received tributaries from the lateral body wall. In sections passing through the mesonephros, the posterior cardinals were seen, but the left posterior cardinal in that area was sometimes small and indistinct. The suboardinals also were present at this stage.









43


Exretory

The mesonephric duct was confluent. with the metanephric duct for a short distance from the urogenital sinus, and they had a common junction with the urogenital sinus. An aggregation of mesenchyme cells which was lying posterior and somewhat dorsal to the mesonephros and surrounding the enlarged terminal end of the ureter represented the developing metanephric kidney. In cross section through this region several tubules could be observed extending out from the ureter into the developing metanephros.


Endocrtne

The thymus and parathyroid complex from the third pouch was still in contact with the pharynx but the thyme had become greatly elongated and its terminal ends were growing medially. The rapidly developing thymus had grown further caudad than the thyroid gland and had passed laterally on each side of the latter. The two thick walled post branchial bodies each of which had a fairly large lumen had separated from the pharynx and moved caudally down the neok. The suprarenal glands were lying dorsal to the genital ridge and medial to the mesonephros. They were simply a mass of glandular cells with no definite shape.


Genital

The genital ridge at this stage was a uniform cellular mass

forming a longitudinal strip paralleling the medial side of the mesonephros. The Mullerian duct was not differentiated. The genital









44

tubercle was prominent enough to appear in sections as a separate structure for several (10 micra) sections between the ventral body wall and the tail.


Skeletal

Although there was no differentiation of skeletal material at this stage, heavily staining concentrations of mesenchyme occurred in the region of the otic capsules, centra, neural arches, girdles, and limb buds, There was also a concentration of mesenchyme around each nasal pit foreshadoving the formation of a paranasal cartilage and of the nasal septum.


Muscular and Int.gumentary

While no muscle fibers had formed the eye muscles were outlined for part of their course as aggregations of mesenchyme cells. On the ventro-lateral side of the trunk, small conical knobs of epidermal cells were present. These were the primordia of the mamae but they were not macroscopically visible at this stage.


Fifteen DaRM


External

By fifteen days the head was bent forward at right angles to the trunk and the mesencephalon still protruded anteriorly, but the back was fairly straight (Pl. IV,E). The lower jaw rested against the dorsal side of the heart prominence. A short snout had developed and it was elevated free fram the underlying organs. The tip of the tail









45

rested against the snout on the right side of the face. The external nares approached their adult form, and with the development of the snout, the mammalian nature of the embryo could be recognized. Vibrissae follicles occurred on each side of the snout and a single large follicle was present between the eye and ear on each side of the body. The mouth had closed somewhat and the tongue was barely visible some distance back in the mouth cavity. In the region of the fourth ventricle, the membranous roof was slightly more opaque although the floor of the fourth ventricle could still be seen and the transparency of the skin over the neural tube continued from the fourth ventricle to the tail. Sagittal and coronal sutures were obvious beneath the skin of the head. The ear pinna was a short, triangular projection from each side of the head. On the ventral side, the mammal were still not discernible macroscopically, but the umbilical hernia had enlarged. The anterior limb buds were directed somewhat caudad, while the hind liPb buds were directed somewhat cephalad. Slight radial ridges in the distal end of the anterior limb buds marked the formation of digits.


Internal


Nervous

The walls of the brain were thicker and more highly vascularised than in the previous stage. The epiphysis was a hollow stalk and its cavity was continuous with the diocoel. Intensive thickening of the walls had greatly reduced the lumen of the mesencephalon and









46

resulted in the formation of the corpora quadrigemina. In the metencephalon the central ialls were quite thick and the lumen was large. The wyeleneephalon had a membranous roof and an even larger lumen. In the anterior region of the myelencephalon the wall had developed a sulcus limitans which divided the lateral plate into the dorsal alar plate and the ventral basal plate. However, caudad to this area, the lateral plates diverged to the extent that the right and left plates were lying almost in the same plane and the tela ohoroidea covered not only the dorsal part of the tube but the dorso-lateral part as well. The choroid plexus had developed in the roof of the myelencephalon and its leaf-like folds pushed down into the fourth ventricle. The tela choroidea of the roof plate had invaginated into the lateral ventricles giving rise to the choroid plexus of this area. From the ventro-lateral wall of each hemisphere, the corpus striatum had developed as a lobular fold bulging into the ventricles. It was quite massive in size but did not show any special cellular differentiation. The foramina of Monroe were reduced to small passages. The thalamus showed considerable increase in size over the previous stage. Olfactory lobes were present on the ventral side of the cerebral hemispheres and received nerve fibers from the olfactory pits. The walls of the neural tube had thickened, and in so doing had constricted the neurocool into a club-shaped cavity which was restricted to the dorsal half of the tube. There was a slight indentation of the floor plate presaging the formation of the ventral median fissure. Ectodermal folds, the forerunners of the lids, were evident









47

on each side of the eye. The most noticable change since the previous stage was the complete obliteration of the lens vesicle by fibers, which were still nucleated. Also, the pigmented layer of the retina had become infiltrated with a black granular pigment. The nervous layer, pars optica, was thicker than in the previous stage. Its inner fibrous layer adjacent to the optic cup cavity had become more dense, and from this layer, fibers constituting the optic nerve extended into the optic stalk and could be traced to the brain. The pars coeca and ora serrata were as yet not recognizable.

The external ear was a large depression in the region of the

old first branchial arch. In cross section, the floor of the external auditory meatus consisted of ridges which were the developing hillocks. The mesodermal band destined to form the tympanic membrane between the invaginating ectoderm and the evaginating entoderm of the first and second pouch was still quite thick. The auditory tube had formed from the narrowed first pharyngeal pouch. At its distal end it appeared to be joined by a tubular extension of the second pouch to produce a cavity which was the beginning of the tympanic cavity. Adjacent to this cavity primordia of the auditory ossicles could be seen as patches of pre-cartilage cells. The inner ear at this stage seemed to have approached its definitive structure. The distal end of the endolymphatic duct had become saccate; the semi-circular canals were formed, and the cochlear canal had camtenced to coil. The nerve fibers of the olfactory nerve which were present in the previous stage were now more dense and had become organized into a definite nerve tract. The medial









48

wall of the vomero-nasal organ was about twice as thick as the opposing wall. Since the tongue had not yet retracted from between the palatine processes, the nasal septum had not fused posterior to the palatine process.


Digestive

The tongue extended well forward between the palatine processes and was almost in contact with the posterior end of the nasal septum. It contained blood vessels, muscle fibers, and nerve fibers. Both the submaxillary and sublingual glands were prominent ard the parotid glands which developed at the angle of the southh were also present. The labio-dental ledge had developed in the oral epithelium of both the upper and lower jaws* The larynx was now an open structure ventral to the pharynx with the space between the two occupied by large arytenoid swellings. The esophagus and trachea were further separated from each other and the concentration of mesenchyme around the trachea had become more dense. No thyroglossal duct was present at this stage. The esophagus was very small in diameter, and in its more distal region the lumen was hardly discernible. The liver had become more massive and was filled throughout with branching sinusoids. The gall bladder was bulbous, and was now superficially embedded in the liver, occupying the same relative position as in the adult. In the intestine the mascularis layer was defined and numerous folds were present in the lining of the duodenum but were hardly discernible in the intestine below this area. The spleen was not detectable at this stage. The dorsal lobe of the pancreas had grown considerably










49

and now appeared as large as the ventral lobe, although due to the irregular shapes an exact comparison was impossible. The anal orifice was present, but was partly occluded by epithelium. Circulation

The auricles of the heart were quite large, but appeared collapsed in the sectioned material. Both atrioventricular canals were greatly restricted and valves were forming in this area. Separating the two atria was the well developed septum primum, but the atria were still in communication through the interatrial foremen. The sinus venosus was greatly reduced in size and its opening into the right atrium was guarded by a well developed valvolae venosae. Fusion of the interventricular septum with the endocardial cushion still had not been accomplished, leaving the two ventricles in communication with each other. The ventricles appeared to be relatively smaller than in the previous stages, the walls having become thicker and the chambers less extensive. There was no change in the veins returning blood from the head region. The common cardinals from each side emptied into the sinus venosus, and there was no sign of an innominate vein. In the fourth aortic arches, the left side was larger than the right which was indicative of their different fates, since the left persists as the systemic arch while the proximal portion of the right becomes the root of the right subclavian artery. There was also a noticeable difference in the size of the right and left sides of the sixth aortic arch. The larger left arch retained its connection with the dorsal aorta by means of the well-developed ductus arteriosus, while the










50

smaller right arch had lost contact with the dorsal aortA. Caudad to the heart coursed the small posterior cardinals along with the supra cardinals. The left supra cardinal was located close to the dorsal aorta and throughout the t runk region the right and left supra cardinals were joined by transverse connecting veins. Excretory

The genital tubercle had continued to increase in length, and appeared for a number of sections between the tail and ventral body wall. The right and left ureters still emptied into the urogenital sinus along with the mesonephric ducts. The mullerian duct appeared at this stage for the first time as a short solid rod lying lateral and parallel to the nesonephric duct for about one fourth of

the length of the latter.


Fadocrine

Differentiation of the pituitary body was nearly complete.

The neural lobe was still tubular, and the anterior wall of Rathke's

pocket had thickened producing a glandular anterior lobe. Issuing anteriorly from the anterior lobe toward the diencephalon was the glandular pars tuberalis, while the pars intermedia existed as an evagintion from the anterior wall of the anterior lobe. Below the pituitary body the old Rathke's pouch persisted as a small solid cord of cells traceable to the roof of the oral cavity.

The thyroid gland had changed in shape from the triangular lobe seer. in the previous stage to a more flattened trough-shaped









51

structure. Each side of the thyroid extended dorsally to the lateral side of the trachea where it fused with a hollow post-branchial body. The thymus bodies were ventro-lateral to the thyroids. They had become much larger, and more lymphoidal in appearance than the thyroids but the two lobes were still broadly separated in the midline. Skeletal

In the cranial region, the nasal septum and Meckel's cartilage were being laid down as dense concentrations of pre-cartilage mesenchyme. Further caudad the centra, neural arches, and ribs were also easily recognizable, and in fact the portion of the centrum around the notochord and dorsal part of the rib appeared somewhat cartilaginous. The bones of the girdles and appendages were likewise represented by concentrations of mesenchyme, with some areas having a cartilaginous appearance.


Muscular and Integumentary

Concentrations of muscle primordia were outlined in the appendages as well as in the region of the intercostal muscles. The eye muscles were also delineated but had not become fibrous, and the muscle layers of the esophagus and intestine were outlined. The mannas were increasing in size.


Sixteen Days

External

By sixteen days the back was almost straight and the abdomen









52

appeared more distended (Pl. VA). The mesencephalon was still visible as a slight hump and a short neck region was more obvious. The eyes and ears were still open, and the mouth was slightly open with the tip of the tongue extending almost to the lips. Vibrissae follicles were more conspicuous on each side of the snout and between the eye and ear than in the previous stage. There were also several follicles located just dorsal to the eye orbit and on the chin, but there were no follicles visible on the body. There was a slight indication of developing mammae. The umbilical hernia had increased in size through more extensive coiling of the gut. The genital tubercle had become quite long, and was covered by the tail as it bent ventrally and was directed anteriorly. The shape of the external nares and general facial features were more adult-like. Digits on the fore and hind limbs were deeply outlined but were still fully webbed and showed dorsal and ventral differences.


Internal


Nervous

The epiphysis was a tubular structure containing a large lumen and situated in the median roof of the diencephalon. In the region of the diencephalon, the neural canal was greatly reduced. The cerebral hemispheres were broadly merged with the diencephalon and fiber tracts passed from the thalamus into the corpus striatum of each hemisphere. In the mesencephalon the corpora quadriganina were still more prominent on the dorso-lateral side while the mesocoele was still further









53

reduced. The walls of the metencepholon were thicker but the cavity was very broad and there was no indication as yet of the cerebellar rudiment. The choroid plexus had proliferated in all directions in the roof of the fourth ventricle. The neurocoel of the spinal cord was compressed laterally producing a slit-like lumen which had a width of about two thirds of the spinal cord. There was a slight depression representing an early stage of the ventral fissure, but at this stage, there was no sign of the dorsal fissure. In the eye, there was a dense mesenchymal sclera on the outside of the pigmented layer and it had not yet begun differentiating into fibers. The pigmented layer of the retina was reduced in width from previous stages. Lacrimal glands and ducts had begun to form. The inner ear showed very little change from the previous stage other than that it was now completely encased in precartilage cells. Digestive

The tongue had further differentiated. Transverse muscle fibers were present in its body; and also, two thick longitudinal bands of muscle extended from the root into the body of the tongue. The submaxillary and sublingual glands had pushed deeper into the gum from their point of origin and had became more lobular. The esophagus was still quite small although a lumen was traceable throughout its length. There was very little change in the lungs other than an increase in size of the lobes and an increase in the number of bronchioles in each lobe. The tissue itself still had a mesenchymatous aspect. The









54

infolding of the duodenal walls had continued but the rest of the gut possessed a smooth lumen. The mesentery suspending the colon had narrowed down so that it resembled a typical mesocolon. The spleen had increased in size and was attached to the mesentery adjacent to the stomach and lateral and parallel to the mesonephros on the left side of the fetus.


Circulatory

The right fourth aortic arch showed further modification toward its adult relationship as it had formed the base of the right subclavian artery. The left subelavian artery had formed from the systemic arch. Valves had developed in the truncus arteriosus. The subclavian veins were connected with the common cardinal veins on each side. No further changes were noted in the circulatory system at this stage.


Excretory

Near the base of the genital tubercle, where it became continuous with the ventral body wall, the urethral groove appeared as a thick ectodermal ridge, which extended for about thirty microns to the urogenital sinus. The latter extended dorsally and branched into two horns which received the mesonephric ducts. The urinary blaQder made its appearance at this stage as the extended end of the urogenital sinus, around which mesenchyme from the ventral body wall condensed forming its walls. From each kidney a ureter extended to join the base of the bladder laterally. This was the first stage in which










55

the mesonephric ducts and ureters emptied separately. Endocrine

The post-branchial bodies had fused with the thyroid gland. The two lobes of the thymus gland lay close together in the midline of the fetus. Both lobes were large and highly vascular, but the left appeared larger than the right. Genital

The mullerian ducts had further differentiated into a tubular structure that could be traced some distance. Caudally they were incomplete and did not extend to the urogenital sinus. Skeletal

This stage marked the first appearance of cartilage in the

cranial region. It was present in all areas of the cranium where cartilage bone develops. Also, the otic capsules were well outlined in cartilage. The areas of dermal bones were represented by concentrations of mesenchyme cells and a small amount of fiber formation by these cells. Centra, neural arches, and the dorsal part of ribs were clearly cartilaginous. The ventral part of the ribs and the sternum were less well defined. The bones of the girdle and the proximal long bones of both forelimbs and hind limbs were preformed in cartilage. The bones in the distal end of the fore and hind limb were represented by mesenchymal concentrations. Meckel's cartilage and the cartilage of the nasal septum were well defined, whereas around the larynx there was









56

a massive concentration of mesenchyme cells. Muscular and Integumentary

The muscles of the eye, tongue, shoulder, upper parts of the appendages, and intercostal musculature were outlined.


Seventeen Davs


External


At seventeen days the crown of the head was smooth and without a mesencephalie.hump (Pl. V,B). The head had become smaller in relation to total body size and was less sharply bent forward so that the chin no longer reached the chest. The digits of the forelimbs were partly webbed, while the digits of the hind limbs were deeply ridged and still fully webbed. Plantar tubercles had made their appearance on the soles of the feet. Hair follicles were present on the body in patches on the dorsal aspect of the fore and hind limbs, and on each dorso-lateral side of the trunk. Follicles were also present on the cheek and around the eyes, but were absent along the mid-dorsal axis of the body, including the top region from the top of the head forward to the nose. The eyes and ears were still fully open although the ear pinna had turned anteriorly. The large distention of the abdomen was reduced but the umbilical hernia remained prominent. The mouth was only partly open with the tip of the tongue extending to the lips.








57


Internal

Nervous

The epiphysis persisted as a tubular organ, and its lumen had become larger than in previous stages. The choroid plexus had formed from the roof of the diencephalon and extended into the cavity of the third ventricle. Cavities had appeared in the corpora quadrigemina. The continued development of the cerebellum had altered the metencephalon, The thickening alar plates in this region of the brain had fused dorsally and were covered with a thin cellular layer. The choroid plexus was relatively better developed in the roof of the fourth ventricle. Fiber tracts between the thalamns and corpus striatum were quite obvious. The neurocoel was roughly elliptical with its greatest diameter extending dorso-ventrally about one third the dorsoventral diameter of the spinal cord. Both dorsal and ventral fissures were present in the spinal cord. The sclerotic coat of the eye had become more fibrous and the choroid coat had further differentiated. The lens eipthelium was separated from the inner layer of the cornea by a space which was continuous posteriorly with the vitreous chamber. The cartilage around the membranous labyrinth was filled with cells which were mesenchymal in appearance. The withdrawal of the tongUW had permitted the palatine processes to fuse mesially and the nasal septum had grown down to the fused palatines and was in the process of fusing with them, thus completely separating the two nasal passages. Jacobson's organ continued as a dominant structure in the nasal septum.










58

In cross section it resembled two kidney shaped tubes with crescentshaped lumens. The epithelium of the convex side of each lumen was several times thicker than on the opposite side.


Digestive

The body of the tongue had become more heavily striated with muscle fibers extending transversely across the tongue. Lingual papillae were discernible although there was as yet no sign of foliate papillae. Incisor tooth buds were also present at this stage. The salivary glands located on each side of the root of the tongue consisted of solid branched lobules which were becoming encapsulated by aggregations of mesenchymal cells. The anal orifice and rectum were occluded with epithelium.


Excretory

The urinary bladder was an extremely large structure with a wide lumen and was still broadly attached to the ventral body wall. The kidney was a massive structure which at this stage presented a more round than kidney-shaped appearance. Numerous tubules and several incompletely formed Bowans capsules were present. The ureters were patent throughout their length. The mesonephric ducts were still intact and emptied into the urinary sinus, which at this stage may be called the urethra.


Endocrine

The thyroid gland had changed considerably in texture. Its tissue consisted of a loosely vacuolated mass in which were forming









59

follicles that appeared to be filled with blood cells. The two thymus bodies were located at the anterior tip of the thoracic cavity and at the external ventral border of the thyroid gland. They were large lymphoid rods which were round in cross section and extended some distance into the thoracic cavity. Genital

The Mullerian ducts had grown down to the urogenital sinus but had not yet joined it; nor had the tubes of the opposite sides yet fused to form the utero-vagina. The genital system was still in an indifferent stage of development. Skeletal

In the cranial region, ossification had begun in the areas of dermal bone formation. Also, numerous blood vessels were present in these areas. The otic capsule, which had previously become cartilaginous, now had mesenchymal cells in the region of the membranous labyrinth. The ear ossicles were faintly outlined in cartilage. Ossification had begun in the mandible. The sternum and the ventral part of the ribs were becoming cartilaginous. In the region of the digits of the fore and hind limbs only dense mesenchymal concentrations were present.


Muscular and Integumentary

All muscles were outlined in their characteristic band or bundle shape, but no definite fibers were present.









60


Eighteen Days


External


In the eighteen-day fetus the head appeared quite similar, to that of a full term fetus (Pl. VC). The pinna of the ear had folded over the opening, but the opening was not tightly sealed. The eye lids had formed and the eyes were half closed. The roof of the hind brain was no longer transparent; however, organs could still be seen through the ventral body wall. The chin was held well above the chest, and the umbilical hernia had almost completely disappeared. Hair follicles were now quite obvious and had extended to the middorsal line, on the dorsal side of the head forward to the nose, and also on the ventral side of the animal, as well as other parts of the body. The tongue had retracted a short distance, and the mouth was almost closed. Both the fore and hind limb digits were conspicuously notched.


Internal


Nervous

The lumen of the epiphysis was still present; the extent of the choroid plexus had increased in the third and fourth ventricles, and the cerebellar rudiment was considerably thicker. In the spinal cord, the neurocoel was a small egg-shaped cavity that was centrally located. The fibrous sclerotic coat of the eye was now clearly evident, and in front of the lens it continued as a roof of the anterior









61

chamber, which formed the inner lining of the cornea. The anterior chamber was fully formed, and the lens was a spheroidal mass of fibers with only a few nuclei, most of which were located in its upper hemisphere. The oval window could be seen in the cartilaginous framework of the inner ear and the preosseus auditory ossicles extended to it. The coils of the cochlea appeared complete. The maxillo-turbinal and several ethmo-turbinals had made their appearance within the nasal cavities.


Digestive

The outer layer of epithelium of the tongue had thickened and now had many taste buds and papillae. Well defined foliate papillae had appeared on each side of the tongue and the longitudinal muscle fibers had become more numerous in the root of the latter. The lumen of the esophagus was greatly compressed dorso-ventrally. In the thoracic cavity the right bronchial tube was larger in diameter than the left. The lobes of the lungs had increased in size and the bronchi were more numerous, although they were smaller in diameter than in earlier stages. There were numerous coils in the intestine and its walls seemed to have approximated definitive form. Mucosal folds were evident in both the small and large intestine but only a few could be seen in the stomach. The mesentery suspending the intestine had become greatly reduced in thickness. The pancreas was quite diffuse and consisted of numerous small lobules connected by cellular cords. In some areas the dorsal and ventral lobes appeared to merge with each other. The gall bladder was superficially connected to the liver and









62

had become more saccate. It closely approached definitive form and had lost the ventral mesentery attachment. The spleen was well developed, occupying the area between the left gonad and the lateral body wall.


Circulatory

Completely enucleated red blood cells appeared at this stage. The atria were still in communication by way of an opening in the septuw primum, the interatrial foramen and the septum secondum had just begun forming as a slight projection of cells from the dorsal walls of the atria. The cavities of the ventricles were reduced in size due to the increase in thickness of the muscular walls. They were now completely separated from each other by the fusion of the interventricular septum to the endocardial cushion. The mitral and tricuspid valves had formed.


Excretory

The bladder was a large round mass with a slit-like lumen and cells of the bladder wall had become differentiated into epithelium. The kidney had acquired its characteristic shape, and had a well-defined darker staining cortex and a lighter staining medullary portion. Definitive tubules were abundant and Bouran' capsules with simple squamous epithelial linings and well formed glomeruli were present. The mesonephros was greatly reduced in size from previous stages. Genital

In the material on hand, sex could not be determined at this









63

stage. The gonad was made up of a mass of round cells which showed many mitotic figures, but development had not progressed far enough to identify this structure as either ovary or testis. The mullerian ducts were still in the indifferent stage but had now fused in the midline distal to the mesonephros forming the utero-vagina. Beyond this point they joined the urethra but they separated again before entering the urethra.


Skeletal

There was an increase in ossification areas in the dermal bones of the skull at this stage, and ossification had started around Meckel's cartilage of the mandible. Tooth germs for both molars and incisors were present but the incisors were farther along in development. The dorsal part of the neural arches and neural spines had still not formed. Ossification had begun in the proximal long bones of the fore and hind limbs, while bones of the phalanges were becoming cartilaginous. Muscular and Integumentary

The muscles of the eye were in the form of fibrous strands, and those of the tongue were also fibrous. Muscles of the heart resembled definitive cardiac muscle. However, in none of the skeletal muscles were transverse striations evident at this stage.













PRENATAL GROWTH


Measurements of Uterine SwellinZs at Implantation Sites


Implantation sites were seen as obvious swellings in the

uterine tubes of pregnant females five days after sperm were found in their vaginal smears. These swellings showed a daily increase in size with the growth of the embryo. Measurements made on fixed material from five to eighteen days development are presented in Table 2.


TABLE 2

MEAN MEASUREMENTS IN MILLIMETERS OF UTERINE SWELLINGS

Age Number of
In Days Swellings Length Width Depth

5 1 3.0 2.5 3.5
6 1 3.5 3.0 4.0
7 3 3.5 3.2 3.5
10 1 5.5 5.5 6.5
12 2 5.5 6.0 7.0
13 3 7.6 7.5 9.0
14 2 8.5 7.5 9.5
15 2 9.2 8.2 10.0
16 2 10.0 9.0 10.0
17 2 12.5 9.0 11.0
18 3 14.5 11.0 12.0

Material representing th4 eighth and ninth days was sectioned before measurements were taken, and there were no embryos obtained representing the eleven-day stage of development. Hence, no conclusion as to growth rate could be drawn from eight through eleven days of


64









65

development. Actually, the implantation sites showed very little difference in size for the first several days, but from twelve through eighteen days of age a daily size increment was quite pronounced. There was only a slight variability in the measurements of swellings in the same uterus.

Since pregnant females are often collected in the field some knowledge of the relative size of uterine swellings for each day of gestation is advantageous in determining the approximate stage of pregnancy. On the basis of the present data, it appears that the age of embryos can be determined from the size of the uterine swellings within a range of several days.


Weights and Volumes of Embryos

After they had been removed from the uterus and stripped of

their fetal membranes, fixed embryos from twelve through eighteen days of age were weighed and their volumes determined. These data are presented in Table 3. Due to their small size embryos younger than twelve days could not be dealt idth by this method.

TABLE 3
MEAN WEIGHTS AND VOLUMES OF EMBRYOS 12-18 DAYS OF AGE

Age of Litter Volume in eights in
Embryo Size Millimeters Grams
12 2 .0100 .0057
13 4 .0232 .0281
14 3 .0453 .0458
15 3 .0800 .0801
16 3 .1390 .1335
17 3 .2276 .2115
18 4 .4092 .3851









66

Weight is one of the most valuable measurements for determining growth, and volume is of value in giving an approximation of size. There was a very close parallel between weights and volumes in each group of embryos measured. A continuous inertass in weight and volume was seen from twelve through eighteen days of age. Relative growth was more rapid after fifteen days of age, the greatest increment occurring from seventeen to eighteen days of development. Since the mean birth weight of this species was found to be 1.61 grams it appears that the greatest increase in weight must take place between eighteen days and parturition, which occurs normally at twenty-three or twenty-four days. Such a rapid increase in growth toward the termination of the gestation period is typical of most mammalian prenatal developmental curves.


Measurements of Certain Body Parts of Embryos from
Twelve Through Eighteen Days of Development

Various linear measurements made on embryos from twelve through eighteen days of development with a pair of fine (bolt-controlled) dividers under 5x magnification included: crown-rump length, head breadth, head length, shoulder breadth, length of foreleg, length of hindleg, and tail length. The averages are given in Table 4, and are represented graphically in Figure 1.

These data indicate that the crown-rump length, head breadth,

head length, shoulder breadth and tail measuremts exhibit approximately the same relative growth trend. In each ease from thirteen through seventeen days of age, growth proceeded at a fairly constant rate.









67

The interval between seventeen and eighteen days of age showed a marked increase in the relative growth rate in crown-rump length, head length, and shoulder breadth; but the increase in head breadth and tail length was less pronounced. There was a marked increase in the cromn-rump length between twelve and thirteen days of age which slightly exceeded the increase seen in this same measurement from seventeen to eighteen days of development. Since the method of age determination used was necessarily only an approximation it is possible that a greater age difference existed between embryos of twelve and thirteen days than between embryos of seventeen and eighteen days of age.


TABLE 4

AVERAGE MEASUREMENTS OF BODY PARTS OF EIBRYOS IN MILLIMETERS Age Number Length Length
in in Crown Head Head Shoulder of of
Days Litter Rump Breadth Length Breadth Foreleg Hindleg Tail
12 2 3.7 * * * * * *
13 4 6.4 1.1 3.5 2.6 1.1 1.0 2.1
14 3 7.2 2.5 4.4 3.7 1.5 1.5 2.8
15 3 9.0 3.1 5.1 4.3 1.8 1.7 3.2
16 3 10.1 3.6 6.0 4.7 2.2 2.1 3.7
17 3 11.3 4.2 6.7 5.2 3.2 3.2 4.0
18 4 13.4 5.1 8.2 7.3 4.5 4.5 4.6

*Too small and undifferentiated to measure at this stage.

The mean lengths of the fore and hind legs were similar. A

fairly uniform growth rate was noticed in both from thirteen to sixteen days of age, however the rate of growth was three times as great for each twenty-four period from sixteen to seventeen days and seventeen to eighteen days of development. The appendages were too small and undifferentiated to measure before thirteen days of age.













PARTURITION

Details of the birth act are known for relatively few wild

mammals. In the genus Peroiyscus Svihla (1932) has described parturition in P. m. artemisiae and Pournefle (1952) described birth of young in 2. gossyylnus. It was possible to make careful observations of the birth of a litter in P. polionotus. A gravid female was isolated around the eighteunth day of gestation and placed in a large gallon jar fitted with a screen cover. On January 1, 1956, at 11 A. M., she was lying on her back with her eyes closed. There were frequent movements of her abdominal wall obviously as a result of the movements of the young in the uterus. Within several minutes she got up on her feet and stretched her body by keeping her back feet in place and pulling forward with her forefeet. At this time, the sides of the abdomen were depressed toward the midorsal line. She then assumed a sitting position and started licking her vulva and scratching it with her forepaws. At U1:10 A. M. the tail and hind feet of the first fetus appeared# She tugged at it with her forefeet and pulled the young mouse out. It took only a minute for this entire process to take place.

The mother placed the young mouse, still connected to her by the very short whitish umbilical cord on the floor of the jar, and maintaining her crouched position began washing her forepaws and face. Several minutes later the placenta appeared. She dislodged it from her vulva with her forepaws, carried it directly to her mouth and began 68









69

eating it. When she had finished she again licked her forepaws and vulva and picked up the young mouse and licked it thoroughly, turning it in various directions with her forepaws as she did so. Upon finishing with the new born she again placed it on the floor of the jar in front of her and seawed to neglect it entirely until after the birth of the next of the litter.

At about two minute intervals the mother got up, faced away

from the new born mouse and stretched in the manner that was previously described. After stretching several times she again resumed a crouching position and began licking the vulva and mashing its sides with her forepaws. Soon another tail and hind leg appeared. This time she simply grasped the young with her forepaws and pulled it free. The placenta followed immediately. She ate the placenta and paid no attention to the new born animl. The second young was born at 11:23 A. M., thirteen minutes after the first. After consuming the second placenta she again washed her forepaws, her face, and the region around the vulva and then returned to the first born and licked it thoroughly. She paid no attention to the second young for about ten minutes, after which she picked it up with her forepaws, cleaned it and placed it with its sibling. She lay down close to them; covered them with her outstretched neck, and remained generally in this position for about thirty minutes. However, she frequently got up, walked away from the two young mice and stretched herself in the manner described previously.

At 11:55 the female again assumed the crouching position, and started licking her vulva and pressing it with her forepaws. Soon the









70

head of another mouse appeared. This time she grasped each side of the head with her forepaws and pulled it out. She also allowed this young one to lie on the floor of the jar beneath her with the cord still intact., She washed her forepaws and licked herself constantly for about five minutes before the placenta appeared. Again she used her forepaws to dislodge it and ate it, after iich she washed her forepaws. Then picking up the last born mouse in her forepaws she turned it in various positions and licked it thoroughly before placing it with the other two mice. Finally the three new born mice were placed under her and she crouched over them and remained still for about one hour. Frequently she extended her head forward and bending it beneath her body licked the three young indiscriminately. Observations were continued for an hour after the birth of the last mouse and the young could be seen lying beneath the mother. They were on their backs with their ventral sides uppermost and they moved their heads back and forth rubbing the ventral side of the mother with their noses as if seeking out the teats. It could not be determined when nursing first started.

The events of parturition in P. polionotus seem to be very

similar to those previously described for other species in the genus. Whether the female out the cord with her teeth or severed it by stretching could not be definitely ascertained. Svihla (1932) describing parturition in P. m. artemisiae, stated that the cord was always broken by stretching. Pournelle (1952) noticed a predominance of morning births in P. gossypinus, for out of twenty-six litters which he recorded seventeen were born between 6:00 A. M. and 12:00 noon. In this study nine









71

out of fourteen litters were born between the hours of 8:00 A. M. and 12:00 noon. However, observations were not made on all of the litters but only on those produced by a group of fourteen females which had been bred at about the same time and produced litters within several days of each other.












LITTER SIZE AND SEX RATIO


Litter Sie

During the period of this study fifty-two litters totalling 174 young, were born in the laboratory. The litter size ranged from one to six with a mode of three mice per litter. The mean litter size was 3.35 mice per litter. These data are summarized in Table 5.


TABLE 5

RELATIVE FREQUENCIES OF DIFFERENT LITTER SIZES IN P. polionotus Size of Litter Frequency

1 1
2 7
3 26
4 11
5 5
6 2
Total 52
Avg. litter size 3.35 Litter size based upon a study of twenty-seven litters totalling ninety-six embryos ranging from the pronuclear stage to approximately twenty days of gestation showed a range of two to six embryos per litter with a mods of three and an average number of 3.55.


Sex Ratios

Sexes of laboratory raised mice were recorded for 128 mice

representing thirty-nine litters and did not depart significantly from .a 1:1 a. Of this number, sixty-five (50.78 per cent) were males 72










73
ad sixty-4hre. (49.22 per ent) were females.












GESTATION


Gestation Length in Non-Lactating FEaales

In this study four records of the length of gestation in nonlactating females were obtained. The beginning of gestation was placed at the time mobile sperm were found in the vaginal smear of the female. Three females produced litters twenty-four days after sperm were recorded in the vagina while gestation in the fourth lasted twenty-three days


Influence of Laotation on Gestation Close observations were made on six females that became pregnant as a result of mating during post-partum estrus, and a record of gestation length was determined for each. There was a range in gestation length from twenty-five to thirty-one days for these individuals with an average of twenty-eight days, which seems to indicate an increase of approximately four days in the gestation period for lae-, tating females. There was no apparent correlation between lengthening of gestation and the number of suckling yomug, although the sample was small. For example, three females with litters of one, three, and four young respeetively all had a twenty-eight day gestation period for the second litter. On the other hand three other females, one with a litter of four and two with litters of three had gestation periods of thirty, twenty-five and thirty-nse days respectively for their second litters.


74










75

Post-Partum Estrus and the Normal Estrous Cycle

In an effort to obtain more accurate data on the time between parturition and post-partum estrus, twelve females in the latter stages of pregnancy were placed in separate cages and checked at frequent intervals until parturition was actually witnessed or until new born yoamg were discovered. As soon as parturition was known to have occurred, a male was placed in each of ten cages and left for twentyfour hours with the feuale. In the remaining two cages, the male was not introduced until twenty-four hours after parturition but was left for forty-eight hours. In every cage the female attacked the male. Svihla (1932) made a similar observation on other species of PEr cus and suggested that the female considered the male an interloper and fought him to protect her young. Seven females out of the ten that were paired within two hours after parturition subsequently produced litters. In the two cases where males were not introduced into the cages with the females until twenty-four hours after parturition, even though the mice remained paired for a period of forty-eight hours neither of the females became pregnant. Although the data do not conclusively prove that heat does not extend more than twenty-four hours beyond parturition, they suggest that post-partum heat generally occurs in this species within twenty-four hours. Asdell (1946) has pointed out that a post-parturition heat does not occur in all species of the genus Peromyscus.

A total of 102 matings were recorded using the procedure previously described for obtaining embryos of known ages. Of this total,









76

twenty-one proved fertile and eighty-one were infertile. Some of the females would apparently accept the male at every heat period for several weeks without booming pregnant. This was determined by placing one male in a cage with several females that had just mated. Vaginal smears were made starting on the fourth day after the first mating and in some instances females had sperm in their vaginal fluid on the fifth day after the first mating. In other cases, sperm were found in the vaginal smear on the seventh day after the first mating. In still other eases no sperm were found and the smear did not indicate an estrous condition. These limited data suggest that the average duration of the estrous cycle in this species may be about five days. They also illustrate the fact that at least under laboratory conditions many of the matings in this species are infertile.













POSTNATAL GROWTH AND DEVELOPMENT


Weights at Birth


Fifty-seven mice representing sixteen litters were weighed

within four hours after birth. Weights ranged from 1.1 to 2.2 grams,, with a mean of 1.61 grams. The lowest mean birth weight was found in a litter of five individuals and the highest mean birth weight in a litter of only two individuals. However, the evidence is not conclusive that there is any correlation between increase in litter size and decrease in mean birth weight. The single individual in one litter had a birth weight of 1.7 grams, and in another litter containing two individuals, a mean birth weight of 2.1 grams was obtained. Six litters had three mice per litter with a mean birth weight of 1.7 grams. In eight other cases including four litters of four, and four litters of five individuals the mean birth weight was 1.5 grams.


Postnatal Growth Measurements


A series of postnatal growth measurements was made on fortyfour mice from thirteen litters, and these data are summarized in Table 6 and presented graphically in Figure 2.

The total length and tail length showed a fairly rapid growth rate up to thirty days of age beyond which time increase in tail length was of little magnitude. Relative total growth in length from thirty to sixty days of age was less than that prior to thirty days of age.


77









78


The hind foot and ear exhibited rapid growth up to twenty days of age, but little beyond this point. The hind foot had attained adult proportions by thirty days of age. There was a fairly constant increase in body weight up to thirty days of age but comparatively little gain thereafter.


TABLE 6


AVERAGE DAILY GROWTH MEASUREMENTS IN
IN GRAMS OF P. polionotus FROM


MILLIMETERS AND WEIGHTS 2-60 DAYS OF AGE


Age in Total Tail Hind Foot Ear Body
Days Length Length Length Length Weight


At Birth
2 3
4 5 6 7
8 9 10
11 12 13
14 16
18 20 22 24 26 28 30
34 36
40 44 48 50 56
60


41.6 48.0 49.8 55.1 58.0
60.4 67.2
68.8
76.4 71.5 83.3
80.8
89.4 84.7 98.8 99.9 110.3
106.6 115.2 109.9
122.0 121.5 126.5
125.4 128.7 131.2 131.0 128.7 130 5


10.8
13.4 13.0 16.3 16.5
18.5
21.1 21.8 25.0
23.3 29.2 27.5 32.6
25.1 36.3 37.5
43.3
40.8 44.7 42.3 50.5
48.3 52.7 50.0
52.2 53.7 53.0 51.3
52.2


6.8 7.7 8.0
9.0 9.7 10.8
12.2 12.3 13.6 13.0
14.6 14.6 15.7 15.3 16.6
16.4 17.7 17.7 18.8
17.4 18.3
18.2 18.6
18.4 18.5 19.0 18.9 18.0 18.6


1.7
2.0 2.3 3.0 3.0 3.5
4.0 4.0 4.0.
*5.0 5.0 5.0
7.0 9.0 9.2

10.0


10.0


12.0

12.0


1.6
2.2 2.2 2.2 2.9 3.0 3.2
4.0 3.9
4.5
4.2 4.9 4.9 5.2 5.2
6.5 6.5
8.1 8.0 9.3 8.9
10.4 11.5 U.8 12.6 12.6 13.2 13.9

14.7{


*From the 12th day on, ear length measurements were made On fixed material.










79
Development

At birth the eyes were closed and the ear pinnas were folded down over the external auditory meatus (Pl. VI). The over-all body color was pink with the dorsum being .slightly darker. The skin covering of the head region was transparent so that the sagittal and coronal sutures could be seen, On the ventral side, small blood vessels, viscera, and ribs could be seen through the transparent skin. Mystacial vibrissae were present at birth and when depressed against the face extended back to the level of the eye.

By the second and third day, the entire dorsal side was pigmented but the region of the head between the ears appeared a shade lighter than the rest of the dorsum. The venter remained pink ant transparent enough for viscera to be seen. The iris ring, which was visible through the somewhat transparent sealed eyelids at birth as a black band surrounding the lighter lens, was obscured by the end of the third day due to the increasing opacity of the eyelids. Upon close inspection with proper light, or under slight magnification, a sparse covering of fine hair could be observed on the dorsal side by the third day. No hair growth could be detected on the ventral side at this age. In a total of forty-two young, the pinna unfolded on the third day in eighteen, on the fourth day in twenty, and on the fifth day in four (P1. VII,A).

