Citation
Effects of maternal hypervitaminosis A on cleft palate formation in rat embryos

Material Information

Title:
Effects of maternal hypervitaminosis A on cleft palate formation in rat embryos
Creator:
Kochhar, D. M ( Devendra M. ), 1938-
Publication Date:
Language:
English
Physical Description:
ix, 112 leaves, : illustrations; 28 cm.

Subjects

Subjects / Keywords:
Vitamins -- pharmacology. ( mesh )
Cleft Palate. ( mesh )
Rats. ( mesh )
Anatomical Sciences thesis Ph. D
Dissertations, Academic -- Anatomical Sciences -- UF
Genre:
Academic theses ( lcgft )
Academic theses ( fast )

Notes

Thesis:
Thesis - University of Florida.
Bibliography:
Includes bibliographical references (leaves 102-109).
General Note:
Manuscript copy.
General Note:
Vita.

Record Information

Source Institution:
University of Florida Libraries
Holding Location:
University of Florida Libraries
Rights Management:
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. This item may be protected by copyright but is made available here under a claim of fair use (17 U.S.C. §107) for non-profit research and educational purposes. 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.
Resource Identifier:
024116907 ( ALEPH )
21351378 ( OCLC )

Downloads

This item has the following downloads:


Full Text
EFFECTS OF MATERNAL HYPERVITAMINOSIS
A ON CLEFT PALATE FORMATION
IN RAT EMBRYOS
By
DEVENDRA MOHAN KOCHHAR
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
August, 1964


DEDICATION
To the memory of my father, Trilok Nath Kochhar, this
dissertation is respectfully dedicated.


ACKNOWLEDGEMENT
The author wishes to express his sincere gratitude to Dr. E.
Marshall Johnson for his supervision and guidance throughout the
progress of this investigation.
The author is greatly indebted to Dr. James G. Wilson for pro
viding an opportunity for a rewarding academic experience and for his
valuable suggestions and ideas conducive to original thoughts.
Deep sense of appreciation is due to the members of the author's
supervisory committee for advice and encouragement. Acknowledgement
is given to Miss Carol E. Ruppert and Mr. Marvin E. McGraw for their
assistance in the preparation of illustrations.
The financial support of Training Grant 3T1 GM 579"0^S1 of the
National Institute of Health is gratefully acknowledged.
For many fruitful discussions and stimulating criticism, and
for assistance in the final preparation of this manuscript, special
acknowledgement is given to author's wife, Omila S. Kochhar.
iii


TABLE 07 CONTENTS
Page
ACKNOWLEDGEMENT iii
LIST 07 TABLES vi
LIST OF 7IGURES vii
INTRODUCTION 1
Cortisone as a Teratogen in Cleft Palate Formation ... 4
Morphological Sequence of Cortisone Induced Cleft
Palate 5
Hypervitaminosis A as a Teratogen in Cleft Palate
Formation 7
Morphological Sequence of Hypervitaminosis A
Induced Cleft Palate 9
Purpose of This Study 13
MATERIALS AND METHODS 15
Breeding and Maintenance of Animals 15
Experiment 1. Incidence of Cleft Palate 15
Experiment 2. Anatomical Morphogenesis of Normal
and Cleft Palate 17
Experiment 3. Radioautographic Studies 19
Experiment 4. Examination of the Effects of Hyper
vitaminosis A on the Epiphyseal Cartilage of
Adult Eats 21
RESULTS 24
Teratogenic Action of Hypervitaminosis A 24
Effects of Maternal Injections of Cortisone and
Papain on Embryos 27
Morphogenesis of Cleft Palate Induced by Hyper
vitaminosis A 31
Effects of Hypervitaminosis A on Adult Hats 79
DISCUSSION 86
Normal Closure of Secondary Palate 86
Cleft Palate and Hypervitaminosis A 88
Embryonic s35 Incorporation Following Maternal
Hypervitaminosis A 94
Interaction Between Hypervitaminosis A and Cortisone 98
SUMMARY
99
iv


Page
LITERATURE CITED 102
APPENDICES 110
Appendix I. Analysis of Rockland Stock Diet for Rats Ill
Appendix II. Composition of Bouin's Fluid 112
v


LIST OF TABLES
Table Page
1. Dosage of Vitamin A Administered to Rats for Dissolution
of Cartilage Matrix 22
2. Teratogenic Action of Hypervitaminosis A Observed in
Terra Fetuses 26
3. Weights of Term Fetuses After Maternal Hypervitaminosis A 29
4. Crown Rump Length and Head Length of Term Fetuses After
Maternal Hypervitaminosis A 30
5. Effects of Maternal Injections of Cortisone and Papain
Observed in Term Fetuses 32
6. Weights of Term Fetuses after Maternal Injections of
Cortisone and Papain 33
7. Teratogenic Effects of Hypervitaminosis A and Cortisone
Observed in Term Fetuses 34
S. Comparison of Control and Kypervitaminctic A Embryos on
Days 15, 16 and 17 of Pregnancy
35


LIST OF FIGURES
Figure Page
1. Fetal resorption as a function of day of gestation
maternal vitamin A treatment commenced 25
2. Frontal freehand sections of heads of day 20 fetuses ... 28
3. Frontal freehand sections of heads of day 15 embryos ... 36
4. Graphs comparing the percentages of day 15 control and
vitamin A treated embryos on the basis of shape and
orientation of their palatine shelves 38
5. Frontal freehand sections of day 16 embryos 40
6. Graphs comparing the percentage of day 16 control and
vitamin A treated embryos on the basis of shape and
orientation of their palatine shelves 42
7. Frontal freehand sections of day 17 embryos 44
S. Graphs comparing the percentages of day 17 control and
vitamin A treated embryos on the basis of shape and
orientation of their palatine shelves 45
9.Frontal sections of anterior one third of palatine shelf
of day 15 control embryo 47
10. Frontal sections of head of day 15 control embryo .... 49
11. Frontal sections of anterior one third of palatine shelf
of day 15 vitamin A treated embryo 50
12. Frontal sections of heads of day 15 vitamin A treated
embryos through middle third of palatine shelf 52
13. Frontal sections of posterior third of palatine shelves
of day 15 embryos 53
14. Radioautographs of day 15 control embryo 55
15. Radioautographs of day 15 vitamin A treated embryo .... 56
16. Frontal sections of head of day 16 control embryo .... 5S
via


Figure Page
17. Frontal sections of heads of day 16 control embryos ... 59
18. Frontal sections of head of day 16 control embryo show
ing movement of palatine shelves to horizontal
position 60
19. Frontal section of head of day 16 control embryo show
ing origin of palatine shelf movement to horizontal
position 61
20. Frontal section of head of day 16 control embryo .... 63
21. Frontal section of head of day 16 vitamin A treated
embryo showing deformed palatine shelves (?S) 63
22. Frontal sections of heads of day 16 vitamin A treated
embryos 64
23. Frontal sections of hea.ds of day 16 vitamin A treated
embryos 65
24. Radioautographs of frontal sections of heads of day
16 embryos; nasal cartilage 67
25. Radioautographs of frontal sections of heads of day
16 embryos; mesenchymal tissue of palatine shelves 68
26. Radioautograph of frontal section of head of day 16
vitamin A treated embryo; heterotopic cartilage
within maxillary osteoblastic tissue 69
27. Frontal sections of head of day 17 control embryo .... 71
28. Comparison of frontal sections of anterior region of
two day 17 vitamin A treated embryos 72
29. Frontal section of posterior region of head of day
17 vitamin A treated embryo 73
30. Serial frontal sections of head of day 17 vitamin A
treated embryo 75
31. Frontal sections of head of day 17 vitamin A treated
embryo 76
32. Radioautographs of frontal sections of heads of day
17 embryos; palatine shelves 77
33. Radioautograph of frontal section of palate of day
17 vitamin A treated embryo 78
viii


Figure Page
34. Radioautographs of frontal sections of heads of day
17 embryos; nasal cartilage 30
35. Radioautographs of sections of day 17 embryos; carti
lage of limb bone 81
36. Toluidine blue stained sections of epiphyseal carti
lages of control and vitamin A treated rats 82


INTRODUCTION
The general opinion of the scientific community concerning the
relative importance of environment and heredity in the production of
congenital malformations has vacillated between these two major causes
through the years. It was known from the studies of experimental
embryologists such as E.B. Wilson, T.II. Morgan, Jacques Loeb, and
Charles Stockard that anatomic defects could be produced in submammalian
embryos by subjecting them to injurious environmental factors (Corner
'60). The mammalian embryos, however, were not thought to be readily
available or susceptible to adverse environmental changes due to
supposed protective influences of the uterus, the embryonic membranes
and their encompassed fluids. Such views permitted the persistance of
the opinion that the presence of congenital defects in mammalian
embryos was predominantly due to genetic factors. In fact, some con
genital malformations in humans, e.g., chondrodystrophy (M^rch, '41)
'lobster-claw' defects of hands and feet (Stiles and Pickard, '43), as
well as many structural defects in experimental animals do have a well-
defined genetic cause (Glueksohn-Waelsh, '54} Zwilling, '56 and Silagi,
62).
An indication that the mammalian embryo may not be completely
guarded against environmental disturbances was reported by Hale ('33,
'35, '37), who noticed the birth of anophthalmic young to vitamin A
deficient pigs. Gregg ('41, *45) described the occurrence of congeni
tal defects in the human fetus after maternal infection with rubella
1


2
(German measles). Warkany and Nelson's ('/+0, >41) findings regarding
the teratological effects of riboflavin deficiency in rats opened the
way for extensive studies on experimentally produced malformations in
mammalian embryos.
A wide variety of teratogenic agents have been discovered which,
when applied to the pregnant female, produce many types of malforma
tions in the developing embryo. Agents discovered to date range
through maternal nutritional deficiencies, vitamin excesses, endocrine
imbalances, irradiation, alkylating agents and viral infections. Such
agents and the resulting malformations in the vertebrate embryos have
been thoroughly reviewed by Kalter and Warkany ('59), Wilson ('59),
and Pitt ('62).
Though it is now known that congenital malformations in mammals
can result from both genetic and environmental causes, it is difficult
to determine at birth which one of the two factors was primarily
operative in producing any particular abnormal fetus. Also important
to determine in the case of an abnormal fetus is how the malformation
originated. One way to begin to understand the possible cause of a
malformation observed at birth is to examine the prospective deformed
embryo during its period of very early development and morphogenesis.
There exists a great disproportion botween the number of studies deal
ing with the production and manifestation of congenital malformations,
on the one hand, and investigations dealing with the morphogenesis of
these malformations during the earlier embryonic period, on the other.
To perform the latter type of studies, however, it is essential that
the malformations under investigation occur in a high percentage of
embryosj this will insure that the embryo being examined during the


3
early developmental period is one which probably will be deformed at
birth. Through the use of certain carefully selected environmental
teratogens, it is now possible to produce in experimental animals, a
high frequency of a particular congenital abnormality which might occur
only sporadically.
The understanding of the teratogenic action of an agent has to
be carried beyond morphological description to the biochemical or other
basis for its action. To be able to do this one must know first some
thing of the physiological and pharmacological effects of the agent on
the tissues of animals in general. Therefore, it is advantageous to
select teratogenic agents which have rather well defined metabolic
functions.
A congenital anomaly which lends itself to such an investigation
is cleft palate. Though this anomaly occurs in response to a very
large number of teratogenic agents, such as nutritional deficiencies
or excesses, hypoxia, hypothermia, drugs, hormonal imbalances, alkylat
ing agents, and x-irradiation (Kalter and Warkany, '59), it occurs
specifically in a particularly high incidence as a result of certain
teratogenic procedures. Three reported procedures are: l) cortisone
injections administered to pregnant mice, as reported by Fraser and
associates ('54) and by Kalter (*54), 2) hypervitaminosis A induced in
rats as reported by Cohlan (53) and Giroud and Martinet ('56), and
3) pteroylglutamic acid deficiency as studied by Nelson et al.('52).
Some of the studies completed to date on the early morphogenesis
of cleft palate reveal that more than one morphologic factor may be
involved in this abnormality. It is yet to be determined whether the
modification of a particular factor is specific to a certain teratogen


4
or if there are common factors in the embryo which when modified by
different teratogens result in a specific defect such as cleft
palate. Answers remain to be found for the following questions: l)
If two teratogens produce a similar defect in the embryo, do they act
through a common biochemical or physical pathway? 2) Do two agents
having similar biochemical properties produce similar malformations?
3) Is there any correlation between morphogenesis of the defect and
the biochemical pathway of action?
Cortisone as a Teratogen in Cleft Palate Formation
The teratogenic action of cortisone in producing cleft palate
without cleft lip or other gross abnormalities in mouse embryos was
first reported by Baxter and Fraser (50)* Administration of 2.5 mg
(milligram) cortisone acetate daily for 4 days beginning at day 11 l/3
of gestation produced the highest frequency of cleft palate with a
minimum incidence of fetal resorption (Fraser and Fainstat, 51). The
incidence of cleft palate varied in different strains of mice; it was
100£ in embryos of A/JAX strain of mice, and 15£ in those of C573L
strain (Fraser et al., 154)
Evans and Clingen (53) reported that cortisone had no teratogenic
influence on the rat embryo, but in 1956 Cost reported the production
of cleft palate in this species by means of cortisone. Costs observa
tions, however, should be considered questionable since he administered
cortisone by way of intraperitoneal injections into the fetuses and
thus introduced another known cleft palate producing factor by piercing
the amnion (Trader et al., 56). Although having no teratogenic


5
action on rat embryo when administered alone, cortisone has been
reported to potentiate the teratogenic action of hypervitaminosis A.
Woollam and Millen (*57) observed that in rat embryos the expected 29%
incidence of cleft palate induced by hypervitaminosis A could be
increased to 100£ if the mothers were treated simultaneously with corti
sone. Such potentiation by cortisone was also observed in the case of
brain malformations produced by hypervitaminosis A. This result, how
ever, could not be repeated by Cohlan and Stone ('6l), who were unable
to alter the incidence of vitamin A induced malformations by means of
cortisone. Fainstat (*54) bas reported that maternal cortisone treat
ment induces cleft palate in offspring of rabbits.
Morphological Sequence of Cortisone Induced
Cleft Palate
On the basis of studies on the palatine shelves of living as
well as fixed embryos from cortisone treated mice, "walker and Fraser
(57) proposed that cleft palate in these embryos developed because of
delay in the initiation of movement of the palatine shelves from a vertical
position at the side of the tongue to a horizontal position superior
to the tongue. During this delay in palatine shelf movement, growth
of the head continued so that when the palatine shelves finally did
become horizontal it was at a time later than normal and they were too
far apart to meet in the midline and fuse. Larsson (60) suggested
that the occurrence of cleft palate in response to cortisone treat
ment could be due to the interference in the synthesis of chondroitin
sulfuric acid. That acid-mucopolysaccharides are present in the
palatine shelves of normal embryos was demonstrated by Walker and


6
Fraser ('56), and Larsson, Bostrom and Carlsoo ('59)* on the basis of
metachromatic staining with toluidine blue and S-^ incorporation
or
respectively. Larsson ('62) employed -radioautography and reported
that in mouse embryos from cortisone treated mothers there was a
reduction in synthesis of sulfo-mucopolysaccharides in the palatine
shelves as well as in other regions of the embryo. On this basis he
concluded that reduced production of acid-mucopolysaccharides during
the time of palatal closure, day 14 in the mouse embryo, was the mech
anism by which cortisone derived its teratogenicity.
It has been reported that cortisone suppresses fibroblast pro
liferation and inhibits wound healing. Ragan et al. ('50) and Plotz
et al. ('50) compared the healing of an artificially produced wound in
normal and cortisone treated rabbits, and observed that following corti
sone treatment, neither new fibroblasts nor new blood vessels appeared
at the site of the wound in contrast to a profuse growth of these com
ponents in the normal. Ground substance, as determined by toluidine
blue metachromasia, was also much reduced in the cortisone-treated
animal. In the presence of cortisone the connective tissue of animals,
both in vivo and in vitro. was depressed in its abilities to incorporate
inorganic sulfate into chondroitin sulfate (Layton 151, 151) Schiller
and Dorfman ('57) demonstrated that cortisone inhibited not only the
35
incorporation of S -labelled sulfate into chondroitin sulfuric acid
but also reduced synthesis of the whole acid-mucopolysaccharide mole-
14
cule as evidenced by depressed incorporation of C -labelled acetate
into both hyaluronic acid and chondroitin sulfuric acid.


7
Hypervitaminosis A as a Teratogen in Cleft Palate Formation
Cohlan (53) reported the teratogenic effect of excess vitamin
A in CF Wistar rats. Pregnant rats were fed 35,000 i.u. of vitamin A
per day by intragastric intubation beginning on either day 2, 3, or 4
of gestation and continued through day 16. Besides exhibiting a 30%
incidence of cleft palate, embryos also displayed excencephaly, cleft
lip, brachygnathia, shortening of maxilla and various eye defects such
as microphthalmia, anophthalmia, open eye, exophthalmos and lenticular
cataract. These results were confirmed by Giroud and Martinet (54)
These authors also obtained a differential incidence of cleft palate
by giving 60,000 i.u. vitamin A per day by oral intubation for 3 con
secutive days beginning at different periods in gestation (C-iroud and
Martinet, '55, 56). That is, 1$ of the embryos had cleft palate when
treatment was started on day 5, 22% had the defect when treatment began
on day 8, 92% when it began on day 11, 49$ on day 14, and none if the
treatment was not started until day 18. Vitamin A acetate and vitamin
A palmitate yielded identical results. Mo malformations were produced,
however, if vitamin A was administered intraperitoneally (Gebauer, 54)
or subcutaneously (Woollam and Millen, '57).
Deuschle, Geiger and Warkany ('59) described an interesting
oculodentofacial pattern of abnormalities in fetuses of rats following
maternal hypervitaminosis A. The outstanding features observed in this
study were exophthalmos, maxillomandibular ankylosis, the presence of
heterotopic cartilage in the maxilla, and the absence of some molar
teeth. Cleft palate, of course, was also encountered. Histologic
sections of the abnormal fetuses revealed that the eyes were almost


8
normal in structure though the lids were absent. Exophthalmia was
explained on the basis of skeletal anomalies of the face. These
investigators observed that the zygomatic extension of the maxilla,
which normally should form the inferior orbital wall, was missing in
the experimental fetuses. The maxilla itself harbored a heterotopic
cartilage which posteriorly seemed to make up the inferior orbital
wall. These authors concluded that in the experimental fetus there
was a shortening of the head, maxilla and mandible. The more proximal
portions of the jaws were most severely affected and it was felt that
this reduction resulted in anomalies of the molar teeth.
Kalter ('60) studied the teratogenic effect of maternal hyper-
vitaminosis A on 3 strains of mice, e.g., A/Jax, DBA/Uax, and C3H/Jax.
A syndrome of dentofacial anomalies essentially similar to that found
in rats was produced. Cleft palate was present and there were abnor
malities of dental structures, such as supernumerary, absent or ectopic
teeth. Heterotopic cartilages were present at the corners of the mouth
in or close to the maxillary bone, aid oral tissue was trapped as
invaginations into buccal cavity. The mouth cavity was drastically
reduced in size. This reduction was thought to result from a condition
whereby the abnormally shaped palatine shelves did not ascend and
instead fused with the lateral buccal surfaces. The tongue also fused
with both palatine shelves and the gingival tissue, thus resulting in
the apparent obliteration of the lateral recesses of the mouth.
Kalter and Warlcany ('6l) reported that some of the abnormalities, such
as ankyloglossia, which were found in mice, were not observed in rats.