At three days of age mice would squeak when disturbed, but

showed very little coordination. When placed on their feet they would rest on the venter for a few seconds and then in trying to move would









80

topple to one side or the other. When they fell over they could not right themselves.

At five days of age the mice still appeared "naked" at a casual glance. However, when inspected under proper light or low magnification the dorsal hairs appeared more dense than in the previous stage. Hair growth continued down the dorsal ridge of the tail for about one half of its length. The ventral side showed a very sparse growth of tiny white hairs but this growth was not nearly as thick as the hairs on the dorsal side. White hairs could also be seen on the dorsal sides of the legs at this stage. The entire dorsal side was a "graphite gray" color, except for the legs which remained pink. The mice began to exhibit slight coordination; and could crawl. When turned over on one side they could eventually right themselves.

By seven days of age, the hair had grown so as to give the dorsal side a uniformly gray appearance. Even the tail was covered with fine hair to its tip. A long, black supra-orbital vibrissa appeared just dorsal to the eye orbit. The skin of the ventral side was quite pink and rugose and appeared naked to the unaided eye, although a sparse growth of hair was present. Abdominal viscera were outlined against the body wall.

Ordinarily the lower incisors began erupting on the sixth day; however, frequently they did not rupture the tissue of the jaw until the seventh day. The upper incisors appeared by the seventh or eighth day. Mice were more active at seven days of age and were able to run, although their ventral sides still dragged along the floor. When they









81

were introduced head first into a container they would react by turning around and attempting to climb up the side of the container. Upon being placed in a jar containing ether fumes, they would show the adult characteristic of face washing with their fore paws.

At eight to nine days the dorsal pelage was dense enough to

conceal the underlying skin. Along the mid-dorsal line the pelage was slightly darker than on the sides, and the venter was covered with fine white hair although the underlying skin could still be seen easily. Ventrally the body was covered with fine white hairs which were dense enough to give a white appearance to the ventral side of the animal and the skin on the ventral side had become so opaque that only a faint outline of underlying organs could be seen. Although the pinna appeared almost bare to the unaided eye, it was covered with a fine growth of hair that could readily be observed under the binocular microscope. The external auditory meatus was deeply furrowed but was not completely open, as the mice did not react to sound at this age. They could, however, nm without dragging the ventral side on the floor.

At eleven days of age (Pl. VII,B) some of the mice showed reaction to sound by flinching when a sharp noise was made. The coordi nation was good, as they ran when touched and tried to bite while being handled. The age at which the eyes opeped was recorded for fortyone individuals. This occurred at fourteen days of age in twenty-four mice, at thirteen days in fourteen others, and, at fifteen days in three.

At thirteen to fourteen days the mice were very alert and

showed ruch adult reactions as digging, huddling when a noise was made,









82

face washing, and nervous urination. By this stage firm fecal pellets with a bright yellowish color had replaced the watery feces of previous stages.

Around fifteen to seventeen days of age the ventral hair was dense enough so that the mammae on the females did not show conspicuously. The young mice were very alert and at night they often left their nesting jars and ventured around the cage. When a sharp noise was made they would jump into the air and run back into the nesting jar. Cheek teeth had erupted the skin in specimens fixed at fifteen and sixteen days of age, but were not yet visible in a specimen fixed at fourteen days of age. Beyond fifteen to sixteen days mice showed very little change other than a gradual increase in size and in length of pelage. Plates VIII and II show stages in development from twentyfour through forty days.

In most females the vagina opened at twenty-nine to thirtyfour days of age. Actual observations were made on fourteen individuals; in six the vagina was open on the twenty-ninth day. The next observation was not made until the thirty-fourth day when only one of the fourteen still had a closed vagina. In males at thirty-one days of age the testes were slightly outlined in the scrotum.

The post juvenile molt began in scer mice as early as thirtytwo days although the majority did not show signs of molt until thirtyfour to thirty-six days of age (Fl. IX). A small patch of buff-colored hairs just beneath each eye, another on the dorsal side of each hind leg in the region of the distal end of the tibia, and a fine line of










83

similar hairs on each side of the body at the zone of junction of the dorsal and ventral pelage marked the beginning of molting. By thirtyeight to forty days of age, the yellowish color had generally spread dorsally over the shoulders and down the back to the rump where it stopped, leaving a crescent-shaped boundary. The concavity of the crescent was directed posteriorly. The pelage in the area over the rump was gray. Ventrally a pattern was also present, in that the neck, chest, and anterior half of the stomach possessed white hairs similar to adult pelage, while the posterior region of the stomach to the anus was grayish white. At around forty-five to forty-eight days of age, these distinctly different color areas had disappeared; the dorsal pelage showed a uniform yellowish color and the ventral pelage was uniformly white as if molting had been completed. In some mice molting was still in progress at fifty-five days of age.














DISCUSSION


As pointed out earlier, the embryology of some laboratory rodents has been studied in considerable detail but this seems not to be true with respect to wild species, even though they may be widespread and generally abundant,, On the other hand, a number of observations have been made on the postnatal development of many species of wild rodents, including several species within the genus Perowyous. The present study represents an attempt to describe the entire developmental period of a single species of wild rodent from conception through to adulthood.

As was to be expected, the development of Peromrscus polionotus resembled that of the related Mus, Rattus, and Mesocricetus in its broad outlines. However, certain differences in embryonic structure and chronology have been revealed and seem worthy of further consideration. Furthermore, information obtained concerning postnatal development and other aspects of reproductive behavior in P. polionotus now permit a more critical comparison with other members of the genus than has heretofore been possible. The significance of certain of the apparent intra- and inter-generic differences in various aspects of prenatal and postnatal development in the oldfield mouse as compared to other species of the same genus on the one hand and to the house mouse, rat, and hamster on the other are not presently clear. Nor will they be clear until more studies of development are made, not only of other species in the genus Peromyscus but of representatives of other genera 84










85

of close and more distant relationship.

A well-developed corona radiata was observed in eggs estimated to be from one to six hours post copulation but by fifteen to eighteen hours this had degenerated into a small mat of mucoid-like strands. Smith (1939) reported that he was unable to find a definitely formed corona radiata in P. polionotus although he did see coronal cells scattered around the eighteen-hour pronuolear stage. Apparently the corona had already degenerated in the earliest stages which he observed. A zona pellucida was present from the pronulear stage through the eight cell stage. In some instances in the later stages it was broken and irregular but this may have been done during histological preparation. Smith (1939) reported the zona pellucida from two, four, six, and seven cell stages but not from the pronuclear stage. On the other hand, Lewis and Wright (1935) found a zona pellucida in all stages of house mouse embryos through the blastocyst stage. It would appear, therefore, that the sona pellucida persists for a longer period of time in the house mouse than in P. poliootus.

The segmentation stages of P. polionotus had ovoid-shaped cells and did not appear to be in direct contact with or under any pressure from the tubal mucosa. This is in agreement %ith the observations of Smith (1939) on this same species. However, Huber (1915) indicated that the segmentation stages of the white rat were compressed between the folds of the tubal mucosa, and commented that they were molded by the tubal mucosa. Either these two forms mst differ in this respect or Huber's material not have become shrunken or distorted during









86


histological preparation.

The relationship between age and chronological development is obviously variable in P. polionotus. For example, embryos of both forty-eight and sixty hours were in the four cell stage of development. This might be due to inaccurate age determination or could be explained by assuming that at forty-eight hours the second cleavage has just been completed and that it takes more than twelve hours for the next cleavage to occur. Smith's (1939) observations lend support to the latter explanation since he found that embryos of forty-seven, or more, hours were in the four cell stage while those of sixty-four, or more, hours ranged from six to eight cells. However, other evidence clearly indicated that different eggs may develop at very different rates. In two litters, both of which were seventy-two hours old, different developmental stages were observed. In the first, the embryos were all in the eight cell stage and were located near the tubo-uterine junction, while in the second they were in the blastocyst stage and were located in the uterine horn. Even more striking was the situation in a single litter of four ninety-sixrhour embryos, all of which were apparently in good condition and healthy. The stages represented in this litter were: two cell, twelve to sixteen cell; morula, and blastocyst. Smith (1939) did not observe such a variation in development in P. polionotus; however, Snell (1941) states that in the house mouse the eggs usually reach the morula stage about sixty hours after fertilization and pass into the uterus some twelve hours later, but adds that this time is subject to considerable variation. In the white rat Griffith and Farris









87

(1942) reported uncleaved, two, three, and four cell stages in the tube of the female forty-eight hours after copulation. They also maintained that a two cell stage or even a single blastomere could be transplanted into the uterus and would develop normally.

In P. polionotus the ectoplacental cone had started to develop on embryos which were five days of age. According to Snell (1941) the ectoplacental cone appears at five to five and a half days in the white mouse, while Huber (1915) found it appearing in the white rat at six days and fourteen hours. In all of these forms the cone is tall, pointed and definitely cone-like in shape. On the other hand, Graves (1945) reported that in the golden hamster the cone appeared near the end of the sixth day and is not tall and pointed but rather fits like a wig on top of the embryonic vesicle. It seems, therefore, that the cone appears almost a day later in the hamster than in P. polionotus despite the fact that the hamster has the shortest gestation period of any known placental mammal, a period which is only about two-thirds as long as that of P. polionotus. Further, the shape of the cone in P. polionotus is similar to that in the m,(use and rat but quite different from that of the hamster. This is of particular interest in view of the fact that the hamster and Peromyscus are both referred to the family Cricetidae, while Mus and Rattus are typical members of the Muridae. On morphological grounds, the distinctions between these two families are not well marked and in fact in a recent classification Ellerman (1940-41) combines all of these rodents under the Yfuridae. It is possible that the shape of the ectoplacental cone may prove to be of some









88

value in determining relationships in this group of rodents. However, additional observations on other species are necessary before it can be determined whether this character is constant or significant.

The so-called "inversion of the germ layers" long known in rodents and previously reported for Peromyscus by Ryder (1887) has been fully verified in this study of P. polionotus. This phenomenon appears to be widespread among the rodents, but there are so many species whose embryological development has not yet been studied that it is impossible to say at present just how extensive it may be.

In one of the nine-day-old embryos there were four cavities in the mesoderm of the amniotic folds (Pl. III,A). Snell (1941) mentions that in the white mouse small cavities appeared in this area and soon coalesced to form the exocoelom. He stated that he did not actually observe the early stages of exocoelom formation in his studies and his description of these cavities was based on the observation of Jolly and Ferester-Tadie. The early stages of exocoelom formation were observed in P. polionotus; the cavities which wero seen appear to be similar to those described by Snell (1941); and presumably exocoelom formation Ulows the same pattern as in Mus.

A depression appeared in the thick base of the notochord of P. polionotue embryos at nine days, and was present only in embryos of this age group. This concavity appeared to correspond to one in the thick base of the notochord in the white mouse which Snell (1941) described as being the archenteron. He noted that this structure which appeared at seven and one-fourth days of age was transitory and









89

disappeared within twelve hours after formation. While the length of time during which it persists was not determined for P. polionotus, it is evident that it is also a transitory structure in this species.

The blood material which was seen in the extra-embryonic and amniotic cavities of embryos of P. polionotus was definitely cellular and appeared to have the same structure as the blood in the sinusoids of the uterine mucosa. Long and Burlingame (1938) described a "coagulated plasm" which was present in white rat embryos as being gelatinous, colorless and lacking not only in blood corpuscles but in all cellular elements. This material was present in the lumen of the yolk sac, in the fore- and hind-gut and in the extra-embryonic and amniotic cavities. They assumed that it had come from the maternal blood stream azu had, with alteration, passed through Reichert's membrane, the yolk sac, and the amnion. As has already been indicated, in P. polionotus the blood appeared to have passed directly into the exocoelom of the embryo from sinusoids in the uterus of the mother by means of at least one definite passage-way or channel formed by the mesodermal layer of the chorion pushing through the ectodermal layer and opening out into the sinusoids (Pl. IIG). However, no means of entry into the amniotic cavity could be found. Since all of the three nine-day-old embryos of P. polionotus which were still in the presomite stage contained this blood and since ?coagulated plasm" has also been observed in white rat embryos such an occurrence of blood or blood products is certainly not rare or unusual. Whether it is the usual condition can not be determined until a good









90

many more species have been studied. According to the literature it does not appear to occur generally in mammals and it may represent a peculiarity of rodents.

Rand and Host (1942) reported a mean litter size of 3.968 and a variation in litter size from three to five individuals for P. polionotus. Their data were obtained from thirty-two litters caught in the field and examinations of thirteen females with embryos. The data obtained in this study from fifty-two litters born in the laboratory showed a variation in litter size from one to six, and a mean litter size of 3.35. In addition, a count of the embryos in twentyseven pregnant females showed a range in litter size of from one to six and an average of 3.55. It seems apparent that two possible conclusions could be. drawn from these data. First, prenatal mortality for this species must have been very low since the mean nwuber of embryos in the pregnant females did not differ significantly from the mean number of young in the litters.

Secondly, since both averages obtained in this study were so similar to the mean reported for this species by Rand and Host (1942) from their study of field litters it would appear that litter size in P. polionotue was very little affected by captive conditions. Further, the mean litter of P. polionotuo is similar to those reported by Svihla (1932), Pournelle (1952) and others for other species of the genus.

In this study, the sex ratio at birth, based on 128 mice, was 50.78 per cent males and 49.22 per cent females which seemed to indicate that if prenatal mortality or captive conditions reduced litter size at all, they had no selective effect insofar as sex was concerned.









91

The sex ratio at birth of P. , ,ossypinus. based on 240 mice was reported by Pournelle (1952) as being 49.48 per cent males and 50.42 per cent females,

The gestation period was from twenty-three to twenty-frow days in the four cases in which it was observed. A greater number of gestation records would have been desirable, but most of the pregnant females, whose approximate time of copulation was known, were sacrificed before reaching full term in order to obtain embryos of known age, Smith (1939) cited a single gestation record for F. polionotus as being twenty-four days. Rand and Host (1942) did not determine the gestation period of P. polionotus, but stated that in other species of Peromyscus it was known to be twenty-two days, Svihla (1932) observed a total of 146 gestation periods for various species in the genus Peromyscus; however, P. polionotus was not included, A strikingly wide variation in length of gestation was found among sub-genera, species, subspecies, and even in individual mice. He gave twenty-two days as the least number and thirty-five days as the greatest number of days for the gestation period in PeroWScu with means ranging from 23,39t .13 to 23,61 .11 in the various subspecies of P, ganiculatus. The length of the gestation period for .P. polionotus here recorded closely approximltes the figure given for P. Maniclatus. This is of particular interest in view of the fact that these two species are not completely reproductively isolated, since they may interbreed and produce viable young under laboratory conditions.

Duration of the gestation period was found to be increased









92

approximately four days for lactating females. Similar increases in gestation length during lactation has been shown for other species of Peromyscus by Svihla (1932). Pournelle (1952) reported a gestation period of 29.6 to 30.8 days for lactating females of P. ,. goosypinus, while gestation for non-lactating females was around twenty-three days. Snell (1941) gave the gestation period for MIus as nineteen to twenty days; however; he stated that one or two young sucklings could prolong gestation up to seven days and with three or more young sucklings prolongation up to twelve or thirteen days was not uncommon. A noticeable lengthening of the gestation periods in lactating females appears, therefore, to be the rule in many rodents although the amount of lengthening may vary. This may well be correlated with differences in the intrauterine environment for the fetuses of lactating and non-lactating individuals.

The time of post-partum heat in Peromyscus has been given in more or less general terms. McNair (1931) stated that P. 1. noveboracensis and P. m. bairdii mated twenty-four to twenty-eight hours after birth of the young. Svihla (1932) working on this same genus stated that the females were usually in heat immediately following the birth of a litter. Also, Pournelle (1952) obtained litters of P. gossypinus aq 4 result of allowing males to remain with females until shortly after parturition. Rand and Host (1942) working with P. polionotus concluded that post-partum heat probably occurred within two to four days after parturition. No doubt the time of occurrence of post-partum heat is variable within the genus, but the data obtained




Full Text
This dissertation was prepared under the direction cf the
chairman of the candidates supervisory committee and has been ap
proved by all members of the coianittee. It was submitted to the
Dean of the College of Arts and Sciences and to the Graduate Coun
cil and was approved as partial fulfillment of the requirements
for the degree of Doctor of Philosophy.
June 3, 1957
Dean, College of Arts and Sciences
Dean, Graduate School
SUPERVISORY COMMITTEE


PLATE IX
A. Dorsal view at thirty days of age; a millimeter
scale is included in photograph.
B* Ventral view of above individual; a millimeter scale
is included in photograph.
C. Side view of a specimen at forty days of age; a milli
meter scale is included in photograph.


45
rested against the snout on the right side of the face. The external
nares approached their adult form, and with the development of the
snout, the mammalian nature of the embryo could be recognized. Vibris-
sae follicles occurred on each side of the snout and a single large
follicle was present between the eye and ear on each side of the body.
The mouth had closed somewhat and the tongue was barely visible some
distance back in the mouth cavity. In the region of the fourth ven
tricle, the membranous roof was slightly more opaque although the
floor of the fourth ventricle could still be seen and the transparency
of the skin over the neural tube continued from the fourth ventricle
to the tail. Sagittal and coronal sutures were obvious beneath the
skin of the head. The ear pinna was a short, triangular projection
from each side of the head. On the ventral side, the mammae were
still not discernible macroscopically, but the umbilical hernia had
enlarged. The anterior limb buds were directed somewhat caudad, while
the hind limb buds were directed somewhat cephalad. Slight radial
ridges in the distal end of the anterior limb buds marked the forma
tion of digits.
Internal
Nervous
The walls of the brain were thicker and more highly vascu
larized than in the previous stage. The epiphysis was a hollow stalk
and its cavity was continuous with the diocoel. Intensive thickening
of the walla had greatly reduced the lumen of the mesencephalon and


18
starting at one end of the embryo. It had a maximum length and width
of 62.0 x 54.25 miera.
The two cell embryo was lying eccentrically in the uterine lumen
toward the antimesometrial side. No discernible difference could be
seen between this embryo and the twenty-four hour old embryos previous
ly described. Each cell measured 31.0 x 31.0 miera.
The twelve to sixteen cell stage embryo was also free uterine.
Only one section of this embryo was found in its proper place on the
slide although several other sections which had been displaced during
staining were seen, hence no detailed description could be made. The
greatest width and length measurements taken from this single section
were 77.5 x 62.0 miera.
Five Days
Embryos at five days of age were implanting, and the implanta
tion sites were discernible upon gross examination as areas of slight
swellings and increased vascularity along the uterine horns. In the
fixed uterus, dimensions of one of these swellings were: length 4 nm.,
width 2,5 mm., depth 3.5 mm. The uterus in the area not directly con
cerned with implantation was almost uniformly round and had an outside
diameter of 2.0 mm. Snell (1941) in his description of implantation
in the white mouse, states that there is an appreciable swelling in
the uterus at the implantation site one day after implantation (five
days after mating).
Of the t hree embryos obtained at this age two were sectioned
for detailed study. The embryos were deeply lodged in the uterine
i


51
structure. Each side of the thyroid extended dorsaUy to the lateral
side of the trachea where it fused with a hollow post-branchial body.
The thymus bodies were ventro-lateral to the thyroids. They had be
come much larger, and more lymphoidal in appearance than the thyroids
but the two lobes were still broadly separated in the midline.
Skeletal
In the cranial region, the nasal septum and Meckel's carti
lage were being laid down as dense concentrations of pre-cartilage
mesenchyme. Further caudad the centra, neural arches, and ribs were
also easily recognizable, and in fact the portion of the centrum
around the notochord and dorsal part of the rib appeared somewhat car
tilaginous. The bones of the girdles and appendages were likewise
represented by concentrations of mesenchyme, with some areas having a
cartilaginous appearance.
Muscular and Integumentary
Concentrations of muscle primordia were outlined in the ap
pendages as well as in the region of the intercostal muscles. The eye
muscles were also delineated but had not become fibrous, and the muscle
layers of the esophagus and intestine were outlined. The mammae were
increasing in size.
Sixteen Days
External
By sixteen days the back was almost straight and the abdomen


100
Huber, G. Carl.
1915. The development of the albino rat, Mus norveglcus
albinus.l. From the pronuclear stage to the stage
of mesoderm anlagej end of first to end of ninth
day. Jour. Morph., 26: 247-356.
Lewis, Warren H. and E. S. Wright.
1935. On the early development of the mouse egg.
Carnegie Inst. Wash. Contrib. Embryol., 25: 115-143.
Long, J. A. and Paul L.. Burlingame.
1936. The development of the external form of the rat,
with some observations on the origin of the extra
embryonic coelom and foetal membranes. Univ. of
Calif.' Pub. in Zool. 43: (8), 143-164.
McNair, George T.
1931. The deer mouse, Peromyscus. a valuable laboratory
animal. Jour. Mamm., 12: 46-52.
Killer, G* S. Jr,, and R. Kellogg.
1955. List of North American Recent Mammals.
O.S.N.M, Bull. 205. 954 pp.
Pournelle, George H.
1952. Reproduction and early post-natal development of
the cotton mouse, Peromyscus gossypinus gossypinus.
Jour, Maram. 33: 1-20.
K Rand, A. L. and Per Host.
1942. Mammal notes from Highland County, Florida. Results
of the Archbold Expeditions, No. 45 Bull. Amer, Mus.
Nat. Hist., 60: 1-21.
Ryder, John A.
1887. The inversion of the germinal layers in Hesperomys.
Amer. Naturalist, 21: 863-664.
^ Schwartz, Albert.
1954. Oldfield mice, Peromyscus pollonotus of South
Carolina. Jour. Mamm., 35: (4)," 5&L-569.
Scott, J. P.
1937.The embryology of the guinea pig. Amer. Jour. Anat.
60: 397-432.
Simpson, G. G.
1945. The principles of classification and a classification
of mammals. Bull. Amer. Mus, of Natl. Hist.
Vol. 85, 350 pp.


LITTER SIZE AND SEX RATIO
Litter Size
During the period of this study fifty-two litters totalling
174 young, were born in the laboratory. The litter size ranged from
one to six with a mode of three mice per litter. The mean litter size
was 3.35 mice per litter. These data are summarized in Table 5.
TABLE 5
RELATIVE FREQUENCIES OF DIFFERENT LITTER
SIZES IN P. polionotus
Size of Litter
1
2
3
4
5
6
Total
Avg. litter size
Frequency
1
7
26
11
5
2
3.35
Litter size based upon a study of twenty-seven litters total
ling ninety-six embryos ranging from the pronuclear stage to approxi
mately twenty days of gestation showed a range of two to six embryos
per litter with a mod of three and an average number of 3.55.
Sex Ratios
Sexes of laboratory raised mice were recorded for 123 mice
representing thirty-nine litters and did not depart significantly from
a 1:1 r*io. Of this number, sixty-five (50.78 per cent) were males
72


24
U-shaped depression similar to that which Snell (1941) termed the ar-
chenteron in the white mouse. The egg cylinder was more elliptical
in shape than in previous stages and its broadest part was at the
region crossed by the amnion. The greatest length and width of this
embryo were 852.5 and 403.0 miera, respectively.
In a third embryo of this litter the allantois extended about
midway into the cavity of the exocoelom. The size of the ectoplacen-
tal cavity was greatly reduced, forming a small crescent-shaped cavity.
The paraxial mesoderm had differentiated as a dense layer, although
segmentation into somites had not begun; a slight embryonic coelom
had formed in the intermediate mesoderm. Measurements of the greatest
length and width were 861.0 x 589.0 miera.
In the most advanced embryo of this litter the head fold was
large and its cavity was filled with head mesenchyme cells. Somite
formation had begun, and five somites could be counted. The archen-
teron still persisted as an entodermal invagination on the ventro-
posterior side of the embryo (PI, III,B).
The three embryos of this litter which were in the presomite
stage and still maintained the egg cylinder form, possessed maternal
blood within their exocoeloms and amniotic cavities. Since this blood
had the same appearance as that surrounding Reichert*s membrane and
filling sinusoids in the uterus, a close search was made of all sec
tions for a possible source of entry into these cavities. In one em
bryo a channel was discovered which appeared to have been formed by
the mesodermal layer of the chorion pushing through the ectodermal


SUMMARY AND CONCLUSION
1. Development of the oldfield mouse, Peromyscus pollonotus. is des
cribed from the time of insemination to the time when the animals
approached adult limits.
2. Recently ovulated eggs were surrounded by a corona radiata which
was, however, largely gone by fifteen hours. The zona pellucida
ms present through the eight cell stage,
3. At twenty-four hours, embryos were in the two cell stage, at forty-
eight and sixty hours in the four cell stage, and by seventy-two
hours in the eight cell stage. By ninety-six hours, they were
generally in the blastocyst stage; implantation occurred on the
fifth day of gestation, and by eight days the amniotic exocoelom
and eetoplacental cavities were present; at nine days the allantois
had started to form; and by ten days the body form had become U-
shaped, the allantoic stalk had joined the chorion and the first
aortic arch was present. On the twelfth day, the body was in the
form of a tightly coiled C; limb buds were present, and the third
aortic arch had formed. By the fifteenth day, vibrissae follicles
occurred on each side of the snout, and the mammalian nature of
the embryo could be recognized. By eighteen days, the fetus had
about the same appearance as at birth. The pinna of the ear had
folded over the opening, eyelids had formed and the eyes were half
closed, the tongue had withdrawn from the lips, the mouth was al
most dosed, and the digits were fully formed.
96


2
cycle." Thus, although embryos or ontogenetic processes may be more
similar than adults in closely related animals, they may also be more
dissimilar. Further, it must be borne in mind that the morphological,
physiological, behavioral and other characteristics exhibited by the
adult and utilized in assessing relationships between species are the
end result of modification of embryonic processes or chronologies
through the action of differing genic constitutions. Therefore diver
gence in adult structure would be expected to be associated with onto
genetic differences, although the latter may be hard to detect. Evi
dence presently available does indicate the existence of significant
differences in embryonic structure or processes among mammals at the
higher levels of classification. Less is known of the kinds and extent
of such differences between lower taxonomic units, and the significance
of those that have been pointed out will not become clear until many
more careful studies are made of the embryology of numbers of related
species.
Modern systematic approach relies upon all aspects of the biol
ogy of species in its attempt to arrive at evolutionary synthesis. The
period of embryonic development is an important stanza in the life cy
cle of any speeies and might be looked upon as an important source of
evidences of relationships. However, since such data are not easy to
obtain, it has played only a relatively minor role in the delimitation
of the lower taxonomic categories. But when such information can be
procured it may prove valuable in augmenting and supplementing the
usually employed adult morphological taxonomic criteria.


71
out of fourteen litters were born between the hours of 8t00 A. M, and
12iCO noon. However, observations were not made on all of the litters
but only on those produced by a group of fourteen females which had
been bred at about the same time and produced litters within several
days of each other.


11
Age
5 days
6 days
7 days
8 days
9 days
10 days
11 days
12 days
13 days
14 days
15 days
16 days
17 days
18 days
TABLE 1 Continued
Number of
Specimens Stage of Development
2 Implanted embryonic vesicle with be
ginning of proamniotic cavity
3 Egg cylinder, proamniotic cavity re
stricted to embryonic ectoderm
3 Proamniotic cavity continuous into
extra-embryonic ectoderm; posterior
amniotic fold beginning
3 Presence of 3 cavities; amniotic,
exocoelom, and ectoplacental
4 Allantois present, Somites present in
one embryo, not yet developed in others
4 Body form U-shape, allantoic stalk joins
chorion, and appearance of first aortic
arch
4 Body form tightly coiled C; limb buds
present
4 Tail distinct; anterior limb buds
fan-shaped
3 Face formed, features not yet discern
ible as mammalian
3 Resemble adult; vibrissae follicles on
each side of snout
4 Digits outlined but fully webbed; mammae
present; no hair follicles on body
3 Digits of forelimbs deeply notched; plan
tar tubercles present; hair follicles
present on body
4 Digits fully formed; eyes half closed
During the course of this investigation data were obtained cm


43
Excretory
The mesonephric duct was confluent with the metanaphric duct
for a short distance from the urogenital 3inus, and they had a common
junction with the urogenital sinus, An aggregation of mesenchyme cells
which was lying posterior and somewhat dorsal to the mesonephros and
surrounding the enlarged terminal end of the ureter represented the de
veloping metanephric kidney. In cross section through this region sev
eral tubules could be observed extending out from the ureter into the
developing metanephros.
Endocrine
The thymus and parathyroid complex from the third pouch was
3till in contact with the pharynx but the thymus had become greatly
elongated and its terminal ends were growing medially. The rapidly de
veloping thymus had grown further caudad than the thyroid gland and had
passed laterally on each side of the latter. The two thick walled post
branchial bodies each of which had a fairly large lumen had separated
from the pharynx and moved caudally down the neck. The suprarenal
glands were lying dorsal to the genital ridge and medial to the meso
nephros. They were simply a mass of glandular cells with no definite
shape.
Genital
The genital ridge at this stage was a uniform cellular mass
forming a longitudinal strip paralleling the medial side of the meso
nephros. The Mullerian duct was not differentiated. The genital


TABLE OF CONTENTSContinued
LITTER SIZE AND SEX RATIO 72
Litter Size 72
Sex Ratios 72
GESTATION 74
Gestation Length in Non-Lactating Females 74
Influence of Lactation on Gestation 74
Post-Partum Estrus and the Normal Estrous Cycle 75
POSTNATAL GROWTH AND DEVELOPMENT 77
Weights at Birth 77
Postnatal Growth Measurements 77
Development 79
DISCUSSION 84
SUMMARY AND CONCLUSION 96
LITERATURE CITED 99
PLATES AND FIGURES 103
BIOGRAPHICAL SKETCH 124
iv


58
In cross section it resembled two kidney shaped tubes vdth crescent
shaped lumens. The epithelium of the convex side of each lumen was
several times thicker than on the opposite side.
Digestive
The body of the tongue had become more heavily striated with
muscle fibers extending transversely across the tongue. Lingual pa
pillae were discernible although there was as yet no sign of foliate
papillae. Incisor tooth buds were also present at this stage. The
salivary glands located on each side of the root of the tongue con
sisted of solid branched lobules which were becoming encapsulated by
aggregations of mesenchymal cells. The anal orifice and rectum were
occluded with epithelium.
Excretory
The urinary bladder was an extremely large structure with a
wide lumen and was still broadly attached to the ventral body wall.
The kidney was a massive structure which at this stage presented a
more round than kidney-shaped appearance. Numerous tubules and sev
eral incompletely formed Bowmans capsules were present. The ureters
were patent throughout their length. The mesonephric ducts were
still intact and emptied into the urinary sinus, which at this stage
may be called the urethra.
Endocrine
The thyroid gland had changed considerably in texture. Its
tissue consisted of a loosely vacuolated mass in which were forming


60
Eighteen Paya
External
In the eighteen-day fetus the head appeared quite similar, to
that of a full tern fetus (PI, V,C). The pinna of the ear had folded
over the opening, but the opening was not tightly sealed. The eye
lids had formed and the eyes were half closed. The roof of the hind
brain was no longer transparentj however, organs could still be seen
through the ventral body wall. The chin was held well above the
chest, and the umbilical hernia had almost completely disappeared.
Hair follicles were now quite obvious and had extended to the mid-
dorsal line, on the dorsal side of the head forward to the nose, and
also on the ventral side of the animal, as well as other parts of the
body. The tongue had retracted a short distance, arid the mouth was
almost closed. Both the fore and hind listo digits were conspicuously
notched,
Internal
Nervous
The lumen of the epiphysis was still present} the extent of
the choroid plexus had increased in the third and fourth ventricles,
and the cerebellar rudiment was considerably thicker. In the spinal
cord, the neurocoel was a small egg-shaped cavity that was centrally
located. The fibrous sclerotic coat of the eye was now clearly evi
dent, and in front of the lens it continued as a roof of the anterior


79
Development
At birth the eyes were closed and the ear pinnae were folded
down over the external auditory meatus (PI. VI). The over-all body
color was pink with the dorsum being slightly darker. The skin cover
ing of the head region was transparent so that the sagittal and coro
nal sutures could be seen. On the ventral side, small blood vessels,
viscera, and ribs could be seen through the transparent skin,
Mystacial vibrissae were present at birth and when depressed against
the face extended back to the level of the eye.
By the second and third day, the entire dorsal side was pig
mented but the region of the head between the ears appeared a shade
lighter than the rest of the dorsum. The venter remained pink and
transparent enough for viscera to be seen. The iris ring, which was
visible through the somewhat transparent sealed eyelids at birth as a
black band surrounding the lighter lens, was obscured by the end of
the third day due to the increasing opacity of the eyelids. Upon close
inspection with proper light, or tinder slight magnification, a sparse
covering of fine hair could be observed on the dorsal side by the third
day. No hair growth could be detected on the ventral side at this age.
In a total of forty-two young, the pinna unfolded on the third day in
eighteen, on the fourth day in twenty, and on the fifth day in four
(PI. VII,A).
At three days of age mice would squeak when disturbed, but
showed very little coordination. When placed on their feet they would
rest on the venter for a few seconds and then in trying to move would


66
Weight is one of the most valuable measurements for deter
mining growth, and volume is of value in giving an approximation of
size. There was a very close parallel between weights and volumes
in each group of embryos measured. A continuous inert ate in weight
and volume was seen from twelve through eighteen days of age. Rela
tive growth was more rapid after fifteen days of age, the greatest
increment occurring from seventeen to eighteen days of development.
Since the mean birth weight of this species was found to be 1.6l
grams it appears that the greatest increase in weight must take place
between eighteen days and parturition, which occurs normally at
twenty-three or twenty-four days. Such a rapid increase in growth to
ward the termination of the gestation period is typical of most mam
malian prenatal developmental curves.
Measurements of Certain Body Parts of Embryos from
Twelve Through Eighteen Days of Development
Various linear measurements made on embryos from twelve through
eighteen days of development with a pair of fine (bolt-controlled) di
viders under 5x magnification included: crown-rump length, head breadth,
head length, shoulder breadth, length of foreleg, length of hindleg, and
tail length. The averages are given in Table 4, and are represented
graphically in Figure 1.
These data indicate that the crown-rump length, head breadth,
head length, shoulder breadth and tail measurements exhibit approximate
ly the same relative growth trend. In eaeh case from thirteen through
f
seventeen days of age, growth proceeded at a fairly constant rate.


26
evaginations. Closure of the neural groove began in the region of the
fifth somite and continued caudally for approximately 150 miera, beyond
i-ihleh the right and left neural folds were closely approximated.
Eleven Paya
As previously noted no embryos of this stage were obtained.
Twelve Days
There were four embryos obtained for this age and one was
sectioned in tero, while the others were removed from the uterus
and fetal membranes before being studied. Giant cells were quite
numerous in the decidua adjacent to the embryo both laterally and
ventrally. Such cells were first seen in the uterus with five-day-old
embryos. It was not determined whether the giant cells persisted be
yond twelve days as embryos were not sectioned in tero beyond this
stage. Snell (1941) reports giant cells as a conspicuous feature ly
ing between Reicherts membrane and the decidua in sections of the
white mouse embryos of six to fourteen days.
External
At the twelve-day stage the body was tightly coiled with the
right side of the head overlying the short tail (PI, IV,B). The mes
encephalon protruded anteriorly as a hump on each 3ide of the dienceph
alon and the optic vesicles were apparent as wide evaginations. An
auditory pit was present on each side in the region of the hind brain
just dorsal to the hyoid arch. Both anterior and posterior limb buds
were present, the anterior being slightly larger than the posterior.


LIST OF ILLUSTRATIONS
Page
103
A* Recently ovulated egg.
B. Pronuclear stage.
C. Two cell stage.
D. Four cell stage.
E. Eight cell stage.
F. Morula stage.
G. Blastocyst stage.
II 105
A. Longitudinal section of embryo five days of age.
B. Longitudinal section of embryo six days of age.
C. Longitudinal section of embryo seven days of age.
D. Longitudinal section of embryo eight days of age.
E. Longitudinal section of embryo nine days of age
showing three cavities.
F. Longitudinal section of embryo nine days of age
showing a break in the chorion allowing a direct
connection of the exocoelom with blood-filled
spaces of the ectoplacental cone.
Ill 107
A. Longitudinal section of embryo nine days of age
showing formation of exocoelom.
B. Sagittal section of embryo nine days of age
showing somite formation.
vi
Plate
I


53
reduced. The walls of the metencepholon were thicker but the cavity
was very broad and there was no indication as yet of the cerebellar
rudiment. The choroid plexus had proliferated in all directions in
the roof of the fourth ventricle. The neurocoel of the spinal cord
was compressed laterally producing a slit-like lumen which had a
width of about two thirds of the spinal cord. There was a slight de
pression representing an early stage of the ventral fissure, but at
this stage, there was no sign of the dorsal fissure. In the eye,
there was a dense mesenchymal sclera on the outside of the pigmented
layer and it had not yet begun differentiating into fibers. The pig
mented layer of the retina was reduced in width from previous stages.
Lacrimal glands and ducts had begun to form. The inner ear showed
very little change from the previous stage other than that it was now
completely encased in precartilage cells.
Digestive
The tongue had further differentiated. Transverse muscle fi
bers were present in its body; and also, two thick longitudinal bands
of muscle extended from the root into the body of the tongue. The
submaxillary and sublingual glands had pushed deeper into the gum from
their point of origin and had become more lobular. The esophagus was
still quite small although a lumen was traceable throughout its length.
There was very little change in the lungs other than an increase in
size of the lobes and an increase in the number of bronchioles in each
lobe. The tissue itself still had a mesenchymatous aspect. The


82
face washing, and nervous urination. By this stage firm fecal pellets
with a bright yellowish color had replaced the watery feces of previous
stages.
Around fifteen to seventeen days of age the ventral hair was
dense enough so that the mammae on the females did not show conspicu
ously. The young mice were very alert and at night they often left
their nesting jars and ventured around the cage. When a sharp noise
was made they would jump into the air and run back into the nesting
jar. Cheek teeth had erupted the skin in specimens fixed at fifteen
and sixteen days of age, but were not yet visible in a specimen fixed
at fourteen days of age* Beyond fifteen to sixteen days mice showed
very little change other than a gradual increase in size and in length
of pelage. Plates VIII and IX show stages in development from twenty-
four through forty days.
In most females the vagina opened at twenty-nine to thirty-
four days of age. Actual observations were made on fourteen individ
uals; in six the vagina was open on the twenty-ninth day. The next
observation was not made until the thirty-fourth day when only one of
the fourteen still had a closed vagina. In males at thirty-one days
of age the testes were slightly outlined in the scrotum.
The post juvenile molt began in some mice as early as thirty-
two days although the majority did not show signs of molt until thirty-
four to thirty-six days of age (PI. IX). A small patch of buff-colored
hairs Just beneath each eye, another on the dorsal side of each hind
leg in the region of the distal end of the tibia, and a fine line of


15
Twenty-Four Hours
Three embryos obtained from a specimen killed twenty-four hours
subsequent to finding sperm in her vaginal fluid had undergone the
first cleavage division and were in the two cell stage. All were in the
isthmus of the same Fallopian tube, while the other contained several
degenerating ova. The cytoplasm of each blastomere was granular and
the nucleus was round and centrally located. The blastameres were
surrounded by the zona pellucida, but there was no evidence of corona
radiata cells at this stage (PI. I,C). The greatest length and width
of one typical two cell embryo were 93*0 and 62.0 raicraj the blastomeres
were equal in size. The nucleus of each blastomere had a diameter of
18 miera.
Forty-Eight Hours
Each of three embryos obtained at this stage was composed of
four cells and was still located in the Fallopian ttibes. They were
distributed two on one side and one on the other. The two embryos in
the isthmus of the same tube were located very close together as they
both appeared in several of the same sections. Smith (1939) obtained
two four cell stage embryos in a female sacrificed 47+ hours after
copulation.
The plane of section did not pass through all four of the
cells in any of these forty-eight hour embryos, so that individual
sections show no more than three cells (PI. I,D). The zona pellucida
surrounding the embryo was still distinct and of the greatest diameter


BIOGRAPHICAL SKETCH
Samuel Kaada Laffoday, born September 13, 1924, in Lamar,
South Carolina, attended Lamar High School and after graduation in
1941, he entered Presbyterian Junior College for Men, Maxton, North
Carolina, From April 1943 to February 1946 he served in the United
States Army, He entered the University of South Carolina in June
1946 and received a Bachelor of Science degree in August 1947, and
a Master of Science degree in June 1949. From 1946 to 1949 he
served as an Instructor in Biology at the University of South
Carolina, Columbia, S, C., and from 1949 until 1951, as Assistant
Professor of Biology at Presbyterian College, Clinton, South
Carolina, Then he began graduate studies at the University of
Florida, He is currently employed as an Assistant Professor in
Biology at the College of Charleston, Charleston, South Carolina,
He holds membership in the South Carolina Academy of Sciences,
Association of Southeastern Biologists, Phi Sigma, and Sigma Xi.
124


PLATE VIII
t
<
117


54
infolding of the duodenal walls had continued but the rest of the
gut possessed a smooth lumen. The mesentery suspending the colon
had narrowed down so that it resembled a typical mesocolon. The
spleen had increased in size and was attached to the mesentery ad
jacent to the stomach and lateral and parallel to the mesonephros
on the left side of the fetus.
Circulatory
The right fourth aortic arch showed further modification to
ward its adult relationship as it had formed the base of the right
subclavian artery. The left subclavian artery had formed from the
systemic arch. Valves had developed in the truncus arteriosus. The
subclavian veins were connected with the common cardinal veins on
each side. No further changes were noted in the circulatory system
at this stage.
Excretory
Near the base of the genital tubercle, where it became con
tinuous with the ventral body wall, the urethral groove appeared as
a thick ectodermal ridge, which extended for about thirty microns to
the urogenital sinus. The latter extended dorsally and branched into
two horns which received the mesonephric ducts. The urinary blaader
made its appearance at this stage as the extended end of the urogeni
tal sinus, around which mesenchyme from the ventral body wall con
densed forming its walls. From each kidney a ureter extended to join
the base of the bladder laterally. This was the first stage in which


35
viere thick showing the primordia of the parathyroid four, thymus four,
and the post-branchial bodies. Where the esophagus and trachea sepa
rated, the lumen of the esophagus was compressed dorso-ventrally while
the trachea was compressed laterally. Mesenchyme surrounding the
trachea was more densely arranged and stained much darker than adja
cent tissue vdiioh suggested the beginning of cartilage formation in
this area. As the esophagus and trachea extended posteriorly, their
lumens became so reduced in size that they were barely detectable.
The trachea remained the larger of the two tubes and increased in size
again before biftreating into right and left bronchi. The right bron
chus remained several times larger than the left until it again divided.
Five lung lobes were seen in sections at this stage, four on the right
and one on the left side. The right medial lobe while connected with
the other lobes of the right side was somewhat distinct and was enclosed
in its own pleural sac. The stomach had increased still more in size,
and the ventral lobe of the pancreas had become a conspicuous globose
mass of tissue just ventral to the stomach. The common bile duct and
gall bladder were discernible at this stage. In following the tail
sections anteriorly a very small tail gut could be seen. The cloacal
membrane was still present.
Circulatory
The greatest change in the heart was an inorease in thickness
of the wall of the ventricle. The four chambered condition was obvious
in the heart as the interatrial septum had extended deeper into the
atrial cavity, and the interventricular septum had become more prominent.


A STUDY OF PRENATAL AND POSTNATAL
DEVELOPMENT IN THE OLDFIELD MOUSE,
Peromyscus polionotus
By
SAMUEL K. LAFFODAY
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
June, 1957

ACKNOWLEDGMENTS
Many individuals have contributed most generously of their
time, knowledge, and experience to this work. Dr, H. B. Sherman, Pro
fessor of Biology at the University of Florida until his retirement in
1955, suggested the problem and guided me through the initial phases
of the work. To him I should like to express my sincere thanks. For
*
their valuable criticisms, suggestions, and interpretations, I should
like to thank Drs. Lewis Berner, J. H. Gregg, G. K. Davis, J. D, Kilby,
Pierce Brodkorb, J, VJ, Brookbank, and J. N. Layne. Dr. Layne helped
especially with the analysis and interpretation of data on postnatal
development. Severa], of my fellow students contributed in various ways
to the work. Robert Haubrich and Claude Adams cared for the laboratory
animals from time to time; John Outterson assisted with the field work,
and Robert Weigel and Joe Davis with the photography. To all of than
I should like to express my appreciation. Dr. E. Ruffin Jones, Jr*, as
chairman of my supervisory committee advised me on my academic program,
guided me in my research, and gave me every consideration and continued
inspiration. It is a pleasure to express to him my sincere appreciation.
Finally, ny wife, Joanne Andrews Laffoday, deserves special acknowledg
ment for the assistance which she has given me. She helped with the
field collections, with the writing and typing of this report, and
throughout has provided moral and financial support. I should like,
therefore, to express to her ny thanks for her many contributions.
ii

TABLE OF CONTENTS
ACKNOWLEDGMENTS ii
LIST OF TABLES v
LIST OF ILLUSTRATIONS vi
INTRODUCTION 1
METHODS AND MATERIALS 6
DESCRIPTION OF PRENATAL DEVELOPMENT 13
Zero Hours ............ ... 13
Fifteen Hours ........ 13
Twenty-Pour Hours 15
Forty-Eight Hours 15
Sixty Hours ........ 16
Seventy-Two Hours .... ..... 16
Ninety-Six Hours 17
Five Days . 18
Six Days 20
Seven Days 20
Eight Days 21
Nine Days 22
Ten Days 25
Eleven Days 28
Twelve Days 28
Thirteen Days 32
Fourteen Days .............. 37
Fifteen Days 44
Sixteen Days 51
Seventeen Days ............. 56
Eighteen Days ................ 60
PRENATAL GROWTH 64
Measurements of Uterine Swellings at Implantation Sites 64
Weights and Volumes of Embryos 65
Measurements of Certain Body Parts of Embryos from
Twelve Through Eighteen Days of Development 66
PARTURITION 68
iii

TABLE OF CONTENTSContinued
LITTER SIZE AND SEX RATIO 72
Litter Size 72
Sex Ratios 72
GESTATION 74
Gestation Length in Non-Lactating Females 74
Influence of Lactation on Gestation 74
Post-Partum Estrus and the Normal Estrous Cycle 75
POSTNATAL GROWTH AND DEVELOPMENT 77
Weights at Birth 77
Postnatal Growth Measurements 77
Development 79
DISCUSSION 84
SUMMARY AND CONCLUSION 96
LITERATURE CITED 99
PLATES AND FIGURES 103
BIOGRAPHICAL SKETCH 124
iv

LIST OF TABLES
Table Page
1. List of Eggs and Embryos Studied , 10
2. Mean Measurements in Millimeters of
Uterine Svrellings 64
3. Mean Weights and Volumes of Embryos
12 to IB Days of Age 65
4. Average Measurements of Body Parts of
Embryos in Millimeters ....... 67
5. Relative Frequencies of Different Litter
Sizes in P polionotus 72
6. Average Daily Growth Measurements in
Millimeters and Weights in Grams of
P. polionotus from 2 to 60 Days of Age ....... 7
v

LIST OF ILLUSTRATIONS
Page
103
A* Recently ovulated egg.
B. Pronuclear stage.
C. Two cell stage.
D. Four cell stage.
E. Eight cell stage.
F. Morula stage.
G. Blastocyst stage.
II 105
A. Longitudinal section of embryo five days of age.
B. Longitudinal section of embryo six days of age.
C. Longitudinal section of embryo seven days of age.
D. Longitudinal section of embryo eight days of age.
E. Longitudinal section of embryo nine days of age
showing three cavities.
F. Longitudinal section of embryo nine days of age
showing a break in the chorion allowing a direct
connection of the exocoelom with blood-filled
spaces of the ectoplacental cone.
Ill 107
A. Longitudinal section of embryo nine days of age
showing formation of exocoelom.
B. Sagittal section of embryo nine days of age
showing somite formation.
vi
Plate
I

LIST OF ILLUSTRATIONSContinued
Plates Page
C.Frontal section of embryo ten days of age
shoving allantois connected to chorion.
IV 109
A. Embryo ten days of age.
B. Embryo twelve days of age.
C. Embryo thirteen days of age.
D. Embryo fourteen days of age.
E. Embryo fifteen days of age.
v in
A. Embryo sixteen days of age.
B. Embryo seventeen days of age.
C. Embryo eighteen days of age.
vi 113
Litter of mice two to four hours old.
VII 115
A. Litter of mice four days of age.
B. Litter of mice eleven days of age.
VIII 117
A. Dorsal view of mouse twenty-four days of age.
B. Side view of mouse twenty-four days of age.
vii

Plates
LIST OF ILLUSTRATIONSContinued
Page
119
IX
A. Dorsal view of mouse thirty days of age.
B. Ventral view of mouse thirty days of age*
C. Side view of mouse forty days of age.
Figure
1 . 121
Graphs showing measurements of certain body
parts of embryos from twelve through
eighteen days of gestation.
2
123
Graphs showing growth rates of selected body
dimensions and weights from birth to sixty
days of age.
viii

INTRODUCTION
The present state of knowledge concerning the embryonic de
velopment and other aspects of reproduction in mammals is markedly
incomplete. Only a small fraction of the more than 3#000 species
of recent mammals have been studied in any detail, and the majority
of these are domestic or laboratory forms. The paucity of develop
mental studies on wild species in large part stems from the diffi
culties attendant upon maintaining many kinds under captive condi
tions, not to mention the problems of inducing breeding in the
laboratory and of obtaining known-age embryologies! stages. Despite
the fragmentary nature of the data, there has been a distinct tendency
to apply the findings based on the study of the embryology of a single
or of a few species to the broader divisions of classification. The
chief justification for such extrapolation seems to lie in the assump
tion that the intrauterine environment is relatively constant in all
mammals, at least in the placental forms, as are the requirements for
maintenance of the embryo. As a consequence the pattern of embryonic
development, freed from the selective processes of differing environ
mental conditions, would be expected to be relatively conservative and
probably closely similar even throughout the higher taxonomic divisions.
However, Simpson (1945) has pointed out that "Embryonic structure is no
more free from the effects of new mutations, from adaptation, conver
gence, divergence, and the like than is any other phase of the life
1

2
cycle." Thus, although embryos or ontogenetic processes may be more
similar than adults in closely related animals, they may also be more
dissimilar. Further, it must be borne in mind that the morphological,
physiological, behavioral and other characteristics exhibited by the
adult and utilized in assessing relationships between species are the
end result of modification of embryonic processes or chronologies
through the action of differing genic constitutions. Therefore diver
gence in adult structure would be expected to be associated with onto
genetic differences, although the latter may be hard to detect. Evi
dence presently available does indicate the existence of significant
differences in embryonic structure or processes among mammals at the
higher levels of classification. Less is known of the kinds and extent
of such differences between lower taxonomic units, and the significance
of those that have been pointed out will not become clear until many
more careful studies are made of the embryology of numbers of related
species.
Modern systematic approach relies upon all aspects of the biol
ogy of species in its attempt to arrive at evolutionary synthesis. The
period of embryonic development is an important stanza in the life cy
cle of any speeies and might be looked upon as an important source of
evidences of relationships. However, since such data are not easy to
obtain, it has played only a relatively minor role in the delimitation
of the lower taxonomic categories. But when such information can be
procured it may prove valuable in augmenting and supplementing the
usually employed adult morphological taxonomic criteria.

3
Among recent mammals the order Rodentia comprises a vast array
of species in which extensive adaptive radiation, convergence, and
other factors often obscure phyletic relationships and clearly provide
a situation where all available data should be eraployed. Various as
pects of reproduction have thus far proved useful in the taxonomy of
various groups of rodents. Among the features that have been employed
are: the developmental history of the fetal membranes, characteristics
of the newborn young, length of gestation, nature of the estrous cycle,
and the anatomy of the genital organs. The value of details of embry
onic development in this connection has not been well explored.
Considerable attention has been given to the classification of
rodents in the families Muridae and Cricetidae, where many of the as
pects of reproduction mentioned above have been utilized in addition
to "classical" taxonomic characters. The status of the two groups is
at present not fully agreed upon by taxonomists. Some workers, Ellerman
(194L) include both under the Muridae, whereas others, Simpson (1945)
afford each full family rank.
A considerable amount of work has been done on the embryology
and development of several species of these rodents extensively used as
laboratory animals. The embryology of the house mouse, Mus muscuius.
and rat, Rattus norvegicus. both murids, is comparatively well known as
the result of studies by Huber (1915), Long and Burlingame (1938) and
Snell (1941). A recent account of the general chronology of prenatal
development in a cricetid, the hamster, Mesocricetus. is that of Boyer
(1953). Differences in the chronology and details of development in

4
these forms are apparent but their importance from a taxonomic stand
point cannot be evaluated until similar data are available for species
of other genera in the two groups. With this consideration in mind
the present investigation was undertaken.
The oldfield mouse, Peromyscus polionotus, was selected for
study. The mice of this genus are typical cricetid types of wide dis
tribution in North America, ranging from Atlantic to Pacific coasts
and from the tree line in the north to Panama, Hamilton (1939). The
genus contains more named forms than any other in North America, 56
species with numerous geographic races being listed by Miller and
Kellogg (1955) Various species of Peromyscus occur in a great vari
ety of habitats and are frequently the commonest of small mammals in
an area, often playing an important role in the ecology of the region.
Their diversity of form and wide geographic and ecological distribu
tion, coupled with the ease by which they can be captured and maintained
under laboratory conditions, have made these mice the subject of numer
ous taxonomic, ecological, physiological, behavioral, and genetic stud
ies. Such investigations have contributed much to our understanding of
differentiation and speciation in mammalian stocks.
Although postnatal development and other details of reproduc
tion have been studied in various members of the genus, it appears that
the only published reference to embryologies! development in the group
3 the mention by Ryder (1887) of "inversion" of the germ layers.
Smith (1939) in an unpublished masters thesis reported upon the early
embryology of P. polionotus from the pronuclear stage to the formation

5
of the blastocyst.
The main objective of the present study was to follow the se
quence of embryological development as completely as possible from
conception to term. Since the general features of embryology in this
species are similar to those of the laboratory mouse, rat, and hamster,
no effort was made to describe in great detail all particulars of dif
ferentiation and organogenesis. Rather, attention was directed princi
pally to those features that seemed to offer a basis for critical com
parisons between the various genera. Since prenatal development is
merely the initial phase in a continuous growth process to adulthood,
observations on postnatal growth and development were considered as
also falling logically within the scope of the investigation. Data
pertaining to other aspects of reproduction, such as length of gesta
tion, parturition, estrous cycle, litter size, and sex ratios of the
young at birth, which were accumulated incidental to the principal ob
jectives of the work are also presented.
I

METHODS AND MATERIALS
The mice used in establishing the laboratory colony were ob
tained from burrows in the Ocala National Forest in Marion County,
twenty-three miles east of Ocala, Florida. Some animals were captured
along the road bed of State Highway 20, from Central Tower east to
Juniper Springs. Collections were also made on the Salt Springs road
from Central Tower north for a distance of approximately six miles.
This population has been referred to as Percatqrscus polionotus subgriseus
by Schwartz (1954)*
Burrows could be seen along the shoulders of the road with lit
tle difficulty and could be dug easily. Only burrows that were plugged
a short distance from the entrance were excavated, as these were usually
occupied by a pair or by a family of mice, while unplugged burrows were
usually unoccupied. After determining the direction of the burrow a
small minnow seine was laid on the ground several feet back from the en
trance in hopes of covering the exit from the escape tunnel. In some
cases, after removal of the plug from the entrance tunnel, the introduc
tion of a piece of rubber tubing into the burrow would cause the mice to
flee by way of the escape tunnel and emerge under the minnow seine.
Often, however, the mice would not leave the nest until it had been com
pletely exposed by digging. If they then ran into the escape tunnel and
remained there instead of coming to the surface the introduction of the
rubber tubing into the escape tunnel would promptly drive them out.
6

7
Mice were housed in the laboratory in cages measuring
9 x 15 x 9 inches in vddth, length, and height, respectively. The top
and bottom were formed of cookie sheets and the sides of l/k inch
hardware cloth. The top3 were secured by two coil springs and were
easily removed. Coarse wood shavings were placed on the floor and a
one quart nesting jar containing a small amount of cotton was kept in
each cage, A water bottle was attached to the outside with a delivery
tube extending into the cage.
The diet consisted chiefly of Purina Laboratory Chow pellets
on which they fed ad lib. Lettuce was put into the cages onee or
twice weekly; and while matings were being made small amounts of beef
liver were offered to the animals and these were readily accepted.
The diet was also supplemented from time to time with whole yellow corn.
To increase the laboratory stocks wild mice taken in the field
as pairs were kept paired in the laboratory. A single pair was kept in
each cage. Usually they would not reproduce for six months or more but
with production of the first litter, there would often result a sustained
littering period for three to five litters. It was soon discovered that
the most productive method of securing litters from laboratory-raised
animals was by placing as many as four females to a cage and introducing
a proven male for a period of about two weeks. Successful pregnancies
could be detected within fifteen days after the date of removing the male
and the gravid animals were then isolated. This method proved quite ef
fective and in several cases when as many as twenty females were used
all of them became pregnant as a result of being with the male for the

3
two-week period#
In an effort to obtain carefully timed embryos, and to measure
the length of the gestation period in non-lactating females, frequent
attempts viere made to observe actual coitus. However, this was soon
abandoned as the presence of an observer discouraged mating. The ma
terial used for this study was obtained by determining the onset of
estrus through vaginal smears. Then a proven male was introduced into
the cage with the female at about 3:30 P. M. and was removed the next
morning at approximately 8:30 A. M. A vaginal smear was then taken
and the slide checked for the presence of sperm. If sperm were found
the mouse was killed with ether after an appropriate period and the
genital tract was removed and fixed in Lavdowskys mixture, Guyer(l947).
By sacrificing pregnant fonales at appropriate intervals a sequence of
embryos was obtained representing each of the first eighteen days of
the gestation period except the eleventh. The ages of embryos in this
paper '¡rere all calculated from the time of finding sperm in the vaginal
fluid. Obviously this method does not provide the exact age of an em
bryo, but it is impossible to determine the exact time of fertilization
vdth any known method. Griffith and Parris (1942) indicated that vari
ous points of reference have been used in the timing of embryos, but
the exact age of the embryos could not be determined by the time of
copulation, ovulation, or fertilization#
The embryos from which sections were to be prepared were pro
cessed by the usual paraffin technique, sectioned at 10 miera, stained
with Standard Alum-Hematoxylin and Triosin (pH 5.4-5.6), and mounted

9
with Perraount. In material representing the first four days post copu
lation, both sides of the Fallopian tubes and the entire uterus were
sectioned. For stages from five through twelve days sections of the
uterus containing implants were excised and the embryos were sectioned
in tero. In each case embryos were sectioned both transversely and
parallel to the long axis of the uterine horn. In addition, embryos
ten days of age and older were removed from the uterus, dissected free
of all fetal membranes and sectioned transversely, A description was
made of external form and the extent of internal development from ten
through eighteen days of age,
A number of measurements was made to determine changes in embryo
size and volume with age. Due to the small size of the specimens no
volumetric or weight measurements were attempted for embryos ten days
of age ard under and all measurements were made with a compound micro
scope and an ocular micrometer. For specimens ranging from twelve
through eighteen days gestation measurements of length end width were
made with the aid of a pair of fine (bolt-controlled) dividers under
5x magnification. Volumetric measurements were also made of this se
ries of embryos. The method employed was to select micro-vials of a
size appropriate for the embryo to be measured. Each of these vials
was ringed with a diamond point marking device, and its volume to that
line was determined. The embryo was placed on filter paper and rolled
around with the aid of a camels hair brush until it appeared to be dry
and then introduced into the previously standardized vial. The vial
was filled up to the marked ring from a 5 ml. Burette that could be

10
read to l/lOO of a ml. The measured amount of water that it took to
fill the vial containing the embryo, subtracted from the known volume
of the vial gave a difference which represented the volume of the em
bryo. Each embryo was measured three times and an average was obtained.
Volume determinations made in this manner were quit consistent, often
yielding identical readings for each of the three measurements.
Weights were also recorded for this series of embryos after
they had been blotted as previously described, placed in stoppered
weighing bottles, and weighed on an analytical balance.
The description of prenatal development i3 based upon a study
of sixteen whole specimens and of fifty-four eggs and embryos which
were sectioned. The stages studied are listed in Table 1.
Age
0 hours
15 hours
24 hours
43 hours
60 hours
72 hours
TABLE 1
LIST OF EGGS AND EMBRYOS STUDIED
Number of
Specimens Stage of Development
5 Newly ovulated eggs surrounded by
corona radiata
4 Pronuclear
3 2-cells
3 4-cells
3 4-cells
5 Morula in lower end of tube, and
blastocysts in uterus
Blastocysts, morula, and 2-cell stage
in uterus
96 hours
4

11
Age
5 days
6 days
7 days
8 days
9 days
10 days
11 days
12 days
13 days
14 days
15 days
16 days
17 days
18 days
TABLE 1 Continued
Number of
Specimens Stage of Development
2 Implanted embryonic vesicle with be
ginning of proamniotic cavity
3 Egg cylinder, proamniotic cavity re
stricted to embryonic ectoderm
3 Proamniotic cavity continuous into
extra-embryonic ectoderm; posterior
amniotic fold beginning
3 Presence of 3 cavities; amniotic,
exocoelom, and ectoplacental
4 Allantois present, Somites present in
one embryo, not yet developed in others
4 Body form U-shape, allantoic stalk joins
chorion, and appearance of first aortic
arch
4 Body form tightly coiled C; limb buds
present
4 Tail distinct; anterior limb buds
fan-shaped
3 Face formed, features not yet discern
ible as mammalian
3 Resemble adult; vibrissae follicles on
each side of snout
4 Digits outlined but fully webbed; mammae
present; no hair follicles on body
3 Digits of forelimbs deeply notched; plan
tar tubercles present; hair follicles
present on body
4 Digits fully formed; eyes half closed
During the course of this investigation data were obtained cm

12
parturition, litter size, sex ratios at birth, gestation, post-partum
estrus, and the normal estrous cycle in P. polionotus.
Observations on postnatal growth and development were made on
thirteen litters of mice totaling forty-four individuals. These were
carefully observed from the time of birth up to fourteen days, with
measurements and weights being taken at two-day intervals. From four
teen to thirty days measurements were made every four days, and from
thirty days to fifty or sixty days, weekly measurements were made.
Weights and measurements were started on some litters at two days of
age and at three days of age on other litters, and all measurements
after four and five days of age were made on anaesthetized animals.
The measurements taken were those generally employed by mammalogists,
that is total length, tail length, hind foot length, and length of
ear from crown.

DESCRIPTION OF PRENATAL DEVELOPMENT
Zero Hours
Two recently ovulated eggs were found in the right Fallopian
tube and three in the left of a specimen killed immediately upon find
ing motile sperm in a vaginal smear. Each egg appeared as a somewhat
spherical, homogeneous mass of lightly staining cytoplasm. There were
no visible nuclei in any of the sections, nor were any polar bodies
present. It could not be ascertained whether or not these eggs were
fertilized, although sperm were present in the Fallopian tubes at the
level of the ova. Each ovum was surrounded by a corona radiata which
consisted of a dense mass of deeply-staining cells, and a wide zona
pellucida was evident between the egg and the corona cells (PI. I,a).
The maximum dimensions in the plane of section of these ova were, in
miera: 62 x 46.5; 69 x 54.2; 62 x 54.2; 62 x 46.5; 62 x 50.
Fifteen Hours
Embryos representing the pronuclear stage were obtained from
a mouse killed fifteen hours after a few non-motile sperm and sperm
fragments were found in the vaginal smear. In most pregnant females
which had mated many motile sperm were found in the vaginal smears.
It seems apparent, therefore, that a longer period of time had elapsed
between copulation and the making of the smear in this particular
specimen than was usually the case. However, under the method of age
determination used in this work, this must nevertheless be recorded

14
as a fifteen-hour stage.
The four embryos found were centrally located in the large
lumen of the ampulla of one Fallopian tube and were not in close as
sociation with the mucosal lining of the tube. A few sperm were also
visible lying in the lumen of the tube. A disintegrated corona radiata
appeared in the form of mucoid-like strands surrounding each embryo.
The embryos were surrounded by a fairly thick zona pellucida and be
tween this membrane and the cytoplasm of the egg was a clear peri-
vitelline space. The sperm centrum appeared as a slight conieal pro
jection. The second polar body was located in the perivitelline space
producing a slight bulge in the encompassing zona pellucida (PI. I,B).
Various degrees of fusion of the pronuclei were evident in the series
of embryos. Fusion was complete in one, in progress in two others,
and had just commenced in another. In the latter case the pronuclei
were in close juxtaposition but the nuclear membranes were still in
tact. The pronuclei in each case were located approximately in the
center of the egg. Size differences were evident in the pronuclei,
the smaller perhaps representing the male pronucleus as suggested by
Huber (1915) and Smith (1939). The larger pronucleus was elliptical
in shape and the smaller one appeared more round. The greatest length
and width, in miera of the larger pronudeus were 18 x 14; and for the
smaller pronucleus 14 x 14.4. One of the embryos measured 77.5 x 77.5
miera in greatest length and width, respectively, while the other
three were the same size, 77.5 x 54.2 miera.

15
Twenty-Four Hours
Three embryos obtained from a specimen killed twenty-four hours
subsequent to finding sperm in her vaginal fluid had undergone the
first cleavage division and were in the two cell stage. All were in the
isthmus of the same Fallopian tube, while the other contained several
degenerating ova. The cytoplasm of each blastomere was granular and
the nucleus was round and centrally located. The blastameres were
surrounded by the zona pellucida, but there was no evidence of corona
radiata cells at this stage (PI. I,C). The greatest length and width
of one typical two cell embryo were 93*0 and 62.0 raicraj the blastomeres
were equal in size. The nucleus of each blastomere had a diameter of
18 miera.
Forty-Eight Hours
Each of three embryos obtained at this stage was composed of
four cells and was still located in the Fallopian ttibes. They were
distributed two on one side and one on the other. The two embryos in
the isthmus of the same tube were located very close together as they
both appeared in several of the same sections. Smith (1939) obtained
two four cell stage embryos in a female sacrificed 47+ hours after
copulation.
The plane of section did not pass through all four of the
cells in any of these forty-eight hour embryos, so that individual
sections show no more than three cells (PI. I,D). The zona pellucida
surrounding the embryo was still distinct and of the greatest diameter

16
of the embryo at this stage was 62 miera.
Sixty Hours
Three embryos from a female sacrificed at sixty hours were
also in the four cell stage and exhibited no discernible advance over
those at forty-eight hours. However, they were slightly larger, with
average maximum diameters of 77.5 and 62.0 miera.
Seventy-Two Hours
Embryos representing the seventy-two hour stage were obtained
from two different females. These two sets of embryos showed sig
nificant differences in development. Three embryos from one female
were located in the distal end of the Fallopian tube and each was in
the eight cell stage of development (PI. I,E). The most distally lo
cated embryo was 120 miera from the tubo-uterine junction. One embryo
measured 62.0 miera in length and width while another was more elongate
and measured 775 x 46.5 miera. The third was damaged in sectioning
and could not be measured. Fragments of the zona pellucida still sur
rounded each embryo. Whether the appearance of the zona pellucida at
this stage of development was natural or due to injury in preparation
could not be determined.
The two embryos of the other set were in the uterine horns
and had already attained the blastocyst stage. Implantation had been
initiated, as the embryos were embedded in pits in the mucosa on the
antimesometrial side of the uterus. In one embryo the inner cell mass

17
was toward the lumen while in the other it was toward the mucosa of
the uterus. The trophoblast consisted of from fourteen to sixteen
cells, and the inner cell mass was composed of from eight to ten cells.
The average dimensions of the two blastocysts were 77*5 x 62.0 miera
in length and width, respectively. The subgerminal cavity was para
bolic in shape and measurements in miera of the greatest length and
width of each cavity were 38.75 and 46,5.
Ninety-Six Hours
A considerable variation in degree of development was exhib
ited by the four embryos secured from a single female at ninety-six
hours. Three embryos representing as many different stages of de
velopment were found in the left horn. The embryo which was located
nearest to the ovary was in the morula stage. Distal to this was an
apparently healthy two cell stage lying in the uterus, and still farther
from the ovary was an embryo of twelve to sixteen cells.
The late blastocyst found at this stage of development was
more elongate than the blastocyst seen in the seventy-two hour stage.
Its greatest length and width measurements were 93.0 x 46.5 miera.
The segmentation cavity measured 62.0 x 31*0 miera, the greatest width
of the cavity being at the level of the base of the inner cell mass.
This embryo was lying free in the uterine lumen near the neck of a mu
cosal pit on the antimesometrial side (PI. I,G).
The morula stage was lying free in the uterus and occupied a
position about equi-distant from either side of the wall (PI. I,F).
It was nearly spherical in shape and a segmentation cavity was

18
starting at one end of the embryo. It had a maximum length and width
of 62.0 x 54.25 miera.
The two cell embryo was lying eccentrically in the uterine lumen
toward the antimesometrial side. No discernible difference could be
seen between this embryo and the twenty-four hour old embryos previous
ly described. Each cell measured 31.0 x 31.0 miera.
The twelve to sixteen cell stage embryo was also free uterine.
Only one section of this embryo was found in its proper place on the
slide although several other sections which had been displaced during
staining were seen, hence no detailed description could be made. The
greatest width and length measurements taken from this single section
were 77.5 x 62.0 miera.
Five Days
Embryos at five days of age were implanting, and the implanta
tion sites were discernible upon gross examination as areas of slight
swellings and increased vascularity along the uterine horns. In the
fixed uterus, dimensions of one of these swellings were: length 4 nm.,
width 2,5 mm., depth 3.5 mm. The uterus in the area not directly con
cerned with implantation was almost uniformly round and had an outside
diameter of 2.0 mm. Snell (1941) in his description of implantation
in the white mouse, states that there is an appreciable swelling in
the uterus at the implantation site one day after implantation (five
days after mating).
Of the t hree embryos obtained at this age two were sectioned
for detailed study. The embryos were deeply lodged in the uterine
i

19
mucosa on the antimesometrial side, and the cellular organization of
the uterine mucosa in the region of the implanted embryo was changed
into cell masses and cords of cells surrounded by sinusoids (PI, II,A).
The mesometrial portion of the pit formed by the embryo was filled
with degenerated uterine epithelial cells.
The embryos of five days of age showed a marked increase in
size over the previous stage examined. The old germinal cavity had
elongated and formed the yolk cavity which was delimited by a thin
layer of trophoectodenn (Reicherts membrane) with scattered distal
entodermal cells applied to its surface. The inner cell mass had be
come differentiated into an egg cylinder extending about half way to
the floor of the yolk cavity. The ectoplacental cone, extra-embryonic
ectoderm, and embryonic ectoderm were easily recognizable. A constric
tion marked the line of junction of the extra-embryonic and embryonic
ectoderm. The latter consisted of a single layer of cuboidal cells
with large round nuclei surrounding a small cleft, the proamniotie
cavity; whereas the former was composed of an irregular mass of cells
with elongate nuclei. A single layer of large cuboidal embryonic en
todermal cells formed the outer layer of the portion of the egg cylin
der that protruded into the yolk cavity. The total length and width
of the egg cylinder, excluding the ectoplacental cone, was 108.5 x 46.5
miera. The ectoplacental cone, a conspicuous conical-shaped mass of
cells extending toward the lumen of the uterus, measured 46.5 miera
in length.

20
Six Day
Three six-day embryos were deeply embedded in the antime-
sometrial wall of the uterus, the lumen of the latter being reduced
to a very narrow slit toward the mesometrial side. The individual
embryos did not differ significantly in their development. The egg
cylinder which extended almost to the ventral wall of the yolk cavity
had increased in size from the condition at five days (PI. II,B).
This was due, apparently, to an increase in the number of cells rather
than to the growth of individual cells* The greatest length and width
measurements of one of the embryos at this age were 155*0 and 62*0
raicra, while the length of the ectoplacental cone was 62.0 miera. The
proamniotic cavity was present as a median slit confined to the embry
onic ectoderm and parallel to the long axis of the egg cylinder.
Seven Days
In three embryos obtained at seven days of age the elongated
egg cylinders had completely invaginated into the yolk cavity (PI. II,C).
The proamniotic cavity was continuous dorsally as far as the ectopla
cental cone, and was considerably wider than the slit-like cavity of
the previous stage. There was an infolding of cells at the junction
of the embryonic ectoderm with the extra-embryonic ectoderm which marked
the beginning of the posterior amniotie fold. Although a few mesodermal
cells viere present in this area, they did not yet constitute a definite
layer. Dorsal to the posterior amniotie fold, another infolding of cells
from one side of the extra-embryonic ectoderm into the proamniotic cavity

21
was apparent. The significance of this invagination could not be de
termined. Snell (1941) considered a similar structure in the white
mouse embryo as being possibly due to very rapid growth occurring in
the posterior wall of the egg cylinder at this stage.
The distal entodermal lining of the trophectoderm was uniform
ly established and consist i of a single layer of spindle-shaped cells.
The embryonic entoderm had changed from the cuboidal shape previously
noted to a thin layer of flattened and elongated cell3. However, the
entodermal cells lining the extra-embryonic ectoderm were still cuboidal
and closely packed, forming a fairly thick layer. The greatest main
axis and width measurements of a representative egg cylinder of this
age, excluding the ectoplacental cone, was 294.0 x 93.0 miera.
Decidual cells had extended across the lumen of the uterus and
had joined the mesometrial side of the uterine wall in these seven day
embryos.
Eight Days
Among the significant advances of embryos obtained at eight
days of age was the presence of a distinct layer of mesoderm that had
been proliferated from the region of the primitive streak (PI. II,D),
From their origin at the junction of the embryonic and extra-embryonic
ectoderm, the mesodermal cells had spread in all directions. The en
larged posterior amniotic fold had almost made contact with the an
terior amniotic fold. The latter consisted of a small projection of
cells into the proaraniotic cavity from the zone of junction of the
extra-embryonic and embryonic ectoderm at the anterior end of the

22
developing embryo. The large cavity in the posterior amniotic fold
was not yet lined with mesoderm..
The fore-gut vas apparent as an Invagination of the embryonic
entoderm on the anterior side of the embryo, at the junction of the
embryonic and extra-embryonic ectoderm. The greatest length and width
measurements of a representative embryo at eight days of age were 620.0
x 201.5 miera,. The ectoplacental cone measured 155*0 miera.
Red blood corpuscles completely filled the spaces between the
cell masses of the uterine mucosa in the region of the ectoplacental
cone, and also surrounded Reichert's membrane. In addition, in some
sections erythrocytes were observed between Reichert's membrane and
the embryo proper and even in the proamniotic cavity.
The uterine lumen was completely obliterated at this stage of
development.
Nine Days
There was marked variation in development among four embryos
of the same litter after nine days of gestation. In the embryo show
ing the earliest stage of development, the amnion had formed thereby
delimiting the amniotic cavity. The exocoelom was in the process of
being formed, as four large cavities appeared in the mesoderm of the
posterior and lateral amniotic folds. In sagittal section a total of
six distinct cavities could be seen in place of the old proamniotic
cavity (PI. Ill,A). The most ventral of these constituted the newly
formed amniotic cavity, the most dorsal the eetoplacental cavity,
and the four smaller cavities marked the developing exocoelom. The

23
head process was a broad layer of cells located at the ventral ex
tremity of the egg cylinder. It appeared to be continuous with the
mesoderm layer from the primitive streak and was directed toward the
anterior end of the developing embryo. The most anterior extension
of the head process was reduced to a very thin layer that appeared
to be continuous with the flattened embryonic entoderm. The total
length and width measurements were 775.0 x 310.0 miera respectively
and the length of the ectoplacental cone was 341.0 miera. The allan
tois had not begun to develop in this embryo.
A second embryo of this same litter was somewhat further de
veloped. Rather than the six cavities seen in the preceding embryo,
this embryo had only three cavitiest the amniotic, the ectoplacental
and the exocoelom (which was fully formed). The ectoplacental cavity
ms much smaller than the other two cavities. The amnion consisted
of a ventral layer of embryonic ectoderm and a dorsal layer of meso
derm while the chorion consisted of a ventral layer of mesoderm and
a dorsal layer of extra-embryonic ectoderm. The allantois was present
as a small projection of mesodermal cells from the posterior end of
the embryo into the cavity of the exocoelom (PI. II,?). The mesoder
mal layer limiting the allantoic projection was continuous with the
mesodermal layer of the amnion. The anterior part of the embryonic
ectoderm adjacent to the invaginating entoderm of the fore-gut was
elevated as the head fold. The notochord had differentiated from
the head process and was located on the ventral side of the egg cylin
der. On the ventral side of the notochord there was visible a slight

24
U-shaped depression similar to that which Snell (1941) termed the ar-
chenteron in the white mouse. The egg cylinder was more elliptical
in shape than in previous stages and its broadest part was at the
region crossed by the amnion. The greatest length and width of this
embryo were 852.5 and 403.0 miera, respectively.
In a third embryo of this litter the allantois extended about
midway into the cavity of the exocoelom. The size of the ectoplacen-
tal cavity was greatly reduced, forming a small crescent-shaped cavity.
The paraxial mesoderm had differentiated as a dense layer, although
segmentation into somites had not begun; a slight embryonic coelom
had formed in the intermediate mesoderm. Measurements of the greatest
length and width were 861.0 x 589.0 miera.
In the most advanced embryo of this litter the head fold was
large and its cavity was filled with head mesenchyme cells. Somite
formation had begun, and five somites could be counted. The archen-
teron still persisted as an entodermal invagination on the ventro-
posterior side of the embryo (PI, III,B).
The three embryos of this litter which were in the presomite
stage and still maintained the egg cylinder form, possessed maternal
blood within their exocoeloms and amniotic cavities. Since this blood
had the same appearance as that surrounding Reichert*s membrane and
filling sinusoids in the uterus, a close search was made of all sec
tions for a possible source of entry into these cavities. In one em
bryo a channel was discovered which appeared to have been formed by
the mesodermal layer of the chorion pushing through the ectodermal

25
layer of the chorion and extending across the ectoplacental cavity
encompassing blood sinusoids in the ectoplacental cons (PI. II,G).
By this means maternal blood was channeled directly into the exoco
elom. No source of entry for blood into the amniotic cavity was
observed.
Ten Days
There were four embryos in the litter taken after ten days of
gestation, and they were all fixed in tero. Two were sectioned in
tero; one was removed from the uterus and fetal membranes and sec
tioned, and the other was removed from the uterus and fetal membranes
and kept as a reference.
External
At this stage the body is U-shaped as the original axis that
was established at the formation of the primitive streak and the head
fold is largely maintained. The head process is at the upper right
side of the U, and the tail region is at the upper left. The dorsal
side of the animal is bent inward making the concavity of the U, and
the posterior part of the embryo is buried to the right. The area of
the future mid-gut forms the bottom of the 0 (PI. IV,A).
The total body length of a single fixed specimen was approxi
mately 2.86 mm. This measurement was taken using a binocular dis
secting microscope and an ocular micrometer. Due to the shape of the
embryo, it was necessary to make several short measurements along the
body and to add these together for the total length. No doubt, this

26
method is subject to error, but with fixed material it was about the
only one which could be employed.
The only conspicuous external feature was the fairly large
bulbous swelling representing the heart on the ventral side of the
animal just beneath the head procese.
Internal
A study of sectioned material showed that the mandibular arch
was fully formed and possessed the first pair of aortic arches. The
stomodeum appeared as a deep groove in sagittal section. The oral
plate had ruptured in one of the embryos, while in another embryo of
this same litter, it was still intact. Cephalic to the stcmodeal
opening was the triangular shaped pre-oral gut. The first pharyngeal
pouches were forming and in the median ventral wall of the pharynx
the thyroid plate had begun to evaginate.
The fore-gut continued posteriorly to about the lower level
of the pericardial chamber where it terminated in the anterior intes
tinal portal. The mid-gut was an open groove thus resembling the con
dition described by Snell (1941) in the white mouse of eight days and
eighteen hours development. At this stage, the length of the hind-gut
approached that of the fore-gut. The somatopleure of the coelom was
continuous with or closely applied to the amnion, while the somato
pleure of the extra-embryonic coelom constituted the wall of the yolk-
sac, The allantoic stalk was a loosely arranged mesodermal projection
extending through the extra-embryonic coelom and joining the chorion
(PI. m,c).