9
Morphological Sequence of Hypervitaminosis A
Induced Cleft Palate
No detailed study for elucidating the morphological sequence of
cleft palate formation in rat embryos after maternal hypervitaminosis
A has yet been performed. Morphogenesis of cleft palate in mouse
embryos from excess vitamin A treated mothers was studied by direct
examination of the palate (Walker and Crain, *60) and by histological
means (Kamei, '62). These authors concluded that the factor responsi
ble for cleft palate formation was similar to that observed after corti
sone treatment; a delay existed in the time of palatine shelf movement
from vertical to horizontal position. No attempt was made to correlate
this deformity with any of the relatively well established effects of
excess vitamin A on tissues.
Investigations into the effects of excessive vitamin A on intact,
non-pregnant animal are relatively recent. Uolbach (47) reported that
the major abnormalities in hypervitaminotic A animals are encountered in
bone and cartilage and noticed that in the skeletal tissue of such
animals maturation of cartilage was accelerated. This investigator also
observed a rapid resorption of bone and cartilage which resulted in
spontaneous fractures.
To determine direct effects of hypervitaminosis A on cartilage
and bone, Pell and Mellanby ('52) cultured the limb bones of day 5 and
6 embryonic chick and fetal mice in a medium containing excess vitamin
A alcohol. In this experiment, bone was quickly resorbed, cartilage
matrix was reduced in mass and lost its characteristic metachromatic
staining, although it retained its affinity for van Giesson's stain.
These findings implied that whereas a dissolution of chondroitin


10
sulfate had occurred, the collagen portion of the matrix -was still
present. That these effects'of excess vitamin A were not directly on
the intercellular material but rather were through the activities of
the chondrocytes was considered to be proven by the fact that no such
effects appeared if the cells were killed beforehand by heating the
cartilage to 45C. Jinother, and quite different effect of vitamin A
was studied on embryonic ectoderm by Fell and Mellanby (*53). If the
embryonic chick ectoderm was cultured in a control medium without the
addition of vitamin A, it developed a keratinized layer but under the
influence of excess vitamin A in the medium, the ectodermal cells
became a ciliated columnar epithelium and secreted mucus.
The above two studies serve to illustrate that vitamin A
influences the metabolism of various tissues such as cartilage and
mucus epithelium which have in common the function of synthesizing
acid-mucopolysaccharides.
On the basis that esterified sulfate is an important component
of the mucopolysaccharide molecule (Dziewiatkowski, *51 and Bostrom,
35
52), and since a major part of parenterally injected S -labelled
inorganic sulfate is recovered from animals as an ester linked to a
mucopolysaccharide molecule (Bostrom, 53), several investigators have
examined the effects of vitamin A deficiency or excess on the incor
poration of the sulfate into cartilaginous tissues and mucus membranes.
Fell, Mellanby and Pele ('56) utilizing radioautographic techniques,
studied the incorporation of by explants of bone from the limb
of embryonic chick cultured in either a normal or an excess vitamin A
medium. These authors concluded that not only did vitamin A cause a
dissolution of the cartilage matrix but it also inhibited further


11
or
synthesis. Dziewiatkowski (54) compared the contents of skeleton
from vitamin A deficient, normal control, and rats treated with vitamin
A that had been previously fed a vitamin A deficient diet. Using
radioautography and biochemical analysis, he observed that in vitamin A
deficient rats the skeleton synthesized less chondroitin sulfate than
normal. Administration of vitamin A to deficient animals was promptly
35
reflected by an increased rate of S uptake. He further reported
that after the administration of vitamin A to deficient rats not only
the rate of synthesis but also the degradation rate of chondroitin
sulfate in the skeleton was accelerated. This was proved by the fact
that vitamin A treated animals accumulated more during the first
24 hours after injection than the untreated, while by 72 and 120 hours
the vitamin A treated had less S-^ than the untreated group. In the
case of vitamin A deficient rats that later had been given vitamin A
there was also an increase in the specific activity of the sulfate-
sulfur of sulfo-mucopolysaccharides isolated from skin.
Wolf and Varandani (60) and Wolf, Varandani and Johnson (*6l)
obtained data which indicated that mucopolysaccharide synthesis by
homogenate of rat and pig colon mucosa was vitamin A dependent.
Radioactivity was incorporated into mucopolysaccharides by incubating
the homogenate with -labelled sulfate or C ^--labelled glucose. The
incorporation of radioactivity into mucopolysaccharides by the colon
homogenate of vitamin A deficient animals was about one half that
encountered in homeogenates from normal animals. When a suspension
containing 10 ug of vitamin A was added to the incubation medium of
the colons from deficient animals, radioisotope incorporation was
elevated to the control level. This stimulation by vitamin A of


12
acid-mucopolysaccharide synthesis is very significant when observed in
the light of results to be reported in this dissertation.
The influence of excess vitamin A on the keratinization of
embryonic skin explants was studied radioautographically by Pele and
Fell (10), who showed that deeper layers of the epidermis in both
control and excess vitamin A culture media liad identical uptake of
S354. In control cultures from older embryos the superficial layers
of the epidermis had only scanty S''3^ uptake, while the corresponding
area in excess vitamin A cultures had intense incorporation of
and, in addition, secreted mucus. This meant that in normal skin the
basal cells synthesize sulfated-mucopolysaccharides until the time when
keratinization begins. This inhibition of synthesis in explants cul
tured in vitamin A containing medium never occurs, hence mucus
epithelium forms instead of keratinized epithelium.
Thomas et al. ('60) reported that bypervitaminosis A in intact
young rabbits resulted in marked depletion of cartilage matrix in the
epiphyseal and articular cartilages. These authors also confirmed the
previous observations of Thomas (56) and McCluskey and Thomas (58)
that injections of small amounts of papain into young rabbits produce
histological changes in the cartilage comparable to those produced by
hypervitaminosis A. Fell and Thomas ('60) described the effects of
crystalline papain protease on embryonic chick cartilage and fetal
mouse bone and compared them with changes observed in these tissues
following treatment with excess vitamin A. Although both vitamin A
and papain removed chondroitin sulfate from the cartilage matrix, only
vitamin A affected the chondroblasts in that they lost their glycogen
and became reduced in size. In contrast to these similarities in vivo,


13
these two agents differ considerably in their effects on fetal nouse
bone in vitro wherein the bone is unaffected by papain but rapidly
disintegrates in the presence of excess vitamin A.
From the work of Fell and her colleagues mentioned in the pro
ceeding pages it appears established that in the presence of excess
vitamin A, chondroitin sulfate is dissolved from cartilage matrix both
in vivo and in vitro. Their conclusion that vitamin A also inhibited
cellular synthesis of acid-mucopolysaccharides is, however, not fully
established. In young rabbits McSlligott ('62) studied radioautograph-
ically the effects of vitamin A treatment on the ability of chondrocytes
to fix radioactive sulfur. He strongly suggested that hypervitaminosis
A primarily inhibits the function of chondrocytes in synthesizing acid-
mucopolysaccharides and that the dissolution of cartilage matrix is a
secondary effect. Frape et al. ('59) reported that addition of vitamin
A to a normal diet fed to piglets considerably reduced the accumulation
35
ol injected S in costochondral junctions and other tissues.
Recently Lucy, Dingle, and Fell (*6l) and Dingle (*61) reported
that under the influence of vitamin A, the embryonic chick limb bones
grown in culture release a proteolytic enzyme from intracellular par
ticles similar in size to mitochondria (probably lysosomes). This
released proteolytic enzyme was thought to act on the mucopolysaccharide-
protein complex of the cartilage matrix and breaks down the protein
moiety thus releasing mucopolysaccharides from the matrix.
Purpose of This Study
This investigation was proposed to examine the early morpho
genesis of cleft palate in rat embryos after maternal hypervitaminosis A


14
in an effort to compare it with that described by Walker and Fraser
('57) and Larsson ('62) in embryos from cortisone treated mice. Such
an approach would demonstrate whether or not these two teratogenic
agents, which in other systems were shown to have an effect on the
acid-mucopolysaccharide content of tissues, induced cleft palate by
bringing about common structural modifications in the embryos.
35
On the basis of his studies on S incorporation into mouse
embryos from control and cortisone treated mothers, Larsson ('62)
suggested that the teratogenic action of cortisone in these embryos
could be correlated with the presence of reduced amounts of acid-
mucopolysaccharides in the affected palate. Results of investigations
attempted to reveal if such a correlation existed in rat embryos after
maternal hypervitaminosis A would also be reported in this disserta
tion. Assuming that the amount of intercellular ground substance was
a measure of acid-mucopolysaccharide content of embryonic tissues, the
former was determined in embryos from normal and hypervitaminotic A
mothers by employing radioautographic techniques coupled with
toluidine blue staining. Finally, if the hypothesis that the terato
genic action of cortisone and hypervitaminosis A was mediated through
their effect on the acid-mucopolysaccharides of tissues was correct,
then treatment of the maternal rat with papain protease might also
result in similar congenital malformation, and, therefore, such
potential teratogenicity would be studied.


MATERIALS AMD METHODS
Breeding and Maintenance of Animals
Black-hooded female rats obtained from Rockland Farms,^ ranging
from 60-90 days of age and weighing 150-200 grams were employed in this
study. The animals were kept in stainless steel cages and given a stock
o
diet and distilled water ad libitum. Every evening the estrus cycle
was diagnosed for each female by microscopic examination of vaginal
smears (Blandau et al., '41) and those females in proestrus were placed
overnight in individual cages with sexually mature male rats. The
presence of a plug or spermatozoa in the vaginal smear at 10:00 A.M.
the next morning indicated that mating had occurred and this day was
considered day 0 of pregnancy.
Experiment 1. Incidence of Cleft Palate
To determine the incidence of cleft palate in these rats, 72
pregnant females were divided into four groups; one group for each of
four experimental protocols.
Group 1. The animals in this group received 60,000 i.u. (inter-
O
national units) of vitamin A acetate"^ daily for 3 consecutive days
^Rockland County, New York.
^See Appendix 1.
^Obtained from Nutritional Biochemical Corporation, Cleveland, Ohio.
15


16
during gestation. The treatment was started on either day 8, 9, 10,
or 11. The vitamin A preparation* was administered orally by means of
a blunted spinal needle attached to a tuberculin syringe. The control
animals were given 1 ml of pure cottonseed oil in the same manner from
day 9 through day 11 of pregnancy.
Group II. Pregnant females in this group received 50 mg corti
sone acetate per day for 5 consecutive days. A commercially available
5
saline suspension of cortisone acetate was injected into the preaxial
thigh muscles beginning on day 9 and continuing through day 13 of gesta
tion. Control animals received a similar volume of physiological saline
from day 9 through 13.
6
Group III. Animals in this group received 50 mg papain daily
by intraperitoneal injection for 5 days beginning on day 9 of gestation.
Saline injected animals served as controls.
Group IV. This group was subdivided into two parts each of which
received combined treatments: a) the pregnant animals were given 60,000
i.u. vitamin A by stomach tube per day on days 10, 11, and 12, and 50 mg
cortisone acetate by intramuscular injections per day on days 9 through
12j b) these animals were treated concurrently with 50 mg cortisone
intramuscularly each day from day 9 through 13 and 50 mg papain intra-
peritoneally each day from day 12 through day 15.
^Vitamin A acetate was dissolved in cottonseed oil to give a
concentration of 60,000 i.u./ml. Though the preparation was kept
refrigerated, a fresh preparation was compounded each week.
^Obtained from Merck, Sharp, and Dohme, West Point, Pennsylvania.
^Obtained as a crude powder from Nutritional Biochemical Cor
poration, Cleveland, Ohio. Crude papain was assayed by biuret method
to contain 1 mg protein per 10 mg sample. The powder was dissolved by
grinding in 0.05M phosphate buffer at pH 7. The solution was then
filtered to remove any insoluble particles (KcCluskey and Thomas, 59).


17
Animals in all groups were sacrificed on day 20 of gestation.
In this procedure the female was anesthetized with ether, laporotomized
and the fetuses removed from the antimesenteric border of the uterus.
7
The young were weighed on a triple beam balance, fixed in Bouin's
fluid and examined grossly for externally detectable malformations.
The crown rump length was measured by means of a Vernier Caliper as was
the length of the head from the tip of the snout to the posterior
extreme of the occiput.
After the fetuses were fixed their heads were studied under the
dissecting microscope after having been sliced with a sharp blade into
frontal sections about 1-2 mm thick. By this method the relative posi
tions of the tongue and palatine shelves could be observed. In some
instances, and as an additional method of observation, before the fetal
head was frontally sectioned the palate was examined from the ventral
aspect by removing the lower jaw and displacing the tongue.
Experiment 2. Anatomical Morohonenesis of Normal
and Cleft Palate
This experiment was performed to study the formation and closure
of the secondary palate in normal embryos and to observe the changes
which occurred in the palatine processes and neighboring tissues lead
ing to the formation of cleft palate in embryos from hypervitaminotic
A rats.
Fifty pregnant rats were divided into two groups. Each animal
in one group received orally 60,000 i.u. vitamin A dissolved in cotton
seed oil per day for 3 successive days beginning on either day 9 or day
7
'See Appendix II.


18
10 of gestation. Previous studies showed that maternal hypervitaminosis
A during both of these gestational periods, i.e. 9-11 day3 or 10-12
days, induced cleft palate in more than 80# of the embryos from treated
mothers. The second group served as controls and the pregnant females
were administered 1 ml. of pure cottonseed oil orally on the appropri
ate days.
The animals from both of these groups were sacrificed at 10 A.M.
on the 14th, 15th, 16th, or 17th days of gestation. The embryos were
removed from the exteriorized uterus, weighed and fixed in Bouin's fluid.
At least 3 embryos from every litter were also fixed in alcohol-forma-
g
lin for 24 hours for histological and histochemical study.
Embryos fixed in Bouin's fluid were measured for crown rump
length and head length, and then the heads were sliced freehand in a
frontal plane and observed under the dissecting microscope. The heads
fixed in alcohol-formalin were cleared in terpineol, embedded in paraf
fin and serially sectioned in frontal plane at 6m on a rotary microtome.
Sections were stained by the following procedures, l) Toluidine blue
for identification of acid-mucopolysaccharides. Toluidine blue was used
as 0.1# solution in 30# ethyl alcohol for 5 minutes (Kramer and Windrum,
'55). Appropriate hyaluronidase controls were also prepared. 2)
Periodic acid-Schiff technique (PAS) for identification of glycogen
(McManus, '48). Negative staining with PAS subsequent to glycogen
digestion by alpha-amylase served as controls. 3) Methyl green pyronin
for detection of ribonucleoproteins (Brachet, '42) with ribonuclease
controls (Pearse, '60). 4) Feulgen stain for deoxyribonucleic acid
g
Three parts 95# alcohol and 1 part 40# formaldehyde.


19
(Feulgen and Rossenbeck, '24, referred bo in Pearse, *60). 5) Iron
hematoxylin stain for routine morphological study and recognition of
individual cell type.
Experiment 3. Radioautographic Studies
Because previous reports indicated that vitamin A was involved
in the metabolism of acid-mucopolysaccharides, S^-labelled sulfate was
administered parenterally to female rats and by radioautographic means
the amount incorporated by various tissues of control embryos and
embryos from vitamin A treated mothers -was examined. For this experi
ment, 10 pregnant females were employed. Five of these were treated on
the 9th, 10th, and 11th days of pregnancy with 60,000 i.u. vitamin A
per day, while the other five served as controls and were given pure
cottonseed oil during the same periods of gestation.
35 9
A single dose of carrier-free S -labelled sodium sulfate'' in
physiological saline was injected intraperitoneally at 10:00 A.M. on
either day 13, 14, or 15 of pregnancy. Two controls and two vitamin A
treated animals received 10 pic (microcuries) body weight on day
13 of pregnancy, also two controls and two vitamin A treated females
received an identical dose on day 14, and one control and one treated
female were injected on the 15th day but with only 5 uc body
weight. All pregnant females were sacrificed 43 hours after the
isotope was administered. The embryos which ranged in age from 15
through 17 days were removed, fixed in alcohol-formalin, dehydrated in
Obtained from Abbott Laboratories, Oak Ridge, Tenn.


20
ethyl alcohol, cleared in terpineol and embedded in paraffin. Serial
sections of the heads were made at 6;u in the frontal plane.
At least 3 embryos from every litter were processed for radio
autography which was done according to the dipping method developed
by Messier and Leblond ('57). Specially prepared 1" x 3" glass
10
slides carrying the paraffin sections of the embryos were taken to
the dark room. The dipping was done in complete darkness except for a
single Wratten series #2 safe light. Kodak IITB-3 emulsion was melted
by placing a small portion of the gelled emulsion in a plastic dipping
container and held for 30 minutes in a water bath at 40-45C. Slides
were dipped singly into the liquid emulsion for 1-2 seconds and then
drained in a vertical position for several minutes. The side of the
slide not bearing the sections was wiped clean of emulsion and the
slide was dried in a horizontal position for one hour before it was
stored in a light-proof plastic box sealed with black electrician's
tape. Plain glass slides were interposed between those bearing radio
active sections in order to prevent back scattering.
The boxes were stored for 12 days under dry ice in such a way
that the slides were oriented horizontally with the emulsion side down.
After their allotted exposure time the radioautographs were developed
at 20C in the dark as follows:
Kodak D-19 Developer 5 minutes
Kodak S3-5a Stop Dath 15 seconds
Kodak Acid Fixer- 10 minutes
Water Rinse 1-15 minutes
^The glass slides used for radioautography were specially pre
pared in order to provide for better adherence between the liquid
emulsion and the tissue preparation. That is, chemically cleaned
slides after having been immersed in a solution of 0.5 gelatin and
0.05$ chrom alum in distilled water were drained and dried at room
temperature in a covered, dust free staining dish (Boyd, '55).


21
Immediately after this processing the slides were stained for 5
minutes in 0.1$ toluidine blue in 30$ ethyl alcohol, dehydrated,
cleared and mounted in HSR^ mounting medium.
The stained radioautographs were studied with the aid of the oil
immersion lens of the light microscope and developed granules were
counted over mesenchymal tissues, cartilage, bone, and oral epithelium.
Experiment 4. Examination of the Effects of Hypervitaminosis A
on the Epiphyseal Cartilage of Adult Hats
This experiment was performed to detect whether any dissolution
of cartilage matrix, similar to that found in rabbits by Thomas,
McCluskey, Potter and Weissmann ('60) and Fell and Thomas ('60)
occurred in the rat after the administration of teratogenic doses of
vitamin A.
Eight non-pregnant and 2 pregnant, 84 bay old female rats
weighing 150-200 grams were treated as shown in Table 1. Immediately
upon sacrifice the tibio-femoral joint was removed from the animal,
freed of skin and muscle, and fixed for 2 days in 10$ buffered forma
lin. Bone was decalcified in a solution of equal parts 2% formic acid
and 20$ sodium citrate as recommended by Hulth and Westerbom ('59).
The joint was cleared in terpineol and embedded in paraffin. Seven
microns thick sections were cut on the rotary microtome and stained for
5 minutes in 0.1$ toluidine blue in 30$ ethyl alcohol. The epiphyseal
cartilages of femur and tibia were examined under the light microscope
-RISE mounting medium was obtained from Hartman-Leddon Company,
Philadelphia, Pennsylvania.


TABLE 1
Dosage of Vitamin A Administered to Rats
for Dissolution of Cartilage Matrix
Experiment
Number
Number and
Condition of
Rats
Treatment Per Day for
3 Consecutive Days by
Oral Incubation
Time Elapsed Between
Start of Treatment
and Sacrifice
A
2 Non-pregnant
1 ml. Cottonseed Oil
4 Days
B
2 Non-pregnant
60,000 i.u. Vitamin A
4 Days
D
2 Non-pregnant
100,000 i.u. Vitamin A
4 Days
E
2 Non-pregnant
200,000 i.u. Vitamin A
4 Days
C
2 Pregnant
60,000 i.u. Vitamin A
on days 9, 10, and 11
of Pregnancy
10 Days


23
to detect the loss of any metachromatically stainable material from
the cartilage matrix under the influence of excess vitamin A.


RESULTS
Teratogenic Action of.Hypervitaminosis A
Administration of 60,000 i.u. vitamin A per day for three con
secutive days to pregnant rats produced a high percentage of intrau
terine mortality. The earlier in pregnancy that the treatment was
instituted, the higher was the incidence of resorption. In Figure 1,
the percentage of resorbed embryos is represented as a function of the
gestational period during which the vitamin A was administered. When
begun on day 8, all of the embryos were resorbed or dead by day 20.
If the three day period of hypervitaminosis A began on day 9 the re
sorption rate was 54% and if the treatment was delayed until day 10 the
resorption rate dropped to 22%, but did not go below this level when the
treatment was not started until day 11. The injected control animals
given pure cottonseed oil on days 9, 10, and 11 had a resorption rate
of 12/o, which is approximately &% above an incidence rate to be
expected in normal controls.
The teratogenic effects of hypervitaminosis A observed on term
fetuses are summarised in Table 2. Among the embryos from vitamin A
treated mothers, 8C$ or more had cleft palate when the treatment was
started on day 9 or day 10 of pregnancy. None of the 21 embryos from
3 females who received treatment from day 11 onwards developed cleft
palate. The incidence of eye defects such as anophthalmia, micro
phthalmia, exophthalmos, and open eye decreased from approximately 50%
24


25
Figure 1. Fetal resorption as a function of day of gestation
maternal vitamin A treatment commenced.


TABLE 2
Teratogenic Action of Hyoervitaminosis A Observed in Term Fetuses
Treatment and Days of
Gestation Treatment
Accorded
Number
of
Litters
Number of
Implanta
tion Sites
p Fetuses
Resorbed
cf
Survivors 1-
lalformed
Cleft
Palate
Eye
Defects
Excen-
cephaly
60,000 i.u. Vitamin A
9-11
13
114
54
80
58
15
K)
60,000 i.u. Vitamin A
10-12
11
112
22
83
48
0
60,000 i.u. Vitamin A
11-13
3
27
22
0
10
0
Cottonseed Oil
9-11
9
87
12
0
0
0


27
to 10/5 as the vitamin A treatment vas delayed from day 9 to day 11.
Microstomia, i.e., reduction in the size of the oral aperture, vas
observed frequently in treated fetuses, though the exact percentage vas
not calculated.
Figure 2 compares the freehand cross-sectional slices from the
heads of typical cottonseed oil injected control (Figure 2A) and vita
min A treated (Figure 2B) fetuses. Except for the incomplete palate
in the treated fetus, most other organs in the heads of the tvo classes
of embryos vere comparable in size and shape.
In order to determine the effect of hypervitaminosis A on the
grovth of the fetus in general, fetuses in all litters vere veighed.
From these veights (Table 3) it vas observed that some decrease in the
mean fetal veight resulted vhen vitamin A treatment vas started on day
9; this disparity from the control mean veight diminished as the treat
ment was delayed until day 10 or day 11. The reduction in body weight
of the treated fetuses was accompanied by a decrease in body size; all
the fetuses from 4 litters picked at random from among the treated
group vere measured for crown rump length and head length, and compared
with those of control rats (Table 4) Though both crown rump length
and head length in treated fetuses vere shorter than controls, only the
head appeared deformed because of the decrease in the length of both
jaws; the trunk, notwithstanding the shortened length, was not detect-
ably malformed.
Effects of Maternal Injections of Cortisone and Papain on Embryos
On the basis of the methods of experimentation and examination
employed, neither cortisone nor papain appeared to be teratogenic.


Figure 2. Frontal freehand sections of heads of day 20 fetuses.
A control; secondary palate (?) is complete; B vitamin A treated;
cleft palate. lf>x.
NS nasal septum. T tongue.