27
The anterior cardinal veins could be seen in the mesoderm of
the head and embryonic blood cells were visible lying in the lumen of
these vessels. The first aortic arch was complete.
The ventricle and sino-atrial regions of the heart were dis
tinct and numerous embryonic blood cells were enclosed by the endo
cardium of the ventricle. The omphalo-mesenteric veins and the allan
toic veins were confluent just posterior to the sino-atrial region of
the heart. The paired dorsal aortae extended posteriorly from the
first aortic arch, which contained embryonic blood cells in its lumen.
Although there were at least eight samites at this stage the exact
number could not be determined. They consisted of uniform masses of
cells without any organization into dermatome, myotome, and sclerotome
regions and lacked a myocoele.
The notochord was an obvious structure extending from the re
gion of the mid-brain to the caudal end of the embryo. Even though
it first differentiated from the head process several days earlier in
development, the notochord appeared to be still joined to the dorsal
entodermal wall of the gut. This is in line with the observations of
Snell (1941) on the white mouse. He found that: "For a considerable
period notochord and gut entoderm remain joined. Eventually, however,
the two halves of the gut entoderm grow across the ventral surface of
the notochord and unite in the mid-ventral line, leaving the notochord
as an axial, rod-like structure between ectoderm and entoderm. In the
head region, the neural groove was still open and the optic vesicles
had been differentiated from the diencephalon as large lateral

26
evaginations. Closure of the neural groove began in the region of the
fifth somite and continued caudally for approximately 150 miera, beyond
i-ihleh the right and left neural folds were closely approximated.
Eleven Paya
As previously noted no embryos of this stage were obtained.
Twelve Days
There were four embryos obtained for this age and one was
sectioned in tero, while the others were removed from the uterus
and fetal membranes before being studied. Giant cells were quite
numerous in the decidua adjacent to the embryo both laterally and
ventrally. Such cells were first seen in the uterus with five-day-old
embryos. It was not determined whether the giant cells persisted be
yond twelve days as embryos were not sectioned in tero beyond this
stage. Snell (1941) reports giant cells as a conspicuous feature ly
ing between Reicherts membrane and the decidua in sections of the
white mouse embryos of six to fourteen days.
External
At the twelve-day stage the body was tightly coiled with the
right side of the head overlying the short tail (PI, IV,B). The mes
encephalon protruded anteriorly as a hump on each 3ide of the dienceph
alon and the optic vesicles were apparent as wide evaginations. An
auditory pit was present on each side in the region of the hind brain
just dorsal to the hyoid arch. Both anterior and posterior limb buds
were present, the anterior being slightly larger than the posterior.

29
The heart as seen from the left side of the animal was a round, bulbous,
membranous swelling lying between the cephalic tip of the telencephalon
and the anterior linto buds. The tail vas well defined although short
and still sharply bent ventrally. Laterally, the prominent mandibular
and hyoid arches were apparent. The third arch was also present, but
was quite small and was somewhat obscured by the hyoid arch. The man
dibular arch had not yet fused to form the mandible. Nasal pits were
present, but the naso-lateral, maxillary, and naso-medial processes
had not yet fused.
Internal
Nervous
The brain had attained the five-vesicle stage and in cross sec
tion through its anterior portion, the lateral telencephalic vesicles
appeared as obvious lateral evaginations. 1 The infundibulum in the floor
of the diencephalon was in contact with the tip end of Rathkes pocket.
The optic vesicles had constricted on each side of the brain to form
the optic stalk. The optic cup had formed and the lens vesicle was in
the optic cup, although the former had not yet separated from the super
ficial ectoderm. Otocysts were well formed at the level of the poste
rior region of the hind brain and had become completely separated from
the overlying ectoderm. In this area the seventh and eighth cranial
nerve ganglia appeared adjacent to the ventro-lateral side of the newly
formed otocyst. The large fifth ganglion was present, as were ganglia
nine and ten and the roots of nerves eleven and twelve. Histologically
the spinal cord was somewhat differentiated into the inner ependymal,

30
middle mantle, and outer marginal layer of cells. Numerous blood
vessels were present in the mantle layer. At the anterolateral mar
gin of the maxillary arch, the superficial ectoderm had thickened
and evaginated slightly to form the olfactory pit.
Digestive
The oral cavity consisted of the stomodeal opening. The
tongue primordio were not recognizable as such. Rathkes pocket ap
peared as a hollow tube extending from the roof of the oral cavity to
the posterior tip of the diencephalon. The first three visceral
arches were present and the hyomandibular cleft on the left side ap
peared to have broken through. The third visceral arch contained
the third aortic arch, and in the floor of the pharynx the thyroid
diverticulum was present. Immediately di3tal to the bifurcation of
the esophagus and trachea, the trachea evaginated laterally to form
tx-ro bronchi. The left bronchus at this stage las larger than the
right. The esophagus was a short structure resembling a slit-like
tube and was roughly 150 miera long. In transverse sections, the
stomach appeared as a long slit-like structure running dorso-ventrally
in the body. A slight curvature of the stomach produced a convexity
toward the left side of the embryo. Pancreatic diverticula were pres
ent at the ventral limits of the stomach. An intestinal loop was
present in the body stalk. The liver was quite unorganized at this
stage, consisting of scattered cords of cells with numerous blood fil
led spaces between the cords. The transverse urorectal fold had di
vided the cloaca into a dorsal rectum and a ventral urogenital sinus.

31
The anal plate still persisted.
Circulatory
The obvious vessels anterior to the heart were the common car
dinals from which the anterior cardinals issued anteriorly and became
elaborately branched in the head region. The posterior cardinals were
well formed and unbranehed, while the dorsal aorta ivas paired in the
trunk region and gave off numerous segmental arteries to the body wall.
The first and second aortic arches had degenerated but the third was
large and still Intact at this stage. The heart was a large structure
and consisted of an auricle and a ventricle which were in broad com
munication with each other. As a result of the formation of trabecu
lae carneae the ventricle and conus arteriosus had became thick walled
in contrast to the thin membranous wall of the auricle. The interven
tricular septum had started to form on the ventral floor of the ven
tricle but at this stage it was merely a raised ridge of fibrous tissue.
The interatrial septum had also begun to form and was present as a
slight conical projection of cells from the dorsal roof of the auricle.
The vitelline veins at this stage were being broken up by the liver
cells and appeared indistinct but could still be recognized emptying
into the sinus venosus. The left umbilical vain had become large al
though it was still connected to the sinus venosus. Also, the vitel
line arteries were still paired channels, and hence had not yet given
rise to the anterior mesenteric artery.

32
Excretory
The mesonephroi were not massive at this stage of development.
The primordium of the metanephric duct which was solid except for it
hollow end paralleled the mesonephric duct for a very short distance.
Endocrine
Rathkes pocket was in contact with the infundibulum presag
ing the formation of the pituitary body and the thyroid primordium
was represented by a tubular mass of cells between the paired hyoid
arches in the floor of the pharynx.
Genital
Medial to the mesonephros vas a concentration of deeply stain
ing cells which constituted the beginning of the genital ridges.
Muscular and Integumentary
The somites had started to differentiate into regions, but
the myocoel was indistinct.
Thirteen Days
By the thirteenth day stage the body was in the form of a C
and was tightly coiled (PI. IV,G). Freso the region of the posterior
limb bud, the caudal part of the trunk and the tail raised up and
curled around so that the tail rested against the ventrolateral side
of the pharyngeal arches on the right side of the animal. The head,
at this stage, constituted at least half of the entire embryo. The
mesencephalon was a large dome-shaped prominence while the hindbrain

33
had a transparent membranous roof. The olfactory pita were more promi
nent than in the previous stage. The naso-lateral and naso-medial pro
cesses were in contact ventrally with the maxillary process, bit were
as yet incompletely fused. The eyes were quite evident and were lo
cated at the dorsal apex of the groove between the naso-lateral process
and the maxillary process. The lens was distinct but there was no sign
of eye lids. There was no pinna visible in the region of the auditory
pit. On the ventral side of the embiyo the heart bulge between the an
terior limb buds was very prominent, and the extreme ventral part of
the ventricular bulge was resting against the naso-medial process. The
large liver protuberance appeared to be continuous caudally with the
heart bulge. On the ventro-lateral side of the trunk between the an
terior and posterior limb buds was a slight ridge produced by the meso
nephros. The anterior limb buds were somewhat fan-shaped and appeared
to be slightly longer than the posterior linda bud, vhose shape was
hemispherical.
Internal
Nervous
The layers of the neural tube were much thicker and the neuro-
coel had become somewhat smaller than in the previous stage. The mar
ginal layer had the appearance of white matter, and surrounded the cord
except on the dorsal side. Numerous mesenchymatous fibers extended
through the white matter of the cord and penetrated deeply into the
mantle layer, forming blood vessels. In the region of the trunk, sym
pathetic ganglia were evident. The olfactory pits were much deeper than

34
in the previous stage and had formed large nasal chambers separated
from each other by a wide naso-medial process with naso-lateral proces
ses on each side. Jacobsen's organ was present as an evagination and
thickening of the ectoderm of the medial side of the nasal chambers.
At this stage there was no trace of the first cranial nerve. The eye
had advanced considerably since the previous stage as the lens had be
come separated from the outer ectodermal layer. It was quite spheroidal
and possessed a central cavity. The optic cup had a very thick inner
sensory layer and a thin outer pigmented layer. The otocyst had in
creased in size and the endolymphatic duct was a conspicuous structure
between the otocyst and the lateral plate of the hind brain. The ecto
dermal wall of the otocyst was much thicker on the ventral side adja
cent to the eighth cranial nerve ganglion than on the dorsal side.
The upper portion of the otocyst had begun to constrict, presaging the
formation of the upper vestibular pouch and the lower cochlear pouch.
Ganglia of the seventh and eighth cranial nerves had separated and the
seventh nerve could be traced down into the hyoid arch.
Digestive and Respiratory
The lateral extensions of the third pharyngeal pouches were
thick masses of cells constituting the primordia of parathyroid three,
and thymus three. The laryngotracheal groove was present in the floor
of the pharynx in the region of the fourth aortic arch. On each side
of the groove, a large arytenoid swelling had pushed up toward the
roof of the pharynx. The fourth pharyngeal pouch had evaginated lat
erally and turned ventrally. The terminal ends of the fourth pouch

35
viere thick showing the primordia of the parathyroid four, thymus four,
and the post-branchial bodies. Where the esophagus and trachea sepa
rated, the lumen of the esophagus was compressed dorso-ventrally while
the trachea was compressed laterally. Mesenchyme surrounding the
trachea was more densely arranged and stained much darker than adja
cent tissue vdiioh suggested the beginning of cartilage formation in
this area. As the esophagus and trachea extended posteriorly, their
lumens became so reduced in size that they were barely detectable.
The trachea remained the larger of the two tubes and increased in size
again before biftreating into right and left bronchi. The right bron
chus remained several times larger than the left until it again divided.
Five lung lobes were seen in sections at this stage, four on the right
and one on the left side. The right medial lobe while connected with
the other lobes of the right side was somewhat distinct and was enclosed
in its own pleural sac. The stomach had increased still more in size,
and the ventral lobe of the pancreas had become a conspicuous globose
mass of tissue just ventral to the stomach. The common bile duct and
gall bladder were discernible at this stage. In following the tail
sections anteriorly a very small tail gut could be seen. The cloacal
membrane was still present.
Circulatory
The greatest change in the heart was an inorease in thickness
of the wall of the ventricle. The four chambered condition was obvious
in the heart as the interatrial septum had extended deeper into the
atrial cavity, and the interventricular septum had become more prominent.

H
Hi chambers of the heart were till broadly connected. the large
left umbilical vein had invaded the liver proper and given rise to
the duelas venosos 'which was continent with the proximal end of the
vitelline veins. In the previous stage, the vitelline veins had al
ready broken up to form the hepatic portal eyetea. The right umbil
ical vein was fairly mall and lay Ineonspieoously in the lateral
liody wall. The persistent distal nd of the vitelline veins drained
blood fro the region of the yolk sac sad intestine into the liver*
dust ventral to the vitelline veins as the superior aesenterie ar
tery which in the previous stage vs the paired vitelline artery. In
the region of the anterior Hob buds, the subclavian artery extended
fro the dorsal aorta into each lisrb bud. The subclavian vein were
drained by the posterior cardinal veins. The stomal iliac arteries
arose from large umbilical arteries on each side and extended into
the posterior limb bude. the iliac veins of the posterior liad buds
were now formed and were drained by the posterior cardinal. Anteri
orly the vertebral artery as located just ventral and closely applied
to the neural tube. The internal carotid arteries lay Just dorsal to
the pharynx and the external carotid arteries were present In the tis
sues of t first visceral arch, the veins of the head region con
sisted of the large anterior cardinals, which were the most conspicu
ous vessels sees in sections, and the external Jugular veins which were
forming la the mandibular arch region. The third, fourth, and sixth
aortic arches were Intact at this stage#

37
Excretory
The mesonephroi were much larger than in the previous stage
and numerous collecting tubules were apparent in cross sections. A
condensation of mesenchyme forming the nephrogenous tissue of the
metanephros was visible around the expanded proximal end of the ure
ter. The combined mesonephric and metanephrie ducts led into the
urogenital sinus near its junction with the rectum.
Endocrine
The thyroid primordia were located between the paired hyoid
arches, and the lateral extensions of the third and fourth pharyngeal
pouches were forming parathyroid three and four, and thymus three and
four. The infundibulum had fused with Rathkes pocket presaging the
formation of the pituitary body.
Genital
The cells of the genital ridge had lost their mesenchymal ap
pearance and were large ovoid cells. A great deal of mitotic activity
was evidenced in tissue of this area.
Fourteen Days
After fourteen days of gestation the body was still C shaped
and torsion was no longer obvious, and the main axis of the embryo was
lying in one plane. Due to cervical flexure, the head was at right
angles to the trunk, and the anterior tip of the snout rested on the
heart prominence (PI. IV,D). The greatest bend in the trunk was in

38
the region of the posterior limb buds, while between the anterior and
posterior limb buds the trunk had straightened slightly from the pre
vious stage. The tail curled to the right and its tip rested on the
right side of the face just cephalad to the eye. The limb buds had
not changed in appearance from the previous day, except that the pos
terior limb buds had become constricted so that they too were fan
shaped. The mesencephalon still protruded as a hump, and the roof of
the medulla of the brain was slightly more opaque than in the previous
age. The mid-dorsal line of superficial ectoderm above the spinal
cord all the way to the caudal end of the trunk was still quite trans
parent. The face was forming, although the features could not be
identified as mammalian. The naso-lateral process had fused with the
medial nasal process, and both had in turn fused with the maxillary
process. The mandible had just barely fused in the mid-ventral line.
Although the mouth was open, the tongue was not apparent in the oral
cavity. The ventral body wall was transparent enough to allow the
visceral organs to be seen. At this stage, the umbilical hernia was
a slight twist of the intestine at the base of the umbilical stalk.
A small genital tubercle was present.
Internal
Nervous
The meninges of the brain appeared as a thin membrane outside
of the marginal layer of the brain. The telencephalic vesicles had
continued evaginatlng both laterally and anteriorly, until they

39
extended far craniad of the original roof of the telencephalon, and
the lamina terminalis was recessed in a deep groove between the cere
bral hemispheres. The cavities of the lateral ventricles were in
broad communication with the third ventricle, and the infundibulum
had continued its evagination from the floor of the diencephalon and
had turned down over the distal tip of Rathke's pocket. The vails
of the neural tube had thickened considerably since the last stage
described, and had compressed the lumen laterally so that it appeared
club-shaped with the narrow end toward the ventral side of the tube.
Ventro-laterally the tube was much thicker than elsewhere represent
ing primordia of the ventral horns. The neural tubes at this stage
were richly supplied with blood vessels.
Several changes were evident in the eyes. There was a cover
ing of superficial ectoderm over the lens, and between the lens and
this covering was a thin band of loose mesenchyme cells. These cells
diverged to each side of the lens and formed an aggregation on the
floor of the optic cup between it and the lens. The cells that had
already reached this position had lost their mesenchymal appearance
and were multinucleated. The lens at this stage was somewhat ellip
tical and its hemispherical cavity was restricted to its upper half.
This cavity was bounded on all sides by lens epithelium, except for
the floor, which consisted of cells from the inner wall of the lens,
among which fiber formation had started. A dorsal and lateral pouch
were forming from the semi-circular canal portion of the otocyst.
Ventral to this area, the otocyst was bent medially and continued as

an elongated cochlear duct which was adjacent to, and parallel with,
the first pharyngeal pouch. This pouch had narrowed presaging its
new role as the auditory tube, but as yet, its distal end showed no
signs of enlarging into a tympanic cavity. The nasal pits had deep
ened and had broken through into the oral cavity, thus establishing
the choanae. The vomero-nasal organ (Jacobsons organ) was now in
the form of tubular sacs extending for a distance through the nasal
septum. At this extreme distal end they were closed, but proximal
to this point a lumen was present. Its ectodermal wall was quite
thick. Fibers representing the olfactory nerve extended from the
floor of the nasal pit to the brain.
Digestive
The proximal end of Rathkes pocket as it extended upward
from the roof of the oral cavity had become greatly reduced in size
and existed as a small solid cord of cells. The tongue was deeply
undercut, and was a wide, flat U-shaped structure projecting from the
pharynx into the mouth cavity. The epithelial layer bordering the
upper and lower jaws was beginning to thicken forming the labio
dental ledge. Salivary glands had started to differentiate on each
side of the oral oavity just ventral to the jaw-cheek groove. The
thyroglossal duct was evident as an evagination from the floor of
the pharynx. The larynx was a solid epithelial plate in the floor
of the pharynx. A short connection existed between the trachea and
esophagus at their cephalic ends. Where they first separate, the
esophagus was larger but further caudadj it was reduced to a minute

Al
tube, and the traehea was somewhat larger. Both of these tubes were
surrounded by a condensation of mesenchyme. Each lobe of the lung
possessed branching bronchi, and cells of this area were still mesen-
chymatous in appearance. The stomach showed a further increase in
size although no folds or rugae had yet formed. The ventral lobe of
the pancreas now appeared to be larger than the dorsal lobe but both
had grown somewhat since the last stage. The liver had filled the
peritoneal cavity to the extent of producing ventro-lateral bulges in
the body wall. Its cellular substance had become more condensed so
that it more closely resembled the adult liver. The gall bladder was
saccate and was closely applied to the ventral side of the liver.
There was only a faint trace of the tail gut in the form of a solid
mass of entodermal cells just ventral to the caudal artery. It per
sisted in the tail region for only a few sections.
Circulatory System
In the head the anterior cardinal vein had become the internal
jugular vein with several tributaries from the brain region and the
short external jugular was present. Paired internal carotid arteries
were evident, also the basilar artery was seen ventral to the myelen
cephalon. More posteriorly, the basilar artery divided into two verte
bral arteries. The hyaloid artery was complete to the eye. The third
aortic arches were partially broken down and the remaining portion was
now the proximal end of the internal carotid artery. Leading from the
ventricle of the heart was a thick walled tube, the bulbous cordis,
which divided into the ascending aortic trunk on the left side, and

A2
the pulmonary trunk on the right. The sixth aortic arches were con
fluent with the pulmonary trunk and the ductus arteriosus was present
in the left side. Posterior to the aortic arches, the dorsal aorta
remained paired for a short distance, then the two descending aortae
fused. The coeliac artery branched off from the aorta and was seen
in the omentum between the stomach and the pancreas. In the heart,
the endocardial cushion of the atrioventricular canal had developed
to the extent that each atrium emptied largely into the ventricle of
the same side. However, since the interventricular septum had not yet
fused to the endocardial cushion the ventricles were still in communi
cation with each other through a small passage just ventral to the
cushion. The right and left atria, although separated by the septum
primum, were also still in communication with each other through the
interatrial foramen. The sinus venosus emptied into the right atrium
and the left common cardinal had swung over across the mid-line and en
tered the sinus venosus. Caudally the sinus venosus received the old
right vitelline vein which had become the inferior vena cava. The
ductus venosus had become a large vessel in the liver. It received
the large left umbilical vein from the body wall and the portal vein
from the liver and emptied into the sinus venosus. The much smaller
right umbilical vein received tributaries from the lateral body wall.
In sections passing through the mesonephros, the posterior cardinals
were seen, but the left posterior cardinal in that area was sometimes
amfti 1 and indistinct. The subcardinals also were present at this stage.

43
Excretory
The mesonephric duct was confluent with the metanaphric duct
for a short distance from the urogenital 3inus, and they had a common
junction with the urogenital sinus, An aggregation of mesenchyme cells
which was lying posterior and somewhat dorsal to the mesonephros and
surrounding the enlarged terminal end of the ureter represented the de
veloping metanephric kidney. In cross section through this region sev
eral tubules could be observed extending out from the ureter into the
developing metanephros.
Endocrine
The thymus and parathyroid complex from the third pouch was
3till in contact with the pharynx but the thymus had become greatly
elongated and its terminal ends were growing medially. The rapidly de
veloping thymus had grown further caudad than the thyroid gland and had
passed laterally on each side of the latter. The two thick walled post
branchial bodies each of which had a fairly large lumen had separated
from the pharynx and moved caudally down the neck. The suprarenal
glands were lying dorsal to the genital ridge and medial to the meso
nephros. They were simply a mass of glandular cells with no definite
shape.
Genital
The genital ridge at this stage was a uniform cellular mass
forming a longitudinal strip paralleling the medial side of the meso
nephros. The Mullerian duct was not differentiated. The genital

44
tubercle was prominent enough to appear in sections as a separate
structure for several (10 miera) sections between the ventral body
wall and the tail.
Skeletal
Although there was no differentiation of skeletal material at
this stage* heavily staining concentrations of mesenchyme occurred in
the region of the otic capsules, centra, neural arches, girdles, and
limb buds. There was also a concentration of mesenchyme around each
nasal pit foreshadowing the formation of a paranasal cartilage and of
the nasal septum.
Muscular and Integumentary
While no muscle fibers had formed the eye muscles were outlin
ed for part of their course as aggregations of mesenchyme cells. On
the ventro-lateral side of the trunk, small conical knobs of epidermal
cells were present. These were the primordia of the mammae but they
were not macroscopically visible at this stage.
Fifteen Days
External
By fifteen days the head was bent forward at right angles to
the trunk and the mesencephalon still protruded anteriorly, but the
back was fairly straight (PI. IV,E). The lower jaw rested against the
dorsal side of the heart prominence. A short snout had developed and
it was elevated free from the underlying organs. The tip of the tail

45
rested against the snout on the right side of the face. The external
nares approached their adult form, and with the development of the
snout, the mammalian nature of the embryo could be recognized. Vibris-
sae follicles occurred on each side of the snout and a single large
follicle was present between the eye and ear on each side of the body.
The mouth had closed somewhat and the tongue was barely visible some
distance back in the mouth cavity. In the region of the fourth ven
tricle, the membranous roof was slightly more opaque although the
floor of the fourth ventricle could still be seen and the transparency
of the skin over the neural tube continued from the fourth ventricle
to the tail. Sagittal and coronal sutures were obvious beneath the
skin of the head. The ear pinna was a short, triangular projection
from each side of the head. On the ventral side, the mammae were
still not discernible macroscopically, but the umbilical hernia had
enlarged. The anterior limb buds were directed somewhat caudad, while
the hind limb buds were directed somewhat cephalad. Slight radial
ridges in the distal end of the anterior limb buds marked the forma
tion of digits.
Internal
Nervous
The walls of the brain were thicker and more highly vascu
larized than in the previous stage. The epiphysis was a hollow stalk
and its cavity was continuous with the diocoel. Intensive thickening
of the walla had greatly reduced the lumen of the mesencephalon and

resulted in the formation of the corpora quadrigemina. In the meten
cephalon the central vails were quite thick and the lumen vas large.
The myelencephalon had a membranous roof and an eren larger lumen.
In the anterior region of the myelencephalon the wall had developed
a sulcus limitans which divided the lateral plate into the dorsal
alar plate and the ventral basal plate. However, eaudad to this area,
the lateral plates diverged to the extent that the right and left
plates were lying almost in the same plane and the tela choroidea
covered not only the dorsal part of the tube but the dorso-lateral
part as well. The choroid plexus had developed in the roof of the
myelencephalon and its leaf-like folds pushed down into the fourth
ventricle. The tela choroidea of the roof plate had invaginated in
to the lateral ventricles giving rise to the choroid plexus of this
area. From the ventro-lateral wall of each hemisphere, the corpus
striatum had developed as a lobular fold bulging into the ventricles.
It was quite massive in size but did not show any special cellular
differentiation. The foramina of Monroe were reduced to small pas
sages. The thalamus showed considerable increase in size over the
previous stage. Olfactory lobes were present on the ventral side of
the cerebral hemispheres and received nerve fibers from the olfactory
pits. The walls of the neural tube had thickened, and in so doing
had constricted the neuroooel into a club-shaped cavity which was re
stricted to the dorsal half of the tube. There was a slight indenta
tion of the floor plate presaging the formation of the ventral median
fissure, Sctodermal folds, the forerunners of the lids, were evident

47
on each side of the eye, The most notloable change since the previous
stage was the complete obliteration of the lens vesicle by fibers,
whioh were still nucleated. Also, the pigmented layer of the retina
had become infiltrated with a black granular pigment. The nervous lay
er, pars optica, was thicker than in the previous stage. Its inner
fibrous layer adjacent to the optic cup cavity had become more dense,
and from this layer, fibers constituting the optic nerve extended in
to the optic stalk and could be traced to the brain. The pars coeca
and ora serrata were as yet not recognizable.
The external ear was a large depression in the region of the
old first branchial arch. In cross section, the floor of the external
auditory meatus consisted of ridges which were the developing hillocks.
The mesodermal band destined to form the tympanic membrane between the
invaginating ectoderm and the evaginating entoderm of the first and
second pouch was still quite thick. The auditory tube had formed fresa
the narrowed first pharyngeal pouch. At its distal end it appeared to
be joined by a tubular extension of the second pouch to produce a cav
ity which was the beginning of the tympanic cavity. Adjacent to this
cavity primordia of the auditory ossicles could be seen as patches of
pre-cartilage cells. The inner ear at this stage seemed to have ap
proached its definitive structure. The distal id of the endolymph
atic duct had become saccate; the semi-circular canals were formed,
and the cochlear canal had ccsnmenced to coil. The nerve fibers of the
olfactory nerve which were present in the previous stage were now more
dense and had become organized into a definite nerve tract. The medial

wall of the vomero-nasal organ was about twice as thick as the oppos
ing wall. Since the tongue had not yet retracted from between the
palatine processes, the nasal septum had not fused posterior to the
palatine process.
Digestive
The tongue extended well forward between the palatine pro
cesses and was almost in contact with the posterior end of the nasal
septum. It contained blood vessels, muscle fibers, and nerve fibers.
Both the submaxillary and sublingual glands were prominent and the
parotid glands which developed at the angle of the mouth were also
present. The labio-dental ledge had developed in the oral epithelium
of both the upper and lower jaws* The larynx was now an open struc
ture ventral to the pharynx with the space between the two occupied
by large arytenoid swellings. The esophagus and trachea were further
separated from each other and the concentration of mesenchyme around
the trachea had become more dense. No thyroglossal duct was present
at this stage. The esophagus was very small in diameter, and in its
more distal region the lumen was hardly discernible. The liver had
become more massive and was filled throughout with branching sinus
oids. The gall bladder was bulbous, and was now superficially embed
ded in the liver, occupying the same relative position as in the adult.
In the intestine the imiscularis layer was defined and numerous folds
were present in the lining of the duodenum but were hardly discernible
in the intestine below this area. The spleen was not detectable at
this stage. The dorsal lobe of the pancreas had grown considerably

49
and now appeared as large as the ventral lobe, although due to the
irregular shapes an exact comparison was impossible. The anal ori
fice was present, but was partly occluded by epithelium.
Circulation
Th8 auricles of the heart were quite large, but appeared col
lapsed in the sectioned material. Both atrioventricular canals were
greatly restricted and valves were forming in this area. Separating
the two atria was the well developed septum primum, but the atria were
still in communication through the interatrial foramen. The sinus
venosus vas greatly reduced in size and its opening into the right
atrium vas guarded by a well developed valvolas venosae. Fusion of
the interventricular septum with the endocardial cushion still had not
been accomplished, leaving the two ventricles in communication with
each other. The ventricles appeared to be relatively smaller than in
the previous stages, the walls having become thicker and the chambers
less extensive. There was no change in the veins returning blood from
the head region. The common cardinals fresa each side emptied into the
sinus venosus, and there was no sign of an innominate vein. In the
fourth aortic arches, the left side was larger than the right which
was indicative of their different fates, since the left persists as
the systemic arch while the proximal portion of the right becomes the
root of the right subclavian artery. There was also a noticeable dif
ference in the size of the right and left sides of the sixth aortic
arch. The larger left arch retained its connection with the dorsal
aorta by means of the well-developed ductus arteriosus, while the

50
smaller right arch had lost contact with the dorsal aorta. Caudad
to the heart coursed the small posterior cardinals along with the
supra cardinals. The left supra cardinal was located close to the
dorsal aorta and throughout the t runk region the right and left
supra cardinals were joined by transverse connecting veins.
Excretory
The genital tubercle had continued to increase in length,
and appeared for a number of sections between the tail and ventral
body wall. The right and left ureters still emptied into the uro
genital sinus along with the mesonephric duct3. The mullerian duct
appeared at this stage for the first time as a short solid rod lying
lateral and parallel to the mesonephric duct for about one fourth of
the length of the latter.
Endocrine
Differentiation of the pituitary body was nearly complete.
The neural lobe was still tubular, and the anterior wall of Rathke18
pocket had thickened producing a glandular anterior lobe. Issuing
anteriorly from the anterior lobe toward the diencephalon was the glan
dular pars tuberalis, while the pars intermedia existed as an evagin-
tion from the anterior wall of the anterior lobe. Below the pituitary
body the old Rathke's pouch persisted as a small solid cord of calls
traceable to the roof of the oral cavity.
The thyroid gland had changed in shape from the triangular
lobe seer, in the previous stage to a more flattened trough-shaped

51
structure. Each side of the thyroid extended dorsaUy to the lateral
side of the trachea where it fused with a hollow post-branchial body.
The thymus bodies were ventro-lateral to the thyroids. They had be
come much larger, and more lymphoidal in appearance than the thyroids
but the two lobes were still broadly separated in the midline.
Skeletal
In the cranial region, the nasal septum and Meckel's carti
lage were being laid down as dense concentrations of pre-cartilage
mesenchyme. Further caudad the centra, neural arches, and ribs were
also easily recognizable, and in fact the portion of the centrum
around the notochord and dorsal part of the rib appeared somewhat car
tilaginous. The bones of the girdles and appendages were likewise
represented by concentrations of mesenchyme, with some areas having a
cartilaginous appearance.
Muscular and Integumentary
Concentrations of muscle primordia were outlined in the ap
pendages as well as in the region of the intercostal muscles. The eye
muscles were also delineated but had not become fibrous, and the muscle
layers of the esophagus and intestine were outlined. The mammae were
increasing in size.
Sixteen Days
External
By sixteen days the back was almost straight and the abdomen

52
appeared more distended (PI. V,A). The mesencephalon was still visible
as a slight hump and a short neck region was more obvious. The eyes
and ears were still open, and the mouth was slightly open with the tip
of the tongue extending almost to the lips. Vibrissae follicles were
more conspicuous on each side of the snout and between the eye and ear
than in the previous stage. There were also several follicles located
just dorsal to the eye orbit and on the chin, but there were no fol
licles visible on the body. There was a slight indication of develop
ing mammae. The umbilical hernia had increased in size through more
extensive coiling of the gut. The genital tubercle had become quite
long, and was covered by the tail as it bent ventrally and was directed
anteriorly. The shape of the external nares and general facial fea
tures were more adult-like. Digits on the fore and hind limbs were
deeply outlined but were still fully webbed and showed dorsal and
ventral differences.
Internal
Nervous
The epiphysis was a tubular structure containing a large lumen
and situated in the median roof of the diencephalon. In the region of
the diencephalon, the neural canal was greatly reduced. The cerebral
hemispheres were broadly merged with the diencephalon and fiber tracts
passed from the thalamus into the corpus striatum of each hemisphere.
In the mesencephalon the corpora quadrigemina were still more promi
nent on the dorso-lateral side while the mesocoele was still further

53
reduced. The walls of the metencepholon were thicker but the cavity
was very broad and there was no indication as yet of the cerebellar
rudiment. The choroid plexus had proliferated in all directions in
the roof of the fourth ventricle. The neurocoel of the spinal cord
was compressed laterally producing a slit-like lumen which had a
width of about two thirds of the spinal cord. There was a slight de
pression representing an early stage of the ventral fissure, but at
this stage, there was no sign of the dorsal fissure. In the eye,
there was a dense mesenchymal sclera on the outside of the pigmented
layer and it had not yet begun differentiating into fibers. The pig
mented layer of the retina was reduced in width from previous stages.
Lacrimal glands and ducts had begun to form. The inner ear showed
very little change from the previous stage other than that it was now
completely encased in precartilage cells.
Digestive
The tongue had further differentiated. Transverse muscle fi
bers were present in its body; and also, two thick longitudinal bands
of muscle extended from the root into the body of the tongue. The
submaxillary and sublingual glands had pushed deeper into the gum from
their point of origin and had become more lobular. The esophagus was
still quite small although a lumen was traceable throughout its length.
There was very little change in the lungs other than an increase in
size of the lobes and an increase in the number of bronchioles in each
lobe. The tissue itself still had a mesenchymatous aspect. The

54
infolding of the duodenal walls had continued but the rest of the
gut possessed a smooth lumen. The mesentery suspending the colon
had narrowed down so that it resembled a typical mesocolon. The
spleen had increased in size and was attached to the mesentery ad
jacent to the stomach and lateral and parallel to the mesonephros
on the left side of the fetus.
Circulatory
The right fourth aortic arch showed further modification to
ward its adult relationship as it had formed the base of the right
subclavian artery. The left subclavian artery had formed from the
systemic arch. Valves had developed in the truncus arteriosus. The
subclavian veins were connected with the common cardinal veins on
each side. No further changes were noted in the circulatory system
at this stage.
Excretory
Near the base of the genital tubercle, where it became con
tinuous with the ventral body wall, the urethral groove appeared as
a thick ectodermal ridge, which extended for about thirty microns to
the urogenital sinus. The latter extended dorsally and branched into
two horns which received the mesonephric ducts. The urinary blaader
made its appearance at this stage as the extended end of the urogeni
tal sinus, around which mesenchyme from the ventral body wall con
densed forming its walls. From each kidney a ureter extended to join
the base of the bladder laterally. This was the first stage in which

55
the mesonephric ducts and ureters emptied separately.
Endocrine
The post-branchial bodies had fused with the thyroid gland.
The two lobes of the thymus gland lay close together in the midline
of the fetus. Both lobes were large and highly vascular, but the
left appeared larger than the right.
Genital
The mullerian ducts had further differentiated into a tubu
lar structure that could be traced some distance. Caudally they were
incomplete and did not extend to the urogenital sinus.
Skeletal
This stage marked the first appearance of cartilage in the
cranial region. It was present in all areas of the cranium where car
tilage bone develops. Also, the otic capsules were well outlined in
cartilage. The areas of dermal bones were represented by concentra
tions of mesenchyme cells and a small amount of fiber formation by
these cells. Centra, neural arches, and the dorsal part of ribs were
clearly cartilaginous. The ventral part of the ribs and the sternum
vrere less well defined. The bones of the girdle and the proximal long
bones of both forelimbs and hind limbs were preformed in cartilage. The
bones in the distal end of the fore and hind limb were represented by
mesenchymal concentrations. Meckels cartilage and the cartilage of
the nasal septum were well defined, whereas around the larynx there was

56
a massive concentration of mesenchyme cells.
Muscular and Integumentary
The muscles of the eye, tongue, shoulder, upper parts of the
appendages, and intercostal musculature were outlined.
Seventeen Days
External
At seventeen days the crown of the head was smooth and with
out a mesencephalic hump (PI. ¥,B). The head had become smaller in
relation to total body size and was less sharply bent forward so that
the chin no longer reached the chest. The digits of the forelimbs
were partly webbed, while the digits of the hind limbs were deeply
ridged and still fully webbed. Plantar tubercles had made their ap
pearance on the soles of the feet. Hair follicles were present on
the body in patches on the dorsal aspect of the fore and hind limbs,
and on each dorso-lateral side of the trunk. Follicles were also
present on the cheek and around the eyes, but were absent along the
mid-dorsal axis of the body, including the top region from the top of
the head forward to the nose. The eyes and ears were still fully open
although the ear pinna had turned anteriorly. The large distention of
the abdomen was reduced but the umbilical hernia remained prominent.
The mouth was only partly open with the tip of the tongue extending
to the lips.

57
Internal
Nervous
The epiphysis persisted as a tubular organ, and its lumen had
become larger than in previous stages. The choroid plexus had formed
from the roof of the diencephalon and extended into the cavity of the
third ventricle. Cavities had appeared in the corpora quadrigemina.
The continued development of the cerebellum had altered the metenceph-
alon. The thickening alar plates in this region of the brain had fus
ed dorsally and were covered with a thin oellular layer. The choroid
plexus was relatively better developed in the roof of the fourth ven
tricle. Fiber tracts between the thalamus and corpus striatum were
quite obvious. The neurocoel was roughly elliptical with its great
est diameter extending dorso-ventrally about one third the dorso-
ventral diameter of the spinal cord. Both dorsal and ventral fissures
were present in the spinal cord. The sclerotic coat of the eye had
become more fibrous and the choroid coat had further differentiated.
The lens eipthelium was separated from the inner layer of the cornea
by a space which was continuous posteriorly with the vitreous chamber.
The cartilage around the membranous labyrinth was filled with cells
which were mesenchymal in appearance. The withdrawal of the tongue
had permitted the palatine processes to fuse mesially and the nasal
septum had grown down to the fused palatines and was in the process of
fusing with them, thus completely separating the two nasal passages.
Jacobson* s organ continued as a dominant structure in the nasal septum.

58
In cross section it resembled two kidney shaped tubes vdth crescent
shaped lumens. The epithelium of the convex side of each lumen was
several times thicker than on the opposite side.
Digestive
The body of the tongue had become more heavily striated with
muscle fibers extending transversely across the tongue. Lingual pa
pillae were discernible although there was as yet no sign of foliate
papillae. Incisor tooth buds were also present at this stage. The
salivary glands located on each side of the root of the tongue con
sisted of solid branched lobules which were becoming encapsulated by
aggregations of mesenchymal cells. The anal orifice and rectum were
occluded with epithelium.
Excretory
The urinary bladder was an extremely large structure with a
wide lumen and was still broadly attached to the ventral body wall.
The kidney was a massive structure which at this stage presented a
more round than kidney-shaped appearance. Numerous tubules and sev
eral incompletely formed Bowmans capsules were present. The ureters
were patent throughout their length. The mesonephric ducts were
still intact and emptied into the urinary sinus, which at this stage
may be called the urethra.
Endocrine
The thyroid gland had changed considerably in texture. Its
tissue consisted of a loosely vacuolated mass in which were forming

59
follicles that appeared to be filled with blood cells. The two thy
mus bodies were located at the anterior tip of the thoracic cavity
and at the external ventral border of the thyroid gland. They were
large lymphoid rods which were round in cross section and extended
some distance into the thoracic cavity.
Genital
The Mullerian ducts had grown down to the urogenital sinus
but had not yet .joined it: nor had the tubes of the opposite sides
yet fused to form the utero-vagina. The genital system was still in
an indifferent stage of development.
Skeletal
In the cranial region, ossification had begun in the areas
of dermal bone formation. Also, numerous blood vessels were present
in these areas. The otic capsule, which had previously become carti
laginous, now had mesenchymal cells in the region of the membranous
labyrinth. The ear ossicles were faintly outlined in cartilage. Os
sification had begun in the mandible. The sternum and the ventral
part of the ribs were becoming cartilaginous. In the region of the
digits of the fore and hind limbs only dense mesenchymal concentra
tions were present.
Muscular and Integumentary
All muscles were outlined in their characteristic band or
bundle shape, but no definite fibers were present.

60
Eighteen Paya
External
In the eighteen-day fetus the head appeared quite similar, to
that of a full tern fetus (PI, V,C). The pinna of the ear had folded
over the opening, but the opening was not tightly sealed. The eye
lids had formed and the eyes were half closed. The roof of the hind
brain was no longer transparentj however, organs could still be seen
through the ventral body wall. The chin was held well above the
chest, and the umbilical hernia had almost completely disappeared.
Hair follicles were now quite obvious and had extended to the mid-
dorsal line, on the dorsal side of the head forward to the nose, and
also on the ventral side of the animal, as well as other parts of the
body. The tongue had retracted a short distance, arid the mouth was
almost closed. Both the fore and hind listo digits were conspicuously
notched,
Internal
Nervous
The lumen of the epiphysis was still present} the extent of
the choroid plexus had increased in the third and fourth ventricles,
and the cerebellar rudiment was considerably thicker. In the spinal
cord, the neurocoel was a small egg-shaped cavity that was centrally
located. The fibrous sclerotic coat of the eye was now clearly evi
dent, and in front of the lens it continued as a roof of the anterior

61
chamber, which formed the inner lining of the cornea. The anterior
chamber was fully formed, and the lens was a spheroidal mass of fibers
with only a few nuclei, most of which were located in its upper hemi
sphere. The oval window could be seen in the cartilaginous framework
of the inner ear and the preosseus auditory ossicles extended to it.
The coils of the cochlea appeared complete. The maxillo-turbinal and
several ethmo-turbinals had made their appearance within the nasal
cavities.
Digestive
The outer layer of epithelium of the tongue had thickened and
now had many taste buds and papillae. Well defined foliate papillae
had appeared cn each side of the tongue and the longitudinal muscle
fibers had become more numerous in the root of the latter. The lumen
of the esophagus was greatly compressed dorso-ventrally. In the tho
racic cavity the right bronchial tube was larger in diameter than the
left. The lobes of the lungs had increased in size and the bronchi
were more numerous, although they were smaller in diameter than in
earlier stages. There were numerous coils in the intestine and its
walls seemed to have approximated definitive form. Mucosal folds were
evident in both the small and large intestine but only a few could be
seen in the stomach. The mesentery suspending the intestine had be
come greatly reduced in thickness. The pancreas was quite diffuse
and consisted of numerous small lobules connected by cellular cords.
In some areas the dorsal and ventral lobes appeared to merge with each
other. The gall bladder was superficially connected to the liver and

62
had become more saccate. It closely approached definitive fora and
had lost the ventral mesentery attachment. The spleen was well de
veloped, occupying the area between the left gonad and the lateral
body wall.
Circulatory
Completely enucleated red blood cells appeared at this stage.
The atria were still in communication by way of an opening in the sep
tum primum, the interatrial foramen and the septum aecondum had just
begun forming as a slight projection of cells from the dorsal walls of
the atria. The cavities of the ventricles were reduced in size due to
the increase in thickness of the muscular walls. They were now com
pletely separated from each other by the fusion of the interventricular
septum to the endocardial cushion. The mitral and tricuspid valves
had formed.
Excretory
The bladder was a large round mass with a slit-like lumen and
cells of the bladder wall had become differentiated into epithelium.
The kidney had acquired its characteristic shape, and had a well-defined
darker staining cortex and a lighter staining medullary portion. De
finitive tubules were abundant and Bovman's capsules with simple squa
mous epithelial linings and well formed glomeruli were present. The
mesonephros was greatly reduced in size from previous stages.
Genital
In the material on hand, sex could not be determined at this

63
stage. The gonad was made up of a mass of round cells which showed
many mitotic figures, but development had not progressed far enough to
identify this structure as either ovary or testis. The mullerian
ducts were still in the indifferent stage but liad now fused in the mid
line distal to the mesonephros forming the utero-vagina. Beyond this
point they joined the urethra but they separated again before entering
the urethra.
Skeletal
There was an increase in ossification areas in the dermal bones
of the skull at this stage, and ossification had started around Meckel's
cartilage of the mandible. Tooth germs for both molars and incisors
were present but the incisors were farther along in development. The
dorsal part of the neural arches and neural spines had still not formed.
Ossification had begun in the proximal long bones of the fore and hind
limbs, while bones of the phalanges were becoming cartilaginous.
Muscular and Integumentary
The muscles of the eye were in the form of fibrous strands, and
those of the tongue were also fibrous. Muscles of the heart resembled
definitive cardiac muscle. However, in none of the skeletal muscles
were transverse striations evident at this stage.

PRENATAL GROWTH
Measurements of Uterine Swellings at
Implantation Sites
Implantation sites were seen as obvious swellings in the
uterine tubes of pregnant females five days after sperm were found in
their vaginal smears. These swellings showed a daily increase in size
with the growth of the embryo. Measurements made on fixed material
from five to eighteen days development are presented in Table 2.
TABLE 2
MEAN MEASUREMENTS IN MILLIMETERS OF
UTERINE SWELLINGS
Age
In Days
Number of
Swellings
Length
Width
Depth
5
1
3.0
2.5
3.5
6
1
3.5
3.0
4.0
7
3
3.5
3.2
3.5
10
1
5.5
5.5
6.5
12
2
5.5
6.0
7.0
13
3
7.6
7.5
9.0
14
2
8.5
7.5
9.5
15
2
9.2
8.2
10.0
16
2
10.0
9.0
10.0
17
2
12.5
9.0
11.0
18
3
14.5
11.0
12.0
Material representing the eighth and ninth days was sectioned
before measurements were taken, and there were no embryos obtained
representing the eleven-day stage of development. Hence, no conclusion
as to growth rate could be drawn from eight through eleven days of
64

65
development. Actually, the implantation sites showed very little dif
ference in size for the first several days, but from twelve through
eighteen days of age a daily size increment was quite pronounced.
There was only a slight variability in the measurements of swellings
in the same uterus.
Since pregnant females are often collected in the field some
knowledge of the relative size of uterine swellings for each day of
gestation is advantageous in determining the approximate stage of preg
nancy. On the basis of the present data, it appears that the age of
embryos can be determined from the size of the uterine swellings with
in a range of several days.
Weights and Volumes of Embryos
After they had been removed from the uterus and stripped of
their fetal membranes, fixed embryos from twelve through eighteen days
of age were weighed and their volumes determined. These data are pre
sented in Table 3. Due to their small size embryos younger than twelve
days could not be dealt vdth by this method.
TABLE 3
MEAN WEIGHTS AND VOLUMES OF EMBRYOS
12-18 DAYS OF AGE
1 Age of
Embryo
tiiier
Size
Volume in
Millimeters
heights in
Grams
12
2
.0100
.0057
13
4
.0232
.0281
14
3
.0453
.0456
15
3
.0800
.0801
16
3
.1390
.1335
17
3
.2276
.2115
18
4
.4092
.3851

66
Weight is one of the most valuable measurements for deter
mining growth, and volume is of value in giving an approximation of
size. There was a very close parallel between weights and volumes
in each group of embryos measured. A continuous inert ate in weight
and volume was seen from twelve through eighteen days of age. Rela
tive growth was more rapid after fifteen days of age, the greatest
increment occurring from seventeen to eighteen days of development.
Since the mean birth weight of this species was found to be 1.6l
grams it appears that the greatest increase in weight must take place
between eighteen days and parturition, which occurs normally at
twenty-three or twenty-four days. Such a rapid increase in growth to
ward the termination of the gestation period is typical of most mam
malian prenatal developmental curves.
Measurements of Certain Body Parts of Embryos from
Twelve Through Eighteen Days of Development
Various linear measurements made on embryos from twelve through
eighteen days of development with a pair of fine (bolt-controlled) di
viders under 5x magnification included: crown-rump length, head breadth,
head length, shoulder breadth, length of foreleg, length of hindleg, and
tail length. The averages are given in Table 4, and are represented
graphically in Figure 1.
These data indicate that the crown-rump length, head breadth,
head length, shoulder breadth and tail measurements exhibit approximate
ly the same relative growth trend. In eaeh case from thirteen through
f
seventeen days of age, growth proceeded at a fairly constant rate.

67
The interval between seventeen and eighteen days of age showed a marked
increase in the relative growth rate in crown-rump length, head length,
and shoulder breadthj but the increase in head breadth and tail length
was less pronounced. There was a marked increase in the crown-rump
length between twelve and thirteen days of age which slightly exceeded
the increase seen in this same measurement from seventeen to eighteen
days of development. Since the method of age determination used was
necessarily only an approximation it is possible that a greater age dif
ference existed between embryos of twelve and thirteen days than between
embryos of seventeen and eighteen days of age.
TABLE 4
AVERAGE MEASUREMENTS OF BODY PARTS OF EMBRYOS IN MILLIMETERS
Age
in
Days
Number
in
Litter
Crown
Rump
Head
Breadth
Head
Length
Shoulder
Breadth
Length
of
Foreleg
Length
of
Hindleg
Tail
12
2
3.7
*
4
*
4
a
13
4
6.4
1.1
3.5
2.6
1.1
1.0
2.1
14
3
7.2
2.5
4.4
3.7
1.5
1.5
2.8
15
3
9.0
3.1
5.1
4.3
1.8
1.7
3.2
16
3
10.1
3.6
6.0
4.7
2.2
2.1
3.7
17
3
11.3
4.2
6.7
5.2
3.2
3.2
4.0
18
4
13.4
5.1
8.2
7.3
4.5
4.5
4*6
*Too small and undifferentiated to measure at this stage.
The mean lengths of the fore and hind legs were similar. A
fairly uniform growth rate was noticed in both from thirteen to six
teen days of age, however the rate of growth was three times as great
for each twenty-four period from sixteen to seventeen days and seven
teen to eighteen days of development. The appendages were too small
and undifferentiated to measure before thirteen days of age.

PARTURITION
Details of the birth act are known for relatively few wild
mammals. In the genus Peromyscua Svihla (1932) has described partu
rition in P. m. artendsiae and. Poumelle (1952) described birth of
young in P, gossyplnus* It was possible to make careful observations
of the birth of a litter in P polionotus. A gravid female was iso
lated around the eighteenth day of gestation and placed in a large
gallon jar fitted with a screen cover* On January 1, 1956, at 11
A, M#, she was lying on her back with her eyes closed* There were
frequent movements of her abdominal wall obviously as a result of the
movements of the young in the uterus. Within several minutes she got
up on her feet and stretched her body by keeping her back feet in
place and pulling forward with her forefeet. At this time, the sides
of the abdomen were depressed toward the midorsal line. She then as
sumed a sitting position and started licking her vulva and scratching
it with her forepaws. At 11:10 A. M. the tail and hind feet of the
first fetus appeared* She tugged at it with her forefeet and pulled
the young mouse out. It took only a minute for this entire process
to take place.
The mother placed the young mouse, still connected to her by
the very short whitish umbilical cord on the floor of the jar, and
maintaining her crouehed position began washing her forepaws and face.
Several minutes later the placenta appeared. She dislodged it from
her vulva with her forepaws, carried it directly to her mouth and began
68

69
eating it. When ahe had finished she again licked her forepaws and
vulva and picked up the young mouse and licked it thoroughly* turning
it in various directions with her forepawa as she did so* Upon fin
ishing with the new bom she again placed it on the floor of the jar
in front of her and seemed to neglect it entirely until after the
birth of the next of the litter.
At about two minute intervals the mother got up, faced away
from the new bom mouse and stretched in the manner that was previous
ly described. After stretching several times she again resumed a
crouching position and began licking the vulva and mashing its sides
with her forepaws. Soon another tail and hind leg appeared. This
time she simply grasped the young with her forepawa and pulled it free.
The placenta followed immediately. She ate the placenta and paid no
attention to the new bora animal. The second young was bom at 11:23
A. thirteen minutes after the first. After consuming the second
placenta she again washed her forepawa, her face, and the region around
the vulva and then returned to the first bom and licked it thoroughly.
She paid no attention to the second young for about ten minutes, after
whieh she picked it up with her forepawa, cleaned it and placed it with
its sibling. She lay down close to them; covered them with her out
stretched neck, and remained generally in this position for about thir
ty minutes. However, she frequently got up, walked away from the two
young mice and stretched herself in the manner described previously.
At 11:55 the female again assumed the crouching position, and
started licking her vulva and pressing it with her forepawa. Soon the

70
head of another mouse appeared. This time she grasped each side of
the head with her forepaws and pulled it out. She also allowed this
young one to lie on the floor of the jar beneath her with the cord
still intact. She washed her forepaws and licked herself constantly
for about five minutes before the placenta appeared. Again she used
her forepaws to dislodge it and ate it, after viiich she washed her
forepaw. Then picking up the last born mouse in her forepaws she
turned it in various positions and licked it thoroughly before placing
it with the other two mice. Finally the three new born mice were plac
ed under her and she crouched over them and remained still fcr about
one hour. Frequently she extended her head forward and bending it be
neath her body licked the three young indiscriminately. Observations
were continued for an hour after the birth of the last mouse and the
young could be seen lying beneath the mother. They were on their backs
with their ventral sides uppermost and they moved their heads back and
forth rubbing the ventral side of the mother with their noses as if
seeking out the teats. It could not be determined when nursing first
started.
The events of parturition in P. polionotus seem to be very
similar to those previously described for other species in the genus.
Whether the female cut the cord with her teeth or severed it by stretch
ing could not be definitely ascertained. Svihla (1932) describing par
turition in P. m. artemisiae, stated that the cord was always broken by
stretching. Poumelle (1952) noticed a predominance of morning births
in P. gossypinus. for out of twenty-six litters which he recorded seven
teen were bora between 6s00 A. M, and 12:00 noon. In this study nine

71
out of fourteen litters were born between the hours of 8t00 A. M, and
12iCO noon. However, observations were not made on all of the litters
but only on those produced by a group of fourteen females which had
been bred at about the same time and produced litters within several
days of each other.

LITTER SIZE AND SEX RATIO
Litter Size
During the period of this study fifty-two litters totalling
174 young, were born in the laboratory. The litter size ranged from
one to six with a mode of three mice per litter. The mean litter size
was 3.35 mice per litter. These data are summarized in Table 5.
TABLE 5
RELATIVE FREQUENCIES OF DIFFERENT LITTER
SIZES IN P. polionotus
Size of Litter
1
2
3
4
5
6
Total
Avg. litter size
Frequency
1
7
26
11
5
2
3.35
Litter size based upon a study of twenty-seven litters total
ling ninety-six embryos ranging from the pronuclear stage to approxi
mately twenty days of gestation showed a range of two to six embryos
per litter with a mod of three and an average number of 3.55.
Sex Ratios
Sexes of laboratory raised mice were recorded for 123 mice
representing thirty-nine litters and did not depart significantly from
a 1:1 r*io. Of this number, sixty-five (50.78 per cent) were males
72

73
and sixfcy-three (49*22 per cent) were females.

GESTATION
Gestation Length in Non-Lactating Females
In this study four records of the length of gestation in non
lactating females were obtained. The beginning of gestation was
placed at the time motile sperm were found in the vaginal smear of
the female. Three females produced litters twenty-four days after
sperm were recorded in the vagina while gestation in the fourth last
ed twenty-three days.
Influence of Lactation on Gestation
Close observations were made on six females that became preg
nant as a result of mating during post-partum estrus, and a record of
gestation length was determined for each. There was a range in ges
tation length from twenty-five to thirty-one days for these individu
als with an average of twenty-eight days, which seems to indicate an
increase of approximately fair days in the gestation period for lac-,
tating females. There was no apparent correlation between lengthen
ing of gestation and the number of suckling young, although the sample
was small. For example, three females with litters of one, three, and
four young respectively all had a twenty-eight day gestation period
for the second litter. On the other hand three other females, one
with a litter of four and two with litter of three had gestation
periods of thirty, twenty-five and thirty-one days respectively for
their second litters.
74

75
Post-Partum Sstrus and the Normal Estrous Cycle
In an effort to obtain more accurate data on the time between
parturition and post-partum estrus, twelve females in the latter
stages of pregnancy were placed in separate cages and checked at fre
quent intervals until parturition was actually witnessed or until new
born young were discovered* As soon as parturition was known to have
occurred, a male was placed in each of ten cages and left for twenty-
four hours with the female. In the remaining two cages, the male was
not introduced until twenty-four hours after parturition but was left
for forty-eight hours. In every cage the female attacked the male.
Svihla (1932) made a similar observation on other species of Peromys-
cus and suggested that the female considered the male an interloper
and fought him to protect her young. Seven fanales out of the ten
that were paired within two hours after parturition subsequently pro
duced litters. In the two cases where males were not introduced into
the cages with the fanales until twenty-four hours after parturition,
even though the mice remained paired for a period of forty-eight hours
neither of the females became pregnant. Although the data do not con
clusively prove that heat does not extend more than twenty-four hours
beyond parturition, they suggest that post-partum heat generally oc
curs in this species within twenty-four hours. Asdell (1946) has
pointed out that a post-parturition heat does not occur in all species
of the genus Peromyscus.
A total of 102 matings were recorded using the procedure pre
viously described for obtaining embryos of known ages. Of this total,

76
twenty-one proved fertile and eighty-one were infertile. Some of the
females would apparently accept the male at every heat period for sev
eral weeks without becoming pregnant. This was determined by placing
one male in a cage with several females that had Just mated. Vaginal
smears were made starting on the fourth day after the first mating
and in some instances females had sperm in their vaginal fluid on the
fifth day after the first mating. In other cases, sperm were found
in the vaginal smear on the seventh day after the first mating. In
still other cases no sperm were found and the smear did not indicate
an estrous condition. These limited data suggest that the average du
ration of the estrous cycle in this species may be about five days.
They also illustrate the fact that at least under laboratory condi
tions many of the matings in this species are infertile.

POSTNATAL GROWTH AND DEVELOPMENT
Weights at Birth
Fifty-seven mice representing sixteen litters were weighed
within four hours after birth. Weights ranged from 1*1 to 2.2 grams,
with a mean of 1.6l grams. The lowest mean birth weight was found in
a litter of five individuals and the highest mean birth weight in a
litter of only two individuals. However, the evidence is not conclu
sive that there is any correlation between increase in litter size
and decrease in mean birth weight. The single individual in one lit
ter had a birth weight of 1.7 grams, and in another litter containing
two individuals, a mean birth weight of 2.1 grams was obtained. Six
litters had three mice per litter with a mean birth weight of 1.7
grams. In eight other cases including four litters of four, and four
litters of five individuals the mean birth weight was 1.5 grams.
Postnatal Growth Measurements
A series of postnatal growth measurements was mads on forty-
four mice from thirteen litters, and these data are summarized in
Table 6 and presented graphically in Figure 2.
The total length and tail length showed a fairly rapid growth
rate up to thirty days of age beyond which time increase in tail length
was of little magnitude. Relative total growth in length from thirty
to sixty days of age was less than that prior to thirty days of age.
77

78
The hind foot and ear exhibited rapid growth up to twenty days of age,
but little beyond this point. The hind foot had attained adult pro
portions by thirty days of age. There was a fairly constant increase
in body weight up to thirty days of age but comparatively little gain
thereafter.
TABLE 6
AVERAGE DAILY GROWTH MEASUREMENTS IN MILLIMETERS AND WEIGHTS
IN GRAMS OF P. polionotus FROM 2-60 DAIS OF AGE
Age in
Days
Total
Length
Tail
Length
Hind Foot
Length
Ear
Length
Body
Weight
At Birth
1.6
2
41.6
10.8
6.8

2.2
3
48.0
13.4
7.7
1.7
2.2
4
49.8
13.0
8.0
2.0
2.2
5
55.1
16.3
9.0
2.3
2.9
6
58.C
16.5
9.7
3.0
3.0
7
60.4
18.5
10.8
3.0
3.2
8
67.2
21.1
12.2
3.5
4.0
9
68.8
21.8
12.3
4.0
3.9
10
76.4
25.0
13.6
4.0
4.5
11
71.5
23.3
13.0
4.0
4.2
12
83.3
29.2
14.6
*5.0
4.9
13
80.8
27.5
14.6
5.0
4.9
14
89.4
32.6
15.7
5.0
5.2
16
84.7
25.1
15.3
7.0
5.2
18
98.8
36.3
16.6
9.0
6.5
20
99.9
37.5
16.4
9.2
6.5
22
110.3
43.3
17.7

8.1
24
106.6
40.8
17.7
10.0
8.0
26
115.2
44.7
18.8

9.3
28
109.9
42.3
17.4

8.9
30
122.0
50.5
18.3
10.0
10.4
34
121.5
48.3
18.2

11.5
36
126.5
52.7
18.6

11.8
40
125.4
50.0
18.4

12.6
44
128.7
52.2
18.5
12.0
12.6
48
131.2
53.7
19.0

13.2
50
131.0
53.0
18.9
12.0
13.9
56
128.7
51.3
18.0


60
120*5
&£
18.6

14J.
From the 12th day on, ear length measurements were made on
fixed material.

79
Development
At birth the eyes were closed and the ear pinnae were folded
down over the external auditory meatus (PI. VI). The over-all body
color was pink with the dorsum being slightly darker. The skin cover
ing of the head region was transparent so that the sagittal and coro
nal sutures could be seen. On the ventral side, small blood vessels,
viscera, and ribs could be seen through the transparent skin,
Mystacial vibrissae were present at birth and when depressed against
the face extended back to the level of the eye.
By the second and third day, the entire dorsal side was pig
mented but the region of the head between the ears appeared a shade
lighter than the rest of the dorsum. The venter remained pink and
transparent enough for viscera to be seen. The iris ring, which was
visible through the somewhat transparent sealed eyelids at birth as a
black band surrounding the lighter lens, was obscured by the end of
the third day due to the increasing opacity of the eyelids. Upon close
inspection with proper light, or tinder slight magnification, a sparse
covering of fine hair could be observed on the dorsal side by the third
day. No hair growth could be detected on the ventral side at this age.
In a total of forty-two young, the pinna unfolded on the third day in
eighteen, on the fourth day in twenty, and on the fifth day in four
(PI. VII,A).
At three days of age mice would squeak when disturbed, but
showed very little coordination. When placed on their feet they would
rest on the venter for a few seconds and then in trying to move would

topple to one side or the other. When they fell over they could not
right themselves.
At five days of age the mice still appeared ''naked at a casual
glance. However, when inspected under proper light or low magnifica
tion the dorsal hairs appeared more dense than in the previous stage.
Hair growth continued down the dorsal ridge of the tail for about one
half of its length. The ventral side showed a very sparse growth of
tiny white hairs but this growth was not nearly as thick as the hairs
on the dorsal side. White hairs could also be seen on the dorsal sides
of the legs at this stage. The entire dorsal side was a "graphite
gray" color, except for the legs which remained pink. The mice began
to exhibit slight coordination; and could crawl. When turned over on
one side they could eventually right themselves.
By seven days of age, the hair had grown so as to give the
dorsal side a uniformly gray appearance. Even the tail was covered
with fine hair to its tip. A long, black supra-orbital vibrissa ap
peared just dorsal to the eye orbit. The skin of the ventral side was
quite pink and rugose and appeared naked to the unaided eye, although
a sparse growth of hair was present. Abdominal viscera were outlined
against the body wall.
Ordinarily the lower incisors began erupting on the sixth day;
however, frequently they did not rupture the tissue of the jaw until
the seventh day. The upper incisors appeared by the seventh or eighth
day. Mice were more active at seven days of age and were able to run,
although their ventral sides still dragged along the floor. When they

81
were introduced head first into a container they would react by turn
ing around and attempting to climb up the side of the container. Upon
being placed in a jar containing ether fumes, they would show the adult
characteristic of face washing with their fore paws.
At eight to nine days the dorsal pelage was dense enough to
conceal the underlying skin. Along the mid-dorsal line the pelage was
slightly darker than on the sides, and the venter was covered with fine
white hair although the underlying skin could still be seen easily.
Vent rally the body was covered with fine white hairs which were dense
enough to give a white appearance to the ventral side of the animal and
the skin on the ventral side had become so opaque that only a faint
outline of underlying organs could be seen. Although the pinna appear
ed almost bare to the unaided eye, it was covered with a fine growth
of hair that could readily be observed under the binocular microscope.
The external auditory meatus was deeply furrowed but was not completely
open, as the mice did not react to sound at this age. They could, how
ever, run without dragging the ventral side on the floor.
At eleven days of age (PI. VII,B) some of the mice showed re
action to sound by flinching when a sharp noise was made. The coordi
nation was good, as they ran when touched and tried to bite while be
ing handled. The age at which the eyes opened was recorded for forty-
one individuals. This occurred at fourteen days of age in twenty-four
mice, at thirteen days in fourteen others, and at fifteen days in three.
At thirteen to fourteen days the mice were very alert and
showed such adult reactions as digging, huddling when a noise was made,

82
face washing, and nervous urination. By this stage firm fecal pellets
with a bright yellowish color had replaced the watery feces of previous
stages.
Around fifteen to seventeen days of age the ventral hair was
dense enough so that the mammae on the females did not show conspicu
ously. The young mice were very alert and at night they often left
their nesting jars and ventured around the cage. When a sharp noise
was made they would jump into the air and run back into the nesting
jar. Cheek teeth had erupted the skin in specimens fixed at fifteen
and sixteen days of age, but were not yet visible in a specimen fixed
at fourteen days of age* Beyond fifteen to sixteen days mice showed
very little change other than a gradual increase in size and in length
of pelage. Plates VIII and IX show stages in development from twenty-
four through forty days.
In most females the vagina opened at twenty-nine to thirty-
four days of age. Actual observations were made on fourteen individ
uals; in six the vagina was open on the twenty-ninth day. The next
observation was not made until the thirty-fourth day when only one of
the fourteen still had a closed vagina. In males at thirty-one days
of age the testes were slightly outlined in the scrotum.
The post juvenile molt began in some mice as early as thirty-
two days although the majority did not show signs of molt until thirty-
four to thirty-six days of age (PI. IX). A small patch of buff-colored
hairs Just beneath each eye, another on the dorsal side of each hind
leg in the region of the distal end of the tibia, and a fine line of

83
similar hairs on each side of the body at the zone of junction of the
dorsal and ventral pelage marked the beginning of molting. By thirty-
eight to forty days of age, the yellowish color had generally spread
dorsally over the shoulders and down the back to the rump where it stop
ped, leaving a crescent-shaped boundary. The concavity of the crescent
was directed posteriorly. The pelage in the area over the rump was
gray. Ventrally a pattern was also present, in that the neck, chest,
and anterior half of the stomach possessed white hairs similar to
adult pelage, while the posterior region of the stomach to the anus
was grayish white. At around forty-five to forty-eight days of age,
these distinctly different color areas had disappeared; the dorsal
pelage showed a uniform yellowish color and the ventral pelage was
uniformly white as if molting had been completed. In some mice molt
ing was still in progress at fifty-five days of age.

DISCUSSION
As pointed out earlier, the embryology of some laboratory ro
dents has been studied in considerable detail but this seems not to be
true with respect to wild species, even though they may be widespread
and generally abundant* On the other hand, a number of observations
have been made on the postnatal development of many species of wild
rodents, including several species within the genus Peromyscus. The
present study represents an attempt to describe the entire develop
mental period of a single speQies of wild rodent from conception through
to adulthood.
As was to be ejected, the development of Peromyscus polionotuB
resembled that of the related Mus, Rattus, and Mesocricetus in its
broad outlines. However, certain differences in embryonic structure
and chronology have been revealed and seem worthy of further consider
ation* Furthermore, information obtained concerning postnatal develop
ment and other aspects of reproductive behavior in P. polionotus now
permit a more critical comparison with other members of the genus than
has heretofore been possible. The significance of certain of the ap
parent intra- and inter-generic differences in various aspects of pre
natal and postnatal development in the oldfield mouse as compared to
other species of the same genus on the one hand and to the house mouse,
rat, and hamster on the other are not presently clear. Nor will they
be clear until more studies of development are made, not only of other
species in the genus Peromyscus but of representatives of other genera
8U

85
of close and more distant relationship,
A well-developed corona radiata was observed in eggs estimated
to be from one to six hours post copulation but by fifteen to eighteen
hours this had degenerated into a small mat of mucoid-like strands.
Smith (1939) reported that he was unable to find a definitely formed
corona radiata in P. polionotus although he did see coronal cells scat
tered around the eighteen-hour pronuclear stage. Apparently the corona
had already degenerated in the earliest stages which he observed. A
zona pellucida was present from the pronuclear stage through the eight
cell stage. In some instances in the later stages it was broken and
irregular but this may have been done during histological preparation.
Smith (1939) reported the zona pellucida from two, four, six, and seven
cell stages but not from the pronuclear stage. On the other hand,
Lewis and Wright (1935) found a zona pellucida in all stages of house
mouse embryos through the blastocyst stage. It would appear, therefore,
that the zona pellucida persists for a longer period of time in the
house mouse than in P. polionotus.
The segmentation stages of P. polionotus had ovoid-shaped cells
and did not appear to be in direct contact with or under any pressure
from the tubal mucosa. This is in agreement vdth the observations of
Smith (1939) on this same species. However, Huber (1915) indicated
that the segmentation stages of the white rat were compressed between
the folds of the tubal mucosa, and commented that they were molded by
the tubal mucosa. Either these two forms must differ in this respect
or Huber*s material must have become shrunken or distorted during

86
histological preparation.
The relationship between age and chronological development is
obviously variable in P. pollonotus. For example, embryos of both
forty-eight and sixty hours were in the four cell stage of development.
This might be due to inaccurate age determination or could be explained
by assuming that at forty-eight hours the second cleavage has just been
completed and that it takes more than twelve hours for the next cleav
age to occur. Smith s (1939) observations lend support to the latter
explanation since he found that embryos of forty-seven, or more, hours
were in the four cell stage while those of sixty-four, or more, hours
ranged from six to eight cells. However, other evidence clearly indi
cated that different eggs may develop at very different rates. In two
litters, both of which were seventy-two hours old, different develop
mental stages were observed. In the first, the embryos were all in
the eight cell stage and were located near the tubo-uterine junction,
while in the second they were in the blastocyst stage and were located
in the uterine horn. Even more striking was the situation in a single
litter of four ninety-six-hour embryos, all of which were apparently
in good condition and healthy. The stages represented in this litter
were: two cell, twelve to sixteen cell; morula, and blastocyst. Smith
(1939) did not observe such a variation in development in P. polionotus;
however, Snell (1941) states that in the house mouse the eggs usually
reach the morula stage about sixty hours after fertilization and pass
into the uterus some twelve hours later, but adds that this time is sub
ject to considerable variation. In the white rat Griffith and Farris

87
(1942) reported uncleaved, two, three, and four cell stages in the
tube of the female forty-eight hours after copulation. They also
maintained that a two cell stage or even a single blastomere could
be transplanted into the uterus and would develop normally.
In P. polionotus the ectoplacental cone had started to develop
on embryos which were five days of age. According to Snell (1941) the
ectoplacental cone appears at five to five and a half days in the
white mouse, while Kuber (1915) found it appearing in the white rat at
six days and fourteen hours. In all of these forms the cone is tall,
pointed and definitely cone-like in shape. On the other hand, Graves
(1945) reported that in the golden hamster the cone appeared near the
end of the sixth day and is not tall and pointed but rather fits like
a wig on top of the embryonic vesicle. It seems, therefore, that the
cone appears almost a day later in the hamster than in F. polionotus
despite the fact that the hamster has the shortest gestation period of
any known placental mammal, a period which is only about two-thirds as
long as that of P, polionotus. Further, the shape of the cone in P.
polionotus is similar to that in the mouse and rat but quite different
from that of the hamster. This is of particular interest in view of
the fact that the hamster and Peromyscus are both referred to the fam
ily Cricetidae, while Mus and Rattus are typical members of the Muridae.
On morphological grounds, the distinctions between these two families
are not well marked and in fact in a recent classification Ellerman
(1940-41) combines all of these rodents under the Muridae. It is pos
sible that the shape of the ectoplacental cone may prove to be of some

88
value in determining relationships in this group of rodents. However,
additional observations on other species are necessary before it can
be determined whether this character is constant or significant.
The so-called "inversion of the germ layers" long known in ro
dents and previously reported for Feromyscus by Ryder (1887) has been
fully verified in this study of P. polionotus. This phenomenon appears
to be widespread among the rodents, but there are so many species whose
embryological development has not yet been studied that it is impos
sible to say at present just how extensive it may be.
In one of the nine-day-old embryos there were four cavities in
the mesoderm of the amniotic folds (Pi. Ill,A), Snell (1941) mentions
that in the white mouse small cavities appeared in this area and soon
coalesced to form the exocoelom. He stated that he did not actually
observe the early stages of exocoelom formation in his studies and his
description of these cavities was based on the observation of Jolly and
Ferester-Tadie. The early stages of exocoelom formation were observed
1x1 £ polionotus; the cavities which were seen appear to be similar to
those described by Snell (1941)i and presumably exocoelom formation
follows the same pattern as in Mus.
A depression appeared in the thick base of the notochord of P.
polionotus embryos at nine days, and was present only in embryos of
this age group. This concavity appeared to correspond to one in the
thick base of the notochord in the white mouse which Snell (1941) des
cribed as being the archenteron. He noted that this structure which
appeared at seven and one-fourth days of age was transitory and

89
disappeared within twelve hours after formation. While the length of
time during which it persists was not determined for P. polionotus. it
is evident that it is also a transitory structure in this species.
The blood material which was seen in the extra-embryonic and
amniotic eavities of embryos of P. polionotus was definitely cellular
and appeared to have the same structure as the blood in the sinusoids
of the uterine mucosa. Long and Burlingame (1938) described a 'coagu
lated plasm" which was present in white rat embryos as being gelatinous,
colorless and lacking not only in blood corpuscles but in all cellular
elements. This material was present in the lumen of the yolk sac, in
the fore- and hind-gut and in the extra-embryonic and amniotic cavities.
They assumed that it had come from the maternal blood stream aid had,
with alteration, passed through Reichert's membrane, the yolk 3ac, and
the amnion. As has already been indicated, in £. polionotus ths blood
appeared to have passed directly into the exocoelom of the embryo from
sinusoids in the uterus of the mother by means of at least one definite
passage-way or channel formed by the mesodermal layer of the chorion
pushing through the ectodermal layer and opening out into the sinusoids
(PI. II,G). However, no means of entry into the amniotic cavity could
be found. Since all of the three nine-day-old embryos of P. polionotus
which were still in the presomite stage contained this blood and sinee
"coagulated plasm" has also been observed in white rat embryos such an
occurrence of blood or blood products is certainly not rare or unusual.
Whether it is the usual condition can not be determined until a good

90
many more species have been studied. According to the literature it
does not appear to occur generally in mammals and it may represent a
peculiarity of rodents.
Rand and Host (1942) reported a mean litter size of 3.968 and
a variation in litter size from three to five individuals for P,
polionotus. Their data were obtained from thirty-two litters caught
in the field and examinations of thirteen females with embryos. The
data obtained in this study from fifty-two litters born in the labora
tory showed a variation in litter size from one to six, and a mean
litter size of 3.35. In addition, a count of the embryos in twenty-
seven pregnant females showed a range in litter size of from one to
six and an average of 3.55. It seems apparent that two possible con
clusions could be drawn from these data. First, prenatal mortality
for this species must have been very low since the mean number of em
bryos in the pregnant females did not differ significantly from the
mean number of young in the litters.
Secondly, since both averages obtained in this study were so
similar to the mean reported for this species by Rand and Host (1942)
from their study of field litters it would appear that litter size in
P. polionotus was very little affected by captive conditions. Further,
the mean litter of P. polionotus is similar to those reported by Svihla
(1932), Pournelle (1952) and others for other species of the genus.
In this study, the sex ratio at birth, based on 128 mice, was
50.78 per cent males and 49.22 per cent females vMch seemed to indi
cate that if prenatal mortality or captive conditions reduced litter
size at all, they had no selective effect insofar as sex was concerned.

91
The sex ratio at birth of P, gossypinua, baaed on 240 mice
was reported by Foumelle (1952) as being 49 #43 per cent males and
50,42 per cent females.
The gestation period was from twenty-three to twenty-four days
in the four cases in which it was observed. A greater number of ges
tation records would have been desirable, but most of the pregnant fe
males, whose approximate time of copulation was known, were sacrificed
before reaching full term in order to obtain embryos of known age.
Smith (1939) cited a single gestation record for F, polionotua as being
twenty-four days. Hand and Host (1942) did not determine the gestation
period of P, polionotus, but stated that in other species of Feroagrscus
it ms known to be twenty-two days. Svihla (1932) observed a total of
146 gestation periods for various species in the genus Peromyscus: how
ever, P, polionotus was not included, A strikingly wide variation in
length of gestation was found among sub-genera, species, subspecies,
and even ir. individual mice. He gave twenty-two days as the least num
ber and thirty-five days as the greatest number of days for the gesta
tion period in Feromrscus. with means ranging from 23.39 .13 to
23.6l .11 in the various subspecies of P, maniculatus. The length
of the gestation period for P, polionotus here recorded closely ap
proximates the figure given for P, maniculatue. This is of particular
interest in view of the fact that these two species are not completely
reproductively Isolated, since they may interbreed and produce viable
young under laboratory conditions.
Duration of the gestation period was found to be increased

92
approximately four days for lactating females* Similar increases in
gestation length during lactation has been shown for other species of
Peromyscus by Svihla (1932). Poumelle (1952) reported a gestation
period of 29.6 to 30.8 days for lactating females of P. g, gossypinus.
while gestation for non-lactating females was around twenty-three days,
Snell (1941) gave the gestation period for Mus a3 nineteen to twenty
daysj however, he stated that one or two young sucklings could pro
long gestation up to seven days and with three or more young sucklings
prolongation up to twelve or thirteen days was not uncommon. A notice
able lengthening of the gestation periods in lactating females appears,
therefore, to be the rule in many rodents although the amount of
lengthening may vary. This may well be correlated with differences in
the intrauterine environment for the fetuses of lactating and non-lac
tating individuals.
The time of post-partum heat in Peromyscus has been given in
more or less general terms. McNair (1931) stated that P. 1.
noveboracensis and P. m. bairdii mated twenty-four to twenty-eight
hours after birth of the young. Svihla (1932) working on this same
genus stated that the females were usually in heat immediately follow
ing the birth of a litter. Also, Pournelle (1952) obtained litters of
P. gossypinua as a result of allowing males to remain with famales un
til shortly after parturition. Rand and Host (1942) working with P.
polionotus concluded that post-partum heat probably occurred within
two to four days after parturition. No doubt the time of occurrence
of post-partum heat is variable within the genus, but the data obtained

93
in this study do not agree with those presented by Rand and Host (1942).
Rather, a study of seven fonales indicated that post-part um heat occur
red within twenty-four hours after parturition.
The fact that some P, pollonotus females used in this study ac
cepted the male on the fifth day following an infertile mating showed
that for this species the length of the estrous cycle was almost identi
cal with the 4.8 day average reported by Clark (1936) for the very
closely related form P. maniculatus. It is also of the same order of
magnitude as that reported for other species of this genus. For example,
Pournelle (1952) reported the average length of the e3trous cycle for P.
gossypinus as being 5.26 days. In the house mouse Allen (1922) reported
that the length of the estrous cycle was from four to six days. Thus
figures for all of these different species are in fairly close agreement.
The fact that the highest mean birth weight of 2.1 grams was
found in a litter containing two mice and the lowest mean birth weight
of 1.5 grams in a litter containing five mice appeared to indicate that
there was a definite relationship between sise of litter and birth weight
of the individual mice. However, since there was only one litter of one
and one litter of two, insufficient data in these two groups made it im
possible to draw any firm conclusions on this subject. Svihla (1932)
recorded the birth weights of several species of Peronqrscus. He found
that for P, maniculatus the birth weight varied from 1.1 to 2.3 grains
with a mean birth weight of 1.72 grams; he concluded that the means of
the various subspecies of P. maniculatus showed marked uniformity and
that there was no significant variation. As has been mentioned, P.