TABLE 3
Weights of Tern
. Fetuses After Maternal
Hyoervitaminosis A
-
Treatment and Days of
Number
Number
Weightsa
Gestation Treatment
of
of
Accorded
Litters
Fetuses
60.000 i.u. Vitamin A
13
53
3.09 0.44
9-11
60,000 i.u. Vitamin A
11
£7
3.20 0.42
10-12
60,000 i.u. Vitamin A
3
21
3.32 0.20
11-13
Cottonseed Oil
9
76
3.64 0.45
9-11
Menn fetal weight in grans the standard deviation


TABLE 4
Crown Hump Length and Head Length of Term Fetuses After Maternal
Hypervitamlnosis A
Treatment and Days of
Gestation Treatment
Accorded
Number
of
Litters
Number
of
Fetuses
Crowna
Rump
Length
Headb
Length
Cottonseed Oil
9-11
4
36
33.6
14.2
60,000 i.u. Vitamin A
9-11
4
17
31.2
13
c'Mean length in millimeters
bIbid.


31
Furthermore, in neither instance was there any increase in the intrau
terine mortality from the control level (Table 5). A combined treat
ment with cortisone and papain simultaneously did not produce any mal
formations though the intrauterine mortality increased from control
rate of 11& to 26$. Also, no marked difference was present in the
mean fetal weight between control fetuses and those from cortisone or
papain treated mothers (Table 6).
To determine whether cortisone would reduce or potentiate the
teratogenic effects of excess vitamin A in producing cleft palate, it
was injected into pregnant rats already receiving teratogenic doses of
vitamin A. Cortisone did not modify the incidence of cleft palate in such
fetuses (Table 7). Although the type of eye defects in the group
receiving the combination of vitamin A and cortisone were essentially
the same as that found in the vitamin A group, they occurred in 33$ of
the fetuses in the former and 48$ of the fetuses in the latter. Simi
larly, the mean fetal weight was higher in fetuses from the combined
treatments than it was in the group treated with vitamin A alone.
Morphogenesis of Cleft Palate Induced by Kypervitaminosis A
I. Macroscopic Observations
15th Day of Gestation.The mean weights of 37 control and 63
treated embryos were comparable, but both the crown rump and head
lengths of vitamin A treated embryos were slightly less than the
controls (Table 8).
The palatine shelves of the control embryos were vertically
oriented, triangular in outline and closely followed the lateral con
tour of the tongue on this day (Figure 3A and 3B). The ventral edge


TABLE 5
Effects of Maternal
Injections 1
of Cortisone and Paoain
Observed
in Terra
Fetuses
Treatment and Days of
Number
Implant-
$
Malforra-
Gestation Treatment
Accorded
of
Litters
ation
Sites
Fetuses
Resorbed
ations
Physiological Saline
9-13
8
76
14
0
50 mg Cortisone
9-13
8
80
15
0
50 mg Paca in
9-13
9
95
13
0
50 rag Cortisone
9-12 and
50 mg Papain
12-15
2
19
26
0
u>
M


TABLE 6
Weights of Term Fetuses after Maternal Injections
of Cortisone and Paoain
Treatment
Number
of
Litters
Number
of
Fetuses
Weights8,
Saline Controls
8
67
3.64 0.40
Cortisone
8
68
3.34 0.39
Papain
9
63
3.50 0.43
aMean fetal weight in grams the standard deviation


TABLE 7
Teratogenic Effects of Hyoervitaminosis A and Cortisone
Observed in Terra Fetuses
Treatment and
Number
Number of
/
Days of Gesta
of
Implanta
Fetuses
tion Treatment
Litters
tion
Resorbed
Accorded
Sites
% Survivors Malformed
Weights8
Cleft
Palate
Eye
Defects
Excen-
cephaly
Physiological Saline
9-13
8
76
14
0
0
0
3.64 1
0.40
50 mg Cortisone
9-13
8
80
15
0
0
0
3.34
0.39
60,000 i.u. Vitamin A
10-12
11
112
22
83
48
0
3.20
0.42
60,000 i.u. Vitamin A
7
72
17
88
33
0
3.64 -
0.26
10-12 and
50 mg Cortisone
9-12
Mean fetal weight in grams the standard deviation


TABLE 8
Comparison of Control and Hypervitaminotic A Embryos
on Days 15. 16 and 17 of Pregnancy
Embryonic
Age
Treatment
Number
of
Litters
$ Fetuses
Resorbed
Number of
Living
Embryos
Weights3-
Crovm
Rump
Length
Head0
Length
Experimental
Control
4
10
37
263 30
10.8
6.1
15th Day
Vitamin A
11
54
63
264 t 29
10.2
5.5
Experimental
Control
7
15
56
468 46
13.5
7.3
16th Day
Vitamin A
16
62
86
467 = 39
13.0
7.2
Experimental
Control
3
7
27
933 26
17.4
9.8
17th Day
Vitamin A
6
56
37
754 62
16.8
8.7
aMean embryonic weight in mg the standard deviation
^Mean length in millimeters
cIbid


Figure 3. Frontal freehand sections of heads of day 15 embryos. A and B -
control; palatine shelves (?S) have triangular outline; C,D and E vitamin A treated;
C and D PS are rounded; E PS partially above the tongue (T). 15x.
NS nasal septum.


37
of the shelf was parallel to the floor of the oral cavity. In other
sections it was observed that posteriorly the palatine shelf instead
of being triangular was platelike.
The usual situation in treated embryos was for the palatine
shelves to be rounded instead of triangular in frontal section
(Figure 3C and 3D). In some of the embryos the shelves actually
appeared to have moved from the lateral side of the tongue to its
dorsal aspect (Figure 32) The shelves viere reduced in sise from those
of the controls and in some embryos they were completely missing'.
Only 28# of the embryos from hypervit amino sis A mothers had
palatine shelves which appeared to possess a morphological relationship
to the tongue similar to that observed in 100# of the control embryos
(Figure 4)* The palatine shelves of 72# of the treated embryos, on the
other hand, were abnormally shaped and their relative position with
respect to the tongue was altered.
Since all the resorption encountered at term (54#) in the
treated rats had already occurred by day 15 (compare Table 2 and Table
S), it may be concluded that all those embryos having deformed pala
tine shelves would have had cleft palate at term. In addition, some
of the treated embryos which on this day show normal palatine shelves
will also acquire cleft palate since more than 80# do show this defect
at term. In 3 of the 63 treated embryos some of the epithelial sur
faces such as those covering median nasal and maxillary processes,
which happened to be opposed to each other, had a tendency to fuse.
loth Day of Gestation .--Fifty six control and 86 treated embryos
were examined on this day. Mean embryonic weight, crown rump length,
and head length were comparable in the two groups (Table 8). The


38
100
"a
.a
6
a
co 75
<0
I
,5 50
0
a
.a
a
s
a
.a
a
£
Normally Shaped
Abnormally Shaped
Orientation of Palatine Shelves
Figure 4. Graphs comparing the percentages of day 15 control and
vitamin A treated embryos on the basis of shape and orientation of their
palatine shelves.


39
palatine shelves of the control embryo were either vertical and, there
fore, still lateral to the tongue as they were on day 15 (Figure 3A),
or horizontal and superior to the tongue (Figure 5A). Shelves in
position such as those in Figure 5A were observed to be both fused and
unfused. All stages of palate development, i.e., vertical, horizontal
unfused, and horizontal fused were observed to occur in different
embryos of the same litter. The tongue was flat, depressed uniformly
and parallel to the palatine shelves.
Though in some treated embryos the palatine shelves appeared
normal, S4/o had abnormal shelves at this day. This abnormality ranged
from deformed shape such as wavy or bent to reduced size including
complete absence. In contrast to the controls the palatine shelves of
treated embryos were oriented in one of these three positions:
1. Vertical on the side of the tongue (represented by Figure 5B).
Though some of the embryos had normal-appearing palatine shelves,
most of the embryos included in this category showed either
reduced or completely missing palatine shelves. Epithelial
fusions between opposing surfaces of median nasal process and
maxilla occurred frequently.
2. Palatine shelves horizontal and superior to the tongue. In
spite of having crossed the barrier of the tongue, the palatine
shelves were located in the corners between the nasal septum
and the tongue (Figure 5C), but had not progressed towards the
midline superior to the tongue. While still separated from each
other, the palatine shelves in some instances had the tendency
to become fused with the ventral edge of the nasal septum on


Figure 5. Frontal freehand sections of day 16 embryos. A control; palatine
shelves (PS) are horizontal, parallel and superior to tongue (T); unfused; B,C,D and E -
vitamin A treated; B PS vertical; C PS horizontal above tongue, but not progressed
towards midline; some fusion of PS with nasal septum (NS) present; D and E PS hori
zontal but abnormally shaped. 15x.


41
their respective sides (Figure 5C). In general the palatine
shelves were abnormally shaped (Figure 52 and 53)
3. Major portion of shelves horizontal, some portions still verti
cal. The posterior portion of the shelves was the part which
was most frequently vertical. The vertical palatine shelf was
not necessarily at the side of the tongue, since due to the
reduction of its size the shelf could be accommodated at a
higher plane than the tongue.
In Figure 6, control and treated embryos are compared with regard
to the shape and orientation of their palatine shelves. In the control
group 65$ of the embryos had vertical palatine shelves and 35$ had
horizontal shelves. In contrast the treated group had only 39$ vertical
and only 20$ horizontal, and 41$ of their embryos had palatine shelves
in intermediate positions.
In summary, these considerations point to the fact that on day
16 of development more treated embryos showed the initiation of pala
tine shelf movement towards the horizontal position than did the control
embryos. However, fewer treated embryos successfully completed the shelf
movement than the controls, as illustrated by a large number of treated
embryos having palatine shelves in an intermediate position (Figure 6).
17th Day of Gestation.Twenty seven control and 37 treated
embryos were studied. It was this day when a significant reduction in
the mean embryonic weight of treated litters became apparent as the
treated embryos averaged about 200 mg lighter than control embryos
(Table 8). Also, while the body size as well as the head in the
treated group was demonstrably shorter than normal, only the head


42
100
, 5
75-
<0
§
b50
-Q
I
I
25
o
.j
Control
| Normally Shaped
Abnormally Shaped
.1
Treated
Orientation of Palatine Shelves
Figure 6. Graphs comparing the percentage of day 16 control and
vitamin A treated embryos on the basis of shape and orientation of their
palatine shelves.


43
appeared malformed; both mandible and maxilla were shorter in their
anteroposterior length, hence the tongue protruded somewhat.
The palate was complete in all control embryos (Figure 7A) in
that the palatine shelves were united throughout their length. In the
predominant number of treated embryos, palatine shelves, though hori
zontally oriented superior to the tongue, were not completely united
with one another, which resulted in a palatine cleft. Observing free
hand slices of the heads of such embryos in a frontal plane it was
noticed that at the anterior level the palatine shelves might either
actually progress to the midline and fuse with each other (Figure 7B),
or they might remain separated and display a deformed outline (Figure 7C).
Posteriorly, however, almost all treated embryos showed a wide gap
intervening across the tongue (Figure 7D).
Figure 8 collates the shape and orientation of the palatine
shelves of the control and treated embryos examined on day 17. In the
treated group a small number of embryos still showed their palatine
shelves oriented in either a vertical or an intermediate position and
these in large part were deformed in shape. From this figure it is
apparent that failure of initiation or completion of the movement of
palatine shelves towards the horizontal plane occurred only in a
relatively small number of treated embryos and that cleft palate in a
large percentage of treated embryos occured in spite of the presence
of horizontally oriented palatine shelves. The immediate cause of the
defect, then, was not so much failure of movement of the shelves as
deficiency of tissue to permit contact with subsequent fusion at the
midline


44
Figure 7. Frontal freehand sections of day 17 embryos. A -
control; palatine shelves (?S) completely united; B, C and D vitamin A
treated; B PS at anterior level progressed to midline and partially
united; C PS abnormally shaped and at anterior level located in corners
between nasal septum (NS) and tongue (T); D PS at posterior level
showing a wide gap across tongue. 15x.


45
| Normally Shaped
Abnormally Shaped
c
o
.N
3:
Treated
Orientation of Palatine Shelves
Figure 8. Graphs comparing the percentages of day 17 control and
vitamin A treated embryos on the basis of shape and orientation of their
palatine shelves.


46
II, Microscopic Observations
Histologic sections of embryos of 14 through 17 days of age were
35
studied by histochemical and S radioautographic means.
14 and 15 Day Old Embryos.'The palatine shelves in 14 and 15 day
old normal control embryos are present within the oral cavity as two
longitudinal ridges on either side of the tongue. Along their entire
length they arose from maxillary mesoderm as two medially directed out-
foldings consisting of mesenchymal tissue covered by columnar epithelium
(Figure 9A). The mesenchymal cells contained large oval nuclei and had
many cytoplasmic processes which were embedded in an intercellular
ground substance. They were not distributed uniformly throughout the
volume of the palatine shelves but on the basis of their density per
unit of volume, the palatine shelves could be divided into three regions.
In an antero-posterior direction these regions were of about equal
length. In the anterior one third of the shelves the mesenchymal cells
were dispersed loosely in the main body of the shelf except for a
slight aggregation on the medial aspect (Figure 9B). The ground sub
stance, which had toluidine blue metachromasia, was abundant in this
anterior part of the shelves which were covered by tall, stratified
columnar epithelium 2-3 cells thick and having a well defined basement
membrane.
It is important to note in passing that in the maxillary mesoderm
there was an aggregation of preosteoblastic cells, which were recog
nizable as such because they characteristically accumulated substantial
amounts of glycogen (indicated by Periodic acid-Schiff positive and
alpha-amylase digestible material) and were strongly basophilic (demon
strated by methyl green pyronin method). The identification of this


A
Figure 9. Frontal sections of anterior one third of palatine shelf of day 15 control embryo. A
anterior level; palatine shelf (PS) consist of mesenchymal tissue covered by columnar epithelium; B -
posterior level; slight aggregation of mesenchymal cells on medial aspect of PS. Toluidine blue. 12Qx.
T tongue. DL dental lamina.


48
tissue was confirmed by the fact that preosteoblastic tissue normally
present in the lower jaw lateral to Meckel's cartilage displayed
identical characteristics (Figure 10A). This preosteoblastic tissue
of the maxilla continued into the palatine shelf.
In the middle third of the palatine shelf the mesenchymal cells
were closely packed, particularly along the medial border. The medial
portion of the palatine shelf was delineated from the rest of the
palatal tissue by an epithelial notch. Ground substance in the shelf
was strongly metachromatic with toluidine blue. Preosteoblastic tissue
extended into both the medial and lateral portions of the palatal
tissue (Figure 10B).
In the posterior third of the palatine shelves the epithelial
notch described above became deeper and consequently separated the
shelf from the more laterally placed maxillary areas. As a result
the shelf appeared plate-like in outline instead of triangular. The
cell density was still higher than in the middle third and metachro
matic ground substance was abtmdant. The preosteoblastic tissue was
now assembled at the dorsomedial edge of the palatine shelves which
were covered by a low, simple columnar epithelium. This epithelium
was continuous laterally with the 2-3 layered columnar epithelium
covering the lateral surface of the shelf as well as the rest of the
oral cavity.
By their histologic pattern 15 day old treated embryos demon
strated several notable differences from normal control embryos of the
same age. Anteriorly, the palatine shelves of the treated embryos had
a slightly rounded contour instead of being triangular (Figure 11A).
One remarkable difference between normal and treated embryos was the


Figure 10. Frontal sections of head of day 15 control embryo,
of palatine shelf (PS); aggregation of preosteoblastic tissue (OST) in
through middle third of PS; preosteoblastic tissue distributed in both
PAS. 4Ox. NS nasal septum. T tongue.
A section through anterior one third
maxilla and mandible; B section
medial and lateral portions of PS.


A
ww
mmm
mmftM
¡pSi
mwMim
iS/i'&S&k
ilJSi
an *
SsS
|if&#
wmmmMmm
graMgane
s -mm
/ (MSsS&aBSBm
VSihiT^ 'jlr>*7>^ ;:;A
Ss
if
vn
o
Figure 11. Frontal sections of anterior one third
embryo. A anterior level; palatine shelf (PS) rounded;
120x. B posterior level; dental lamina for upper molar
with Figure 9B). Iron hematoxylin. 120x. T tongue.
of palatine shelf of day 15 vitamin A treated
heterotopic chondrogenesis (KCT) present. PAS.
(DL) exists more medially than in control (compare


51
reduced size in the treated embryos of the maxillary preosteoblastic
area on the lateral aspect of the palatine shelf. This reduction in
size of the maxillary bone primordium was brought about not only by
the differentiation of a lesser number of mesenchymal cells into
preosteoblasts, but also by another phenomenon. That is, some of the
cells within the bone primordium differentiated into chondroblasts
and chondrocytes, as evidenced by the accumulation around these cells
of a typically metachromatic cartilage matrix (Figure 11A). At this
level it was also observed that the dental laminae for the upper
molars in some of the treated embryos arose more medially (Figure 113)
than in the controls (Figure 9B), and, therefore, the mesenchymal
tissue of palatine shelves and alveolar ridge was reduced in amount.
At a level slightly posterior to the one just described, the
palatine shelves might be either of normal size or somewhat smaller
than normal. In Figure 12A, the palatine shelves were quite similar
to those of control embryos, except that the epithelial notch was not
evident. In some embryos the palatine shelves at this level were almost
completely absent (Figure 123). In this photomicrograph the maxillary
preosteoblastic area was extensively replaced by the previously mentioned
heterotopic chondrogenic tissue.
The greatest difference between control and treated embryos
existed in the process of outfolding by which the posterior region of
palatine shelves in treated embryos took origin from maxillary tissue
(compare Figure 13A with Figure 133). A considerably lesser amount of
mesenchymal tissue formed this portion of the shelf in the treated than
in the control embryo. Due to the reduced length of both jaws in the
treated embryo, the anteroposterior length of the palatine shelves was


Figure 12. Frontal sections of heads of day 15 vitamin A treated embryos through middle third of
palatine shelf. A epithelial notch in palatine shelf (PS) is absent. PAS counterstained with Celestine
blue. 4Qx. B palatine shelves almost completely absent; maxilla contains heterotopic cartilage (HOT) .
PAS. 40x.


Figure 13. Frontal sections of
embryo; palatal tissue (PS) outfolding
folding abnormal; less palatal tissue
posterior third of palatine shelves of day 15 embryos. A control
from maxillary tissue; B vitamin A treated embryo; process of out-
(PS) outfolds from maxilla. Iron hematoxylin. I20x. T tongue.


54
also reduced. With toluidine blue staining one fact, however, became
quito clear. The metachromatic intensity of the ground substance in
the palatine shelves of treated embryos was not decreased from that in
the controls.
Radioautographs of 15 day old control embryos revealed wide-
spread S''"^ activity after injection of labelled sulfate into the mother.
The activity was observed largely over mesenchymal tissues or other
tissues derived from mesenchyme, e.g., cartilage, bone. The distribu-
35
tion of S -labelled material in these tissues coincided with the
presence of toluidine blue stainable metachromatic component.
Palatine shelves and mesenchymal tissue in other regions of
control embryos of this age showed very slight radioactivity (Figure
144), while aggregations of cells in precartilaginous tissue of nasal
35
septum and Meckel's cartilage showed moderate S activity. A higher
density of developed particles was observed in the cartilage of the
limb bud, where considerable metachromatic matrix was also present
(Figure MB).
3 e
Kasai and limb cartilages in the treated embryo showed S J
incorporation comparable to that observed in controls, although
mesenchymal tissue in the treated.embryos, including that of palatine
shelves, revealed a very much higher incorporation than was seen in
comparable areas of control embryos (Figure 15A and 153. Compare with
Figure 14A and 143). Although the maxillary preosteoblastic tissue in
35
treated embryos had S activity similar to controls, some regions
within this tissue showed a very high radioactivity. Such regions
also revealed metachromasia with toluidine blue, indicating the presence
of heterotopic cartilage.


0

Figure 14.
cartilage of limb.
Radioautographs of day 15 control embryo. A frontal section of palatine shelf; B -
Toluidine blue. 1800x.


Figure 15. Radioautographs of day 15 vitamin A treated embryo. A frontal section of palatine shelf
(compare with Figure 14A)j B cartilage of limb. Toluidine blue. lBOQx.
\


57
16 Day Old Embryos.In 16 day old control embryos in which the
process of shelf movement had not yet occurred, the palatine shelves were
oriented vertically, as observed on day 15. However, their dimensions
had increased from the level of the previous day. The distribution of
histological components along the anteroposterior axis of the palatine
shelves was still typical for each of the three regions described for
day 15 control embryos (Figures 16A, 16B and 17A).
Evidence of movement by the palatine shelves from the vertical
to the horizontal position was first observed at the junction of the
middle third and posterior third of the palatine shelf (Figure 17B).
At this point a medially directed extension arose from the medial
surface of the palatine shelf just superior to the dorsalmost level
of the tongue. This extension consisted of mesenchymal cells, ground
substance and preosteoblasts, and it progressively enlarged at the
expense of the ventral and vertical portions of the shelf, which
appeared to be gradually withdrawn into the body of the shelf (Figure
ISA). This movement of the palatine shelves to the horizontal plane
then extended to the anterior portion, which in a similar manner
crossed the barrier of the tongue (Figure 1SB).
It is of particular importance that the movement from the
vertical to the horizontal plane was initiated at a point where
osteogenic tissue was present (Figure 19A) and already forming bone
matrix. This osteogenic tissue was seen to execute a bend or curve
at the point where the process of horizontal movement was under way
(Figure 19B).
The horizontal, unfused palatine shelf possessed a core of mes
enchymal tissue similar to that present in the vertical shelf. The


Figure 16. Frontal sections of head of day 16 control embryo. A section through anterior one third
of palatine shelf (PS); PS contains a medially directed extension (OP) from maxillary preosteoblastic tissue
(OST). PAS. B section through middle third of PS; epithelial notch (N) is present; denser accumula
tion of mesenchymal cells and preosteoblasts occurs in medial and lateral portions of PS. Toluidine blue.
40x. T tongue. M mandible.


Figure 17. Frontal sections of heads of day 16 control embryos. A section through posterior third
of palatine shelf (PS); aggregation of preosteoblastic tisstie (OST) at dorsalmost aspect of PS; B section
through PS shoving beginning of movement to horizontal position at junction of middle and posterior third;
medial extension of PS is present. Toluidine blue. /¡.Ox.


Figure 18. Frontal sections of head of day 16 control embryo shoving movement
horizontal position. A palatine shelves (PS) are almost completely horizontal; B -
completely above tongue (T). Toluidine blue. 40k.
NS nasal septum.
of palatine shelves to
palatine shelves are


Figure 19. Frontal section of head of day 16 control embryo showing origin of palatine shelf move
ment to horizontal position. A preosteoblastic tissue (OST) accumulates on medial aspect of palatine
shelf; B a bend occurs in this preosteoblastic tissue. PAS. 40x.


62
epithelium covering the two apposing palatine shelves was simple
cuboidal or low columnar and became stratified columnar as it continued
laterally over the palatine shelf (Figure 20). The assemblage of
preosteoblasts, which in the vertical palatine shelf was observed on
its medial aspect, was now seen towards the superior aspect of the
horizontal palatine shelf (Figure 20). This palatal preosteoblastic
area extended medially to the midline of the shelf and laterally was
continuous with preosteoblastic tissue of the maxilla, which had some
bone trabeculae present. The zygomatic process of the maxilla projected
towards the inferior margin of the orbit (Figure 183) and the mandible
was developing around and lateral to Meckels cartilage and was in a
slightly more advanced stage of morphological differentiation than the
maxillary bone (Figure 183). The dental laminae for the upper and
lower molars were lodged into maxillary and mandibular bone respectively
(Figure 20).
In the treated embryos on this day the reduction in size of the
palatine shelves became more apparent. Anteriorly they did not reach
the inferior boundary of the tongue as the control did (compare Figure
21 with Figure 16a), and in addition their medial boundary was
irregular. The dental laminae for the upper molars were more medial
in position than they were in controls (compare Figure 22A with Figure
16b). The maxillary osteoblastic tissue was invaded by heterotopic
cartilage and was less extensive (Figure 223). The horizontal palatine
shelves in the treated embryos were widely separated (Figure 23A) and
had an irregular outline. Arrest of further development in those
dental laminae which did not encounter osteogenic tissue was observed.
Since the osteogenesis in the maxilla was more retarded than in the


Figure 20. Frontal section of head of day 16 control embryo. Palatine shelves (PS) horizontal; pre-
osteoblastic tissue (0?) present on superior aspect of PS; dental laminae (DL) for molars are lodging into
maxilla and mandible. PAS. 40x.
Figure 21. Frontal section of head of day 16 vitamin A treated embryo shovjing deformed palatine
shelves (PS). Toluidine blue. 40x.


Figure 22. Frontal sections of heads of day 16
arise more medially than controls (compare with Figure
maxillary osteoblastic tissue. Toluidine blue. /^Ox.
vitamin
16B): B
A trea.ted embryos. A dental laminae
- heterotopic cartilage (KCT) replaces
(DL)


O'
U1
A
B
Figure 23. Frontal sections of heads of day 16 vitamin A treated embryos. A palatine shelves (?S)
are horizontal and deformed; maxillary bone is replaced by cartilage (HOT) and further development of upper
dental lamina (DL) is arrested. PAS. 40x. B insufficient tissue is outfolded from maxilla to form PS.
Toluidine blue. 4-Ox.


mandible, the upper molars did not develop any further than the
laminar stage, while the lower molars were developing almost normally
(Fuguro 23A). While the palatal preosteoblastic tissue was located
normally and the epithelium covering the shelf was similar to that of
controls, the mesenchymal tissue in the palatine shelves was reduced
in amount, however, the staining intensity of the ground substance by
toluidine blue was not affected. Posteriorly the palatine shelves had
not outfolded sufficiently from the maxillary process and were reduced
in size (Figure 233. Compare with Figure 17A).
35
The incorporation of S -sulfate into 16 day control embryos
increased markedly from what it was on the previous day. The most
active component was the cartilage as could be readily visualized
from radioautographs of nasal septum (Fugure 24A) and limb cartilage.
Incorporation into palatine shelves and mesenchymal tissue of other
areas was also increased in embryos of this age. The medial boundary
of the shelf had a greater incorporation than the lateral portion of
the shelf.
Studies of S-35 incorporation into treated embryos demonstrated
that the nasal (Figure 243) and limb cartilages of treated embryos had
intense activity which was very much greater than that exhibited by
controls (compare with Figure 24A). Higher S-^ incorporation was
also observed in the mesenchymal tissue of the palatine shelves in
treated embryos (Figure 253) than in the controls (Figure 25A). The
maxillary osteoblastic area and the osteoid matrix in the mandible showed
identical incorporation in control and treated embryos. However, the
heterotopic cartilage (Figure 26) in maxilla had a very high radio
activity just as cartilage of the nasal septum and Meckel's cartilage.


Figure 24. Radioautographs of frontal sections of heads of day 16 embryos; nasal cartilage. A -
control; B vitamin A treated. Toluidine blue. 2300x.


Figure 25
palatine shelves
Radioautographs of frontal sections of heads of day 16 embryos; mesenchymal tissue of
A control; B vitamin A treated. Toluidine blue. ISOQx.


69
Rigure 26. Radioautograph of frontal section of head
of day 16 vitamin A treated embryo; heterotopic cartilage within
maxillary osteoblastic tissue. Toluidine blue. ISOOx.


70
17 Day Old Embryos.The horizontal palatine shelves fused with
one another in the control embryos by day 17, and the nasal and oral
cavities became separated from each other and communicated only through
the nasopalatine foramina. In the anterior portion of the palate there
was slight accumulation of preosteoblasts in the midline region (Figure
27A) but the rest of the palate at this anterior level had loose mesen
chymal cells.
At a slightly more posterior level the palate became deeply
arched (Figure 273). The maxillar5r bone occupied a small triangular
area in the lateral half of the palate (Figure 27B). The medial angle
of the triangle was now continuous with the palatal preosteoblastic
area, the latter had split into right and left halves (Figure 273). The
stage of osteogenesis in the mandible was identical with that observed
in the maxilla and the dental primordia for the upper and lower molars had
advanced considerably in their development and were deeply lodged in their
respective alveoli (Figure 27B).
Figure 28 compares the anterior palatal regions of two embryos
from the same vitamin A treated mother. In one embryo, to be referred
to hereafter as embryo I (Figure 23A), the palate was incomplete on one
side. In the other embryo, embryo C (Figure 283), the palate was complete
at this level. Posteriorly in embryo I, the palatine shelves had failed
to meet and were very thick and abnormally shaped (Figure 29A and 293).
Though abundant tissue was present, the maxillary bone was much reduced
in size and also was partially replaced by heterotopic cartilage
(Figure 29A). Through the intervention of this cartilage the reduced
maxilla was put in contact with the almost normal mandible (maxillo
mandibular ankylosis, Figure 29A). In the dental lamina of the upper


Figure 27. Frontal sections of head of day 17 control embryo. A section through anterior region
of palate; maxillary bone (OST) is large and laterally gives rise to zygcmatic process; slight accumulation
of preosteoblasts in center of palate (0?); B section through a level posterior to that of A; maxilla
(OST) on each side associates with palatal osteoblastic tissue (OP). Toluidine blue. 40x.


Figure 28. Comparison of frontal sections of anterior region of tv/o day 17 vitamin A treated embryos.
A palate (?) is incomplete; maxillary cartilage present. Toluidine blue. 40x. B palate (?) is complete;
lines of fusions between palatine shelves and nasal septum (NS) are present. Toluidine blue. 12Ox.


Figure 29. Frontal section of posterior region of head of day 17 vitamin A treated embryo. A pala
tine shelves (PS) are thick and deformed; maxilla is replaced by cartilage (HCT); maxillomandibular ankylosis
present; B dental lamina (DL) is not lodged in maxilla; its two limbs diverge. PAS. 40x. M mandible.


74
molars (Figure 293), which did not encounter maxillary bone and hence
did not get lodged into it, the two limbs of the lamina opened ana
thus separated the palatal tissue from maxilla (Figure 293).
The maxillary bone in embryo C was similarly reduced in size
(Figure 304) from the controls (Figure 273) and though the secondary
palate was complete, it was very thin compared to the control.
Palatal preosteoblastic tissue in embryo C was present in normal size
and location (Figure 304).
Comparing the posterior level of a control embryo with embryo I
and embryo C, it was observed that maxillary bone in both embryos I and
C at this level was almost absent. 'While embryo I had a cleft palate,
the completed palate in embryo C was very thin and in its middle there
was an area which revealed rarefied mesenchymal tissue and breaks in
the epithelial membrane (Figure 30B). More posteriorly this embryo
did have an incomplete palate (Figure 314.).
Similar to embryo C, many treated embryos had abnormal infoldings
of oral epithelium (Figure 304) which contained a considerable amount
of palatal tissue. Keterotopic cartilages were often observed in the
neighborhood of such infoldings (Figure 31B).
Comparing the radioautographs of the 17 day old control and
treated embryos, the mesenchymal tissue in palatine shelves and neighboring
*2 r
area of the control embryo showed higher incorporation (Figure 324)
than palatine shelves of the incomplete palate in the treated embryo
(Figure 323). But in the treated embryo palatal mesenchymal tissue
which had been entrapped by infoldings of oral epithelium showed a
greater S incorporation than other regions of the palate (Figure 334
and 333); its density appeared to be comparable with the density in the


Figure 30. Serial frontal sections of head of day 17 vitamin A treated embryo. A maxilla (OST)
is reduced in size; palatal preosteoblastic tissue (OP) present; B palate (?) is narrow; its center shows
breaks in epithelium and rarefied mesenchymal tissue. Toluidine blue. 40x.


Figure 31. Frontal sections of head of day 17 vitamin A treated embryo. A section through
posterior level of palate; palate (P) is incomplete. Toluidine blue. 40x. B infoldings of oral
epithelium have entrapped palatal mesenchymal tissue (IMF); heterotopic maxillary cartilage (HCT) is
present. PAS. 120x.


Figure 32. Radioautographs of frontal .sections of heads of day 17 embryos; palatine shelves. A -
control; B vitamin A treated. Toluidine blue. 1800x.
o


Figure 33. Radioautograph of frontal section of palate of day 17 vitamin A treated embryo. A low
power view of infolding (IMF) of palatal tissue. Toluidine blue. 120x. B radioautograph of infolding.
Toluidine blue. 1800x.


79
normal palate. Radioactivity in the nasal (Figure 34A and 343) and
limb (Figure 354 and 353) cartilage of control embryos was also greater
than in the treated embryos.
Effects of Kyoervitaminosis 4 on Adult Rats
In order to investigate if the teratogenic dose of vitamin 4
employed to produce cleft palate in embryos vas also derogatory to the
pregnant animals, the latter were weighed during the three day period
of vitamin A treatment in pregnancy. It was found that while the
control mothers treated for three days with pure cottonseed oil gained
an average of 9 grams during this three day period, the vitamin A
treated rats lost an average of 9 grams during the same period. This
weight loss could possibly be due to an effect reported in rabbits by
Thomas et al. (* 60). These authors noticed the dissolution of cartilage
matrix in articular and epiphyseal plates of rabbits treated with a
single dose of one million international units of vitamin A. To deter
mine if it was so, toluidine blue stained sections of epiphyseal carti
lage from the distal end of femur and proximal end of tibia were exam
ined from adult rats treated with varying doses of vitamin A (described
in Table 1). Figure 36 compares the proximal epiphyseal plate of
tibia from rats belonging to all five treatment groups (Table l).
Epiphyseal cartilage in the control animals gave a metachromatic
reaction with toluidine blue (Figure 36A). This reaction was increased
in animals given three doses of 60,000 i.u. vitamin A each day whether
pregnant or non-pregnant and whether examined four days after vitamin A
administration (Figure 363) or 10 days after treatment (Figure 360).


CO
o
A
B
Figure 34. Radioautographs of frontal sections of heads of day 17 embryos; nasal cartilage,
control} B vitamin A treated. Toluidine blue. 230Qx.


A
B
03-
Figure 35. Radioautographs of sections of day 17 embryos; cartilage of limb bone
vitamin A treated. Toluidine blue. 1800x.
A*.
control; B


Figure 36. Toluidine blue stained sections of epiphyseal cartilages of control and vitamin A treated
rats. A section from control rat in experiment A (see Table 1 for experiment designations); cartilage
matrix is metachromatic; B section from treated rat in experiment B; metachromasia in matrix is increased.
12 Ox.


Figure 36 continued. C section from vitamin A treated rat in experiment Cj increased metachromasia;
D section from vitamin A treated rat in experiment Dj cartilage is depleted of stainable matrix. I20x.


84
Figure 36 continued. E section from vitamin A
treated rat in group Ej cartilage is depleted of stainable
matrix. 12Gx.


35
Epiphyseal plates of rats treated with larger doses of vitamin A,
e.g. 100,000 and 200,000 i.u. per day for three days, however,
demonstrated a considerable loss of metachromasia from the cartilage
matrix (figure 3&D and 3E). This loss of metachromatic material was
not uniform, and isolated patches of cartilage still retained some
stainable matrix.


DISCUSSION
Normal Closure of Secondary Palate
Normal formation of the secondary palate in mammalian embryos
involves a fundamental morphogenetic movement by means of which the
palatine processes of mamillary mesoderm reorient themselves from a
vertical position at the side of tongue to a horizontal position
superior to tongue. This reorientation takes place rapidly and does
not seem to be accompanied by a correspondingly rapid growth within the
shelves (Walker and Fraser, '56). In an effort to investigate the mor
phological or chemical basis for this movement, Walker and Fraser ('56),
Walker ('60, '61) and Larsson (62) made histological and histochemical
studies of the palatine shelves of young embryos, and concluded that the
only components making up the palatine shelves were a core of loose
mesenchymal tissue covered by an epithelium. The mesenchymal cells had
long cytoplasmic processes embedded in a ground substance which was
metachromatically stainable with toluidine blue. It was suggested that
the factor which was responsible for initiating palatine shelf movement
probably resided in the acid-mucopolysaccharides of the intercellular
ground substance.
In the present investigation it was shown that the mesenchymal
cells within the palatine shelves were not uniformly distributed in the
anteroposterior axis of the shelf. They were found to be sparse and
loosely arranged anteriorly, while posteriorly where the shelf movement
86


87
originated they were very densely aggregated. The intercellular ground
substance of the mesenchymal tissue stained metachromatically with
35
toluidine blue and had S uptake which was most marked in the medial
portion of the posterior palatine shelf area.
Another important histological component, hitherto undetected in
the vertically disposed palatine shelf was preosteoblastic tissue of
maxilla which sent a branch of similarly differentiated cells into the
medial portion of the palatine shelves. This primordium of the palatine
bones extended into the vertical palatine shelves. Walker and Fraser
(56) did not report this component but thoy did describe the occurrence
of osteogenesis in tissue proximal to the base of the shelves (maxillary
bone), and commented that no bony projection entered the palatine shelves
in their material.
The presence of this preosteoblastic tissue acquires significance
when one observes the region of palatine shelf where the movement to the
horizontal position is first indicated. That is, the preosteoblastic
tissue, along with the densely populated mesenchymal cells and their
strongly matachromatic ground substance, was one of the first components
to extend medially into an outfolding which indicated the beginning of
movement from the vertical to the horizontal position. Though one is
unable to draw any far reaching conclusions from this observation as it
is on the fixed and sectioned embryo instead of living material, it is
indicated that the presence of preosteoblastic tissue as a continuous
tissue from maxilla into the palatine shelf may have some normal
directive influence on the initiation of shelf movement.


Cleft Palate and Hypervitamlnosis A
In a comprehensive review Wilson (59) proposed that an agent
capable of cansing malformations also causes an increase in intrauter
ine death and that both the intrauterine mortality and the malformations
may be different degrees of expression of the same general effect of the
teratogen. In the present investigation it was observed that maternal
hypervitaminosis A instituted on day 9 resulted in death of over half of
the embryos. The death rate decreased to 22% when the treatment was
started on day 10. The incidence of cleft palate, however, did not
change. In other words there appeared to be an independent variation
between the death rate and the malformation rate as 80$ of the surviv
ing embryos developed cleft palate whether the death rate was 54$ or
22$ (Table 2). These facts, however, do not rule out the possibility
that one and the same toxic effect of excess vitamin A may be operative
in the two manifestations. Rather one additional observation supports
Wilsons hypothesis. That is, when animals viere administered excess
vitamin A on day 9, all the intrauterine mortality which could be
expected to be encountered at term (54$) was already present on day 15
(Table S). That is, the factor that caused embryonic death in hyper-
vitaminosis A produced its effect before day 15. Also, on this day
all those embryos that would develop palatal clefts already showed the
early stages of abnormal development.
The labile period during which cleft palate could be produced in
these embryos was quite narrow because treatment of the mother begun on
day 11 did not result in cleft palate but treatment begun on day 8
resulted in 100$ embryonic death. It is important to realise that


89
though vitamin A treatment was instituted on day 9 of pregnancy, the
morphological factors that lead to cleft palate in the embryo do not
become apparent until day 15. Some subtle event or component must be
susceptible to the teratogen during days 9 and 10; since females pre
sented with excess vitamin A on day 11 do not give birth to offspring
with cleft palate.
There was observed a definite, though not extensive, general
retardation of growth in fetuses after maternal hypervitaminosis A.
The extent of retardation of fetal growth depended on the gestational
day when the three day period of vitamin A treatment of the mother was
commenced. The growth was most affected when treatment began on day
9, less so when treatment was started on day 10 and still less if
delayed until day 11.
Cleft palate in vitamin A treated embryos, however, was not con
sidered to be the result of such an over-all growth retardation. The
embryonic growth, reflected as embryonic weight, was first detected to
be retarded in 17 day old treated embryos; the latter were on an
average 200 mg lighter in weight than day 17 control embryos. The
teratogenic effect of maternal hypervitaminosis A was, however,
detected much earlier than the effect on total body growth; on day 15
in treated embryo it was noticed that the development of the head was
stunted in an anteroposterior direction; consequently both maxilla and
mandible were shorter in length than those of control embryos of the
same age and accordingly were clearly regions of localised growth
retardation.
From the standpoint of anatomical morphogenesis, cleft palate can
be considered to develop in several possible ways which have been
summarized by Burston (59), and by Fraser ('55, 60).


90
Since the correct positioning of the left and right palatine
shelves on the superior aspect of tongue is essential for the normal
closure of the secondary palate (reviews by Peter, '24 and Lazzaro
40, referred to in Larsson, 62), it was considered possible that the
failure on the part of the tongue to descend from between the vertically
disposed palatine shelves may lead to cleft palate. In support of this
concept Trasler et al. ('56) reported that cleft palate resulted in
mouse embryos whose amniotic sacs were punctured before the time of
palatal closure; the loss of amniotic fluid and consequent pressure of
the uterus resulted in the head of the fetus being forcibly pressed
against the chest, and hence the descent of the tongue was prevented.
Asling et al. (*60) described the occurrence of cleft palate in the
young born to pteroylglutanic acid deficient rats, and ascribed these
clefts to retarded growth of mandible to an extent which precluded
sufficient room for descent of the tongue to permit the palatine
shelves to move to horizontal position. Similar observations were reported
by Pitch (*6l) who studied the development of cleft palate in mouse
embryos homozygous for the short-head mutation, wherein the palatine
shelves appeared to remain vertical due to mechanical interference by the
mass of tongue.
Another closely related hypothesis for the origin of cleft
palate, which encompassed both morphology and some of the biochemical
phenomena, was proposed by Walker and Fraser ('57). These authors
studied the production of cleft palate in mouse embryos from cortisone
treated mothers and were able to present the concept that in contrast
to the previous theories the failure of palatine shelves to change
their position from the vertical to the horizontal plane at the normal


91
tine, did not depend on the tongue but was dependent upon some factor
residing within the palatine shelves themselves. Walker and Fraser
(150 conceived of a force which steadily built up in the normal
palatine shelves and which at a certain time became strong enough to
cause them to move towards the horizontal plane. Cleft palate in the
cortisone treated embryos was held to arise due to the interference
with the build up of this sheIf-force' whereupon the palatine shelves
failed to initiate movement from the vertical position to a horizontal
plane at the normal time (Walker and Fraser, '57). This delay in
initiation of movement was compounded by the fact that head growth
continued while the shelves were still in vertical position and,
therefore, when they ultimately did become horizontal above the tongue,
growth of the head had taken them so far apart that they were unable
to meet and fuse. Such a delay in shelf movement was also reported in
the development of cleft palate in mouse embryos under the teratogenic
influence of such agents as maternal hypervitaminosis A (Walker and
Crain, 160; Kamei, '62), riboflavin deficiency (Walker and Crain '61)
and X-irradiation (Callas and Walker, >63). There are other possible
means by which cleft palate may develop, but these have not been
observed in experimental teratogenesis. One would be failure of the
palatine shelves, which though horizontal and above the tongue at
normal time, to contact each other due to either: a) too narrow
palatine shelves, or b) excessive width of the head. At least one
example of each faulty process exists in the scientific literature.
Fitch ('57) described the effects of a recessive gene in the mouse which
caused cleft palate resultant from overly narrow palatine shelves.