94
polionotus is more closely related to this species than to the other
species of this genus ana they are also similar in size as adults. It
is interesting, therefore, that the range of variation of birth weights
of the two was almost identical, Svihla (1932) obtained a mean birth
weight of 1*72 grams for P. maniculatus, and in this study a mean birth
weight of 1.6l grams was obtained for P. polionotus. It seems probable
that this difference is correlated with the fact that the adults of the
various subspecies of P, maniculatus average larger than the adults of
P. polionotus. and this difference is probably reflected in the birth
wights.
Rand and Host (1942) reported of P, polionotus that growth in
six litters (twenty-three individuals) was rapid for about the first
twenty-eight days at which time the rate slowed down, and at sixty days
the mice were well within the limits of variation of the adults. The
observations made in the present study are in essential agreement with
their observations.
The condition of the young at birth, and the general sequence
of development of P. polionotus were similar to those described for
this same species by Rand and Host (1942), to those given for various
subspecies of P. maniculatus by Svihla (1932), and also to those given
for P. gossypinus by Poumelle (1952). Clark (1938) reported that some
P. polionotus females exhibited a comified smear at twenty-three days
of age. Rand and Host (1942) cited the case of one female P. polionotus
having an open vagina at twenty-six days of age. In the fourteen young
P. polionotus females observed in this study the vagina opened between

95
twenty-nine and thirty-five days of age. Obviously, therefore, there
is a good deal of variation in the time of opening of the vagina.
This may be due in part to differences in the environment but is un
doubtedly in part simply a matter of individual variation.

SUMMARY AND CONCLUSION
1. Development of the oldfield mouse, Peromyscus pollonotus. is des
cribed from the time of insemination to the time when the animals
approached adult limits.
2. Recently ovulated eggs were surrounded by a corona radiata which
was, however, largely gone by fifteen hours. The zona pellucida
ms present through the eight cell stage,
3. At twenty-four hours, embryos were in the two cell stage, at forty-
eight and sixty hours in the four cell stage, and by seventy-two
hours in the eight cell stage. By ninety-six hours, they were
generally in the blastocyst stage; implantation occurred on the
fifth day of gestation, and by eight days the amniotic exocoelom
and eetoplacental cavities were present; at nine days the allantois
had started to form; and by ten days the body form had become U-
shaped, the allantoic stalk had joined the chorion and the first
aortic arch was present. On the twelfth day, the body was in the
form of a tightly coiled C; limb buds were present, and the third
aortic arch had formed. By the fifteenth day, vibrissae follicles
occurred on each side of the snout, and the mammalian nature of
the embryo could be recognized. By eighteen days, the fetus had
about the same appearance as at birth. The pinna of the ear had
folded over the opening, eyelids had formed and the eyes were half
closed, the tongue had withdrawn from the lips, the mouth was al
most dosed, and the digits were fully formed.
96

97
4. There is obviously considerable variation in development of embryos
of the same gestation period in this species, since in a single
litter of ninety-six hours gestation four embryos were found repre
senting a two cell stage, a twelve to sixteen cell stage, a morula,
and a blastocyst, respectively,
5* The so called "inversion of the germ layers" has been confirmed for
P. polionotus,
6. Blood observed in the extra-embryonic and amniotic cavities of the
embryo apparently represents maternal blood from uterine sinusoids
which has found its way into the embryonic vesicle through passage
ways formed in the chorion,
7. Parturition was observed and is described in detail. In non-
lactating females the gestation period was twenty-three to twenty-
four days, but in lactating females It was twenty-five to thirty
days, Post-partum heat occurred within twenty-four hours after
parturition and the average duration of the estrous cycle was about
five days.
8. The average size of fifty-two litters was 3.35, while the average
number of embryos per litter in twenty-seven pregnant females was
3.55. The sex ratio of the litters was 50.78 per cent males and
49.22 per cent females. Mean birth weights of the litters varied
from 1.1 to 2.1 grams with an overall mean of 1.6l grams,
9. At birth the eyes were closed, the ear pinna were folded dowi over
the external auditory meatus, and the overall body color was pink
with the dorsum being slightly darker. Postnatal growth was rapid

/
98
for the first thirty days after birth, but then slowed down con
siderably and virtually ceased after fifty days.
10. The ear pinna unfolded on the third or fourth day and incisor
teeth errupted the gum from the sixth to eighth day. Young mice
i
reacted to sound at around eleven days of age. The eyes opened
between thirteen and fifteen days, usually on the fourteenth day.
The vagina of females opened between twenty-nine and thirty-five
days and the post juvenile molt started at thirty-four to thirty-
six days of age,
11. In general both prenatal and postnatal development of P, polionotus
resembled in broad outlines the development of Mus. Rattus. and
Mesocricetus, whose development has been adequately described. In
the time of appearance and the shape and structure of the ectopia-
cental cone, Peromyscus resembles Mus and Rattus rather than
Mesocricetus. to which it is generally considered to be more close
ly allied. It is possible that the nature of the ectoplacental
cone might be of some significance in the taxonomy of the rodents
in the murid and cricetid groups.

LITERATURE CITED
Allen, E.
1922. The oestrus cycle in the mouse. Am* Jour. Anat.,
30: 297-3A8
Asdell, 3, A.
1946. Patterns of mammalian reproduction, Comstock
Pub. Co., Inc., p. 437.
Boyer, Charles C.
1953. Chronology of development for the golden hamster.
Jour. Morph., 92: 1-37.
Clark, Frank H.
1936. The estrous cycle of the deer-mouse, Peromysous
maniculatus. Contrib. Lab. Vert. Genet, lx 1-7.
^ Clark, Frank H,
1938. Age of sexual maturity in mice of the genus
Feromyscus, Jour. Manan,, 19: 230-234.
Dice, Lee R. and Robert M. Bradley.
1942. Growth in the deer-mouse, Peromyscus maniculatus.
Jour. Mamm., 23: 416-427.
Ellerman, T, R,
1940-41. The families and genera of living rodents. London,
British Mus. (Natural History). Vol. 1, 689 pp;
Vol. 2, 690 pp. Muridae Vol. 2.
Graves, A. P.
1945. Development of the golden hamster, Cricetus auratus
during the first nine days. Amer. Jour. Anat.
77: (2), 219-251.
Griffith, John Q., and E. J. Farris.
1942. The rat in laboratory investigation. J. B.
Lippincott Co., xiv 488.
Guyer, MichaeD F.
1947. Animal micrology. Univ. of Chicago Press.,
Chicago, Ill. p 331.
Hamilton, h. J. Jr.
1939. American mammals. McGraw-Hill Book Co., Inc.,
p. 434.
99

100
Huber, G. Carl.
1915. The development of the albino rat, Mus norveglcus
albinus.l. From the pronuclear stage to the stage
of mesoderm anlagej end of first to end of ninth
day. Jour. Morph., 26: 247-356.
Lewis, Warren H. and E. S. Wright.
1935. On the early development of the mouse egg.
Carnegie Inst. Wash. Contrib. Embryol., 25: 115-143.
Long, J. A. and Paul L.. Burlingame.
1936. The development of the external form of the rat,
with some observations on the origin of the extra
embryonic coelom and foetal membranes. Univ. of
Calif.' Pub. in Zool. 43: (8), 143-164.
McNair, George T.
1931. The deer mouse, Peromyscus. a valuable laboratory
animal. Jour. Mamm., 12: 46-52.
Killer, G* S. Jr,, and R. Kellogg.
1955. List of North American Recent Mammals.
O.S.N.M, Bull. 205. 954 pp.
Pournelle, George H.
1952. Reproduction and early post-natal development of
the cotton mouse, Peromyscus gossypinus gossypinus.
Jour, Maram. 33: 1-20.
K Rand, A. L. and Per Host.
1942. Mammal notes from Highland County, Florida. Results
of the Archbold Expeditions, No. 45 Bull. Amer, Mus.
Nat. Hist., 60: 1-21.
Ryder, John A.
1887. The inversion of the germinal layers in Hesperomys.
Amer. Naturalist, 21: 863-664.
^ Schwartz, Albert.
1954. Oldfield mice, Peromyscus pollonotus of South
Carolina. Jour. Mamm., 35: (4)," 5&L-569.
Scott, J. P.
1937.The embryology of the guinea pig. Amer. Jour. Anat.
60: 397-432.
Simpson, G. G.
1945. The principles of classification and a classification
of mammals. Bull. Amer. Mus, of Natl. Hist.
Vol. 85, 350 pp.

101
Smith, William Kenneth.
1939. An investigation into the early embryology and
associated phenomena of Peromyscus polionotus.
Master's thesis, Univ. of Florida, 1-35.
Snell, George D.
1941* Biology of the laboratory mouse. Dover Pub., Inc.,
New York, t. Y. p. 497.
Svihla, Arthur.
1932. A comparative life history study of the mice of
the genus Peromyscus. Mise. Pub. Mus. Zool.,
Univ. of Mich., No. 24: 5-39.

PLATE I
/
A. Recent ovulated egg vdth corona radiata; X 100.
B. Two eggs in pronuclear stage of.development;
fifteen hours^ld; X 100,
C. Two cell stage; twenty-four hours old; X 100.
D. Four cell stage; forty-eight hours old; X 100.
E. Eight cell stage; seventy-two hours old; X 100.
F. Morula stage; ninety-six hours old; X 100.
G. Blastocyst stage; ninety-six hours old; X 100.

FUTE I
103

PLATE II
A.Longitudinal section of implanted embryo at five days;
X 100; exc, extra-embryonic ectoderm; ee, embryonic
ectoderm; pe, proximal entoderm; yc, yolk cavity;
tr, trophectoderm.
B.Longitudinal section of embryo at six days; X 100.
C. Longitudinal section of embryo at seven days; X 100.
D. Longitudinal section of embryo at eight days; X 100;
epc, ectoplacental cone; af, posterior amniotic fold;
e, entoderm; m, mesoderm; ec, embryonic ectoderm;
pc, proamniotic cavity; rm, Reicherts membrane.
E. Longitudinal section of embryo at nine days showing
three cavities and allantois; X 100; ect, ectoplacental
cavity; c, chorion; exc, exocoelom; a, amnion; ac, amni-
otic cavity; al, allantois.
F.Longitudinal section of mouse embryo at nine days show
ing a break in the chorion allowing a connection of the
ectoplacental cavity to blood-filled spaces of the ecto
placental cone; X 100; ect, ectoplacental cavity;
c, chorion; exc, exocoelom; a, amnion.
iv

PLATE II
105

PLATE III
A.Longitudinal section of nine-day-old embryo showing
early formation of the extra-embryonic coelom which
is represented by four small eavities in the mesoderm
of the posterior and lateral amniotic folds; X 100.
B.Sagittal section through nine-day-old embryo showing
somite formation; X 100.
C.Frontal section of a ten-day-old embryo showing the
allantois connecting with the chorion; X 100.

PLATE III
A
*

PLATE IV
A. Embryo ten days of agej a millimeter scale in photograph.
B. Embryo twelve days of agej X 7*
C. Embryo thirteen days of agej X 8..
D. Embryo fourteen days of agej X 8.
E. Embryo fifteen days of agej X 75.

PLATE IV
109

PLATS V
A. Embryo sixteen days of age; X 8.
B. Embryo seventeen days of age; X 7.
C. Embryo eighteen days of age, X 5.5.

PLATE V
111

PLATE VI
A litter of P. polionotua two to four hours after birth
a millimeter scale is included in photograph.

PIATE VI
113

'**
PLATE VII
A, A litter at four days of age; a millimeter scale is
included in photograph.
B, A litter at eleven days of age; a millimeter scale is
included in photograph.

FUTE VII

#
PLATE VIII
A. Oldfield mouse at twenty-four days of age; a milli
meter scale is included in photograph*
B. Lateral aspect of specimen in Fig. A; a millimeter
scale is included in photograph.

PLATE VIII
t
<
117

PLATE IX
A. Dorsal view at thirty days of age; a millimeter
scale is included in photograph.
B* Ventral view of above individual; a millimeter scale
is included in photograph.
C. Side view of a specimen at forty days of age; a milli
meter scale is included in photograph.

PLATE IX
119

FIGURE 1
Relative growth ratea of certain body parts frcm twelve
through eighteen days of gestation.

FIGURE 1
Crown Rump
Shoulder Breadth
Tail Length
Head Length
Head Breadth
Length of Foreleg
121

FIGURE 2
Graphs showing growth rates of selected body dimensions
(upper figure) and weights (lower figure) from birth to
sixty days of age based on forty-four mice from thirteen
litters

grams millimeters
FIGURE 2
123

BIOGRAPHICAL SKETCH
Samuel Kaada Laffoday, born September 13, 1924, in Lamar,
South Carolina, attended Lamar High School and after graduation in
1941, he entered Presbyterian Junior College for Men, Maxton, North
Carolina, From April 1943 to February 1946 he served in the United
States Army, He entered the University of South Carolina in June
1946 and received a Bachelor of Science degree in August 1947, and
a Master of Science degree in June 1949. From 1946 to 1949 he
served as an Instructor in Biology at the University of South
Carolina, Columbia, S, C., and from 1949 until 1951, as Assistant
Professor of Biology at Presbyterian College, Clinton, South
Carolina, Then he began graduate studies at the University of
Florida, He is currently employed as an Assistant Professor in
Biology at the College of Charleston, Charleston, South Carolina,
He holds membership in the South Carolina Academy of Sciences,
Association of Southeastern Biologists, Phi Sigma, and Sigma Xi.
124

This dissertation was prepared under the direction cf the
chairman of the candidates supervisory committee and has been ap
proved by all members of the coianittee. It was submitted to the
Dean of the College of Arts and Sciences and to the Graduate Coun
cil and was approved as partial fulfillment of the requirements
for the degree of Doctor of Philosophy.
June 3, 1957
Dean, College of Arts and Sciences
Dean, Graduate School
SUPERVISORY COMMITTEE



H
Hi chambers of the heart were till broadly connected. the large
left umbilical vein had invaded the liver proper and given rise to
the duelas venosos 'which was continent with the proximal end of the
vitelline veins. In the previous stage, the vitelline veins had al
ready broken up to form the hepatic portal eyetea. The right umbil
ical vein was fairly mall and lay Ineonspieoously in the lateral
liody wall. The persistent distal nd of the vitelline veins drained
blood fro the region of the yolk sac sad intestine into the liver*
dust ventral to the vitelline veins as the superior aesenterie ar
tery which in the previous stage vs the paired vitelline artery. In
the region of the anterior Hob buds, the subclavian artery extended
fro the dorsal aorta into each lisrb bud. The subclavian vein were
drained by the posterior cardinal veins. The stomal iliac arteries
arose from large umbilical arteries on each side and extended into
the posterior limb bude. the iliac veins of the posterior liad buds
were now formed and were drained by the posterior cardinal. Anteri
orly the vertebral artery as located just ventral and closely applied
to the neural tube. The internal carotid arteries lay Just dorsal to
the pharynx and the external carotid arteries were present In the tis
sues of t first visceral arch, the veins of the head region con
sisted of the large anterior cardinals, which were the most conspicu
ous vessels sees in sections, and the external Jugular veins which were
forming la the mandibular arch region. The third, fourth, and sixth
aortic arches were Intact at this stage#


9
with Perraount. In material representing the first four days post copu
lation, both sides of the Fallopian tubes and the entire uterus were
sectioned. For stages from five through twelve days sections of the
uterus containing implants were excised and the embryos were sectioned
in tero. In each case embryos were sectioned both transversely and
parallel to the long axis of the uterine horn. In addition, embryos
ten days of age and older were removed from the uterus, dissected free
of all fetal membranes and sectioned transversely, A description was
made of external form and the extent of internal development from ten
through eighteen days of age,
A number of measurements was made to determine changes in embryo
size and volume with age. Due to the small size of the specimens no
volumetric or weight measurements were attempted for embryos ten days
of age ard under and all measurements were made with a compound micro
scope and an ocular micrometer. For specimens ranging from twelve
through eighteen days gestation measurements of length end width were
made with the aid of a pair of fine (bolt-controlled) dividers under
5x magnification. Volumetric measurements were also made of this se
ries of embryos. The method employed was to select micro-vials of a
size appropriate for the embryo to be measured. Each of these vials
was ringed with a diamond point marking device, and its volume to that
line was determined. The embryo was placed on filter paper and rolled
around with the aid of a camels hair brush until it appeared to be dry
and then introduced into the previously standardized vial. The vial
was filled up to the marked ring from a 5 ml. Burette that could be


25
layer of the chorion and extending across the ectoplacental cavity
encompassing blood sinusoids in the ectoplacental cons (PI. II,G).
By this means maternal blood was channeled directly into the exoco
elom. No source of entry for blood into the amniotic cavity was
observed.
Ten Days
There were four embryos in the litter taken after ten days of
gestation, and they were all fixed in tero. Two were sectioned in
tero; one was removed from the uterus and fetal membranes and sec
tioned, and the other was removed from the uterus and fetal membranes
and kept as a reference.
External
At this stage the body is U-shaped as the original axis that
was established at the formation of the primitive streak and the head
fold is largely maintained. The head process is at the upper right
side of the U, and the tail region is at the upper left. The dorsal
side of the animal is bent inward making the concavity of the U, and
the posterior part of the embryo is buried to the right. The area of
the future mid-gut forms the bottom of the 0 (PI. IV,A).
The total body length of a single fixed specimen was approxi
mately 2.86 mm. This measurement was taken using a binocular dis
secting microscope and an ocular micrometer. Due to the shape of the
embryo, it was necessary to make several short measurements along the
body and to add these together for the total length. No doubt, this


FIGURE 1
Relative growth ratea of certain body parts frcm twelve
through eighteen days of gestation.


PLATE IV
109


76
twenty-one proved fertile and eighty-one were infertile. Some of the
females would apparently accept the male at every heat period for sev
eral weeks without becoming pregnant. This was determined by placing
one male in a cage with several females that had Just mated. Vaginal
smears were made starting on the fourth day after the first mating
and in some instances females had sperm in their vaginal fluid on the
fifth day after the first mating. In other cases, sperm were found
in the vaginal smear on the seventh day after the first mating. In
still other cases no sperm were found and the smear did not indicate
an estrous condition. These limited data suggest that the average du
ration of the estrous cycle in this species may be about five days.
They also illustrate the fact that at least under laboratory condi
tions many of the matings in this species are infertile.


Plates
LIST OF ILLUSTRATIONSContinued
Page
119
IX
A. Dorsal view of mouse thirty days of age.
B. Ventral view of mouse thirty days of age*
C. Side view of mouse forty days of age.
Figure
1 . 121
Graphs showing measurements of certain body
parts of embryos from twelve through
eighteen days of gestation.
2
123
Graphs showing growth rates of selected body
dimensions and weights from birth to sixty
days of age.
viii


97
4. There is obviously considerable variation in development of embryos
of the same gestation period in this species, since in a single
litter of ninety-six hours gestation four embryos were found repre
senting a two cell stage, a twelve to sixteen cell stage, a morula,
and a blastocyst, respectively,
5* The so called "inversion of the germ layers" has been confirmed for
P. polionotus,
6. Blood observed in the extra-embryonic and amniotic cavities of the
embryo apparently represents maternal blood from uterine sinusoids
which has found its way into the embryonic vesicle through passage
ways formed in the chorion,
7. Parturition was observed and is described in detail. In non-
lactating females the gestation period was twenty-three to twenty-
four days, but in lactating females It was twenty-five to thirty
days, Post-partum heat occurred within twenty-four hours after
parturition and the average duration of the estrous cycle was about
five days.
8. The average size of fifty-two litters was 3.35, while the average
number of embryos per litter in twenty-seven pregnant females was
3.55. The sex ratio of the litters was 50.78 per cent males and
49.22 per cent females. Mean birth weights of the litters varied
from 1.1 to 2.1 grams with an overall mean of 1.6l grams,
9. At birth the eyes were closed, the ear pinna were folded dowi over
the external auditory meatus, and the overall body color was pink
with the dorsum being slightly darker. Postnatal growth was rapid


34
in the previous stage and had formed large nasal chambers separated
from each other by a wide naso-medial process with naso-lateral proces
ses on each side. Jacobsen's organ was present as an evagination and
thickening of the ectoderm of the medial side of the nasal chambers.
At this stage there was no trace of the first cranial nerve. The eye
had advanced considerably since the previous stage as the lens had be
come separated from the outer ectodermal layer. It was quite spheroidal
and possessed a central cavity. The optic cup had a very thick inner
sensory layer and a thin outer pigmented layer. The otocyst had in
creased in size and the endolymphatic duct was a conspicuous structure
between the otocyst and the lateral plate of the hind brain. The ecto
dermal wall of the otocyst was much thicker on the ventral side adja
cent to the eighth cranial nerve ganglion than on the dorsal side.
The upper portion of the otocyst had begun to constrict, presaging the
formation of the upper vestibular pouch and the lower cochlear pouch.
Ganglia of the seventh and eighth cranial nerves had separated and the
seventh nerve could be traced down into the hyoid arch.
Digestive and Respiratory
The lateral extensions of the third pharyngeal pouches were
thick masses of cells constituting the primordia of parathyroid three,
and thymus three. The laryngotracheal groove was present in the floor
of the pharynx in the region of the fourth aortic arch. On each side
of the groove, a large arytenoid swelling had pushed up toward the
roof of the pharynx. The fourth pharyngeal pouch had evaginated lat
erally and turned ventrally. The terminal ends of the fourth pouch


10
read to l/lOO of a ml. The measured amount of water that it took to
fill the vial containing the embryo, subtracted from the known volume
of the vial gave a difference which represented the volume of the em
bryo. Each embryo was measured three times and an average was obtained.
Volume determinations made in this manner were quit consistent, often
yielding identical readings for each of the three measurements.
Weights were also recorded for this series of embryos after
they had been blotted as previously described, placed in stoppered
weighing bottles, and weighed on an analytical balance.
The description of prenatal development i3 based upon a study
of sixteen whole specimens and of fifty-four eggs and embryos which
were sectioned. The stages studied are listed in Table 1.
Age
0 hours
15 hours
24 hours
43 hours
60 hours
72 hours
TABLE 1
LIST OF EGGS AND EMBRYOS STUDIED
Number of
Specimens Stage of Development
5 Newly ovulated eggs surrounded by
corona radiata
4 Pronuclear
3 2-cells
3 4-cells
3 4-cells
5 Morula in lower end of tube, and
blastocysts in uterus
Blastocysts, morula, and 2-cell stage
in uterus
96 hours
4


52
appeared more distended (PI. V,A). The mesencephalon was still visible
as a slight hump and a short neck region was more obvious. The eyes
and ears were still open, and the mouth was slightly open with the tip
of the tongue extending almost to the lips. Vibrissae follicles were
more conspicuous on each side of the snout and between the eye and ear
than in the previous stage. There were also several follicles located
just dorsal to the eye orbit and on the chin, but there were no fol
licles visible on the body. There was a slight indication of develop
ing mammae. The umbilical hernia had increased in size through more
extensive coiling of the gut. The genital tubercle had become quite
long, and was covered by the tail as it bent ventrally and was directed
anteriorly. The shape of the external nares and general facial fea
tures were more adult-like. Digits on the fore and hind limbs were
deeply outlined but were still fully webbed and showed dorsal and
ventral differences.
Internal
Nervous
The epiphysis was a tubular structure containing a large lumen
and situated in the median roof of the diencephalon. In the region of
the diencephalon, the neural canal was greatly reduced. The cerebral
hemispheres were broadly merged with the diencephalon and fiber tracts
passed from the thalamus into the corpus striatum of each hemisphere.
In the mesencephalon the corpora quadrigemina were still more promi
nent on the dorso-lateral side while the mesocoele was still further


87
(1942) reported uncleaved, two, three, and four cell stages in the
tube of the female forty-eight hours after copulation. They also
maintained that a two cell stage or even a single blastomere could
be transplanted into the uterus and would develop normally.
In P. polionotus the ectoplacental cone had started to develop
on embryos which were five days of age. According to Snell (1941) the
ectoplacental cone appears at five to five and a half days in the
white mouse, while Kuber (1915) found it appearing in the white rat at
six days and fourteen hours. In all of these forms the cone is tall,
pointed and definitely cone-like in shape. On the other hand, Graves
(1945) reported that in the golden hamster the cone appeared near the
end of the sixth day and is not tall and pointed but rather fits like
a wig on top of the embryonic vesicle. It seems, therefore, that the
cone appears almost a day later in the hamster than in F. polionotus
despite the fact that the hamster has the shortest gestation period of
any known placental mammal, a period which is only about two-thirds as
long as that of P, polionotus. Further, the shape of the cone in P.
polionotus is similar to that in the mouse and rat but quite different
from that of the hamster. This is of particular interest in view of
the fact that the hamster and Peromyscus are both referred to the fam
ily Cricetidae, while Mus and Rattus are typical members of the Muridae.
On morphological grounds, the distinctions between these two families
are not well marked and in fact in a recent classification Ellerman
(1940-41) combines all of these rodents under the Muridae. It is pos
sible that the shape of the ectoplacental cone may prove to be of some


an elongated cochlear duct which was adjacent to, and parallel with,
the first pharyngeal pouch. This pouch had narrowed presaging its
new role as the auditory tube, but as yet, its distal end showed no
signs of enlarging into a tympanic cavity. The nasal pits had deep
ened and had broken through into the oral cavity, thus establishing
the choanae. The vomero-nasal organ (Jacobsons organ) was now in
the form of tubular sacs extending for a distance through the nasal
septum. At this extreme distal end they were closed, but proximal
to this point a lumen was present. Its ectodermal wall was quite
thick. Fibers representing the olfactory nerve extended from the
floor of the nasal pit to the brain.
Digestive
The proximal end of Rathkes pocket as it extended upward
from the roof of the oral cavity had become greatly reduced in size
and existed as a small solid cord of cells. The tongue was deeply
undercut, and was a wide, flat U-shaped structure projecting from the
pharynx into the mouth cavity. The epithelial layer bordering the
upper and lower jaws was beginning to thicken forming the labio
dental ledge. Salivary glands had started to differentiate on each
side of the oral oavity just ventral to the jaw-cheek groove. The
thyroglossal duct was evident as an evagination from the floor of
the pharynx. The larynx was a solid epithelial plate in the floor
of the pharynx. A short connection existed between the trachea and
esophagus at their cephalic ends. Where they first separate, the
esophagus was larger but further caudadj it was reduced to a minute


93
in this study do not agree with those presented by Rand and Host (1942).
Rather, a study of seven fonales indicated that post-part um heat occur
red within twenty-four hours after parturition.
The fact that some P, pollonotus females used in this study ac
cepted the male on the fifth day following an infertile mating showed
that for this species the length of the estrous cycle was almost identi
cal with the 4.8 day average reported by Clark (1936) for the very
closely related form P. maniculatus. It is also of the same order of
magnitude as that reported for other species of this genus. For example,
Pournelle (1952) reported the average length of the e3trous cycle for P.
gossypinus as being 5.26 days. In the house mouse Allen (1922) reported
that the length of the estrous cycle was from four to six days. Thus
figures for all of these different species are in fairly close agreement.
The fact that the highest mean birth weight of 2.1 grams was
found in a litter containing two mice and the lowest mean birth weight
of 1.5 grams in a litter containing five mice appeared to indicate that
there was a definite relationship between sise of litter and birth weight
of the individual mice. However, since there was only one litter of one
and one litter of two, insufficient data in these two groups made it im
possible to draw any firm conclusions on this subject. Svihla (1932)
recorded the birth weights of several species of Peronqrscus. He found
that for P, maniculatus the birth weight varied from 1.1 to 2.3 grains
with a mean birth weight of 1.72 grams; he concluded that the means of
the various subspecies of P. maniculatus showed marked uniformity and
that there was no significant variation. As has been mentioned, P.


67
The interval between seventeen and eighteen days of age showed a marked
increase in the relative growth rate in crown-rump length, head length,
and shoulder breadthj but the increase in head breadth and tail length
was less pronounced. There was a marked increase in the crown-rump
length between twelve and thirteen days of age which slightly exceeded
the increase seen in this same measurement from seventeen to eighteen
days of development. Since the method of age determination used was
necessarily only an approximation it is possible that a greater age dif
ference existed between embryos of twelve and thirteen days than between
embryos of seventeen and eighteen days of age.
TABLE 4
AVERAGE MEASUREMENTS OF BODY PARTS OF EMBRYOS IN MILLIMETERS
Age
in
Days
Number
in
Litter
Crown
Rump
Head
Breadth
Head
Length
Shoulder
Breadth
Length
of
Foreleg
Length
of
Hindleg
Tail
12
2
3.7
*
4
*
4
a
13
4
6.4
1.1
3.5
2.6
1.1
1.0
2.1
14
3
7.2
2.5
4.4
3.7
1.5
1.5
2.8
15
3
9.0
3.1
5.1
4.3
1.8
1.7
3.2
16
3
10.1
3.6
6.0
4.7
2.2
2.1
3.7
17
3
11.3
4.2
6.7
5.2
3.2
3.2
4.0
18
4
13.4
5.1
8.2
7.3
4.5
4.5
4*6
*Too small and undifferentiated to measure at this stage.
The mean lengths of the fore and hind legs were similar. A
fairly uniform growth rate was noticed in both from thirteen to six
teen days of age, however the rate of growth was three times as great
for each twenty-four period from sixteen to seventeen days and seven
teen to eighteen days of development. The appendages were too small
and undifferentiated to measure before thirteen days of age.


PLATE I
/
A. Recent ovulated egg vdth corona radiata; X 100.
B. Two eggs in pronuclear stage of.development;
fifteen hours^ld; X 100,
C. Two cell stage; twenty-four hours old; X 100.
D. Four cell stage; forty-eight hours old; X 100.
E. Eight cell stage; seventy-two hours old; X 100.
F. Morula stage; ninety-six hours old; X 100.
G. Blastocyst stage; ninety-six hours old; X 100.


70
head of another mouse appeared. This time she grasped each side of
the head with her forepaws and pulled it out. She also allowed this
young one to lie on the floor of the jar beneath her with the cord
still intact. She washed her forepaws and licked herself constantly
for about five minutes before the placenta appeared. Again she used
her forepaws to dislodge it and ate it, after viiich she washed her
forepaw. Then picking up the last born mouse in her forepaws she
turned it in various positions and licked it thoroughly before placing
it with the other two mice. Finally the three new born mice were plac
ed under her and she crouched over them and remained still fcr about
one hour. Frequently she extended her head forward and bending it be
neath her body licked the three young indiscriminately. Observations
were continued for an hour after the birth of the last mouse and the
young could be seen lying beneath the mother. They were on their backs
with their ventral sides uppermost and they moved their heads back and
forth rubbing the ventral side of the mother with their noses as if
seeking out the teats. It could not be determined when nursing first
started.
The events of parturition in P. polionotus seem to be very
similar to those previously described for other species in the genus.
Whether the female cut the cord with her teeth or severed it by stretch
ing could not be definitely ascertained. Svihla (1932) describing par
turition in P. m. artemisiae, stated that the cord was always broken by
stretching. Poumelle (1952) noticed a predominance of morning births
in P. gossypinus. for out of twenty-six litters which he recorded seven
teen were bora between 6s00 A. M, and 12:00 noon. In this study nine


POSTNATAL GROWTH AND DEVELOPMENT
Weights at Birth
Fifty-seven mice representing sixteen litters were weighed
within four hours after birth. Weights ranged from 1*1 to 2.2 grams,
with a mean of 1.6l grams. The lowest mean birth weight was found in
a litter of five individuals and the highest mean birth weight in a
litter of only two individuals. However, the evidence is not conclu
sive that there is any correlation between increase in litter size
and decrease in mean birth weight. The single individual in one lit
ter had a birth weight of 1.7 grams, and in another litter containing
two individuals, a mean birth weight of 2.1 grams was obtained. Six
litters had three mice per litter with a mean birth weight of 1.7
grams. In eight other cases including four litters of four, and four
litters of five individuals the mean birth weight was 1.5 grams.
Postnatal Growth Measurements
A series of postnatal growth measurements was mads on forty-
four mice from thirteen litters, and these data are summarized in
Table 6 and presented graphically in Figure 2.
The total length and tail length showed a fairly rapid growth
rate up to thirty days of age beyond which time increase in tail length
was of little magnitude. Relative total growth in length from thirty
to sixty days of age was less than that prior to thirty days of age.
77


81
were introduced head first into a container they would react by turn
ing around and attempting to climb up the side of the container. Upon
being placed in a jar containing ether fumes, they would show the adult
characteristic of face washing with their fore paws.
At eight to nine days the dorsal pelage was dense enough to
conceal the underlying skin. Along the mid-dorsal line the pelage was
slightly darker than on the sides, and the venter was covered with fine
white hair although the underlying skin could still be seen easily.
Vent rally the body was covered with fine white hairs which were dense
enough to give a white appearance to the ventral side of the animal and
the skin on the ventral side had become so opaque that only a faint
outline of underlying organs could be seen. Although the pinna appear
ed almost bare to the unaided eye, it was covered with a fine growth
of hair that could readily be observed under the binocular microscope.
The external auditory meatus was deeply furrowed but was not completely
open, as the mice did not react to sound at this age. They could, how
ever, run without dragging the ventral side on the floor.
At eleven days of age (PI. VII,B) some of the mice showed re
action to sound by flinching when a sharp noise was made. The coordi
nation was good, as they ran when touched and tried to bite while be
ing handled. The age at which the eyes opened was recorded for forty-
one individuals. This occurred at fourteen days of age in twenty-four
mice, at thirteen days in fourteen others, and at fifteen days in three.
At thirteen to fourteen days the mice were very alert and
showed such adult reactions as digging, huddling when a noise was made,


16
of the embryo at this stage was 62 miera.
Sixty Hours
Three embryos from a female sacrificed at sixty hours were
also in the four cell stage and exhibited no discernible advance over
those at forty-eight hours. However, they were slightly larger, with
average maximum diameters of 77.5 and 62.0 miera.
Seventy-Two Hours
Embryos representing the seventy-two hour stage were obtained
from two different females. These two sets of embryos showed sig
nificant differences in development. Three embryos from one female
were located in the distal end of the Fallopian tube and each was in
the eight cell stage of development (PI. I,E). The most distally lo
cated embryo was 120 miera from the tubo-uterine junction. One embryo
measured 62.0 miera in length and width while another was more elongate
and measured 775 x 46.5 miera. The third was damaged in sectioning
and could not be measured. Fragments of the zona pellucida still sur
rounded each embryo. Whether the appearance of the zona pellucida at
this stage of development was natural or due to injury in preparation
could not be determined.
The two embryos of the other set were in the uterine horns
and had already attained the blastocyst stage. Implantation had been
initiated, as the embryos were embedded in pits in the mucosa on the
antimesometrial side of the uterus. In one embryo the inner cell mass


LITERATURE CITED
Allen, E.
1922. The oestrus cycle in the mouse. Am* Jour. Anat.,
30: 297-3A8
Asdell, 3, A.
1946. Patterns of mammalian reproduction, Comstock
Pub. Co., Inc., p. 437.
Boyer, Charles C.
1953. Chronology of development for the golden hamster.
Jour. Morph., 92: 1-37.
Clark, Frank H.
1936. The estrous cycle of the deer-mouse, Peromysous
maniculatus. Contrib. Lab. Vert. Genet, lx 1-7.
^ Clark, Frank H,
1938. Age of sexual maturity in mice of the genus
Feromyscus, Jour. Manan,, 19: 230-234.
Dice, Lee R. and Robert M. Bradley.
1942. Growth in the deer-mouse, Peromyscus maniculatus.
Jour. Mamm., 23: 416-427.
Ellerman, T, R,
1940-41. The families and genera of living rodents. London,
British Mus. (Natural History). Vol. 1, 689 pp;
Vol. 2, 690 pp. Muridae Vol. 2.
Graves, A. P.
1945. Development of the golden hamster, Cricetus auratus
during the first nine days. Amer. Jour. Anat.
77: (2), 219-251.
Griffith, John Q., and E. J. Farris.
1942. The rat in laboratory investigation. J. B.
Lippincott Co., xiv 488.
Guyer, MichaeD F.
1947. Animal micrology. Univ. of Chicago Press.,
Chicago, Ill. p 331.
Hamilton, h. J. Jr.
1939. American mammals. McGraw-Hill Book Co., Inc.,
p. 434.
99


FIGURE 2
Graphs showing growth rates of selected body dimensions
(upper figure) and weights (lower figure) from birth to
sixty days of age based on forty-four mice from thirteen
litters


FIGURE 1
Crown Rump
Shoulder Breadth
Tail Length
Head Length
Head Breadth
Length of Foreleg
121


92
approximately four days for lactating females* Similar increases in
gestation length during lactation has been shown for other species of
Peromyscus by Svihla (1932). Poumelle (1952) reported a gestation
period of 29.6 to 30.8 days for lactating females of P. g, gossypinus.
while gestation for non-lactating females was around twenty-three days,
Snell (1941) gave the gestation period for Mus a3 nineteen to twenty
daysj however, he stated that one or two young sucklings could pro
long gestation up to seven days and with three or more young sucklings
prolongation up to twelve or thirteen days was not uncommon. A notice
able lengthening of the gestation periods in lactating females appears,
therefore, to be the rule in many rodents although the amount of
lengthening may vary. This may well be correlated with differences in
the intrauterine environment for the fetuses of lactating and non-lac
tating individuals.
The time of post-partum heat in Peromyscus has been given in
more or less general terms. McNair (1931) stated that P. 1.
noveboracensis and P. m. bairdii mated twenty-four to twenty-eight
hours after birth of the young. Svihla (1932) working on this same
genus stated that the females were usually in heat immediately follow
ing the birth of a litter. Also, Pournelle (1952) obtained litters of
P. gossypinua as a result of allowing males to remain with famales un
til shortly after parturition. Rand and Host (1942) working with P.
polionotus concluded that post-partum heat probably occurred within
two to four days after parturition. No doubt the time of occurrence
of post-partum heat is variable within the genus, but the data obtained


METHODS AND MATERIALS
The mice used in establishing the laboratory colony were ob
tained from burrows in the Ocala National Forest in Marion County,
twenty-three miles east of Ocala, Florida. Some animals were captured
along the road bed of State Highway 20, from Central Tower east to
Juniper Springs. Collections were also made on the Salt Springs road
from Central Tower north for a distance of approximately six miles.
This population has been referred to as Percatqrscus polionotus subgriseus
by Schwartz (1954)*
Burrows could be seen along the shoulders of the road with lit
tle difficulty and could be dug easily. Only burrows that were plugged
a short distance from the entrance were excavated, as these were usually
occupied by a pair or by a family of mice, while unplugged burrows were
usually unoccupied. After determining the direction of the burrow a
small minnow seine was laid on the ground several feet back from the en
trance in hopes of covering the exit from the escape tunnel. In some
cases, after removal of the plug from the entrance tunnel, the introduc
tion of a piece of rubber tubing into the burrow would cause the mice to
flee by way of the escape tunnel and emerge under the minnow seine.
Often, however, the mice would not leave the nest until it had been com
pletely exposed by digging. If they then ran into the escape tunnel and
remained there instead of coming to the surface the introduction of the
rubber tubing into the escape tunnel would promptly drive them out.
6


86
histological preparation.
The relationship between age and chronological development is
obviously variable in P. pollonotus. For example, embryos of both
forty-eight and sixty hours were in the four cell stage of development.
This might be due to inaccurate age determination or could be explained
by assuming that at forty-eight hours the second cleavage has just been
completed and that it takes more than twelve hours for the next cleav
age to occur. Smith s (1939) observations lend support to the latter
explanation since he found that embryos of forty-seven, or more, hours
were in the four cell stage while those of sixty-four, or more, hours
ranged from six to eight cells. However, other evidence clearly indi
cated that different eggs may develop at very different rates. In two
litters, both of which were seventy-two hours old, different develop
mental stages were observed. In the first, the embryos were all in
the eight cell stage and were located near the tubo-uterine junction,
while in the second they were in the blastocyst stage and were located
in the uterine horn. Even more striking was the situation in a single
litter of four ninety-six-hour embryos, all of which were apparently
in good condition and healthy. The stages represented in this litter
were: two cell, twelve to sixteen cell; morula, and blastocyst. Smith
(1939) did not observe such a variation in development in P. polionotus;
however, Snell (1941) states that in the house mouse the eggs usually
reach the morula stage about sixty hours after fertilization and pass
into the uterus some twelve hours later, but adds that this time is sub
ject to considerable variation. In the white rat Griffith and Farris


4
these forms are apparent but their importance from a taxonomic stand
point cannot be evaluated until similar data are available for species
of other genera in the two groups. With this consideration in mind
the present investigation was undertaken.
The oldfield mouse, Peromyscus polionotus, was selected for
study. The mice of this genus are typical cricetid types of wide dis
tribution in North America, ranging from Atlantic to Pacific coasts
and from the tree line in the north to Panama, Hamilton (1939). The
genus contains more named forms than any other in North America, 56
species with numerous geographic races being listed by Miller and
Kellogg (1955) Various species of Peromyscus occur in a great vari
ety of habitats and are frequently the commonest of small mammals in
an area, often playing an important role in the ecology of the region.
Their diversity of form and wide geographic and ecological distribu
tion, coupled with the ease by which they can be captured and maintained
under laboratory conditions, have made these mice the subject of numer
ous taxonomic, ecological, physiological, behavioral, and genetic stud
ies. Such investigations have contributed much to our understanding of
differentiation and speciation in mammalian stocks.
Although postnatal development and other details of reproduc
tion have been studied in various members of the genus, it appears that
the only published reference to embryologies! development in the group
3 the mention by Ryder (1887) of "inversion" of the germ layers.
Smith (1939) in an unpublished masters thesis reported upon the early
embryology of P. polionotus from the pronuclear stage to the formation


83
similar hairs on each side of the body at the zone of junction of the
dorsal and ventral pelage marked the beginning of molting. By thirty-
eight to forty days of age, the yellowish color had generally spread
dorsally over the shoulders and down the back to the rump where it stop
ped, leaving a crescent-shaped boundary. The concavity of the crescent
was directed posteriorly. The pelage in the area over the rump was
gray. Ventrally a pattern was also present, in that the neck, chest,
and anterior half of the stomach possessed white hairs similar to
adult pelage, while the posterior region of the stomach to the anus
was grayish white. At around forty-five to forty-eight days of age,
these distinctly different color areas had disappeared; the dorsal
pelage showed a uniform yellowish color and the ventral pelage was
uniformly white as if molting had been completed. In some mice molt
ing was still in progress at fifty-five days of age.