Full Text
EFFECTS OF MATERNAL HYPERVITAMINOSIS
A ON CLEFT PALATE FORMATION
IN RAT EMBRYOS
By
DEVENDRA MOHAN KOCHHAR
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
August, 1964

DEDICATION
To the memory of my father, Trilok Nath Kochhar, this
dissertation is respectfully dedicated.

ACKNOWLEDGEMENT
The author wishes to express his sincere gratitude to Dr. E.
Marshall Johnson for his supervision and guidance throughout the
progress of this investigation.
The author is greatly indebted to Dr. James G. Wilson for pro¬
viding an opportunity for a rewarding academic experience and for his
valuable suggestions and ideas conducive to original thoughts.
Deep sense of appreciation is due to the members of the author's
supervisory committee for advice and encouragement. Acknowledgement
is given to Miss Carol E. Ruppert and Mr. Marvin E. McGraw for their
assistance in the preparation of illustrations.
The financial support of Training Grant 3T1 GM 579"0^S1 of the
National Institute of Health is gratefully acknowledged.
For many fruitful discussions and stimulating criticism, and
for assistance in the final preparation of this manuscript, special
acknowledgement is given to author's wife, Omila S. Kochhar.
iii

TABLE 07 CONTENTS
Page
ACKNOWLEDC-EKEKT iii
LIST 07 TABLES vi
LIST OF 7IGURES vii
INTRODUCTION 1
Cortisone as a Teratogen in Cleft Palate Formation ... 4
Morphological Sequence of Cortisone Induced Cleft
Palate 5
Hypervitaminosis A as a Teratogen in Cleft Palate
Formation 7
Morphological Sequence of Hypervitaminosis A
Induced Cleft Palate 9
Purpose of This Study 13
MATERIALS AND METHODS 15
Breeding and Maintenance of Animals 15
Experiment 1. Incidence of Cleft Palate 15
Experiment 2. Anatomical Morphogenesis of Normal
and Cleft Palate 17
Experiment 3. Radioautographic Studies 19
Experiment 4. Examination of the Effects of Hyper¬
vitaminosis A on the Epiphyseal Cartilage of
Adult Rats 21
RESULTS 24
Teratogenic Action of Hypervitaminosis A 24
Effects of Maternal Injections of Cortisone and
Papain on Embryos 27
Morphogenesis of Cleft Palate Induced by Hyper¬
vitaminosis A 31
Effects of Hypervitaminosis A on Adult Rats 79
DISCUSSION 86
Normal Closure of Secondary Palate 86
Cleft Palate and Hypervitaminosis A 88
Embryonic s35 Incorporation Following Maternal
Hypervitaminosis A 94
Interaction Between Hypervitaminosis A and Cortisone . . 98
SUMMARY
99
iv

Page
LITERATURE CITED 102
APPENDICES 110
Appendix I. Analysis of Rockland Stock Diet for Rats . Ill
Appendix II. Composition of Bouin1s Fluid 112
v

LIST 0? TABLES
Table Page
1. Dosage of Vitamin A Administered to Rats for Dissolution
of Cartilage Matrix 22
2. Teratogenic Action of Hypervitaminosis A Observed in
Term Fetuses 26
3. Weights of Term Fetuses After Maternal Hypervitaminosis A . 29
4. Crown Rump Length and Head Length of Term Fetuses After
Maternal Hypervitaminosis A 30
5. Effects of Maternal Injections of Cortisone and Papain
Observed in Term Fetuses 32
6. Weights of Term Fetuses after Maternal Injections of
Cortisone and Papain 33
7. Teratogenic Effects of Hypervitaminosis A and Cortisone
Observed in Term Fetuses 34
S. Comparison of Control and Kypervitaminctic A Embryos on
Days 15, 16 and 17 of Pregnancy 35
vi

LIST OF FIGURES
Figure
rage
1. Fetal resorption as a function of day of gestation
maternal vitamin A treatment commenced 25
2. Frontal freehand sections of heads of day 20 fetuses ... 28
3. Frontal freehand sections of heads of day 15 embryos ... 36
4. Graphs comparing the percentages of day 15 control and
vitamin A treated embryos on the basis of shape and
orientation of their palatine shelves 38
5. Frontal freehand sections of day 16 embryos 40
6. Graphs comparing the percentage of day 16 control and
vitamin A treated embryos on the basis of shape and
orientation of their palatine shelves . 42
7. Frontal freehand sections of day 17 embryos 44
8. Graphs comparing the percentages of day 17 control and
vitamin A treated embryos on the basis of shape and
orientation of their palatine shelves 45
9. Frontal sections of anterior one third of palatine shelf
of day 15 control embryo 47
10. Frontal sections of head of day 15 control embryo .... 49
11. Frontal sections of anterior one third of palatine shelf
of day 15 vitamin A treated embryo 50
12. Frontal sections of heads of day 15 vitamin A treated
embryos through middle third of palatine shelf 52
13. Frontal sections of posterior third of palatine shelves
of day 15 embryos 53
14. Radioautographs of day 15 control embryo 55
15. Radioautographs of day 15 vitamin A treated embryo .... 56
16. Frontal sections of head of day 16 control embryo .... 5S
vmi

Figure Page
17. Frontal sections of heads of day 16 control embryos ... 59
18. Frontal sections of head of day 16 control embryo show¬
ing movement of palatine shelves to horizontal
position 60
19. Frontal section of head of day 16 control embryo show¬
ing origin of palatine shelf movement to horizontal
position 61
20. Frontal section of head of day 16 control embryo .... 63
21. Frontal section of head of day 16 vitamin A treated
embryo showing deformed palatine shelves (?S) 63
22. Frontal sections of heads of day 16 vitamin A treated
embryos 64
23. Frontal sections of heads of day 16 vitamin A treated
embryos 65
24. Radioautographs of frontal sections of heads of day
16 embryos; nasal cartilage 67
25. Radioautographs of frontal sections of heads of day
16 embryos; mesenchymal tissue of palatine shelves . . 68
26. Radioautograph of frontal section of head of day 16
vitamin A treated embryo; heterotopic cartilage
within maxillary osteoblastic tissue . 69
27. Frontal sections of head of day 17 control embryo .... 71
28. Comparison of frontal sections of anterior region of
two day 17 vitamin A treated embryos 72
29. Frontal section of posterior region of head of day
17 vitamin A treated embryo 73
30. Serial frontal sections of head of day 17 vitamin A
treated embryo . 75
31. Frontal sections of head of day 17 vitamin A treated
embryo 76
32. Radioautographs of frontal sections of heads of day
17 embryos; palatine shelves 77
33. Radioautograph of frontal section of palate of day
17 vitamin A treated embryo 78
viii

Figure Page
34. Radioautographs of frontal sections of heads of day
17 embryos; nasal cartilage 80
35. Radioautographs of sections of day 17 embryos; carti¬
lage of limb bone 81
36. Toluidine blue stained sections of epiphyseal carti¬
lages of control and vitamin A treated rats 82

INTRODUCTION
The general opinion of the scientific community concerning the
relative importance of environment and heredity in the production of
congenital malformations has vacillated between these two major causes
through the years. It was known from the studies of experimental
embryologists such as E.B. Wilson, T.II. Morgan, Jacques Loeb, and
Charles Stockard that anatomic defects could be produced in submammalian
embryos by subjecting them to injurious environmental factors (Corner
•60). The mammalian embryos, however, were not thought to be readily
available or susceptible to adverse environmental changes due to
supposed protective influences of the uterus, the embryonic membranes
and their encompassed fluids. Such views permitted the persistance of
the opinion that the presence of congenital defects in mammalian
embryos was predominantly due to genetic factors. In fact, some con¬
genital malformations in humans, e.g., chondrodystrophy (M#rch, '41),
'lobster-claw' defects of hands and feet (Stiles and Pickard, '43), as
well as many structural defects in experimental animals do have a well-
defined genetic cause (Glueksohn-Waelsh, '54} Zwilling, '56 and Silagi,
â– 62).
An indication that the mammalian embryo may not be completely
guarded against environmental disturbances was reported by Kale ('33,
'35, '37), who noticed the birth of anophthalmic young to vitamin A
deficient pigs. Gregg ('41, '45) described the occurrence of congeni¬
tal defects in the human fetus after maternal infection with rubella
1

2
(German measles). Warkany and Nelson's ('40> '41) findings regarding
the teratological effects of riboflavin deficiency in rats opened the
way for extensive studies on experimentally produced malformations in
mammalian embryos.
A wide variety of teratogenic agents have been discovered which,
when applied to the pregnant female, produce many types of malforma¬
tions in the developing embryo. Agents discovered to date range
through maternal nutritional deficiencies, vitamin excesses, endocrine
imbalances, irradiation, alkylating agents and viral infections. Such
agents and the resulting malformations in the vertebrate embryos have
been thoroughly reviewed by Kalter and Warkany (’59), Wilson ('59),
and Pitt ('62).
Though it is now known that congenital malformations in mammals
can result from both genetic and environmental causes, it is difficult
to determine at birth which one of the two factors was primarily
operative in producing any particular abnormal fetus. Also important
to determine in the case of an abnormal fetus is how the malformation
originated. One way to begin to understand the possible cause of a
malformation observed at birth is to examine the prospective deformed
embryo during its period of very early development and morphogenesis.
There exists a great disproportion botween the number of studies deal¬
ing with the production and manifestation of congenital malformations,
on the one hand, and investigations dealing with the morphogenesis of
these malformations during the earlier embryonic period, on the other.
To perform the latter type of studies, however, it is essential that
the malformations under investigation occur in a high percentage of
embryosj this will insure that the embryo being examined during the

3
early developmental period is one â– which probably will be deformed at
birth. Through the use of certain carefully selected environmental
teratogens, it is now possible to produce in experimental animals, a
high frequency of a particular congenital abnormality which might occur
only sporadically.
The understanding of the teratogenic action of an agent has to
be carried beyond morphological description to the biochemical or other
basis for its action. To be able to do this one must know first some¬
thing of the physiological and pharmacological effects of the agent on
the tissues of animals in general. Therefore, it is advantageous to
select teratogenic agents which have rather well defined metabolic
functions.
A congenital anomaly which lends itself to such an investigation
is cleft palate. Though this anomaly occurs in response to a very
large number of teratogenic agents, such as nutritional deficiencies
or excesses, hypoxia, hypothermia, drugs, hormonal imbalances, alkylat¬
ing agents, and x-irradiation (Kalter and Warkany, '59), it occurs
specifically in a particularly high incidence as a result of certain
teratogenic procedures. Three reported procedures are: l) cortisone
injections administered to pregnant mice, as reported by Fraser and
associates ('54) and by Kalter (*54)» 2) hypervitaminosis A induced in
rats as reported by Cohlan (’53) and Giroud and Martinet ('56), and
3) pteroylglutamic acid deficiency as studied by Kelson et al.('52).
Some of the studies completed to date on the early morphogenesis
of cleft palate reveal that more than one morphologic factor may be
involved in this abnormality. It is yet to be determined whether the
modification of a particular factor is specific to a certain teratogen

4
or if there are common factors in the embryo which v/hen modified by
different teratogens result in a specific defect such as cleft
palate. Answers remain to be found for the following questions: l)
If two teratogens produce a similar defect in the embryo, do they act
through a common biochemical or physical pathway? 2) Do two agents
having similar biochemical properties produce similar malformations?
3) Is there any correlation between morphogenesis of the defect and
the biochemical pathway of action?
Cortisone as a Teratogen in Cleft Palate Formation
The teratogenic action of cortisone in producing cleft palate
without cleft lip or other gross abnormalities in mouse embryos was
first reported by Baxter and Fraser ('50). Administration of 2.5 mg
(milligram) cortisone acetate daily for 4 days beginning at day 11 l/3
of gestation produced the highest frequency of cleft palate with a
minimum incidence of fetal resorption (Fraser and Fainstat, 1 pi). The
incidence of cleft palate varied in different strains of mice; it was
100¡o in embryos of A/JAX strain of mice, and Yi% in those of C573L
strain (Fraser et al., ’54).
Evans and Clingen (’53) reported that cortisone had no teratogenic
influence on the rat embryo, but in 1956 lost reported the production
of cleft palate in this species by means of cortisone. Jost's observa¬
tions, however, should be considered questionable since he administered
cortisone by way of intraperitoneal injections into the fetuses and
thus introduced another known cleft palate producing factor by piercing
the amnion (Trasler et al., ’56). Although having no teratogenic

5
action on rat embryo when administered alone, cortisone has been
reported to potentiate the teratogenic action of hypervitaminosis A.
Woo11am and Millen (157) observed that in rat embryos the expected 29%
incidence of cleft palate induced by hypervitaminosis A could be
increased to 100£ if the mothers were treated simultaneously with corti¬
sone. Such potentiation by cortisone was also observed in the case of
brain malformations produced by hypervitaminosis A. This result, how¬
ever, could not be repeated by Cohlan and Stone ('6l), who were unable
to alter the incidence of vitamin A induced malformations by means of
cortisone. Fainstat (*54) bas reported that maternal cortisone treat¬
ment induces cleft palate in offspring of rabbits.
Morphological Sequence of Cortisone Induced
Cleft Palate
On the basis of studies on the palatine shelves of living as
well as fixed embryos from cortisone treated mice, Walker and Fraser
('57) proposed that cleft palate in these embryos developed because of
delay in the initiation of movement of the palatine shelves from a vertical
position at the side of the tongue to a horizontal position superior
to the tongue. During this delay in palatine shelf movement, growth
of the head continued so that when the palatine shelves finally did
become horizontal it was at a time later than normal and they were too
far apart to meet in the midline and fuse. Larsson (*60) suggested
that the occurrence of cleft palate in response to cortisone treat¬
ment could be due to the interference in the synthesis of chondroitin
sulfuric acid. That acid-mucopolysaccharides are present in the
palatine shelves of normal embryos was demonstrated by Walker and

6
Fraser ('56), and Larsson, Bostrom and Carlsoo ('59), on the basis of
metachromatic staining with toluidine blue and S-^ incorporation
or
respectively. Larsson ('62) employed -radioautography and reported
that in mouse embryos from cortisone treated mothers there was a
reduction in synthesis of sulfo-mucopolysaccharides in the palatine
shelves as well as in other regions of the embryo. On this basis he
concluded that reduced production of acid-mucopolysaccharides during
the time of palatal closure, day 14 in the mouse embryo, was the mech¬
anism by which cortisone derived its teratogenicity.
It has been reported that cortisone suppresses fibroblast pro¬
liferation and inhibits wound healing. Ragan et al. (’50) and Plotz
et al. (’50) compared the healing of an artificially produced wound in
normal and cortisone treated rabbits, and observed that following corti¬
sone treatment, neither new fibroblasts nor new blood vessels appeared
at the site of the wound in contrast to a profuse growth of these com¬
ponents in the normal. Ground substance, as determined by toluidine
blue metachromasia, was also much reduced in the cortisone-treated
animal. In the presence of cortisone the connective tissue of animals,
both in vivo and in vitro, was depressed in its abilities to incorporate
inorganic sulfate into chondroitin sulfate (Layton 151, 151). Schiller
and Dorfman (’57) demonstrated that cortisone inhibited not only the
35
incorporation of S -labelled sulfate into chondroitin sulfuric acid
but also reduced synthesis of the whole acid-mucopolysaccharide mole-
14
cule as evidenced by depressed incorporation of C -labelled acetate
into both hyaluronic acid and chondroitin sulfuric acid.

7
Hypervitaminosis A as a Teratogen in Cleft Palate Formation
Cohlan (’53) reported the teratogenic effect of excess vitamin
A in CF Wistar rats. Pregnant rats were fed 35,000 i.u. of vitamin A
per day by intragastric intubation beginning on either day 2, 3, or 4
of gestation and continued through day 16. Besides exhibiting a 30%
incidence of cleft palate, embryos also displayed excencephaly, cleft
lip, brachygnathia, shortening of maxilla and various eye defects such
as microphthalmia, anophthalmia, open eye, exophthalmos and lenticular
cataract. These results were confirmed by Giroud and Martinet ('54).
These authors also obtained a differential incidence of cleft palate
by giving 60,000 i.u. vitamin A per day by oral intubation for 3 con¬
secutive days beginning at different periods in gestation (Giroud and
Martinet, '55, ’56). That is, i& of the embryos had cleft palate when
treatment was started on day 5, 22% had the defect when treatment began
on day 8, 92% when it began on day 11, 49^ on day 14, and none if the
treatment was not started until day 18. Vitamin A acetate and vitamin
A palmitate yielded identical results. Mo malformations were produced,
however, if vitamin A was administered intraperitoneally (Gebauer, ’54)
or subcutaneously (Woollam and Millen, '57).
Deuschle, Geiger and Warkany ('59) described an interesting
oculodentofacial pattern of abnormalities in fetuses of rats following
maternal hypervitaminosis A. The outstanding features observed in this
study were exophthalmos, maxillomandibular ankylosis, the presence of
heterotopic cartilage in the maxilla, and the absence of some molar
teeth. Cleft palate, of course, was also encountered. Histologic
sections of the abnormal fetuses revealed that the eyes were almost

8
normal in structure though the lids were absent. Exophthalmia was
explained on the basis of skeletal anomalies of the face. These
investigators observed that the zygomatic extension of the maxilla,
which normally should form the inferior orbital wall, was missing in
the experimental fetuses. The maxilla itself harbored a heterotopic
cartilage which posteriorly seemed to make up the inferior orbital
wall. These authors concluded that in the experimental fetus there
was a shortening of the head, maxilla and mandible. The more proximal
portions of the jaws were most severely affected and it was felt that
this reduction resulted in anomalies of the molar teeth.
Kalter ('60) studied the teratogenic effect of maternal hyper-
vitaminosis A on 3 strains of mice, e.g., A/Jax, DBA/Uax, and C3H/Jax.
A syndrome of dentofacial anomalies essentially similar to that found
in rats was produced. Cleft palate was present and there were abnor¬
malities of dental structures, such as supernumerary, absent or ectopic
teeth. Heterotopic cartilages were present at the corners of the mouth
in or close to the maxillary bone, aid oral tissue was trapped as
invaginations into buccal cavity. The mouth cavity was drastically
reduced in size. This reduction was thought to result from a condition
whereby the abnormally shaped palatine shelves did not ascend and •
instead fused with the lateral buccal surfaces. The tongue also fused
with both palatine shelves and the gingival tissue, thus resulting in
the apparent obliteration of the lateral recesses of the mouth.
Kalter and Warkany ('6l) reported that some of the abnormalities, such
as ankyloglossia, which were found in mice, were not observed in rats.

9
Morphological Sequence of Hypervitamlnosis A
Induced Cleft Palate
No detailed study for elucidating the morphological sequence of
cleft palate formation in rat embryos after maternal hypervitaminosis
A has yet been performed. Morphogenesis of cleft palate in mouse
embryos from excess vitamin A treated mothers was studied by direct
examination of the palate (Walker and Crain, *60) and by histological
means (Kamei, '62). These authors concluded that the factor responsi¬
ble for cleft palate formation was similar to that observed after corti¬
sone treatment! a delay existed in the time of palatine shelf movement
from vertical to horizontal position. No attempt was made to correlate
this deformity with any of the relatively well established effects of
excess vitamin A on tissues.
Investigations into the effects of excessive vitamin A on intact,
non-pregnant animal are relatively recent. Wolbach (’47) reported that
the major abnormalities in hypervitaminotic A animals are encountered in
bone and cartilage and noticed that in the skeletal tissue of such
animals maturation of cartilage was accelerated. This investigator also
observed a rapid resorption of bone and cartilage which resulted in
spontaneous fractures.
To determine direct effects of hypervitaminosis A on cartilage
and bone, Fell and Mellanby ('52) cultured the limb bones of day 5 and
6 embryonic chick and fetal mice in a medium containing excess vitamin
A alcohol. In this experiment, bone was quickly resorbed, cartilage
matrix was reduced in mass and lost its characteristic metachromatic
staining, although it retained its affinity for van Giesson's stain.
These findings implied that whereas a dissolution of chondroitin

10
sulfato had occurred, the collagen portion of the matrix -was still
present. That these effects'of excess vitamin A were not directly on
the intercellular material but rather were through the activities of
the chondrocytes was considered to be proven by the fact that no such
effects appeared if the cells were killed beforehand by heating the
cartilage to 45°C. Another, and quite different effect of vitamin A
was studied on embryonic ectoderm by Fell and Mellanby (* 53). If the
embryonic chick ectoderm was cultured in a control medium without the
addition of vitamin A, it developed a keratinized layer but under the
influence of excess vitamin A in the medium, the ectodermal cells
became a ciliated columnar epithelium and secreted mucus.
The above two studies serve to illustrate that vitamin A
influences the metabolism of various tissues such as cartilage and
mucus epithelium which have in common the function of synthesizing
acid-mucopolysaccharides.
On the basis that esterified sulfate is an important component
of the mucopolysaccharide molecule (Dziewiatkowski, *51 and Bostrom,
35
’52), and since a major part of parenterally injected S -labelled
inorganic sulfate is recovered from animals as an ester linked to a
mucopolysaccharide molecule (Bostrom, ’53), several investigators have
examined the effects of vitamin A deficiency or excess on the incor¬
poration of the sulfate into cartilaginous tissues and mucus membranes.
Fell, Mellanby and Pele (’56) utilizing radioautographic techniques,
studied the incorporation of by explants of bone from the limb
of embryonic chick cultured in either a normal or an excess vitamin A
medium. These authors concluded that not only did vitamin A cause a
dissolution of the cartilage matrix but it also inhibited further

11
o c
synthesis. Dziewiatkowski (’54) compared the contents of skeleton
from vitamin A deficient, normal control, and rats treated with vitamin
A that had been previously fed a vitamin A deficient diet. Using
radioautography and biochemical analysis, he observed that in vitamin A
deficient rats the skeleton synthesized less chondroitin sulfate than
normal. Administration of vitamin A to deficient animals was promptly
35
reflected by an increased rate of S uptake. He further reported
that after the administration of vitamin A to deficient rats not only
the rate of synthesis but also the degradation rate of chondroitin
sulfate in the skeleton was accelerated. This vías proved by the fact
that vitamin A treated animals accumulated more S during the first
24 hours after injection than the untreated, while by 72 and 120 hours
the vitamin A treated had less S^ than the untreated group. In the
case of vitamin A deficient rats that later had been given vitamin A
there was also an increase in the specific activity of the sulfate-
sulfur of sulfo-mucopolysaccharides isolated from skin.
Wolf and Varandani ('60) and Wolf, Varandani and Johnson (’61)
obtained data which indicated that mucopolysaccharide synthesis by
homogenate of rat and pig colon mucosa was vitamin A dependent.
Radioactivity was incorporated into mucopolysaccharides by incubating
the homogenate with -labelled sulfate or C^-labelled glucose. The
incorporation of radioactivity into mucopolysaccharides by the colon
homogenate of vitamin A deficient animals was about one half that
encountered in homeogenates from normal animals. When a suspension
containing 10 ug of vitamin A was added to the incubation medium of
the colons from deficient animals, radioisotope incorporation was
elevated to the control level. This stimulation by vitamin A of

12
acid-mucopolysaccharide synthesis is very significant when observed in
the light of results to be reported in this dissertation.
The influence of excess vitamin A on the keratinization of
embryonic skin explants was studied radioautographically by Pele and
Fell (’60), who showed that deeper layers of the epidermis in both
control and excess vitamin A culture media liad identical uptake of
S35°4. In control cultures from older embryos the superficial layers
of the epidermis had only scanty S'/’3o4 uptake, while the corresponding
area in excess vitamin A cultures had intense incorporation of
and, in addition, secreted mucus. This meant that in normal skin the
basal cells synthesize sulfated-mucopolysaccharides until the time when
keratinization begins. This inhibition of synthesis in explants cul¬
tured in vitamin A containing medium never occurs, hence mucus
epithelium forms instead of keratinized epithelium.
Thomas et al. (160) reported that bypervitaminosis A in intact
young rabbits resulted in marked depletion of cartilage matrix in the
epiphyseal and articular cartilages. These authors also confirmed the
previous observations of Thomas (’56) and McCluskey and Thomas (’58)
that injections of small amounts of papain into young rabbits produce
histological changes in the cartilage comparable to those produced by
hypervitaminosis A. Fell and Thomas (’60) described the effects of
crystalline papain protease on embryonic chick cartilage and fetal
mouse bone and compared them with changes observed in these tissues
following treatment with excess vitamin A. Although both vitamin A
and papain removed chondroitin sulfate from the cartilage matrix, only
vitamin A affected the chondroblasts in that they lost their glycogen
and became reduced in size. In contrast to these similarities in vivo.

13
these two agents differ considerably in their effects on fetal mouse
bone in vitro wherein the bone is unaffected by papain but rapidly
disintegrates in the presence of excess vitamin A.
From the work of Fell and her colleagues mentioned in the pro¬
ceeding pages it appears established that in the presence of excess
vitamin A, chondroitin sulfate is dissolved from cartilage matrix both
in vivo and in vitro. Their conclusion that vitamin A also inhibited
cellular synthesis of acid-mucopolysaccharides is, however, not fully
established. In young rabbits McSlligott ('62) studied radioautograph-
ically the effects of vitamin A treatment on the ability of chondrocytes
to fix radioactive sulfur. He strongly suggested that hypervitaminosis
A primarily inhibits the function of chondrocytes in synthesizing acid-
mucopolysaccharides and that the dissolution of cartilage matrix is a
secondary effect. Frame et al. (’59) reported that addition of vitamin
A to a normal diet fed to piglets considerably reduced the accumulation
35
ol injected S in costochondral junctions and other tissues.
Recently Lucy, Dingle, and Fell (*6l) and Dingle ('61) reported
that under the influence of vitamin A, the embryonic chick limb bones
grown in culture release a proteolytic enzyme from intracellular par¬
ticles similar in size to mitochondria (probably lysosomes). This
released proteolytic enzyme was thought to act on the mucopolysaccharide-
protein complex of the cartilage matrix and breaks down the protein
moiety thus releasing mucopolysaccharides from the matrix.
Purpose of This Study
This investigation was proposed to examine the early morpho¬
genesis of cleft palate in rat embryos after maternal hypervitaminosis A

14
in an effort to compare it with that described by Walker and Fraser
('57) and Larsson ('62) in embryos from cortisone treated mice. Such
an approach would demonstrate whether or not these two teratogenic
agents, which in other systems were shown to have an effect on the
acid-mucopolysaccharide content of tissues, induced cleft palate by
bringing about common structural modifications in the embryos.
35
On the basis of his studies on S incorporation into mouse
embryos from control and cortisone treated mothers, Larsson (’62)
suggested that the teratogenic action of cortisone in these embryos
could be correlated with the presence of reduced amounts of acid-
mucopolysaccharides in the affected palate. Results of investigations
attempted to reveal if such a correlation existed in rat embryos after
maternal hypervitaminosis A would also be reported in this disserta¬
tion. Assuming that the amount of intercellular ground substance was
a measure of acid-mucopolysaccharide content of embryonic tissues, the
former was determined in embryos from normal and hypervitaminotic A
mothers by employing radioautographic techniques coupled with
toluidine blue staining. Finally, if the hypothesis that the terato¬
genic action of cortisone and hypervitaminosis A was mediated through
their effect on the acid-mucopolysaccharides of tissues was correct,
then treatment of the maternal rat with papain protease might also
result in similar congenital malformation, and, therefore, such
potential teratogenicity would be studied.

MATERIALS AND METHODS
Breeding and Maintenance of Animals
Black-hooded female rats obtained from Rockland Farms, ranging
from 60-90 days of age and weighing 150-200 grams were employed in this
study. The animals were kept in stainless steel cages and given a stock
o
diet and distilled water ad libitum. Every evening the estrus cycle
was diagnosed for each female by microscopic examination of vaginal
smears (Blandau et al., '41) and those females in proestrus were placed
overnight in individual cages with sexually mature male rats. The
presence of a plug or spermatozoa in the vaginal smear at 10:00 A.M.
the next morning indicated that mating had occurred and this day was
considered day 0 of pregnancy.
Experiment 1, Incidence of Cleft Palate
To determine the incidence of cleft palate in these rats, 72
pregnant females were divided into four groups; one group for each of
four experimental protocols.
Group 1. The animals in this group received 60,000 i.u. (inter-
national units) of vitamin A acetate"^ daily for 3 consecutive days
^Rockland County, New York.
^See Appendix 1.
^Obtained from Nutritional Biochemical Corporation, Cleveland, Ohio.
15

16
during gestation. The treatment was started on either day 8, 9, 10,
or 11. The vitamin A preparation* was administered orally by means of
a blunted spinal needle attached to a tuberculin syringe. The control
animals were given 1 ml of pure cottonseed oil in the same manner from
day 9 through day 11 of pregnancy.
Group II. Pregnant females in this group received 50 mg corti¬
sone acetate per day for 5 consecutive days. A commercially available
5
saline suspension of cortisone acetate was injected into the preaxial
thigh muscles beginning on day 9 and continuing through day 13 of gesta¬
tion. Control animals received a similar volume of physiological saline
from day 9 through 13.
6
Group III. Animals in this group received 50 mg papain daily
by intraperitoneal injection for 5 days beginning on day 9 of gestation.
Saline injected animals served as controls.
Group IV. This group was subdivided into two parts each of which
received combined treatments: a) the pregnant animals were given 60,000
i.u. vitamin A by stomach tube per day on days 10, 11, and 12, and 50 mg
cortisone acetate by intramuscular injections per day on days 9 through
12j b) these animals were treated concurrently with 50 mg cortisone
intramuscularly each day from day 9 through 13 and 50 mg papain intra-
peritoneally each day from day 12 through day 15.
^Vitamin A acetate was dissolved in cottonseed oil to give a
concentration of 60,000 i.u./ml. Though the preparation was kept
refrigerated, a fresh preparation was compounded each week.
^Obtained from Merck, Sharp, and Dohme, West Point, Pennsylvania.
^Obtained as a crude powder from Nutritional Biochemical Cor¬
poration, Cleveland, Ohio. Crude papain was assayed by biuret method
to contain 1 mg protein per 10 mg sample. The powder was dissolved by
grinding in 0.05M phosphate buffer at pH 7. The solution was then
filtered to remove any insoluble particles (McCluskey and Thomas, *59).

17
Animals in all groups were sacrificed on day 20 of gestation.
In this procedure the female was anesthetized with ether, laporotomized
and the fetuses removed from the antimesenteric border of the uterus.
7
The young were weighed on a triple beam balance, fixed in Bouin's
fluid and examined grossly for externally detectable malformations.
The crown rump length was measured by means of a Vernier Caliper as was
the length of the head from the tip of the snout to the posterior
extreme of the occiput.
After the fetuses were fixed their heads were studied under the
dissecting microscope after having been sliced with a sharp blade into
frontal sections about 1-2 mm thick. By this method the relative posi¬
tions of the tongue and palatine shelves could be observed. In some
instances, and as an additional method of observation, before the fetal
head was frontally sectioned the palate was examined from the ventral
aspect by removing the lower jaw and displacing the tongue.
Experiment 2. Anatomical Morphogenesis of Normal
and Cleft Palate
This experiment was performed to study the formation and closure
of the secondary palate in normal embryos and to observe the changes
which occurred in the palatine processes and neighboring tissues lead¬
ing to the formation of cleft palate in embryos from hypervitaminotic
A rats.
Fifty pregnant rats were divided into two groups. Each animal
in one group received orally 60,000 i.u. vitamin A dissolved in cotton¬
seed oil per day for 3 successive days beginning on either day 9 or day
7
'See Appendix II.

18
10 of gestation. Previous studies showed that maternal hypervitaminosis
A during both of these gestational periods, i.e. 9-11 days or 10-12
days, induced cleft palate in more than 80# of the embryos from treated
mothers. The second group served as controls and the pregnant females
were administered 1 ml. of pure cottonseed oil orally on the appropri¬
ate days.
The animals from both of these groups were sacrificed at 10 A.M.
on the 14th, 15th, 16th, or 17th days of gestation. The embryos were
removed from the exteriorized uterus, weighed and fixed in Bouin's fluid.
At least 3 embryos from every litter were also fixed in alcohol-forma-
g
lin for 24 hours for histological and histochemical study.
Embryos fixed in Bouin's fluid were measured for crown rump
length and head length, and then the heads were sliced freehand in a
frontal plane and observed under the dissecting microscope. The heads
fixed in alcohol-formalin were cleared in terpineol, embedded in paraf¬
fin and serially sectioned in frontal plane at 6m on a rotary microtome.
Sections were stained by the following procedures, l) Toluidine blue
for identification of acid-mucopolysaccharides. Toluidine blue was used
as 0.1# solution in 30# ethyl alcohol for 5 minutes (Kramer and Windrum,
'55). Appropriate hyaluronidase controls were also prepared. 2)
Periodic acid-Schiff technique (PAS) for identification of glycogen
(McManus, '42). Negative staining with PAS subsequent to glycogen
digestion by alpha-amylase served as controls. 3) Methyl green pyronin
for detection of ribonucleoproteins (Brachet, '42) with ribonuclease
controls (Pearse, '60). 4) Feulgen stain for deoxyribonucleic acid
g
Three parts 95# alcohol and 1 part 40# formaldehyde.

19
(Feulgen and Rossenbeck, '24, referred bo in Pearse, *60). 5) Iron
hematoxylin stain for routine morphological study and recognition of
individual cell type.
Experiment 3. Radioautographic Studies
Because previous reports indicated that vitamin A was involved
in the metabolism of acid-mucopolysaccharides, S^-labelled sulfate was
administered parenterally to female rats and by radioautographic means
the amount incorporated by various tissues of control embryos and
embryos from vitamin A treated mothers -was examined. For this experi¬
ment, 10 pregnant females were employed. Five of these were treated on
the 9th, 10th, and 11th days of pregnancy with 60,000 i.u. vitamin A
per day, while the other five served as controls and were given pure
cottonseed oil during the same periods of gestation.
35 9
A single dose of carrier-free S -labelled sodium sulfate in
physiological saline was injected intraperitoneally at 10:00 A.M. on
either day 13, 14> or 15 of pregnancy. Two controls and two vitamin A
treated animals received 10 >ic (microcuries} S^^/gm body weight on day
13 of pregnancy, also two controls and two vitamin A treated females
received an identical dose on day 14, and one control and one treated
female were injected on the 15th day but with only 5 he S^Vo111 body
weight. All pregnant females were sacrificed 43 hours after the
isotope was administered. The embryos which ranged in age from 15
through 17 days were removed, fixed in alcohol-formalin, dehydrated in
Obtained from Abbott Laboratories, Oak Ridge, Tenn.

20
ethyl alcohol, cleared in terpineol and embedded in paraffin. Serial
sections of the heads were made at 6;li in the frontal plane.
At least 3 embryos from every litter were processed for radio-
autography which was done according to the dipping method developed
by Messier and Leblond ('57). Specially prepared 1" x 3" glass
10
slides carrying the paraffin sections of the embryos were taken to
the dark room. The dipping was done in complete darkness except for a
single Wratten series if2 safe light. Kodak NIB-3 emulsion was melted
by placing a small portion of the gelled emulsion in a plastic dipping
container and held for 30 minutes in a water bath at 40-45°C. Slides
were dipped singly into the liquid emulsion for 1-2 seconds and then
drained in a vertical position for several minutes. The side of the
slide not bearing the sections was wiped clean of emulsion and the
slide was dried in a horizontal position for one hour before it was
stored in a light-proof plastic box sealed with black electrician's
tape. Plain glass slides were interposed between those bearing radio¬
active sections in order to prevent back scattering.
The boxes were stored for 12 days under dry ice in such a way
that the slides were oriented horizontally with the emulsion side down.
After their allotted exposure time the radioautographs were developed
at 20°C in the dark as follows:
Kodak D-19 Developer-
Kodak S3-5a Stop Bath'
Kodak Acid Fixer
hater Rinse
•5 minutes
â– 15 seconds
•10 minutes
â– 1-15 minutes
pared in order to provide for better adherence between the liquid
emulsion and the tissue preparation. That is, chemically cleaned
slides after having been immersed in a solution of 0.5Í» gelatin and
0.05Jb chrom alum in distilled water were drained and dried at room
temperature in a covered, dust free staining dish (Boyd, '55).

21
Immediately after this processing the slides were stained for 5
minutes in 0,1% toluidine blue in 30% ethyl alcohol, dehydrated,
cleared and mounted in HSR"^ mounting medium.
The stained radioautographs were studied with the aid of the oil
immersion lens of the light microscope and developed granules were
counted over mesenchymal tissues, cartilage, bone, and oral epithelium.
Experiment 4. Examination of the Effects of Hypervibaminosis A
on the Epiphyseal Cartilage of Adult Rats
This experiment was performed to detect whether any dissolution
of cartilage matrix, similar to that found in rabbits by Thomas,
McCluskey, Potter and Weissmann ('60) and Fell and Thomas (160)
occurred in the rat after the administration of teratogenic doses of
vitamin A.
Eight non-pregnant and 2 pregnant, 84 day old female rats
weighing 150-200 grams were treated as shown in Table 1. Immediately
upon sacrifice the tibio-femoral joint was removed from the animal,
freed of skin and muscle, and fixed for 2 days in ICbS buffered forma¬
lin. Bone was decalcified in a solution of equal parts 2% formic acid
and 20% sodium citrate as recommended by Hulth and Westerbom ('59).
The joint was cleared in terpineol and embedded in paraffin. Seven
microns thick sections were cut on the rotary microtome and stained for
5 minutes in 0.l£ toluidine blue in 30% ethyl alcohol. The epiphyseal
cartilages of femur and tibia were examined under the light microscope
liSH mounting medium was obtained from Hartraan-Leddon Company,
Philadelphia, Pennsylvania.

TABLE 1
Dosage of Vitamin A Administered to Rats
for Dissolution of Cartilage Matrix
Experiment
Number
Number and
Condition of
Rats
Treatment Per Day for
3 Consecutive Days by
Oral Incubation
Time Elapsed Between
Start of Treatment
and Sacrifice
A
2 Non-pregnant
1 ml. Cottonseed Oil
4 Days
B
2 Non-pregnant
60,000 i.u. Vitamin A
4 Days
D
2 Non-pregnant
100,000 i.u. Vitamin A
4 Days
E
2 Non-pregnant
200,000 i.u. Vitamin A
4 Days
C
2 Pregnant
60,000 i.u. Vitamin A
on days 9» 10, and 11
of Pregnancy
10 Days

23
to detect the loss of any metachromatically stainable material from
the cartilage matrix under the influence of excess vitamin A.

RESULTS
Teratogenic Action of Hypervitaminosis A
Administration of 60,000 i.u. vitamin A per day for three con¬
secutive days to pregnant rats produced a high percentage of intrau¬
terine mortality. The earlier in pregnancy that the treatment was
instituted, the higher was the incidence of resorption. In Figure 1,
the percentage of resorbed embryos is represented as a function of the
gestational period during which the vitamin A was administered. When
begun on day 8, all of the embryos were resorbed or dead by day 20.
If the three day period of hypervitaminosis A began on day 9 the re¬
sorption rate was 54$ and if the treatment was delayed until day 10 the
resorption rate dropped to 22$, but did not go below this level when the
treatment was not started until day 11. The injected control animals
given pure cottonseed oil on days 9, 10, and 11 had a resorption rate
of 12%, which is approximately 8$ above an incidence rate to be
expected in normal controls.
The teratogenic effects of hypervitaminosis A observed on term
fetuses are summarised in Table 2. Among the embryos from vitamin A
treated mothers, 80$ or more had cleft palate when the treatment was
started on day 9 or day 10 of pregnancy. None of the 21 embryos from
3 females who received treatment from day 11 onwards developed cleft
palate. The incidence of eye defects such as anophthalmia, micro¬
phthalmia, exophthalmos, and open eye decreased from approximately 50$
24

25
Figure 1. Fetal resorption as a function of day of gestation
maternal vitamin A treatment commenced.

TABLE 2
Teratogenic Action of Hyoervitaminosis A Observed in Term Fetuses
Treatment and Days of
Gestation Treatment
Accorded
Number
of
Litters
Number of
Implanta¬
tion Sites
cp Fetuses
Resorbed
C?
P
Cleft
Palate
Survivors I-
Eye
Defects
lalxormed
Excen-
ceph&ly
60,000 i.u. Vitamin A
9-11
M
O'
13
114
54
80
58
15
60,000 i.u. Vitamin A
10-12
11
112
22
83
48
0
60,000 i.u. Vitamin A
11-13
3
27
22
0
10
0
Cottonseed Oil
9-11
9
87
12
0
0
0

27
to 10/S as the vitamin A treatment was delayed from day 9 to day 11.
Microstomia, i.e., reduction in the size of the oral aperture, was
observed frequently in treated fetuses, though the exact percentage was
not calculated.
Figure 2 compares the freehand cross-sectional slices from the
heads of typical cottonseed oil injected control (Figure 2A) and vita¬
min A treated (Figure 2B) fetuses. Except for the incomplete palate
in the treated fetus, most other organs in the heads of the two classes
of embryos Mere comparable in size and shape.
In order to determine the effect of hypervitaminosis A on the
growth of the fetus in general, fetuses in all litters were weighed.
From these weights (Table 3) it was observed that some decrease in the
mean fetal weight resulted when vitamin A treatment was started on day
9; this disparity from the control mean weight diminished as the treat¬
ment was delayed until day 10 or day 11. The reduction in body weight
of the treated fetuses was accompanied by a decrease in body size; all
the fetuses from 4 litters picked at random from among the treated
group were measured for crown rump length and head length, and compared
with those of control rats (Table 4)• Though both crown rump length
and head length in treated fetuses were shorter than controls, only the
head appeared deformed because of the decrease in the length of both
jaws; the trunk, notwithstanding the shortened length, was not detect-
ably malformed.
Effects of Maternal Injections of Cortisone and Papain on Embryos
On the basis of the methods of experimentation and examination
employed, neither cortisone nor papain appeared to be teratogenic.

Figure 2. Frontal freehand sections of heads of day 20 fetuses.
A - control; secondary palate (?) is complete; B - vitamin A treated;
cleft palate. 15x.
NS - nasal septum. T - tongue.

TABLE 3
Weights of Tern
. Fetuses After Maternal
Hyoervitaminosis A
-
Treatment and Days of
Number
Number
Weightsa
Gestation Treatment
of
of
Accorded
Litters
Fetuses
60.000 i.u. Vitamin A
13
53
3.09 ± 0.44
9-11
60,000 i.u. Vitamin A
11
87
3.20 - 0.42
10-12
60,000 i.u. Vitamin A
3
21
3.32 - 0.20
11-13
Cottonseed Oil
9
76
3.64 ± 0.45
9-11
aHean fetal weight in grans - the standard deviation

TABLE 4
Crown Hump Length and Bead Length of Term Fetuses After Maternal
Hypervitamlnosis A
Treatment and Days of
Gestation Treatment
Accorded
Number
of
Litters
Number
of
Fetuses
Crown0'
Rump
Length
Headb
Length
Cottonseed Oil
9-11
4
36
33.6
14.2
60,000 i.u. Vitamin A
9-11
4
17
31.2
13
c,Mean length in millimeters
bIbid.