ACKNOWLEDGMENTS
Many individuals have contributed most generously of their
time, knowledge, and experience to this work. Dr, H. B. Sherman, Pro
fessor of Biology at the University of Florida until his retirement in
1955, suggested the problem and guided me through the initial phases
of the work. To him I should like to express my sincere thanks. For
*
their valuable criticisms, suggestions, and interpretations, I should
like to thank Drs. Lewis Berner, J. H. Gregg, G. K. Davis, J. D, Kilby,
Pierce Brodkorb, J, VJ, Brookbank, and J. N. Layne. Dr. Layne helped
especially with the analysis and interpretation of data on postnatal
development. Severa], of my fellow students contributed in various ways
to the work. Robert Haubrich and Claude Adams cared for the laboratory
animals from time to time; John Outterson assisted with the field work,
and Robert Weigel and Joe Davis with the photography. To all of than
I should like to express my appreciation. Dr. E. Ruffin Jones, Jr*, as
chairman of my supervisory committee advised me on my academic program,
guided me in my research, and gave me every consideration and continued
inspiration. It is a pleasure to express to him my sincere appreciation.
Finally, ny wife, Joanne Andrews Laffoday, deserves special acknowledg
ment for the assistance which she has given me. She helped with the
field collections, with the writing and typing of this report, and
throughout has provided moral and financial support. I should like,
therefore, to express to her ny thanks for her many contributions.
ii


3
Among recent mammals the order Rodentia comprises a vast array
of species in which extensive adaptive radiation, convergence, and
other factors often obscure phyletic relationships and clearly provide
a situation where all available data should be eraployed. Various as
pects of reproduction have thus far proved useful in the taxonomy of
various groups of rodents. Among the features that have been employed
are: the developmental history of the fetal membranes, characteristics
of the newborn young, length of gestation, nature of the estrous cycle,
and the anatomy of the genital organs. The value of details of embry
onic development in this connection has not been well explored.
Considerable attention has been given to the classification of
rodents in the families Muridae and Cricetidae, where many of the as
pects of reproduction mentioned above have been utilized in addition
to "classical" taxonomic characters. The status of the two groups is
at present not fully agreed upon by taxonomists. Some workers, Ellerman
(194L) include both under the Muridae, whereas others, Simpson (1945)
afford each full family rank.
A considerable amount of work has been done on the embryology
and development of several species of these rodents extensively used as
laboratory animals. The embryology of the house mouse, Mus muscuius.
and rat, Rattus norvegicus. both murids, is comparatively well known as
the result of studies by Huber (1915), Long and Burlingame (1938) and
Snell (1941). A recent account of the general chronology of prenatal
development in a cricetid, the hamster, Mesocricetus. is that of Boyer
(1953). Differences in the chronology and details of development in


56
a massive concentration of mesenchyme cells.
Muscular and Integumentary
The muscles of the eye, tongue, shoulder, upper parts of the
appendages, and intercostal musculature were outlined.
Seventeen Days
External
At seventeen days the crown of the head was smooth and with
out a mesencephalic hump (PI. ¥,B). The head had become smaller in
relation to total body size and was less sharply bent forward so that
the chin no longer reached the chest. The digits of the forelimbs
were partly webbed, while the digits of the hind limbs were deeply
ridged and still fully webbed. Plantar tubercles had made their ap
pearance on the soles of the feet. Hair follicles were present on
the body in patches on the dorsal aspect of the fore and hind limbs,
and on each dorso-lateral side of the trunk. Follicles were also
present on the cheek and around the eyes, but were absent along the
mid-dorsal axis of the body, including the top region from the top of
the head forward to the nose. The eyes and ears were still fully open
although the ear pinna had turned anteriorly. The large distention of
the abdomen was reduced but the umbilical hernia remained prominent.
The mouth was only partly open with the tip of the tongue extending
to the lips.


PLATS V
A. Embryo sixteen days of age; X 8.
B. Embryo seventeen days of age; X 7.
C. Embryo eighteen days of age, X 5.5.


47
on each side of the eye, The most notloable change since the previous
stage was the complete obliteration of the lens vesicle by fibers,
whioh were still nucleated. Also, the pigmented layer of the retina
had become infiltrated with a black granular pigment. The nervous lay
er, pars optica, was thicker than in the previous stage. Its inner
fibrous layer adjacent to the optic cup cavity had become more dense,
and from this layer, fibers constituting the optic nerve extended in
to the optic stalk and could be traced to the brain. The pars coeca
and ora serrata were as yet not recognizable.
The external ear was a large depression in the region of the
old first branchial arch. In cross section, the floor of the external
auditory meatus consisted of ridges which were the developing hillocks.
The mesodermal band destined to form the tympanic membrane between the
invaginating ectoderm and the evaginating entoderm of the first and
second pouch was still quite thick. The auditory tube had formed fresa
the narrowed first pharyngeal pouch. At its distal end it appeared to
be joined by a tubular extension of the second pouch to produce a cav
ity which was the beginning of the tympanic cavity. Adjacent to this
cavity primordia of the auditory ossicles could be seen as patches of
pre-cartilage cells. The inner ear at this stage seemed to have ap
proached its definitive structure. The distal id of the endolymph
atic duct had become saccate; the semi-circular canals were formed,
and the cochlear canal had ccsnmenced to coil. The nerve fibers of the
olfactory nerve which were present in the previous stage were now more
dense and had become organized into a definite nerve tract. The medial


DISCUSSION
As pointed out earlier, the embryology of some laboratory ro
dents has been studied in considerable detail but this seems not to be
true with respect to wild species, even though they may be widespread
and generally abundant* On the other hand, a number of observations
have been made on the postnatal development of many species of wild
rodents, including several species within the genus Peromyscus. The
present study represents an attempt to describe the entire develop
mental period of a single speQies of wild rodent from conception through
to adulthood.
As was to be ejected, the development of Peromyscus polionotuB
resembled that of the related Mus, Rattus, and Mesocricetus in its
broad outlines. However, certain differences in embryonic structure
and chronology have been revealed and seem worthy of further consider
ation* Furthermore, information obtained concerning postnatal develop
ment and other aspects of reproductive behavior in P. polionotus now
permit a more critical comparison with other members of the genus than
has heretofore been possible. The significance of certain of the ap
parent intra- and inter-generic differences in various aspects of pre
natal and postnatal development in the oldfield mouse as compared to
other species of the same genus on the one hand and to the house mouse,
rat, and hamster on the other are not presently clear. Nor will they
be clear until more studies of development are made, not only of other
species in the genus Peromyscus but of representatives of other genera
8U


LIST OF ILLUSTRATIONSContinued
Plates Page
C.Frontal section of embryo ten days of age
shoving allantois connected to chorion.
IV 109
A. Embryo ten days of age.
B. Embryo twelve days of age.
C. Embryo thirteen days of age.
D. Embryo fourteen days of age.
E. Embryo fifteen days of age.
v in
A. Embryo sixteen days of age.
B. Embryo seventeen days of age.
C. Embryo eighteen days of age.
vi 113
Litter of mice two to four hours old.
VII 115
A. Litter of mice four days of age.
B. Litter of mice eleven days of age.
VIII 117
A. Dorsal view of mouse twenty-four days of age.
B. Side view of mouse twenty-four days of age.
vii


12
parturition, litter size, sex ratios at birth, gestation, post-partum
estrus, and the normal estrous cycle in P. polionotus.
Observations on postnatal growth and development were made on
thirteen litters of mice totaling forty-four individuals. These were
carefully observed from the time of birth up to fourteen days, with
measurements and weights being taken at two-day intervals. From four
teen to thirty days measurements were made every four days, and from
thirty days to fifty or sixty days, weekly measurements were made.
Weights and measurements were started on some litters at two days of
age and at three days of age on other litters, and all measurements
after four and five days of age were made on anaesthetized animals.
The measurements taken were those generally employed by mammalogists,
that is total length, tail length, hind foot length, and length of
ear from crown.


37
Excretory
The mesonephroi were much larger than in the previous stage
and numerous collecting tubules were apparent in cross sections. A
condensation of mesenchyme forming the nephrogenous tissue of the
metanephros was visible around the expanded proximal end of the ure
ter. The combined mesonephric and metanephrie ducts led into the
urogenital sinus near its junction with the rectum.
Endocrine
The thyroid primordia were located between the paired hyoid
arches, and the lateral extensions of the third and fourth pharyngeal
pouches were forming parathyroid three and four, and thymus three and
four. The infundibulum had fused with Rathkes pocket presaging the
formation of the pituitary body.
Genital
The cells of the genital ridge had lost their mesenchymal ap
pearance and were large ovoid cells. A great deal of mitotic activity
was evidenced in tissue of this area.
Fourteen Days
After fourteen days of gestation the body was still C shaped
and torsion was no longer obvious, and the main axis of the embryo was
lying in one plane. Due to cervical flexure, the head was at right
angles to the trunk, and the anterior tip of the snout rested on the
heart prominence (PI. IV,D). The greatest bend in the trunk was in


PLATE VI
A litter of P. polionotua two to four hours after birth
a millimeter scale is included in photograph.


89
disappeared within twelve hours after formation. While the length of
time during which it persists was not determined for P. polionotus. it
is evident that it is also a transitory structure in this species.
The blood material which was seen in the extra-embryonic and
amniotic eavities of embryos of P. polionotus was definitely cellular
and appeared to have the same structure as the blood in the sinusoids
of the uterine mucosa. Long and Burlingame (1938) described a 'coagu
lated plasm" which was present in white rat embryos as being gelatinous,
colorless and lacking not only in blood corpuscles but in all cellular
elements. This material was present in the lumen of the yolk sac, in
the fore- and hind-gut and in the extra-embryonic and amniotic cavities.
They assumed that it had come from the maternal blood stream aid had,
with alteration, passed through Reichert's membrane, the yolk 3ac, and
the amnion. As has already been indicated, in £. polionotus ths blood
appeared to have passed directly into the exocoelom of the embryo from
sinusoids in the uterus of the mother by means of at least one definite
passage-way or channel formed by the mesodermal layer of the chorion
pushing through the ectodermal layer and opening out into the sinusoids
(PI. II,G). However, no means of entry into the amniotic cavity could
be found. Since all of the three nine-day-old embryos of P. polionotus
which were still in the presomite stage contained this blood and sinee
"coagulated plasm" has also been observed in white rat embryos such an
occurrence of blood or blood products is certainly not rare or unusual.
Whether it is the usual condition can not be determined until a good


20
Six Day
Three six-day embryos were deeply embedded in the antime-
sometrial wall of the uterus, the lumen of the latter being reduced
to a very narrow slit toward the mesometrial side. The individual
embryos did not differ significantly in their development. The egg
cylinder which extended almost to the ventral wall of the yolk cavity
had increased in size from the condition at five days (PI. II,B).
This was due, apparently, to an increase in the number of cells rather
than to the growth of individual cells* The greatest length and width
measurements of one of the embryos at this age were 155*0 and 62*0
raicra, while the length of the ectoplacental cone was 62.0 miera. The
proamniotic cavity was present as a median slit confined to the embry
onic ectoderm and parallel to the long axis of the egg cylinder.
Seven Days
In three embryos obtained at seven days of age the elongated
egg cylinders had completely invaginated into the yolk cavity (PI. II,C).
The proamniotic cavity was continuous dorsally as far as the ectopla
cental cone, and was considerably wider than the slit-like cavity of
the previous stage. There was an infolding of cells at the junction
of the embryonic ectoderm with the extra-embryonic ectoderm which marked
the beginning of the posterior amniotie fold. Although a few mesodermal
cells viere present in this area, they did not yet constitute a definite
layer. Dorsal to the posterior amniotie fold, another infolding of cells
from one side of the extra-embryonic ectoderm into the proamniotic cavity


23
head process was a broad layer of cells located at the ventral ex
tremity of the egg cylinder. It appeared to be continuous with the
mesoderm layer from the primitive streak and was directed toward the
anterior end of the developing embryo. The most anterior extension
of the head process was reduced to a very thin layer that appeared
to be continuous with the flattened embryonic entoderm. The total
length and width measurements were 775.0 x 310.0 miera respectively
and the length of the ectoplacental cone was 341.0 miera. The allan
tois had not begun to develop in this embryo.
A second embryo of this same litter was somewhat further de
veloped. Rather than the six cavities seen in the preceding embryo,
this embryo had only three cavitiest the amniotic, the ectoplacental
and the exocoelom (which was fully formed). The ectoplacental cavity
ms much smaller than the other two cavities. The amnion consisted
of a ventral layer of embryonic ectoderm and a dorsal layer of meso
derm while the chorion consisted of a ventral layer of mesoderm and
a dorsal layer of extra-embryonic ectoderm. The allantois was present
as a small projection of mesodermal cells from the posterior end of
the embryo into the cavity of the exocoelom (PI. II,?). The mesoder
mal layer limiting the allantoic projection was continuous with the
mesodermal layer of the amnion. The anterior part of the embryonic
ectoderm adjacent to the invaginating entoderm of the fore-gut was
elevated as the head fold. The notochord had differentiated from
the head process and was located on the ventral side of the egg cylin
der. On the ventral side of the notochord there was visible a slight


14
as a fifteen-hour stage.
The four embryos found were centrally located in the large
lumen of the ampulla of one Fallopian tube and were not in close as
sociation with the mucosal lining of the tube. A few sperm were also
visible lying in the lumen of the tube. A disintegrated corona radiata
appeared in the form of mucoid-like strands surrounding each embryo.
The embryos were surrounded by a fairly thick zona pellucida and be
tween this membrane and the cytoplasm of the egg was a clear peri-
vitelline space. The sperm centrum appeared as a slight conieal pro
jection. The second polar body was located in the perivitelline space
producing a slight bulge in the encompassing zona pellucida (PI. I,B).
Various degrees of fusion of the pronuclei were evident in the series
of embryos. Fusion was complete in one, in progress in two others,
and had just commenced in another. In the latter case the pronuclei
were in close juxtaposition but the nuclear membranes were still in
tact. The pronuclei in each case were located approximately in the
center of the egg. Size differences were evident in the pronuclei,
the smaller perhaps representing the male pronucleus as suggested by
Huber (1915) and Smith (1939). The larger pronucleus was elliptical
in shape and the smaller one appeared more round. The greatest length
and width, in miera of the larger pronudeus were 18 x 14; and for the
smaller pronucleus 14 x 14.4. One of the embryos measured 77.5 x 77.5
miera in greatest length and width, respectively, while the other
three were the same size, 77.5 x 54.2 miera.


101
Smith, William Kenneth.
1939. An investigation into the early embryology and
associated phenomena of Peromyscus polionotus.
Master's thesis, Univ. of Florida, 1-35.
Snell, George D.
1941* Biology of the laboratory mouse. Dover Pub., Inc.,
New York, t. Y. p. 497.
Svihla, Arthur.
1932. A comparative life history study of the mice of
the genus Peromyscus. Mise. Pub. Mus. Zool.,
Univ. of Mich., No. 24: 5-39.


PLATE IV
A. Embryo ten days of agej a millimeter scale in photograph.
B. Embryo twelve days of agej X 7*
C. Embryo thirteen days of agej X 8..
D. Embryo fourteen days of agej X 8.
E. Embryo fifteen days of agej X 75.


Al
tube, and the traehea was somewhat larger. Both of these tubes were
surrounded by a condensation of mesenchyme. Each lobe of the lung
possessed branching bronchi, and cells of this area were still mesen-
chymatous in appearance. The stomach showed a further increase in
size although no folds or rugae had yet formed. The ventral lobe of
the pancreas now appeared to be larger than the dorsal lobe but both
had grown somewhat since the last stage. The liver had filled the
peritoneal cavity to the extent of producing ventro-lateral bulges in
the body wall. Its cellular substance had become more condensed so
that it more closely resembled the adult liver. The gall bladder was
saccate and was closely applied to the ventral side of the liver.
There was only a faint trace of the tail gut in the form of a solid
mass of entodermal cells just ventral to the caudal artery. It per
sisted in the tail region for only a few sections.
Circulatory System
In the head the anterior cardinal vein had become the internal
jugular vein with several tributaries from the brain region and the
short external jugular was present. Paired internal carotid arteries
were evident, also the basilar artery was seen ventral to the myelen
cephalon. More posteriorly, the basilar artery divided into two verte
bral arteries. The hyaloid artery was complete to the eye. The third
aortic arches were partially broken down and the remaining portion was
now the proximal end of the internal carotid artery. Leading from the
ventricle of the heart was a thick walled tube, the bulbous cordis,
which divided into the ascending aortic trunk on the left side, and


94
polionotus is more closely related to this species than to the other
species of this genus ana they are also similar in size as adults. It
is interesting, therefore, that the range of variation of birth weights
of the two was almost identical, Svihla (1932) obtained a mean birth
weight of 1*72 grams for P. maniculatus, and in this study a mean birth
weight of 1.6l grams was obtained for P. polionotus. It seems probable
that this difference is correlated with the fact that the adults of the
various subspecies of P, maniculatus average larger than the adults of
P. polionotus. and this difference is probably reflected in the birth
wights.
Rand and Host (1942) reported of P, polionotus that growth in
six litters (twenty-three individuals) was rapid for about the first
twenty-eight days at which time the rate slowed down, and at sixty days
the mice were well within the limits of variation of the adults. The
observations made in the present study are in essential agreement with
their observations.
The condition of the young at birth, and the general sequence
of development of P. polionotus were similar to those described for
this same species by Rand and Host (1942), to those given for various
subspecies of P. maniculatus by Svihla (1932), and also to those given
for P. gossypinus by Poumelle (1952). Clark (1938) reported that some
P. polionotus females exhibited a comified smear at twenty-three days
of age. Rand and Host (1942) cited the case of one female P. polionotus
having an open vagina at twenty-six days of age. In the fourteen young
P. polionotus females observed in this study the vagina opened between


5
of the blastocyst.
The main objective of the present study was to follow the se
quence of embryological development as completely as possible from
conception to term. Since the general features of embryology in this
species are similar to those of the laboratory mouse, rat, and hamster,
no effort was made to describe in great detail all particulars of dif
ferentiation and organogenesis. Rather, attention was directed princi
pally to those features that seemed to offer a basis for critical com
parisons between the various genera. Since prenatal development is
merely the initial phase in a continuous growth process to adulthood,
observations on postnatal growth and development were considered as
also falling logically within the scope of the investigation. Data
pertaining to other aspects of reproduction, such as length of gesta
tion, parturition, estrous cycle, litter size, and sex ratios of the
young at birth, which were accumulated incidental to the principal ob
jectives of the work are also presented.
I


GESTATION
Gestation Length in Non-Lactating Females
In this study four records of the length of gestation in non
lactating females were obtained. The beginning of gestation was
placed at the time motile sperm were found in the vaginal smear of
the female. Three females produced litters twenty-four days after
sperm were recorded in the vagina while gestation in the fourth last
ed twenty-three days.
Influence of Lactation on Gestation
Close observations were made on six females that became preg
nant as a result of mating during post-partum estrus, and a record of
gestation length was determined for each. There was a range in ges
tation length from twenty-five to thirty-one days for these individu
als with an average of twenty-eight days, which seems to indicate an
increase of approximately fair days in the gestation period for lac-,
tating females. There was no apparent correlation between lengthen
ing of gestation and the number of suckling young, although the sample
was small. For example, three females with litters of one, three, and
four young respectively all had a twenty-eight day gestation period
for the second litter. On the other hand three other females, one
with a litter of four and two with litter of three had gestation
periods of thirty, twenty-five and thirty-one days respectively for
their second litters.
74


INTRODUCTION
The present state of knowledge concerning the embryonic de
velopment and other aspects of reproduction in mammals is markedly
incomplete. Only a small fraction of the more than 3#000 species
of recent mammals have been studied in any detail, and the majority
of these are domestic or laboratory forms. The paucity of develop
mental studies on wild species in large part stems from the diffi
culties attendant upon maintaining many kinds under captive condi
tions, not to mention the problems of inducing breeding in the
laboratory and of obtaining known-age embryologies! stages. Despite
the fragmentary nature of the data, there has been a distinct tendency
to apply the findings based on the study of the embryology of a single
or of a few species to the broader divisions of classification. The
chief justification for such extrapolation seems to lie in the assump
tion that the intrauterine environment is relatively constant in all
mammals, at least in the placental forms, as are the requirements for
maintenance of the embryo. As a consequence the pattern of embryonic
development, freed from the selective processes of differing environ
mental conditions, would be expected to be relatively conservative and
probably closely similar even throughout the higher taxonomic divisions.
However, Simpson (1945) has pointed out that "Embryonic structure is no
more free from the effects of new mutations, from adaptation, conver
gence, divergence, and the like than is any other phase of the life
1


29
The heart as seen from the left side of the animal was a round, bulbous,
membranous swelling lying between the cephalic tip of the telencephalon
and the anterior linto buds. The tail vas well defined although short
and still sharply bent ventrally. Laterally, the prominent mandibular
and hyoid arches were apparent. The third arch was also present, but
was quite small and was somewhat obscured by the hyoid arch. The man
dibular arch had not yet fused to form the mandible. Nasal pits were
present, but the naso-lateral, maxillary, and naso-medial processes
had not yet fused.
Internal
Nervous
The brain had attained the five-vesicle stage and in cross sec
tion through its anterior portion, the lateral telencephalic vesicles
appeared as obvious lateral evaginations. 1 The infundibulum in the floor
of the diencephalon was in contact with the tip end of Rathkes pocket.
The optic vesicles had constricted on each side of the brain to form
the optic stalk. The optic cup had formed and the lens vesicle was in
the optic cup, although the former had not yet separated from the super
ficial ectoderm. Otocysts were well formed at the level of the poste
rior region of the hind brain and had become completely separated from
the overlying ectoderm. In this area the seventh and eighth cranial
nerve ganglia appeared adjacent to the ventro-lateral side of the newly
formed otocyst. The large fifth ganglion was present, as were ganglia
nine and ten and the roots of nerves eleven and twelve. Histologically
the spinal cord was somewhat differentiated into the inner ependymal,


7
Mice were housed in the laboratory in cages measuring
9 x 15 x 9 inches in vddth, length, and height, respectively. The top
and bottom were formed of cookie sheets and the sides of l/k inch
hardware cloth. The top3 were secured by two coil springs and were
easily removed. Coarse wood shavings were placed on the floor and a
one quart nesting jar containing a small amount of cotton was kept in
each cage, A water bottle was attached to the outside with a delivery
tube extending into the cage.
The diet consisted chiefly of Purina Laboratory Chow pellets
on which they fed ad lib. Lettuce was put into the cages onee or
twice weekly; and while matings were being made small amounts of beef
liver were offered to the animals and these were readily accepted.
The diet was also supplemented from time to time with whole yellow corn.
To increase the laboratory stocks wild mice taken in the field
as pairs were kept paired in the laboratory. A single pair was kept in
each cage. Usually they would not reproduce for six months or more but
with production of the first litter, there would often result a sustained
littering period for three to five litters. It was soon discovered that
the most productive method of securing litters from laboratory-raised
animals was by placing as many as four females to a cage and introducing
a proven male for a period of about two weeks. Successful pregnancies
could be detected within fifteen days after the date of removing the male
and the gravid animals were then isolated. This method proved quite ef
fective and in several cases when as many as twenty females were used
all of them became pregnant as a result of being with the male for the


TABLE OF CONTENTS
ACKNOWLEDGMENTS ii
LIST OF TABLES v
LIST OF ILLUSTRATIONS vi
INTRODUCTION 1
METHODS AND MATERIALS 6
DESCRIPTION OF PRENATAL DEVELOPMENT 13
Zero Hours ............ ... 13
Fifteen Hours ........ 13
Twenty-Pour Hours 15
Forty-Eight Hours 15
Sixty Hours ........ 16
Seventy-Two Hours .... ..... 16
Ninety-Six Hours 17
Five Days . 18
Six Days 20
Seven Days 20
Eight Days 21
Nine Days 22
Ten Days 25
Eleven Days 28
Twelve Days 28
Thirteen Days 32
Fourteen Days .............. 37
Fifteen Days 44
Sixteen Days 51
Seventeen Days ............. 56
Eighteen Days ................ 60
PRENATAL GROWTH 64
Measurements of Uterine Swellings at Implantation Sites 64
Weights and Volumes of Embryos 65
Measurements of Certain Body Parts of Embryos from
Twelve Through Eighteen Days of Development 66
PARTURITION 68
iii


PIATE VI
113


PRENATAL GROWTH
Measurements of Uterine Swellings at
Implantation Sites
Implantation sites were seen as obvious swellings in the
uterine tubes of pregnant females five days after sperm were found in
their vaginal smears. These swellings showed a daily increase in size
with the growth of the embryo. Measurements made on fixed material
from five to eighteen days development are presented in Table 2.
TABLE 2
MEAN MEASUREMENTS IN MILLIMETERS OF
UTERINE SWELLINGS
Age
In Days
Number of
Swellings
Length
Width
Depth
5
1
3.0
2.5
3.5
6
1
3.5
3.0
4.0
7
3
3.5
3.2
3.5
10
1
5.5
5.5
6.5
12
2
5.5
6.0
7.0
13
3
7.6
7.5
9.0
14
2
8.5
7.5
9.5
15
2
9.2
8.2
10.0
16
2
10.0
9.0
10.0
17
2
12.5
9.0
11.0
18
3
14.5
11.0
12.0
Material representing the eighth and ninth days was sectioned
before measurements were taken, and there were no embryos obtained
representing the eleven-day stage of development. Hence, no conclusion
as to growth rate could be drawn from eight through eleven days of
64


50
smaller right arch had lost contact with the dorsal aorta. Caudad
to the heart coursed the small posterior cardinals along with the
supra cardinals. The left supra cardinal was located close to the
dorsal aorta and throughout the t runk region the right and left
supra cardinals were joined by transverse connecting veins.
Excretory
The genital tubercle had continued to increase in length,
and appeared for a number of sections between the tail and ventral
body wall. The right and left ureters still emptied into the uro
genital sinus along with the mesonephric duct3. The mullerian duct
appeared at this stage for the first time as a short solid rod lying
lateral and parallel to the mesonephric duct for about one fourth of
the length of the latter.
Endocrine
Differentiation of the pituitary body was nearly complete.
The neural lobe was still tubular, and the anterior wall of Rathke18
pocket had thickened producing a glandular anterior lobe. Issuing
anteriorly from the anterior lobe toward the diencephalon was the glan
dular pars tuberalis, while the pars intermedia existed as an evagin-
tion from the anterior wall of the anterior lobe. Below the pituitary
body the old Rathke's pouch persisted as a small solid cord of calls
traceable to the roof of the oral cavity.
The thyroid gland had changed in shape from the triangular
lobe seer, in the previous stage to a more flattened trough-shaped


75
Post-Partum Sstrus and the Normal Estrous Cycle
In an effort to obtain more accurate data on the time between
parturition and post-partum estrus, twelve females in the latter
stages of pregnancy were placed in separate cages and checked at fre
quent intervals until parturition was actually witnessed or until new
born young were discovered* As soon as parturition was known to have
occurred, a male was placed in each of ten cages and left for twenty-
four hours with the female. In the remaining two cages, the male was
not introduced until twenty-four hours after parturition but was left
for forty-eight hours. In every cage the female attacked the male.
Svihla (1932) made a similar observation on other species of Peromys-
cus and suggested that the female considered the male an interloper
and fought him to protect her young. Seven fanales out of the ten
that were paired within two hours after parturition subsequently pro
duced litters. In the two cases where males were not introduced into
the cages with the fanales until twenty-four hours after parturition,
even though the mice remained paired for a period of forty-eight hours
neither of the females became pregnant. Although the data do not con
clusively prove that heat does not extend more than twenty-four hours
beyond parturition, they suggest that post-partum heat generally oc
curs in this species within twenty-four hours. Asdell (1946) has
pointed out that a post-parturition heat does not occur in all species
of the genus Peromyscus.
A total of 102 matings were recorded using the procedure pre
viously described for obtaining embryos of known ages. Of this total,


A STUDY OF PRENATAL AND POSTNATAL
DEVELOPMENT IN THE OLDFIELD MOUSE,
Peromyscus polionotus
By
SAMUEL K. LAFFODAY
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
June, 1957


PLATE IX
119


63
stage. The gonad was made up of a mass of round cells which showed
many mitotic figures, but development had not progressed far enough to
identify this structure as either ovary or testis. The mullerian
ducts were still in the indifferent stage but liad now fused in the mid
line distal to the mesonephros forming the utero-vagina. Beyond this
point they joined the urethra but they separated again before entering
the urethra.
Skeletal
There was an increase in ossification areas in the dermal bones
of the skull at this stage, and ossification had started around Meckel's
cartilage of the mandible. Tooth germs for both molars and incisors
were present but the incisors were farther along in development. The
dorsal part of the neural arches and neural spines had still not formed.
Ossification had begun in the proximal long bones of the fore and hind
limbs, while bones of the phalanges were becoming cartilaginous.
Muscular and Integumentary
The muscles of the eye were in the form of fibrous strands, and
those of the tongue were also fibrous. Muscles of the heart resembled
definitive cardiac muscle. However, in none of the skeletal muscles
were transverse striations evident at this stage.


27
The anterior cardinal veins could be seen in the mesoderm of
the head and embryonic blood cells were visible lying in the lumen of
these vessels. The first aortic arch was complete.
The ventricle and sino-atrial regions of the heart were dis
tinct and numerous embryonic blood cells were enclosed by the endo
cardium of the ventricle. The omphalo-mesenteric veins and the allan
toic veins were confluent just posterior to the sino-atrial region of
the heart. The paired dorsal aortae extended posteriorly from the
first aortic arch, which contained embryonic blood cells in its lumen.
Although there were at least eight samites at this stage the exact
number could not be determined. They consisted of uniform masses of
cells without any organization into dermatome, myotome, and sclerotome
regions and lacked a myocoele.
The notochord was an obvious structure extending from the re
gion of the mid-brain to the caudal end of the embryo. Even though
it first differentiated from the head process several days earlier in
development, the notochord appeared to be still joined to the dorsal
entodermal wall of the gut. This is in line with the observations of
Snell (1941) on the white mouse. He found that: "For a considerable
period notochord and gut entoderm remain joined. Eventually, however,
the two halves of the gut entoderm grow across the ventral surface of
the notochord and unite in the mid-ventral line, leaving the notochord
as an axial, rod-like structure between ectoderm and entoderm. In the
head region, the neural groove was still open and the optic vesicles
had been differentiated from the diencephalon as large lateral


PLATE III
A.Longitudinal section of nine-day-old embryo showing
early formation of the extra-embryonic coelom which
is represented by four small eavities in the mesoderm
of the posterior and lateral amniotic folds; X 100.
B.Sagittal section through nine-day-old embryo showing
somite formation; X 100.
C.Frontal section of a ten-day-old embryo showing the
allantois connecting with the chorion; X 100.


FUTE I
103


55
the mesonephric ducts and ureters emptied separately.
Endocrine
The post-branchial bodies had fused with the thyroid gland.
The two lobes of the thymus gland lay close together in the midline
of the fetus. Both lobes were large and highly vascular, but the
left appeared larger than the right.
Genital
The mullerian ducts had further differentiated into a tubu
lar structure that could be traced some distance. Caudally they were
incomplete and did not extend to the urogenital sinus.
Skeletal
This stage marked the first appearance of cartilage in the
cranial region. It was present in all areas of the cranium where car
tilage bone develops. Also, the otic capsules were well outlined in
cartilage. The areas of dermal bones were represented by concentra
tions of mesenchyme cells and a small amount of fiber formation by
these cells. Centra, neural arches, and the dorsal part of ribs were
clearly cartilaginous. The ventral part of the ribs and the sternum
vrere less well defined. The bones of the girdle and the proximal long
bones of both forelimbs and hind limbs were preformed in cartilage. The
bones in the distal end of the fore and hind limb were represented by
mesenchymal concentrations. Meckels cartilage and the cartilage of
the nasal septum were well defined, whereas around the larynx there was


33
had a transparent membranous roof. The olfactory pita were more promi
nent than in the previous stage. The naso-lateral and naso-medial pro
cesses were in contact ventrally with the maxillary process, bit were
as yet incompletely fused. The eyes were quite evident and were lo
cated at the dorsal apex of the groove between the naso-lateral process
and the maxillary process. The lens was distinct but there was no sign
of eye lids. There was no pinna visible in the region of the auditory
pit. On the ventral side of the embiyo the heart bulge between the an
terior limb buds was very prominent, and the extreme ventral part of
the ventricular bulge was resting against the naso-medial process. The
large liver protuberance appeared to be continuous caudally with the
heart bulge. On the ventro-lateral side of the trunk between the an
terior and posterior limb buds was a slight ridge produced by the meso
nephros. The anterior limb buds were somewhat fan-shaped and appeared
to be slightly longer than the posterior linda bud, vhose shape was
hemispherical.
Internal
Nervous
The layers of the neural tube were much thicker and the neuro-
coel had become somewhat smaller than in the previous stage. The mar
ginal layer had the appearance of white matter, and surrounded the cord
except on the dorsal side. Numerous mesenchymatous fibers extended
through the white matter of the cord and penetrated deeply into the
mantle layer, forming blood vessels. In the region of the trunk, sym
pathetic ganglia were evident. The olfactory pits were much deeper than


61
chamber, which formed the inner lining of the cornea. The anterior
chamber was fully formed, and the lens was a spheroidal mass of fibers
with only a few nuclei, most of which were located in its upper hemi
sphere. The oval window could be seen in the cartilaginous framework
of the inner ear and the preosseus auditory ossicles extended to it.
The coils of the cochlea appeared complete. The maxillo-turbinal and
several ethmo-turbinals had made their appearance within the nasal
cavities.
Digestive
The outer layer of epithelium of the tongue had thickened and
now had many taste buds and papillae. Well defined foliate papillae
had appeared cn each side of the tongue and the longitudinal muscle
fibers had become more numerous in the root of the latter. The lumen
of the esophagus was greatly compressed dorso-ventrally. In the tho
racic cavity the right bronchial tube was larger in diameter than the
left. The lobes of the lungs had increased in size and the bronchi
were more numerous, although they were smaller in diameter than in
earlier stages. There were numerous coils in the intestine and its
walls seemed to have approximated definitive form. Mucosal folds were
evident in both the small and large intestine but only a few could be
seen in the stomach. The mesentery suspending the intestine had be
come greatly reduced in thickness. The pancreas was quite diffuse
and consisted of numerous small lobules connected by cellular cords.
In some areas the dorsal and ventral lobes appeared to merge with each
other. The gall bladder was superficially connected to the liver and


78
The hind foot and ear exhibited rapid growth up to twenty days of age,
but little beyond this point. The hind foot had attained adult pro
portions by thirty days of age. There was a fairly constant increase
in body weight up to thirty days of age but comparatively little gain
thereafter.
TABLE 6
AVERAGE DAILY GROWTH MEASUREMENTS IN MILLIMETERS AND WEIGHTS
IN GRAMS OF P. polionotus FROM 2-60 DAIS OF AGE
Age in
Days
Total
Length
Tail
Length
Hind Foot
Length
Ear
Length
Body
Weight
At Birth
1.6
2
41.6
10.8
6.8

2.2
3
48.0
13.4
7.7
1.7
2.2
4
49.8
13.0
8.0
2.0
2.2
5
55.1
16.3
9.0
2.3
2.9
6
58.C
16.5
9.7
3.0
3.0
7
60.4
18.5
10.8
3.0
3.2
8
67.2
21.1
12.2
3.5
4.0
9
68.8
21.8
12.3
4.0
3.9
10
76.4
25.0
13.6
4.0
4.5
11
71.5
23.3
13.0
4.0
4.2
12
83.3
29.2
14.6
*5.0
4.9
13
80.8
27.5
14.6
5.0
4.9
14
89.4
32.6
15.7
5.0
5.2
16
84.7
25.1
15.3
7.0
5.2
18
98.8
36.3
16.6
9.0
6.5
20
99.9
37.5
16.4
9.2
6.5
22
110.3
43.3
17.7

8.1
24
106.6
40.8
17.7
10.0
8.0
26
115.2
44.7
18.8

9.3
28
109.9
42.3
17.4

8.9
30
122.0
50.5
18.3
10.0
10.4
34
121.5
48.3
18.2

11.5
36
126.5
52.7
18.6

11.8
40
125.4
50.0
18.4

12.6
44
128.7
52.2
18.5
12.0
12.6
48
131.2
53.7
19.0

13.2
50
131.0
53.0
18.9
12.0
13.9
56
128.7
51.3
18.0


60
120*5
&£
18.6

14J.
From the 12th day on, ear length measurements were made on
fixed material.


91
The sex ratio at birth of P, gossypinua, baaed on 240 mice
was reported by Foumelle (1952) as being 49 #43 per cent males and
50,42 per cent females.
The gestation period was from twenty-three to twenty-four days
in the four cases in which it was observed. A greater number of ges
tation records would have been desirable, but most of the pregnant fe
males, whose approximate time of copulation was known, were sacrificed
before reaching full term in order to obtain embryos of known age.
Smith (1939) cited a single gestation record for F, polionotua as being
twenty-four days. Hand and Host (1942) did not determine the gestation
period of P, polionotus, but stated that in other species of Feroagrscus
it ms known to be twenty-two days. Svihla (1932) observed a total of
146 gestation periods for various species in the genus Peromyscus: how
ever, P, polionotus was not included, A strikingly wide variation in
length of gestation was found among sub-genera, species, subspecies,
and even ir. individual mice. He gave twenty-two days as the least num
ber and thirty-five days as the greatest number of days for the gesta
tion period in Feromrscus. with means ranging from 23.39 .13 to
23.6l .11 in the various subspecies of P, maniculatus. The length
of the gestation period for P, polionotus here recorded closely ap
proximates the figure given for P, maniculatue. This is of particular
interest in view of the fact that these two species are not completely
reproductively Isolated, since they may interbreed and produce viable
young under laboratory conditions.
Duration of the gestation period was found to be increased


17
was toward the lumen while in the other it was toward the mucosa of
the uterus. The trophoblast consisted of from fourteen to sixteen
cells, and the inner cell mass was composed of from eight to ten cells.
The average dimensions of the two blastocysts were 77*5 x 62.0 miera
in length and width, respectively. The subgerminal cavity was para
bolic in shape and measurements in miera of the greatest length and
width of each cavity were 38.75 and 46,5.
Ninety-Six Hours
A considerable variation in degree of development was exhib
ited by the four embryos secured from a single female at ninety-six
hours. Three embryos representing as many different stages of de
velopment were found in the left horn. The embryo which was located
nearest to the ovary was in the morula stage. Distal to this was an
apparently healthy two cell stage lying in the uterus, and still farther
from the ovary was an embryo of twelve to sixteen cells.
The late blastocyst found at this stage of development was
more elongate than the blastocyst seen in the seventy-two hour stage.
Its greatest length and width measurements were 93.0 x 46.5 miera.
The segmentation cavity measured 62.0 x 31*0 miera, the greatest width
of the cavity being at the level of the base of the inner cell mass.
This embryo was lying free in the uterine lumen near the neck of a mu
cosal pit on the antimesometrial side (PI. I,G).
The morula stage was lying free in the uterus and occupied a
position about equi-distant from either side of the wall (PI. I,F).
It was nearly spherical in shape and a segmentation cavity was


30
middle mantle, and outer marginal layer of cells. Numerous blood
vessels were present in the mantle layer. At the anterolateral mar
gin of the maxillary arch, the superficial ectoderm had thickened
and evaginated slightly to form the olfactory pit.
Digestive
The oral cavity consisted of the stomodeal opening. The
tongue primordio were not recognizable as such. Rathkes pocket ap
peared as a hollow tube extending from the roof of the oral cavity to
the posterior tip of the diencephalon. The first three visceral
arches were present and the hyomandibular cleft on the left side ap
peared to have broken through. The third visceral arch contained
the third aortic arch, and in the floor of the pharynx the thyroid
diverticulum was present. Immediately di3tal to the bifurcation of
the esophagus and trachea, the trachea evaginated laterally to form
tx-ro bronchi. The left bronchus at this stage las larger than the
right. The esophagus was a short structure resembling a slit-like
tube and was roughly 150 miera long. In transverse sections, the
stomach appeared as a long slit-like structure running dorso-ventrally
in the body. A slight curvature of the stomach produced a convexity
toward the left side of the embryo. Pancreatic diverticula were pres
ent at the ventral limits of the stomach. An intestinal loop was
present in the body stalk. The liver was quite unorganized at this
stage, consisting of scattered cords of cells with numerous blood fil
led spaces between the cords. The transverse urorectal fold had di
vided the cloaca into a dorsal rectum and a ventral urogenital sinus.


62
had become more saccate. It closely approached definitive fora and
had lost the ventral mesentery attachment. The spleen was well de
veloped, occupying the area between the left gonad and the lateral
body wall.
Circulatory
Completely enucleated red blood cells appeared at this stage.
The atria were still in communication by way of an opening in the sep
tum primum, the interatrial foramen and the septum aecondum had just
begun forming as a slight projection of cells from the dorsal walls of
the atria. The cavities of the ventricles were reduced in size due to
the increase in thickness of the muscular walls. They were now com
pletely separated from each other by the fusion of the interventricular
septum to the endocardial cushion. The mitral and tricuspid valves
had formed.
Excretory
The bladder was a large round mass with a slit-like lumen and
cells of the bladder wall had become differentiated into epithelium.
The kidney had acquired its characteristic shape, and had a well-defined
darker staining cortex and a lighter staining medullary portion. De
finitive tubules were abundant and Bovman's capsules with simple squa
mous epithelial linings and well formed glomeruli were present. The
mesonephros was greatly reduced in size from previous stages.
Genital
In the material on hand, sex could not be determined at this


59
follicles that appeared to be filled with blood cells. The two thy
mus bodies were located at the anterior tip of the thoracic cavity
and at the external ventral border of the thyroid gland. They were
large lymphoid rods which were round in cross section and extended
some distance into the thoracic cavity.
Genital
The Mullerian ducts had grown down to the urogenital sinus
but had not yet .joined it: nor had the tubes of the opposite sides
yet fused to form the utero-vagina. The genital system was still in
an indifferent stage of development.
Skeletal
In the cranial region, ossification had begun in the areas
of dermal bone formation. Also, numerous blood vessels were present
in these areas. The otic capsule, which had previously become carti
laginous, now had mesenchymal cells in the region of the membranous
labyrinth. The ear ossicles were faintly outlined in cartilage. Os
sification had begun in the mandible. The sternum and the ventral
part of the ribs were becoming cartilaginous. In the region of the
digits of the fore and hind limbs only dense mesenchymal concentra
tions were present.
Muscular and Integumentary
All muscles were outlined in their characteristic band or
bundle shape, but no definite fibers were present.


topple to one side or the other. When they fell over they could not
right themselves.
At five days of age the mice still appeared ''naked at a casual
glance. However, when inspected under proper light or low magnifica
tion the dorsal hairs appeared more dense than in the previous stage.
Hair growth continued down the dorsal ridge of the tail for about one
half of its length. The ventral side showed a very sparse growth of
tiny white hairs but this growth was not nearly as thick as the hairs
on the dorsal side. White hairs could also be seen on the dorsal sides
of the legs at this stage. The entire dorsal side was a "graphite
gray" color, except for the legs which remained pink. The mice began
to exhibit slight coordination; and could crawl. When turned over on
one side they could eventually right themselves.
By seven days of age, the hair had grown so as to give the
dorsal side a uniformly gray appearance. Even the tail was covered
with fine hair to its tip. A long, black supra-orbital vibrissa ap
peared just dorsal to the eye orbit. The skin of the ventral side was
quite pink and rugose and appeared naked to the unaided eye, although
a sparse growth of hair was present. Abdominal viscera were outlined
against the body wall.
Ordinarily the lower incisors began erupting on the sixth day;
however, frequently they did not rupture the tissue of the jaw until
the seventh day. The upper incisors appeared by the seventh or eighth
day. Mice were more active at seven days of age and were able to run,
although their ventral sides still dragged along the floor. When they


LIST OF TABLES
Table Page
1. List of Eggs and Embryos Studied , 10
2. Mean Measurements in Millimeters of
Uterine Svrellings 64
3. Mean Weights and Volumes of Embryos
12 to IB Days of Age 65
4. Average Measurements of Body Parts of
Embryos in Millimeters ....... 67
5. Relative Frequencies of Different Litter
Sizes in P polionotus 72
6. Average Daily Growth Measurements in
Millimeters and Weights in Grams of
P. polionotus from 2 to 60 Days of Age ....... 7
v


57
Internal
Nervous
The epiphysis persisted as a tubular organ, and its lumen had
become larger than in previous stages. The choroid plexus had formed
from the roof of the diencephalon and extended into the cavity of the
third ventricle. Cavities had appeared in the corpora quadrigemina.
The continued development of the cerebellum had altered the metenceph-
alon. The thickening alar plates in this region of the brain had fus
ed dorsally and were covered with a thin oellular layer. The choroid
plexus was relatively better developed in the roof of the fourth ven
tricle. Fiber tracts between the thalamus and corpus striatum were
quite obvious. The neurocoel was roughly elliptical with its great
est diameter extending dorso-ventrally about one third the dorso-
ventral diameter of the spinal cord. Both dorsal and ventral fissures
were present in the spinal cord. The sclerotic coat of the eye had
become more fibrous and the choroid coat had further differentiated.
The lens eipthelium was separated from the inner layer of the cornea
by a space which was continuous posteriorly with the vitreous chamber.
The cartilage around the membranous labyrinth was filled with cells
which were mesenchymal in appearance. The withdrawal of the tongue
had permitted the palatine processes to fuse mesially and the nasal
septum had grown down to the fused palatines and was in the process of
fusing with them, thus completely separating the two nasal passages.
Jacobson* s organ continued as a dominant structure in the nasal septum.


21
was apparent. The significance of this invagination could not be de
termined. Snell (1941) considered a similar structure in the white
mouse embryo as being possibly due to very rapid growth occurring in
the posterior wall of the egg cylinder at this stage.
The distal entodermal lining of the trophectoderm was uniform
ly established and consist i of a single layer of spindle-shaped cells.
The embryonic entoderm had changed from the cuboidal shape previously
noted to a thin layer of flattened and elongated cell3. However, the
entodermal cells lining the extra-embryonic ectoderm were still cuboidal
and closely packed, forming a fairly thick layer. The greatest main
axis and width measurements of a representative egg cylinder of this
age, excluding the ectoplacental cone, was 294.0 x 93.0 miera.
Decidual cells had extended across the lumen of the uterus and
had joined the mesometrial side of the uterine wall in these seven day
embryos.
Eight Days
Among the significant advances of embryos obtained at eight
days of age was the presence of a distinct layer of mesoderm that had
been proliferated from the region of the primitive streak (PI. II,D),
From their origin at the junction of the embryonic and extra-embryonic
ectoderm, the mesodermal cells had spread in all directions. The en
larged posterior amniotic fold had almost made contact with the an
terior amniotic fold. The latter consisted of a small projection of
cells into the proaraniotic cavity from the zone of junction of the
extra-embryonic and embryonic ectoderm at the anterior end of the


3
two-week period#
In an effort to obtain carefully timed embryos, and to measure
the length of the gestation period in non-lactating females, frequent
attempts viere made to observe actual coitus. However, this was soon
abandoned as the presence of an observer discouraged mating. The ma
terial used for this study was obtained by determining the onset of
estrus through vaginal smears. Then a proven male was introduced into
the cage with the female at about 3:30 P. M. and was removed the next
morning at approximately 8:30 A. M. A vaginal smear was then taken
and the slide checked for the presence of sperm. If sperm were found
the mouse was killed with ether after an appropriate period and the
genital tract was removed and fixed in Lavdowskys mixture, Guyer(l947).
By sacrificing pregnant fonales at appropriate intervals a sequence of
embryos was obtained representing each of the first eighteen days of
the gestation period except the eleventh. The ages of embryos in this
paper '¡rere all calculated from the time of finding sperm in the vaginal
fluid. Obviously this method does not provide the exact age of an em
bryo, but it is impossible to determine the exact time of fertilization
vdth any known method. Griffith and Parris (1942) indicated that vari
ous points of reference have been used in the timing of embryos, but
the exact age of the embryos could not be determined by the time of
copulation, ovulation, or fertilization#
The embryos from which sections were to be prepared were pro
cessed by the usual paraffin technique, sectioned at 10 miera, stained
with Standard Alum-Hematoxylin and Triosin (pH 5.4-5.6), and mounted


39
extended far craniad of the original roof of the telencephalon, and
the lamina terminalis was recessed in a deep groove between the cere
bral hemispheres. The cavities of the lateral ventricles were in
broad communication with the third ventricle, and the infundibulum
had continued its evagination from the floor of the diencephalon and
had turned down over the distal tip of Rathke's pocket. The vails
of the neural tube had thickened considerably since the last stage
described, and had compressed the lumen laterally so that it appeared
club-shaped with the narrow end toward the ventral side of the tube.
Ventro-laterally the tube was much thicker than elsewhere represent
ing primordia of the ventral horns. The neural tubes at this stage
were richly supplied with blood vessels.
Several changes were evident in the eyes. There was a cover
ing of superficial ectoderm over the lens, and between the lens and
this covering was a thin band of loose mesenchyme cells. These cells
diverged to each side of the lens and formed an aggregation on the
floor of the optic cup between it and the lens. The cells that had
already reached this position had lost their mesenchymal appearance
and were multinucleated. The lens at this stage was somewhat ellip
tical and its hemispherical cavity was restricted to its upper half.
This cavity was bounded on all sides by lens epithelium, except for
the floor, which consisted of cells from the inner wall of the lens,
among which fiber formation had started. A dorsal and lateral pouch
were forming from the semi-circular canal portion of the otocyst.
Ventral to this area, the otocyst was bent medially and continued as


#
PLATE VIII
A. Oldfield mouse at twenty-four days of age; a milli
meter scale is included in photograph*
B. Lateral aspect of specimen in Fig. A; a millimeter
scale is included in photograph.


19
mucosa on the antimesometrial side, and the cellular organization of
the uterine mucosa in the region of the implanted embryo was changed
into cell masses and cords of cells surrounded by sinusoids (PI, II,A).
The mesometrial portion of the pit formed by the embryo was filled
with degenerated uterine epithelial cells.
The embryos of five days of age showed a marked increase in
size over the previous stage examined. The old germinal cavity had
elongated and formed the yolk cavity which was delimited by a thin
layer of trophoectodenn (Reicherts membrane) with scattered distal
entodermal cells applied to its surface. The inner cell mass had be
come differentiated into an egg cylinder extending about half way to
the floor of the yolk cavity. The ectoplacental cone, extra-embryonic
ectoderm, and embryonic ectoderm were easily recognizable. A constric
tion marked the line of junction of the extra-embryonic and embryonic
ectoderm. The latter consisted of a single layer of cuboidal cells
with large round nuclei surrounding a small cleft, the proamniotie
cavity; whereas the former was composed of an irregular mass of cells
with elongate nuclei. A single layer of large cuboidal embryonic en
todermal cells formed the outer layer of the portion of the egg cylin
der that protruded into the yolk cavity. The total length and width
of the egg cylinder, excluding the ectoplacental cone, was 108.5 x 46.5
miera. The ectoplacental cone, a conspicuous conical-shaped mass of
cells extending toward the lumen of the uterus, measured 46.5 miera
in length.


wall of the vomero-nasal organ was about twice as thick as the oppos
ing wall. Since the tongue had not yet retracted from between the
palatine processes, the nasal septum had not fused posterior to the
palatine process.
Digestive
The tongue extended well forward between the palatine pro
cesses and was almost in contact with the posterior end of the nasal
septum. It contained blood vessels, muscle fibers, and nerve fibers.
Both the submaxillary and sublingual glands were prominent and the
parotid glands which developed at the angle of the mouth were also
present. The labio-dental ledge had developed in the oral epithelium
of both the upper and lower jaws* The larynx was now an open struc
ture ventral to the pharynx with the space between the two occupied
by large arytenoid swellings. The esophagus and trachea were further
separated from each other and the concentration of mesenchyme around
the trachea had become more dense. No thyroglossal duct was present
at this stage. The esophagus was very small in diameter, and in its
more distal region the lumen was hardly discernible. The liver had
become more massive and was filled throughout with branching sinus
oids. The gall bladder was bulbous, and was now superficially embed
ded in the liver, occupying the same relative position as in the adult.
In the intestine the imiscularis layer was defined and numerous folds
were present in the lining of the duodenum but were hardly discernible
in the intestine below this area. The spleen was not detectable at
this stage. The dorsal lobe of the pancreas had grown considerably


95
twenty-nine and thirty-five days of age. Obviously, therefore, there
is a good deal of variation in the time of opening of the vagina.
This may be due in part to differences in the environment but is un
doubtedly in part simply a matter of individual variation.


PLATE V
111


A2
the pulmonary trunk on the right. The sixth aortic arches were con
fluent with the pulmonary trunk and the ductus arteriosus was present
in the left side. Posterior to the aortic arches, the dorsal aorta
remained paired for a short distance, then the two descending aortae
fused. The coeliac artery branched off from the aorta and was seen
in the omentum between the stomach and the pancreas. In the heart,
the endocardial cushion of the atrioventricular canal had developed
to the extent that each atrium emptied largely into the ventricle of
the same side. However, since the interventricular septum had not yet
fused to the endocardial cushion the ventricles were still in communi
cation with each other through a small passage just ventral to the
cushion. The right and left atria, although separated by the septum
primum, were also still in communication with each other through the
interatrial foramen. The sinus venosus emptied into the right atrium
and the left common cardinal had swung over across the mid-line and en
tered the sinus venosus. Caudally the sinus venosus received the old
right vitelline vein which had become the inferior vena cava. The
ductus venosus had become a large vessel in the liver. It received
the large left umbilical vein from the body wall and the portal vein
from the liver and emptied into the sinus venosus. The much smaller
right umbilical vein received tributaries from the lateral body wall.
In sections passing through the mesonephros, the posterior cardinals
were seen, but the left posterior cardinal in that area was sometimes
amfti 1 and indistinct. The subcardinals also were present at this stage.


69
eating it. When ahe had finished she again licked her forepaws and
vulva and picked up the young mouse and licked it thoroughly* turning
it in various directions with her forepawa as she did so* Upon fin
ishing with the new bom she again placed it on the floor of the jar
in front of her and seemed to neglect it entirely until after the
birth of the next of the litter.
At about two minute intervals the mother got up, faced away
from the new bom mouse and stretched in the manner that was previous
ly described. After stretching several times she again resumed a
crouching position and began licking the vulva and mashing its sides
with her forepaws. Soon another tail and hind leg appeared. This
time she simply grasped the young with her forepawa and pulled it free.
The placenta followed immediately. She ate the placenta and paid no
attention to the new bora animal. The second young was bom at 11:23
A. thirteen minutes after the first. After consuming the second
placenta she again washed her forepawa, her face, and the region around
the vulva and then returned to the first bom and licked it thoroughly.
She paid no attention to the second young for about ten minutes, after
whieh she picked it up with her forepawa, cleaned it and placed it with
its sibling. She lay down close to them; covered them with her out
stretched neck, and remained generally in this position for about thir
ty minutes. However, she frequently got up, walked away from the two
young mice and stretched herself in the manner described previously.
At 11:55 the female again assumed the crouching position, and
started licking her vulva and pressing it with her forepawa. Soon the


85
of close and more distant relationship,
A well-developed corona radiata was observed in eggs estimated
to be from one to six hours post copulation but by fifteen to eighteen
hours this had degenerated into a small mat of mucoid-like strands.
Smith (1939) reported that he was unable to find a definitely formed
corona radiata in P. polionotus although he did see coronal cells scat
tered around the eighteen-hour pronuclear stage. Apparently the corona
had already degenerated in the earliest stages which he observed. A
zona pellucida was present from the pronuclear stage through the eight
cell stage. In some instances in the later stages it was broken and
irregular but this may have been done during histological preparation.
Smith (1939) reported the zona pellucida from two, four, six, and seven
cell stages but not from the pronuclear stage. On the other hand,
Lewis and Wright (1935) found a zona pellucida in all stages of house
mouse embryos through the blastocyst stage. It would appear, therefore,
that the zona pellucida persists for a longer period of time in the
house mouse than in P. polionotus.
The segmentation stages of P. polionotus had ovoid-shaped cells
and did not appear to be in direct contact with or under any pressure
from the tubal mucosa. This is in agreement vdth the observations of
Smith (1939) on this same species. However, Huber (1915) indicated
that the segmentation stages of the white rat were compressed between
the folds of the tubal mucosa, and commented that they were molded by
the tubal mucosa. Either these two forms must differ in this respect
or Huber*s material must have become shrunken or distorted during


44
tubercle was prominent enough to appear in sections as a separate
structure for several (10 miera) sections between the ventral body
wall and the tail.
Skeletal
Although there was no differentiation of skeletal material at
this stage* heavily staining concentrations of mesenchyme occurred in
the region of the otic capsules, centra, neural arches, girdles, and
limb buds. There was also a concentration of mesenchyme around each
nasal pit foreshadowing the formation of a paranasal cartilage and of
the nasal septum.
Muscular and Integumentary
While no muscle fibers had formed the eye muscles were outlin
ed for part of their course as aggregations of mesenchyme cells. On
the ventro-lateral side of the trunk, small conical knobs of epidermal
cells were present. These were the primordia of the mammae but they
were not macroscopically visible at this stage.
Fifteen Days
External
By fifteen days the head was bent forward at right angles to
the trunk and the mesencephalon still protruded anteriorly, but the
back was fairly straight (PI. IV,E). The lower jaw rested against the
dorsal side of the heart prominence. A short snout had developed and
it was elevated free from the underlying organs. The tip of the tail


PLATE III
A
*


49
and now appeared as large as the ventral lobe, although due to the
irregular shapes an exact comparison was impossible. The anal ori
fice was present, but was partly occluded by epithelium.
Circulation
Th8 auricles of the heart were quite large, but appeared col
lapsed in the sectioned material. Both atrioventricular canals were
greatly restricted and valves were forming in this area. Separating
the two atria was the well developed septum primum, but the atria were
still in communication through the interatrial foramen. The sinus
venosus vas greatly reduced in size and its opening into the right
atrium vas guarded by a well developed valvolas venosae. Fusion of
the interventricular septum with the endocardial cushion still had not
been accomplished, leaving the two ventricles in communication with
each other. The ventricles appeared to be relatively smaller than in
the previous stages, the walls having become thicker and the chambers
less extensive. There was no change in the veins returning blood from
the head region. The common cardinals fresa each side emptied into the
sinus venosus, and there was no sign of an innominate vein. In the
fourth aortic arches, the left side was larger than the right which
was indicative of their different fates, since the left persists as
the systemic arch while the proximal portion of the right becomes the
root of the right subclavian artery. There was also a noticeable dif
ference in the size of the right and left sides of the sixth aortic
arch. The larger left arch retained its connection with the dorsal
aorta by means of the well-developed ductus arteriosus, while the


31
The anal plate still persisted.
Circulatory
The obvious vessels anterior to the heart were the common car
dinals from which the anterior cardinals issued anteriorly and became
elaborately branched in the head region. The posterior cardinals were
well formed and unbranehed, while the dorsal aorta ivas paired in the
trunk region and gave off numerous segmental arteries to the body wall.
The first and second aortic arches had degenerated but the third was
large and still Intact at this stage. The heart was a large structure
and consisted of an auricle and a ventricle which were in broad com
munication with each other. As a result of the formation of trabecu
lae carneae the ventricle and conus arteriosus had became thick walled
in contrast to the thin membranous wall of the auricle. The interven
tricular septum had started to form on the ventral floor of the ven
tricle but at this stage it was merely a raised ridge of fibrous tissue.
The interatrial septum had also begun to form and was present as a
slight conical projection of cells from the dorsal roof of the auricle.
The vitelline veins at this stage were being broken up by the liver
cells and appeared indistinct but could still be recognized emptying
into the sinus venosus. The left umbilical vain had become large al
though it was still connected to the sinus venosus. Also, the vitel
line arteries were still paired channels, and hence had not yet given
rise to the anterior mesenteric artery.


22
developing embryo. The large cavity in the posterior amniotic fold
was not yet lined with mesoderm..
The fore-gut vas apparent as an Invagination of the embryonic
entoderm on the anterior side of the embryo, at the junction of the
embryonic and extra-embryonic ectoderm. The greatest length and width
measurements of a representative embryo at eight days of age were 620.0
x 201.5 miera,. The ectoplacental cone measured 155*0 miera.
Red blood corpuscles completely filled the spaces between the
cell masses of the uterine mucosa in the region of the ectoplacental
cone, and also surrounded Reichert's membrane. In addition, in some
sections erythrocytes were observed between Reichert's membrane and
the embryo proper and even in the proamniotic cavity.
The uterine lumen was completely obliterated at this stage of
development.
Nine Days
There was marked variation in development among four embryos
of the same litter after nine days of gestation. In the embryo show
ing the earliest stage of development, the amnion had formed thereby
delimiting the amniotic cavity. The exocoelom was in the process of
being formed, as four large cavities appeared in the mesoderm of the
posterior and lateral amniotic folds. In sagittal section a total of
six distinct cavities could be seen in place of the old proamniotic
cavity (PI. Ill,A). The most ventral of these constituted the newly
formed amniotic cavity, the most dorsal the eetoplacental cavity,
and the four smaller cavities marked the developing exocoelom. The


38
the region of the posterior limb buds, while between the anterior and
posterior limb buds the trunk had straightened slightly from the pre
vious stage. The tail curled to the right and its tip rested on the
right side of the face just cephalad to the eye. The limb buds had
not changed in appearance from the previous day, except that the pos
terior limb buds had become constricted so that they too were fan
shaped. The mesencephalon still protruded as a hump, and the roof of
the medulla of the brain was slightly more opaque than in the previous
age. The mid-dorsal line of superficial ectoderm above the spinal
cord all the way to the caudal end of the trunk was still quite trans
parent. The face was forming, although the features could not be
identified as mammalian. The naso-lateral process had fused with the
medial nasal process, and both had in turn fused with the maxillary
process. The mandible had just barely fused in the mid-ventral line.
Although the mouth was open, the tongue was not apparent in the oral
cavity. The ventral body wall was transparent enough to allow the
visceral organs to be seen. At this stage, the umbilical hernia was
a slight twist of the intestine at the base of the umbilical stalk.
A small genital tubercle was present.
Internal
Nervous
The meninges of the brain appeared as a thin membrane outside
of the marginal layer of the brain. The telencephalic vesicles had
continued evaginatlng both laterally and anteriorly, until they


grams millimeters
FIGURE 2
123


88
value in determining relationships in this group of rodents. However,
additional observations on other species are necessary before it can
be determined whether this character is constant or significant.
The so-called "inversion of the germ layers" long known in ro
dents and previously reported for Feromyscus by Ryder (1887) has been
fully verified in this study of P. polionotus. This phenomenon appears
to be widespread among the rodents, but there are so many species whose
embryological development has not yet been studied that it is impos
sible to say at present just how extensive it may be.
In one of the nine-day-old embryos there were four cavities in
the mesoderm of the amniotic folds (Pi. Ill,A), Snell (1941) mentions
that in the white mouse small cavities appeared in this area and soon
coalesced to form the exocoelom. He stated that he did not actually
observe the early stages of exocoelom formation in his studies and his
description of these cavities was based on the observation of Jolly and
Ferester-Tadie. The early stages of exocoelom formation were observed
1x1 £ polionotus; the cavities which were seen appear to be similar to
those described by Snell (1941)i and presumably exocoelom formation
follows the same pattern as in Mus.
A depression appeared in the thick base of the notochord of P.
polionotus embryos at nine days, and was present only in embryos of
this age group. This concavity appeared to correspond to one in the
thick base of the notochord in the white mouse which Snell (1941) des
cribed as being the archenteron. He noted that this structure which
appeared at seven and one-fourth days of age was transitory and


26
method is subject to error, but with fixed material it was about the
only one which could be employed.
The only conspicuous external feature was the fairly large
bulbous swelling representing the heart on the ventral side of the
animal just beneath the head procese.
Internal
A study of sectioned material showed that the mandibular arch
was fully formed and possessed the first pair of aortic arches. The
stomodeum appeared as a deep groove in sagittal section. The oral
plate had ruptured in one of the embryos, while in another embryo of
this same litter, it was still intact. Cephalic to the stcmodeal
opening was the triangular shaped pre-oral gut. The first pharyngeal
pouches were forming and in the median ventral wall of the pharynx
the thyroid plate had begun to evaginate.
The fore-gut continued posteriorly to about the lower level
of the pericardial chamber where it terminated in the anterior intes
tinal portal. The mid-gut was an open groove thus resembling the con
dition described by Snell (1941) in the white mouse of eight days and
eighteen hours development. At this stage, the length of the hind-gut
approached that of the fore-gut. The somatopleure of the coelom was
continuous with or closely applied to the amnion, while the somato
pleure of the extra-embryonic coelom constituted the wall of the yolk-
sac, The allantoic stalk was a loosely arranged mesodermal projection
extending through the extra-embryonic coelom and joining the chorion
(PI. m,c).


73
and sixfcy-three (49*22 per cent) were females.


90
many more species have been studied. According to the literature it
does not appear to occur generally in mammals and it may represent a
peculiarity of rodents.
Rand and Host (1942) reported a mean litter size of 3.968 and
a variation in litter size from three to five individuals for P,
polionotus. Their data were obtained from thirty-two litters caught
in the field and examinations of thirteen females with embryos. The
data obtained in this study from fifty-two litters born in the labora
tory showed a variation in litter size from one to six, and a mean
litter size of 3.35. In addition, a count of the embryos in twenty-
seven pregnant females showed a range in litter size of from one to
six and an average of 3.55. It seems apparent that two possible con
clusions could be drawn from these data. First, prenatal mortality
for this species must have been very low since the mean number of em
bryos in the pregnant females did not differ significantly from the
mean number of young in the litters.
Secondly, since both averages obtained in this study were so
similar to the mean reported for this species by Rand and Host (1942)
from their study of field litters it would appear that litter size in
P. polionotus was very little affected by captive conditions. Further,
the mean litter of P. polionotus is similar to those reported by Svihla
(1932), Pournelle (1952) and others for other species of the genus.
In this study, the sex ratio at birth, based on 128 mice, was
50.78 per cent males and 49.22 per cent females vMch seemed to indi
cate that if prenatal mortality or captive conditions reduced litter
size at all, they had no selective effect insofar as sex was concerned.


PARTURITION
Details of the birth act are known for relatively few wild
mammals. In the genus Peromyscua Svihla (1932) has described partu
rition in P. m. artendsiae and. Poumelle (1952) described birth of
young in P, gossyplnus* It was possible to make careful observations
of the birth of a litter in P polionotus. A gravid female was iso
lated around the eighteenth day of gestation and placed in a large
gallon jar fitted with a screen cover* On January 1, 1956, at 11
A, M#, she was lying on her back with her eyes closed* There were
frequent movements of her abdominal wall obviously as a result of the
movements of the young in the uterus. Within several minutes she got
up on her feet and stretched her body by keeping her back feet in
place and pulling forward with her forefeet. At this time, the sides
of the abdomen were depressed toward the midorsal line. She then as
sumed a sitting position and started licking her vulva and scratching
it with her forepaws. At 11:10 A. M. the tail and hind feet of the
first fetus appeared* She tugged at it with her forefeet and pulled
the young mouse out. It took only a minute for this entire process
to take place.
The mother placed the young mouse, still connected to her by
the very short whitish umbilical cord on the floor of the jar, and
maintaining her crouehed position began washing her forepaws and face.
Several minutes later the placenta appeared. She dislodged it from
her vulva with her forepaws, carried it directly to her mouth and began
68


32
Excretory
The mesonephroi were not massive at this stage of development.
The primordium of the metanephric duct which was solid except for it
hollow end paralleled the mesonephric duct for a very short distance.
Endocrine
Rathkes pocket was in contact with the infundibulum presag
ing the formation of the pituitary body and the thyroid primordium
was represented by a tubular mass of cells between the paired hyoid
arches in the floor of the pharynx.
Genital
Medial to the mesonephros vas a concentration of deeply stain
ing cells which constituted the beginning of the genital ridges.
Muscular and Integumentary
The somites had started to differentiate into regions, but
the myocoel was indistinct.
Thirteen Days
By the thirteenth day stage the body was in the form of a C
and was tightly coiled (PI. IV,G). Freso the region of the posterior
limb bud, the caudal part of the trunk and the tail raised up and
curled around so that the tail rested against the ventrolateral side
of the pharyngeal arches on the right side of the animal. The head,
at this stage, constituted at least half of the entire embryo. The
mesencephalon was a large dome-shaped prominence while the hindbrain


resulted in the formation of the corpora quadrigemina. In the meten
cephalon the central vails were quite thick and the lumen vas large.
The myelencephalon had a membranous roof and an eren larger lumen.
In the anterior region of the myelencephalon the wall had developed
a sulcus limitans which divided the lateral plate into the dorsal
alar plate and the ventral basal plate. However, eaudad to this area,
the lateral plates diverged to the extent that the right and left
plates were lying almost in the same plane and the tela choroidea
covered not only the dorsal part of the tube but the dorso-lateral
part as well. The choroid plexus had developed in the roof of the
myelencephalon and its leaf-like folds pushed down into the fourth
ventricle. The tela choroidea of the roof plate had invaginated in
to the lateral ventricles giving rise to the choroid plexus of this
area. From the ventro-lateral wall of each hemisphere, the corpus
striatum had developed as a lobular fold bulging into the ventricles.
It was quite massive in size but did not show any special cellular
differentiation. The foramina of Monroe were reduced to small pas
sages. The thalamus showed considerable increase in size over the
previous stage. Olfactory lobes were present on the ventral side of
the cerebral hemispheres and received nerve fibers from the olfactory
pits. The walls of the neural tube had thickened, and in so doing
had constricted the neuroooel into a club-shaped cavity which was re
stricted to the dorsal half of the tube. There was a slight indenta
tion of the floor plate presaging the formation of the ventral median
fissure, Sctodermal folds, the forerunners of the lids, were evident


PLATE II
105


FUTE VII


65
development. Actually, the implantation sites showed very little dif
ference in size for the first several days, but from twelve through
eighteen days of age a daily size increment was quite pronounced.
There was only a slight variability in the measurements of swellings
in the same uterus.
Since pregnant females are often collected in the field some
knowledge of the relative size of uterine swellings for each day of
gestation is advantageous in determining the approximate stage of preg
nancy. On the basis of the present data, it appears that the age of
embryos can be determined from the size of the uterine swellings with
in a range of several days.
Weights and Volumes of Embryos
After they had been removed from the uterus and stripped of
their fetal membranes, fixed embryos from twelve through eighteen days
of age were weighed and their volumes determined. These data are pre
sented in Table 3. Due to their small size embryos younger than twelve
days could not be dealt vdth by this method.
TABLE 3
MEAN WEIGHTS AND VOLUMES OF EMBRYOS
12-18 DAYS OF AGE
1 Age of
Embryo
tiiier
Size
Volume in
Millimeters
heights in
Grams
12
2
.0100
.0057
13
4
.0232
.0281
14
3
.0453
.0456
15
3
.0800
.0801
16
3
.1390
.1335
17
3
.2276
.2115
18
4
.4092
.3851


/
98
for the first thirty days after birth, but then slowed down con
siderably and virtually ceased after fifty days.
10. The ear pinna unfolded on the third or fourth day and incisor
teeth errupted the gum from the sixth to eighth day. Young mice
i
reacted to sound at around eleven days of age. The eyes opened
between thirteen and fifteen days, usually on the fourteenth day.
The vagina of females opened between twenty-nine and thirty-five
days and the post juvenile molt started at thirty-four to thirty-
six days of age,
11. In general both prenatal and postnatal development of P, polionotus
resembled in broad outlines the development of Mus. Rattus. and
Mesocricetus, whose development has been adequately described. In
the time of appearance and the shape and structure of the ectopia-
cental cone, Peromyscus resembles Mus and Rattus rather than
Mesocricetus. to which it is generally considered to be more close
ly allied. It is possible that the nature of the ectoplacental
cone might be of some significance in the taxonomy of the rodents
in the murid and cricetid groups.


PLATE II
A.Longitudinal section of implanted embryo at five days;
X 100; exc, extra-embryonic ectoderm; ee, embryonic
ectoderm; pe, proximal entoderm; yc, yolk cavity;
tr, trophectoderm.
B.Longitudinal section of embryo at six days; X 100.
C. Longitudinal section of embryo at seven days; X 100.
D. Longitudinal section of embryo at eight days; X 100;
epc, ectoplacental cone; af, posterior amniotic fold;
e, entoderm; m, mesoderm; ec, embryonic ectoderm;
pc, proamniotic cavity; rm, Reicherts membrane.
E. Longitudinal section of embryo at nine days showing
three cavities and allantois; X 100; ect, ectoplacental
cavity; c, chorion; exc, exocoelom; a, amnion; ac, amni-
otic cavity; al, allantois.
F.Longitudinal section of mouse embryo at nine days show
ing a break in the chorion allowing a connection of the
ectoplacental cavity to blood-filled spaces of the ecto
placental cone; X 100; ect, ectoplacental cavity;
c, chorion; exc, exocoelom; a, amnion.
iv


'**
PLATE VII
A, A litter at four days of age; a millimeter scale is
included in photograph.
B, A litter at eleven days of age; a millimeter scale is
included in photograph.


DESCRIPTION OF PRENATAL DEVELOPMENT
Zero Hours
Two recently ovulated eggs were found in the right Fallopian
tube and three in the left of a specimen killed immediately upon find
ing motile sperm in a vaginal smear. Each egg appeared as a somewhat
spherical, homogeneous mass of lightly staining cytoplasm. There were
no visible nuclei in any of the sections, nor were any polar bodies
present. It could not be ascertained whether or not these eggs were
fertilized, although sperm were present in the Fallopian tubes at the
level of the ova. Each ovum was surrounded by a corona radiata which
consisted of a dense mass of deeply-staining cells, and a wide zona
pellucida was evident between the egg and the corona cells (PI. I,a).
The maximum dimensions in the plane of section of these ova were, in
miera: 62 x 46.5; 69 x 54.2; 62 x 54.2; 62 x 46.5; 62 x 50.
Fifteen Hours
Embryos representing the pronuclear stage were obtained from
a mouse killed fifteen hours after a few non-motile sperm and sperm
fragments were found in the vaginal smear. In most pregnant females
which had mated many motile sperm were found in the vaginal smears.
It seems apparent, therefore, that a longer period of time had elapsed
between copulation and the making of the smear in this particular
specimen than was usually the case. However, under the method of age
determination used in this work, this must nevertheless be recorded