31
Furthermore, in neither instance was there any increase in the intrau¬
terine mortality from the control level (Table 5). A combined treat¬
ment with cortisone and papain simultaneously did not produce any mal¬
formations though the intrauterine mortality increased from control
rate of 14$ to 26$. Also, no marked difference was present in the
mean fetal weight between control fetuses and those from cortisone or
papain treated mothers (Table 6).
To determine whether cortisone would reduce or potentiate the
teratogenic effects of excess vitamin A in producing cleft palate, it
was injected into pregnant rats already receiving teratogenic doses of
vitamin A. Cortisone did not modify the incidence of cleft palate in such
fetuses (Table 7). Although the type of eye defects in the group
receiving the combination of vitamin A and cortisone were essentially
the same as that found in the vitamin A group, they occurred in 33$ of
the fetuses in the former and 4&$ of the fetuses in the latter. Simi¬
larly, the mean fetal weight was higher in fetuses from the combined
treatments than it was in the group treated with vitamin A alone.
Morphogenesis of Cleft Palate Induced by Kypervitaminosis A
I. Macroscopic Observations
15th Day of Gestation.—The mean weights of 37 control and 63
treated embryos were comparable, but both the crown rump and head
lengths of vitamin A treated embryos were slightly less than the
controls (Table 8).
The palatine shelves of the control embryos were vertically
oriented, triangular in outline and closely followed the lateral con¬
tour of the tongue on this day (Figure 3A and 3B). The ventral edge

TABLE 5
Effects of Maternal Injections of Cortisone and Paoain Observed
in Term Fetuses
Treatment and Days of Number
Gestation Treatment of
Accorded Litters
Physiological Saline 8
9-13
50 mg Cortisone 8
9-13
50 mg Papain 9
9-13
50 rag Cortisone 2
9-12 and
50 mg Papain
12-15
Implant¬
ation
Sites
a
Fetuses
Resorbed
Malform¬
ations
76
14
0
80
15
0
95
13
0
19
26
0

TABLE 6
Weights of Terra Fetuses after Maternal Injections
of Cortisone and Paoain
Treatment
Number
of
Litters
Number
of
Fetuses
Weightsa
Saline Controls
8
67
3.64 - 0.40
Cortisone
8
68
3.34 * 0.39
Papain
9
63
3.50 ± 0.43
aMean fetal weight in grams - the standard deviation

TABLE 7
Teratogenic Effects of Hyoervitaminosis A and Cortisone
Observed in Terra Fetuses
Treatment and
Number
Number of
Cf
/°
% Survivors Malformed
Days of Gesta¬
of
Implanta¬
Fetuses
tion Treatment
Litters
tion
Resorbed
Accorded Sites
Weights8
Cleft
Palate
Eye
Defects
Excen-
cephaly
Physiological Saline
9-13
8
76
14
0
0
0
3.64 1
0.40
50 mg Cortisone
9-13
8
80
15
0
0
0
3.34 ±
0.39
60,000 i.u. Vitamin A
10-12
11
112
22
83
48
0
3.20 ±
0.42
60,000 i.u. Vitamin A
7
72
17
88
33
0
3.64 -
0.26
10-12 and
50 mg Cortisone
9-12
V.O
â– [>-
®Mean fetal weight in grams - the standard deviation

TABLE 8
Comparison of Control and Hypervitaminotic A Embryos
on Days 15. 16 and 17 of Pregnancy
Embryonic
Age
Treatment
Number
of
Litters
% Fetuses
Resorbed
Number of
Living
Embryos
Weights8-
Crovmu
Rump
Length
Head0
Length
Experimental
Control
4
10
37
263 ± 30
10.8
6.1
15th Day
Vitamin A
11
54
63
264 t 29
10.2
5.5
Experimental
Control
7
15
56
468 ± 46
13.5
7.3
16th Day
Vitamin A
16
62
86
467 = 39
13.0
7.2
Experimental
Control
3
7
27
933 - 26
17.4
9.8
17th Day
Vitamin A
6
56
37
754 ¿ 62
16.8
8.7
aMean embryonic weight in mg - the standard deviation
kj'iean length in millimeters
cIbid

Figure 3. Frontal freehand sections of heads of day 15 embryos. A and B -
control; palatine shelves (PS) have triangular outline; C,D and E - vitamin A treated;
C and D - PS are rounded; E - PS partially above the tongue (T). 15x.
NS - nasal septum.

37
of the shelf was parallel to the floor of the oral cavity. In other
sections it was observed that posteriorly the palatine shelf instead
of being triangular was platelike.
The usual situation in treated embryos was for the palatine
shelves to be rounded instead of triangular in frontal section
(Figure 3C and 3D). In some of the embryos the shelves actually
appeared to have moved from the lateral side of the tongue to its
dorsal aspect (Figure 32)• The shelves were reduced in size from those
of the controls and in some embryos they were completely missing.
Only 284 of the embryos from hypervitarninosis A mothers had
palatine shelves which appeared to possess a morphological relationship
to the tongue similar to that observed in 100/4 of the control embryos
(Figure 4). The palatine shelves of 72/5 of the treated embryos, on the
other hand, were abnormally shaped and their relative position with
respect to the tongue was altered.
Since all the resorption encountered at term (544) in the
treated rats had already occurred by day 15 (compare Table 2 and Table
S), it may be concluded that all those embryos having deformed pala¬
tine shelves would have had cleft palate at term. In addition, some
of the treated embryos which on this day show normal palatine shelves
will also acquire cleft palate since more than 80/5 do show this defect
at term. In 3 of the 63 treated embryos some of the epithelial sur¬
faces such as those covering median nasal and maxillary processes,
which happened to be opposed to each other, had a tendency to fuse.
loth Day of Gestation .--Fifty six control and 86 treated embryos
were examined on this day. Mean embryonic weight, crown rump length,
and head length were comparable in the two groups (Table 8). The

38
100
ti
-5
a
<0
I
.5 50
Uj
0
o
o
.Vj
Normally Shaped
Abnormally Shaped
Orientation of Palatine Shelves
Figure 4. Graphs comparing the percentages of day 15 control and
vitamin A treated embryos on the basis of shape and orientation of their
palatine shelves.

39
palatine shelves of the control embryo were either vertical and, there¬
fore, still lateral to the tongue as they were on day 15 (Figure 3A),
or horizontal and superior to the tongue (Figure 5A). Shelves in
position such as those in Figure 5A were observed to be both fused and
unfused. All stages of palate development, i.e., vertical, horizontal
unfused, and horizontal fused were observed to occur in different
embryos of the same litter. The tongue was flat, depressed uniformly
and parallel to the palatine shelves.
Though in some treated embryos the palatine shelves appeared
normal, 84/o had abnormal shelves at this day. This abnormality ranged
from deformed shape such as wavy or bent to reduced size including
complete absence. In contrast to the controls the palatine shelves of
treated embryos were oriented in one of these three positions:
1. Vertical on the side of the tongue (represented by Figure 5B).
Though some of the embryos had normal-appearing palatine shelves,
most of the embryos included in this category showed either
reduced or completely missing palatine shelves. Epithelial
fusions between opposing surfaces of median nasal process and
maxilla occurred frequently.
2. Palatine shelves horizontal and superior to the tongue. In
spite of having crossed the barrier of the tongue, the palatine
shelves were located in the corners between the nasal septum
and the tongue (Figure 5C), but had not progressed towards the
midline superior to ti» tongue. While still separated from each
other, the palatine shelves in some instances had the tendency
to become fused with the ventral edge of the nasal septum on

Figure 5. Frontal freehand sections of day 16 embryos. A - control; palatine
shelves (PS) are horizontal, parallel and superior to tongue (T); unfused; B,C,D and E -
vitamin A treated; B - PS vertical; C - PS horizontal above tongue, but not progressed
towards midline; some fusion of PS with nasal septum (NS) present; D and E - PS hori¬
zontal but abnormally shaped. 15x.

41
their respective sides (Figure 5C). In general the palatine
shelves were abnormally shaped (Figure 50 and 53)•
3. Major portion of shelves horizontal, some portions still verti¬
cal. The posterior portion of the shelves was the part which
was most frequently vertical. The vertical palatine shelf was
not necessarily at the side of the tongue, since due to the
reduction of its size the shelf could be accommodated at a
higher plane than the tongue.
In Figure 6, control and treated embryos are compared with regard
to the shape and orientation of their palatine shelves. In the control
group 65$ of the embryos had vertical palatine shelves and 35$ had
horizontal shelves. In contrast the treated group had only 39$ vertical
and only 20$ horizontal, and 41$ of their embryos had palatine shelves
in intermediate positions.
In summary, these considerations point to the fact that on day
16 of development more treated embryos showed the initiation of pala¬
tine shelf movement towards the horizontal position than did the control
embryos. However, fewer treated embryos successfully completed the shelf
movement than the controls, as illustrated by a large number of treated
embryos having palatine shelves in an intermediate position (Figure 6).
17th Day of Gestation.—Twenty seven control and 37 treated
embryos were studied. It was this day when a significant reduction in
the mean embryonic weight of treated litters became apparent as the
treated embryos averaged about 200 mg lighter than control embryos
(Table S). Also, while the body size as well as the head in the
treated group was demonstrably shorter than normal, only the head

42
100
, 75-
b50
<5
a
I
25
0
CJ
.o
Control
J Normally Shaped
Abnormally Shaped
£
â– s
Treated
Orientation of Palatine Shelves
Figure 6. Graphs comparing the percentage of day 16 control and
vitamin A treated embryos on the basis of shape and orientation of their
palatine shelves.

43
appeared malformed; both mandible and maxilla were shorter in their
anteroposterior length, hence the tongue protruded somewhat.
The palate was complete in all control embryos (Figure 7A) in
that the palatine shelves were united throughout their length. In the
predominant number of treated embryos, palatine shelves, though hori¬
zontally oriented superior to the tongue, were not completely united
with one another, which resulted in a palatine cleft. Observing free¬
hand slices of the heads of such embryos in a frontal plane it was
noticed that at the anterior level the palatine shelves might either
actually progress to the midline and fuse with each other (Figure 7B),
or they might remain separated and display a deformed outline (Figure 7C).
Posteriorly, however, almost all treated embryos showed a wide gap
intervening across the tongue (Figure 7D).
Figure 8 collates the shape and orientation of the palatine
shelves of the control and treated embryos examined on day 17. In the
treated group a small number of embryos still showed their palatine
shelves oriented in either a vertical or an intermediate position and
these in large part were deformed in shape. From this figure it is
apparent that failure of initiation or completion of the movement of
palatine shelves towards the horizontal plane occurred only in a
relatively small number of treated embryos and that cleft palate in a
large percentage of treated embryos occured in spite of the presence
of horizontally oriented palatine shelves. The immediate cause of the
defect, then, was not so much failure of movement of the shelves as
deficiency of tissue to permit contact with subsequent fusion at the
midline

44
Figure 7. Frontal freehand sections of day 17 embryos. A -
control; palatine shelves (?S) completely united; B, C and D - vitamin A
treated; B - PS at anterior level progressed to midline and partially
united; C - PS abnormally shaped and at anterior level located in corners
between nasal septum (NS) and tongue (T); D - PS at posterior level
showing a wide gap across tongue. 15x.

45
Normally Shaped
Abnormally Shaped
o
N
Treated
Orientation of Palatine Shelves
Figure 8. Graphs comparing the percentages of day 17 control and
vitamin A treated embryos on the basis of shape and orientation of their
palatine shelves.

46
II. Microscopic Observations
Histologic sections of embryos of 14 through 17 days of age were
35
studied by histochemical and S radioautographic means.
14 and 15 Day Old Embryos.—'The palatine shelves in 14 and 15 day
old normal control embryos are present -within the oral cavity as two
longitudinal ridges on either side of the tongue. Along their entire
length they arose from maxillary mesoderm as two medially directed out-
foldings consisting of mesenchymal tissue covered by columnar epithelium
(Figure 9A). The mesenchymal cells contained large oval nuclei and had
many cytoplasmic processes which were embedded in an intercellular
ground substance. They were not distributed uniformly throughout the
volume of the palatine shelves but on the basis of their density per
unit of volume, the palatine shelves could be divided into three regions.
In an antero-posterior direction these regions were of about equal
length. In the anterior one third of the shelves the mesenchymal cells
were dispersed loosely in the main body of the shelf except for a
slight aggregation on the medial aspect (Figure 9B). The ground sub¬
stance, which had toluidine blue metachromasia, was abundant in this
anterior part of the shelves which were covered by tall, stratified
columnar epithelium 2-3 cells thick and having a well defined basement
membrane.
It is important to note in passing that in the maxillary mesoderm
there was an aggregation of preosteoblastic cells, which were recog¬
nizable as such because they characteristically accumulated substantial
amounts of glycogen (indicated by Periodic acid-Schiff positive and
alpha-amylase digestible material) and were strongly basophilic (demon¬
strated by methyl green pyronin method). The identification of this

A
B
Figure 9. Frontal sections of anterior one third of palatine shelf of day 15 control embryo. A -
anterior level; palatine shelf (PS) consist of mesenchymal tissue covered by columnar epithelium; B -
posterior level; slight aggregation of mesenchymal cells on medial aspect of PS. Toluidine blue. 120x.
T - tongue. DL - dental lamina.
t5

48
tissue was confirmed by the fact that preosteoblastic tissue normally
present in the lower jaw lateral to Meckel's cartilage displayed
identical characteristics (Figure 10A). This preosteoblastic tissue
of the maxilla continued into the palatine shelf.
In the middle third of the palatine shelf the mesenchymal cells
were closely packed, particularly along the medial border. The medial
portion of the palatine shelf was delineated from the rest of the
palatal tissue by an epithelial notch. Ground substance in the shelf
was strongly metachromatic with toluidine blue. Preosteoblastic tissue
extended into both the medial and lateral portions of the palatal
tissue (Figure 10B).
In the posterior third of the palatine shelves the epithelial
notch described above became deeper and consequently separated the
shelf from the more laterally placed maxillary areas. As a result
the shelf appeared plate-like in outline instead of triangular. The
cell density was still higher than in the middle third and metachro¬
matic ground substance was abtmdant. The preosteoblastic tissue was
now assembled at the dorsomedial edge of the palatine shelves which
were covered by a low, simple columnar epithelium. This epithelium
was continuous laterally with the 2-3 layered columnar epithelium
covering the lateral surface of the shelf as well as the rest of the
oral cavity.
By their histologic pattern 15 day old treated embryos demon¬
strated several notable differences from normal control embryos of the
same age. Anteriorly, the palatine shelves of the treated embryos had
a slightly rounded contour instead of being triangular (Figure 11A).
One remarkable difference between normal and treated embryos was the

Figure 10. Frontal sections of head of day 15 control embryo,
of palatine shelf (PS); aggregation of preosteoblastic tissue (OST) in
through middle third of PS; preosteoblastic tissue distributed in both
PAS. 40x. NS ~ nasal septum. T - tongue.
A - section through anterior one third
maxilla and mandible; B - section
medial and lateral portions of PS.

A
’f'tyyy'
&'&^A$JSSSS«1
■^'■‘i'tVt-’&’SfcW
ln - •-> ¡i >>*? '*•••>.' Á V-
¡¡Ési®
IIISiiiB;
wém;
Hü
T'il
BHfiH
v.-'-'af
9
RtsaJBiii
$MÍ
y*V'3¿+
wm;tm
ÉÉllÉÉ
§W5$t|fe
fetes» i
i»»
¡¡¡fee
IS
a»
B
vn
o
Figure 11. Frontal sections of anterior one third
embryo. A - anterior level; palatine shelf (PS) rounded;
120x. B - posterior level; dental lamina for upper molar
with Figure 9B). Iron hematoxylin. 120x. T - tongue.
of palatine shelf of day 15 vitamin A treated
heterotopic chondrogenesis (KCT) present. PAS.
(DL) exists more medially than in control (compare

51
reduced size in the treated embryos of the maxillary preosteoblastic
area on the lateral aspect of the palatine shelf. This reduction in
size of the maxillary bone primordium was brought about not only by
the differentiation of a lesser number of mesenchymal cells into
preosteoblasts, but also by another phenomenon. That is, some of the
cells within the bone primordium differentiated into chondroblasts
and chondrocytes, as evidenced by the accumulation around these cells
of a typically metachromatic cartilage matrix (Figure 11A). At this
level it was also observed that the dental laminae for the upper
molars in some of the treated embryos arose more medially (Figure 113)
than in the controls (Figure 9B), and, therefore, the mesenchymal
tissue of palatine shelves and alveolar ridge was reduced in amount.
At a level slightly posterior to the one just described, the
palatine shelves might be either of normal size or somewhat smaller
than normal. In Figure 12A, the palatine shelves were quite similar
to those of control embryos, except that the epithelial notch was not
evident. In some embryos the palatine shelves at this level were almost
completely absent (Figure 123). In this photomicrograph the maxillary
preosteoblastic area was extensively replaced by the previously mentioned
heterotopic chondrogenic tissue.
The greatest difference between control and treated embryos
existed in the process of outfolding by which the posterior region of
palatine shelves in treated embryos took origin from maxillary tissue
(compare Figure 13A with Figure 133). A considerably lesser amount of
mesenchymal tissue formed this portion of the shelf in the treated than
in the control embryo. Due to the reduced length of both jaws in the
treated embryo, the anteroposterior length of the palatine shelves was

Figure 12. Frontal sections of heads of day 15 vitamin A treated embryos through middle third of
palatine shelf. A - epithelial notch in palatine shelf (PS) is absent. PAS counterstained with Celestine
blue. 4Qx. B - palatine shelves almost completely absent; maxilla contains heterotopic cartilage (HOT) .
PAS.

Figure 13. Frontal sections of
embryo; palatal tissue (PS) outfolding
folding abnormal; less palatal tissue
posterior third of palatine shelves of day 15 embryos. A - control
from maxillary tissue; B - vitamin A treated embryo; process of out-
(PS) outfolds from maxilla. Iron hematoxylin. 120x. T - tongue.

54
also reduced. With toluidine blue staining one fact, however, became
quito clear. The metachromatic intensity of the ground substance in
the palatine shelves of treated embryos was not decreased from that in
the controls.
Radioautographs of 15 day old control embryos revealed wide-
spread activity after injection of labelled sulfate into the mother.
The activity was observed largely over mesenchymal tissues or other
tissues derived from mesenchyme, e.g., cartilage, bone. The distribu-
35
tion of S -labelled material in these tissues coincided with the
presence of toluidine blue stainable metachromatic component.
Palatine shelves and mesenchymal tissue in other regions of
control embryos of this age showed very slight radioactivity (Figure
144), while aggregations of cells in precartilaginous tissue of nasal
35
septum and Meckel's cartilage showed moderate S activity. A higher
density of developed particles was observed in the cartilage of the
limb bud, where considerable metachromatic matrix was also present
(Figure 14b).
Kasai and limb cartilages in the treated embryo showed S J
incorporation comparable to that observed in controls, although
mesenchymal tissue in the treated.embryos, including that of palatine
shelves, revealed a very much higher incorporation than was seen in
comparable areas of control embryos (Figure 15A and 153. Compare with
Figure 14A and 143). Although the maxillary preosteoblastic tissue in
35
treated embryos had S activity similar to controls, some regions
within this tissue showed a very high radioactivity. Such regions
also revealed metachromasia with toluidine blue, indicating the presence
of heterotopic cartilage.

Figure 14. Radioautographs of day 15 control embryo. A - frontal section of palatine shelf; B
cartilage of limb. Toluidine blue. lSOOx.
r

Figure 15. Radioautographs of day 15 vitamin A treated embryo. A - frontal section of palatine shelf
(compare with Figure 14A); B -- cartilage of limb. Toluidine blue. 180Qx.
\

57
16 Day Old Embryos.—In 16 day old control embryos in which the
process of shelf movement had not yet occurred, the palatine shelves were
oriented vertically, as observed on day 15. However, their dimensions
had increased from the level of the previous day. The distribution of
histological components along the anteroposterior axis of the palatine
shelves was still typical for each of the three regions described for
day 15 control embryos (Figures 16A, 16B and 17A).
Evidence of movement by the palatine shelves from the vertical
to the horizontal position was first observed at the junction of the
middle third and posterior third of the palatine shelf (Figure 17B).
At this point a medially directed extension arose from the medial
surface of the palatine shelf just superior to the dorsalmost level
of the tongue. This extension consisted of mesenchymal cells, ground
substance and preosteoblasts, and it progressively enlarged at the
expense of the ventral and vertical portions of the shelf, which
appeared to be gradually withdrawn into the body of the shelf (Figure
ISA). This movement of the palatine shelves to the horizontal plane
then extended to the anterior portion, which in a similar manner
crossed the barrier of the tongue (Figure IBB).
It is of particular importance that the movement from the
vertical to the horizontal plane was initiated at a point where
osteogenic tissue was present (Figure 19A) and already forming bone
matrix. This osteogenic tissue was seen to execute a bend or curve
at the point where the process of horizontal movement was under way
(Figure 19B).
The horizontal, unfused palatine shelf possessed a core of mes¬
enchymal tissue similar to that present in the vertical shelf. The

Figure 16. Frontal sections of head of day 16 control embryo. A - section through anterior one third
of palatine shelf (PS); PS contains a medially directed extension (OP) from maxillary preosteoblastic tissue
(OST). PAS. 40x. B - section through middle third of PS; epithelial notch (N) is present; denser accumula¬
tion of mesenchymal cells and preosteoblasts occurs in medial and lateral portions of PS. Toluidine blue.
40x. T - tongue. M - mandible.

Figure 17. Frontal sections of heads of day 16 control embryos. A - section through posterior third
of palatine shelf (PS); aggregation of preosteoblastic tissue (OST) at dorsalmost aspect of PS; B - section
through PS shoving beginning of movement to horizontal position at junction of middle and posterior third;
medial extension of PS is present. Toluidine blue. /¡.Ox,

Figure 18. Frontal sections of head of day 16 control embryo showing movement of palatine shelves to
horizontal position. A - palatine shelves (?S) are almost completely horizontal] B - palatine shelves are
completely above tongue (T). Toluidine blue. 4Ox.
NS - nasal septum.

Figuro 19. Frontal section of head of day 16 control embryo showing origin of palatine shelf move¬
ment to horizontal position. A - preosteoblastic tissue (OST) accumulates on medial aspect of palatine
shelf; B - a bend occurs in this preosteoblastic tissue. PAS. 4Ox.

62
epithelium covering the two apposing palatine shelves was simple
cuboidal or low columnar and. became stratified columnar as it continued
laterally over the palatine shelf (Figure 20). The assemblage of
preosteoblasts, which in the vertical palatine shelf was observed on
its medial aspect, was now seen towards the superior aspect of the
horizontal palatine shelf (Figure 20). This palatal preosteoblastic
area extended medially to the midline of the shelf and laterally was
continuous with preosteoblastic tissue of the maxilla, which had some
bone trabeculae present. The zygomatic process of the maxilla projected
towards the inferior margin of the orbit (Figure 183) and the mandible
was developing around and lateral to Meckel’s cartilage and was in a
slightly more advanced stage of morphological differentiation than the
maxillary bone (Figure 183). The dental laminae for the upper and
lower molars were lodged into maxillary and mandibular bone respectively
(Figure 20).
In the treated embryos on this day the reduction in size of the
palatine shelves became more apparent. Anteriorly they did not reach
the inferior boundary of the tongue as the control did (compare Figure
21 with Figure 16a), and in addition their medial boundary was
irregular. The dental laminae for the upper molars were more medial
in position than they were in controls (compare Figure 22A with Figure
16b). The maxillary osteoblastic tissue was invaded by heterotopic
cartilage and was less extensive (Figure 223). The horizontal palatine
shelves in the treated embryos were widely separated (Figure 23A) and
had an irregular outline. Arrest of further development in those
dental laminae which did not encounter osteogenic tissue was observed.
Since the osteogenesis in the maxilla was more retarded than in the

Figure 20. Frontal section of head of day 16 control embryo. Palatine shelves (PS) horizontal; pre-
osteoblastic tissue (0?) present on superior aspect of PS; dental laminae (DL) for molars are lodging into
maxilla and mandible. PAS. 40x.
Figure 21. Frontal section of head of day 16 vitamin A treated embryo showing deformed palatine
shelves (PS). Toluidine blue. 40x.

Figure 22. Frontal sections of heads of day 16 vitamin A treated embryos. A - dental laminae (DL)
arise more medially than controls (compare with Figure 16B): B - heterotopic cartilage (HCT) replaces
maxillary osteoblastic tissue. Toluidine blue. 40x.

o
A
B
Figure 23. Frontal sections of heads of day 16 vitamin A treated embryos. A - palatine shelves (PS)
are horizontal and deformed] maxillary bone is replaced by cartilage (HOT) and further development of upper
dental lamina (DL) is arrested. PAS. 40x. B - insufficient tissue is outfolded from maxilla to form PS.
T oluidine blue. ¿fix.

mandible, the upper molars did not develop any further than the
laminar stage, while the lower molars were developing almost normally
(Fuguro 23A). While the palatal preosteoblastic tissue was located
normally and the epithelium covering the shelf was similar to that of
controls, the mesenchymal tissue in the palatine shelves was reduced
in amount. However, the staining intensity of the ground substance by
toluidine blue was not affected. Posteriorly the palatine shelves had
not outfolded sufficiently from the maxillary process and were reduced
in size (Figure 233. Compare with Figure 17A).
35
The incorporation of S -sulfate into 16 day control embryos
increased markedly from what it was on the previous day. The most
active component was the cartilage as could be readily visualized
from radioautographs of nasal septum (Fugure 24A) and limb cartilage.
Incorporation into palatine shelves and mesenchymal tissue of other
areas was also increased in embryos of this age. The medial boundary
of the shelf had a greater incorporation than the lateral portion of
the shelf.
Studies of S-35 incorporation into treated embryos demonstrated
that the nasal (Figure 243) and limb cartilages of treated embryos had
intense activity which was very much greater than that exhibited by
controls (compare with Figure 24A). Higher S-^ incorporation was
also observed in the mesenchymal tissue of the palatine shelves in
treated embryos (Figure 253) than in the controls (Figure 25A). The
maxillary osteoblastic area and the osteoid matrix in the mandible showed
identical incorporation in control and treated embryos. However, the
heterotopic cartilage (Figure 26) in maxilla had a very high radio¬
activity just as cartilage of the nasal septum and Meckel's cartilage.

• •
A
Figure 24. Radioautographs of frontal sections of
control; B - vitamin A treated. Toluidine blue. 2300x.
heads of day 16 embryos; nasal cartilage. A -

A
Figure 25.
palatine shelves.
Radioautographs of frontal sections of heads of day 16 embryos; mesenchymal tissue of
A - control; B - vitamin A treated. Toluidine blue. lSOQx.

69
Figure 26. Radioautograph of frontal section of head
of day 16 vitamin A treated embryo; heterotopic cartilage within
maxillary osteoblastic tissue. Toluidine blue. 180Qx.

70
17 Day Old Embryos»—The horizontal palatine shelves fused with
one another in the control embryos by day 17, and the nasal and oral
cavities became separated from each other and communicated only through
the nasopalatine foramina. In the anterior portion of the palate there
â– was slight accumulation of preosteoblasts in the midline region (Figure
27A) but the rest of the palate at this anterior level had loose mesen¬
chymal cells.
At a slightly more posterior level the palate became deeply
arched (Figure 273). The maxillary bone occupied a small triangular
area in the lateral half of the palate (Figure 27B). The medial angle
of the triangle was now continuous with the palatal preosteoblastic
area, the latter had split into right and left halves (Figure 273). The
stage of osteogenesis in the mandible vías identical with that observed
in the maxilla and the dental primordia for the upper and lower molars had
advanced considerably in their development and were deeply lodged in their
respective alveoli (Figure 27B).
Figure 28 compares the anterior palatal regions of two embryos
from the same vitamin A treated mother. In one embryo, to be referred
to hereafter as embryo I (Figure 23A), the palate was incomplete on one
side. In the other embryo, embryo C (Figure 283), the palate was complete
at this level. Posteriorly in embryo I, the palatine shelves had failed
to meet and were very thick and abnormally shaped (Figure 29A and 293).
Though abundant tissue was present, the maxillary bone was much reduced
in size and also was partially replaced by heterotopic cartilage
(Figure 29A). Through the intervention of this cartilage the reduced
maxilla was put in contact with the almost normal mandible (maxillo¬
mandibular ankylosis, Figure 29A). In the dental lamina of the upper

Figure 27. Frontal sections of head of day 17 control embryo. A - section through anterior region
of palate; maxillary bone (OST) is large and laterally gives rise to zygcnxatic process; slight accumulation
of preosteoblasts in center of palate (0?); B - section through a level posterior to that of A; maxilla
(OST) on each side associates with palatal osteoblastic tissue (OP). Toluidine blue. 40x.

Figure 28. Comparison of frontal sections of anterior region of two day 17 vitamin A treated embryos.
A - palate (?) is incomplete; maxillary cartilage present. Toluidine blue. 40x. B - palate (?) is complete;
lines of fusions between palatine shelves and nasal septum (NS) are present. Toluidine blue. 120x.

Figure 29. Frontal section of posterior region of head of day 17 vitamin A treated embryo. A - pala¬
tine shelves (PS) are thick and deformed; maxilla is replaced by cartilage (KCl); maxillomandibular ankylosis
present; B - dental lamina (DL) is not lodged in maxilla; its two limbs diverge. PAS. 40x. M - mandible.

74
molars (Figure 293), which did not encounter maxillary bone and hence
did not get lodged into it, the two limbs of the lamina opened ana
thus separated the palatal tissue from maxilla (Figure 293).
The maxillary bone in embryo C was similarly reduced in size
(Figure 30A) from the controls (Figure 27B) and though the secondary
palate was complete, it was very thin compared to the control.
Palatal preosteoblastic tissue in embryo G was present in normal size
and location (Figure 3QA).
Comparing the posterior level of a control embryo with embryo I
and embryo C, it was observed that maxillary bone in both embryos I and
C at this level was almost absent. While embryo I had a cleft palate,
the completed palate in embryo G was very thin and in its middle there
was an area which revealed rarefied mesenchymal tissue and breaks in
the epithelial membrane (Figure 30B). More posteriorly this embryo
did have an incomplete palate (Figure 3Li).
Similar to embryo C, many treated embryos had abnormal infoldings
of oral epithelium (Figure 30A) which contained a considerable amount
of palatal tissue. Heterotopic cartilages were often observed in the
neighborhood of such infoldings (Figure 31B).
Comparing the radioautographs of the 17 day old control and
treated embryos, the mesenchymal tissue in palatine shelves and neighboring
q r
area of the control embryo showed higher S"^ incorporation (Figure 32A)
than palatine shelves of the incomplete palate in the treated embryo
(Figure 323). But in the treated embryo palatal mesenchymal tissue
which had been entrapped by infoldings of oral epithelium showed a
greater S incorporation than other regions of the palate (Figure 33A
and 333); its density appeared to be comparable with the density in the

Figure 30. Serial frontal sections of head of day 17 vitamin A treated embryo. A - maxilla (OST)
is reduced in size; palatal preosteoblastic tissue (OP) present; B - palate (P) is narrow; its center shews
breaks in epithelium and rarefied mesenchymal tissue. Toluidine blue. 40x.

*"•3
On
Figure 31. Frontal sections of head of day 17 vitamin A treated embryo. A - section through
posterior level of palate; palate (P) is incomplete. Toluidine blue. 40x« B - infoldings of oral
epithelium have entrapped palatal mesenchymal tissue (lilF); heterotopic maxillary cartilage (ECT) is
present. PAS. 120x.

Figure 32. Radioautographs of frontal sections of heads of day 17 embryos; palatine shelves. A -
control; B ~ vitamin A treated. Toluidine blue. I800x.
<}

Figure 33. Radioautograph of frontal section of palate of day 17
pov;er view of infolding (INF) of palatal tissue. Toluidine blue. 12Qx.
Toluidine blue. 1800x.
vitamin A treated embryo. A - low
B - radioautograph of infolding.

79
normal palate. Radioactivity in the nasal (Figuro 34A and 343) and
limb (Figure 35A and 35B) cartilage of control embryos was also greater
than in the treated embryos.
Effects of Kyoervitarainosis A on Adult Rats
In order to investigate if the teratogenic dose of vitamin A
employed to produce cleft palate in embryos was also derogatory to the
pregnant animals, the latter were weighed during the three day period
of vitamin A treatment in pregnancy. It was found that while the
control mothers treated for three days with pure cottonseed oil gained
an average of 9 grams during this three day period, the vitamin A
treated rats lost an average of 9 grams during the same period. This
weight loss could possibly be due to an effect reported in rabbits by
Thomas et al. ('60). These authors noticed the dissolution of cartilage
matrix in articular and epiphyseal plates of rabbits treated with a
single dose of one million international units of vitamin A. To deter¬
mine if it was so, toluidine blue stained sections of epiphyseal carti¬
lage from the distal end of femur and proximal end of tibia were exam¬
ined from adult rats treated with varying doses of vitamin A (described
in Table 1). Figure 36 compares the proximal epiphyseal plate of
tibia from rats belonging to all five treatment groups (Table i).
Epiphyseal cartilage in the control animals gave a metachromatic
reaction with toluidine blue (Figure 36a). This reaction was increased
in animals given three doses of 60,000 i.u. vitamin A each day whether
pregnant or non-pregnant and whether examined four days after vitamin A
administration (Figure 36B) or 10 days after treatment (Figure 360).

Figure 34. Radioautographs of frontal sections of heads of day 17 embryos$ nasal cartilage,
control) B - vitamin A treated. Toluidine blue. 230Qx.

Figure 35. Radioautographs of sections of day 17 embryos; cartilage of limb bone. A - control; B -
vitamin A treated. Toluidine blue. 1800x.
05-

Figure 36. Toluidine blue stained sections of epiphyseal cartilages of control and vitamin A treated
rats. A - section from control rat in experiment A (see Table 1 for experiment designations); cartilage
matrix is metachrornatic; B - section from treated rat in experiment B; metachromasia in matrix is increased.
12Qx.

Figure 36 continued. C - section from vitamin A treated rat in experiment Cj increased metachromasiaj
D - section from vitamin A treated rat in experiment D; cartilage is depleted of stainable matrix. 12Qx.

84
Figure 36 continued. E - section from vitamin A
treated rat in group Ej cartilage is depleted of stainable
matrix. 120x.

85
Epiphyseal plates of rats treated with larger doses of vitamin A,
e.g. 100,000 and 200,000 i.tu per day for three days, however,
demonstrated a considerable loss of metachromasia from the cartilage
matrix (figure 36D and 3ÓE). This loss of metachromatic material was
not uniform, and isolated patches of cartilage still retained some
stainable matrix.

DISCUSSION
Normal Closure of Secondary Palate
Normal formation of the secondary palate in mammalian embryos
involves a fundamental morphogenetic movement by means of which the
palatine processes of mamillary mesoderm reorient themselves from a
vertical position at the side of tongue to a horizontal position
superior to tongue. This reorientation takes place rapidly and does
not seem to be accompanied by a correspondingly rapid growth within the
shelves (Walker and Fraser, '56). In an effort to investigate the mor¬
phological or chemical basis for this movement, Walker and Fraser ('56),
Walker ('60, '61) and Larsson ('62) made histological and histochemical
studies of the palatine shelves of young embryos, and concluded that the
only components making up the palatine shelves were a core of loose
mesenchymal tissue covered by an epithelium. The mesenchymal cells had
long cytoplasmic processes embedded in a ground substance which was
metachromatically stainable with toluidine blue. It was suggested that
the factor which was responsible for initiating palatine shelf movement
probably resided in the acid-mucopolysaccharides of the intercellular
ground substance.
In the present investigation it was shown that the mesenchymal
cells within the palatine shelves were not uniformly distributed in the
anteroposterior axis of the shelf. They were found to be sparse and
loosely arranged anteriorly, while posteriorly where the shelf movement
86

87
originated they were very densely aggregated. The intercellular ground
substance of the mesenchymal tissue stained metachromatically with
35
toluidine blue and had S uptake which was most marked in the medial
portion of the posterior palatine shelf area.
Another important histological component, hitherto undetected in
the vertically disposed palatine shelf was preosteoblastic tissue of
maxilla which sent a branch of similarly differentiated cells into the
medial portion of the palatine shelves. This primordium of the palatine
bones extended into the vertical palatine shelves. V/alker and Fraser
(’56) did not report this component but thoy did describe the occurrence
of osteogenesis in tissue proximal to the base of the shelves (maxillary
bone), and commented that no bony projection entered the palatine shelves
in their material.
The presence of this preosteoblastic tissue acquires significance
when one observes the region of palatine shelf where the movement to the
horizontal position is first indicated. That is, the preosteoblastic
tissue, along with the densely populated mesenchymal cells and their
strongly matachromatic ground substance, was one of the first components
to extend medially into an outfolding which indicated the beginning of
movement from the vertical to the horizontal position. Though one is
unable to draw any far reaching conclusions from this observation as it
is on the fixed and sectioned embryo instead of living material, it is
indicated that the presence of preosteoblastic tissue as a continuous
tissue from maxilla into the palatine shelf may have some normal
directive influence on the initiation of shelf movement.

ss
Cleft Palate and Hypervitaminosis A
In a comprehensive review Wilson (’59) proposed that an agent
capable of cansing malformations also causes an increase in intrauter¬
ine death and that both the intrauterine mortality and the malformations
may be different degrees of expression of the same general effect of the
teratogen. In the present investigation it was observed that maternal
hypervitaminosis A instituted on day 9 resulted in death of over half of
the embryos. The death rate decreased to 22% when the treatment was
started on day 10. The incidence of cleft palate, however, did not
change. In other words there appeared to be an independent variation
between the death rate and the malformation rate as S0> of the surviv¬
ing embryos developed cleft palate whether the death rate was 54/* or
22% (Table 2). These facts, however, do not rule out the possibility
that one and the same toxic effect of excess vitamin A may be operative
in the two manifestations. Rather one additional observation supports
Wilson’s hypothesis. That is, when animals viere administered excess
vitamin A on day 9, all the intrauterine mortality which could be
expected to be encountered at term (54/0 was already present on day 15
(Table 8). That is, the factor that caused embryonic death in hyper¬
vitaminosis A produced its effect before day 15. Also, on this day
all those embryos that would develop palatal clefts already showed the
early stages of abnormal development.
The labile period during which cleft palate could be produced in
these embryos was cuite narrow because treatment of the mother begun on
day 11 did not result in cleft palate but treatment begun on day 8
resulted in lOGjo embryonic death. It is important to realise that

though vitamin A treatment was instituted on day 9 of pregnancy, the
morphological factors that lead to cleft palate in the embryo do not
become apparent until day 15. Some subtle event or component must be
susceptible to the teratogen during days 9 and 10; since females pre¬
sented with excess vitamin A on day 11 do not give birth to offspring
â– with cleft palate.
There was observed a definite, though not extensive, general
retardation of growth in fetuses after maternal hypervitaminosis A.
The extent of retardation of fetal growth depended on the gestational
day when the three day period of vitamin A treatment of the mother was
commenced. The growth was most affected when treatment began on day
9, less so when treatment was started on day 10 and still less if
delayed until day 11.
Cleft palate in vitamin A treated embryos, however, was not con¬
sidered to be the result of such an over-all growth retardation. The
embryonic growth, reflected as embryonic weight, was first detected to
be retarded in 17 day old treated embryos; the latter were on an
average 200 mg lighter in weight than day 17 control embryos. The
teratogenic effect of maternal hypervitaminosis A was, however,
detected much earlier than the effect on total body growth; on day 15
in treated embryo it was noticed that the development of the head was
stunted in an anteroposterior direction; consequently both maxilla and
mandible were shorter in length than those of control embryos of the
same age and accordingly were clearly regions of localised growth
retardation.
from the standpoint of anatomical morphogenesis, cleft palate can
be considered to develop in several possible ways which have been
summarized by Burston (159), and by Fraser ('55, *60).

90
Since the correct positioning of the left and right palatine
shelves on the superior aspect of tongue is essential for the normal
closure of the secondary palate (reviews by Peter, '24 and Lazzaro
’40, referred to in Larsson, ’62), it was considered possible that the
failure on the part of the tongue to descend from between the vertically
disposed palatine shelves may lead to cleft palate. In support of this
concept Trasler et al. (’56) reported that cleft palate resulted in
mouse embryos whose amniotic sacs were punctured before the time of
palatal closure; the loss of amniotic fluid and consequent pressure of
the uterus resulted in the head of the fetus being forcibly pressed
against the chest, and hence the descent of the tongue was prevented.
Asling et al. (160) described the occurrence of cleft palate in the
young born to pteroylglutamic acid deficient rats, and ascribed these
clefts to retarded growth of mandible to an extent which precluded
sufficient room for descent of the tongue to permit the palatine
shelves to move to horizontal position. Similar observations were reported
by Pitch (16l) who studied the development of cleft palate in mouse
embryos homozygous for the short-head mutation, wherein the palatine
shelves appeared to remain vertical due to mechanical interference by the
mass of tongue.
Another closely related hypothesis for the origin of cleft
palate, which encompassed both morphology and some of the biochemical
phenomena, was proposed by Walker and Fraser ('57). These authors
studied the production of cleft palate in mouse embryos from cortisone
treated mothers and were able to present the concept that in contrast
to the previous theories the failure of palatine shelves to change
their position from the vertical to the horizontal plane at the normal

91
time, did not depend on the tongue but was dependent upon some factor
residing within the palatine shelves themselves. Walker and Fraser
(156) conceived of a force which steadily built up in the normal
palatine shelves and which at a certain time became strong enough to
cause them to move towards the horizontal plane. Cleft palate in the
cortisone treated embryos was held to arise due to the interference
with the build up of this 'shelf-force1 whereupon the palatine shelves
failed to initiate movement from the vertical position to a horizontal
plane at the normal time (Walker and Fraser, '57). This delay in
initiation of movement was compounded by the fact that head growth
continued while the shelves were still in vertical position and,
therefore, when they ultimately did become horizontal above the tongue,
growth of the head had taken them so far apart that they were unable
to meet and fuse. Such a delay in shelf movement was also reported in
the development of cleft palate in mouse embryos under the teratogenic
influence of such agents as maternal hypervitaminosis A (Walker and
Crain, 160; Kamei, '62), riboflavin deficiency (Walker and Crain '61)
and X-irradiation (Callas and Walker, '63). There are other possible
means by which cleft palate may develop, but these have not been
observed in experimental teratogenesis. One would be failure of the
palatine shelves, which though horizontal and above the tongue at
normal time, to contact each other due to either: a) too narrow
palatine shelves, or b) excessive width of the head. At least one
example of each faulty process exists in the scientific literature.
Fitch ('57) described the effects of a recessive gene in the mouse which
caused cleft palate resultant from overly narrow palatine shelves.

92
Stark and Ehrmann (*53) reported excessive width of the head to be
the explanation of cleft palate existent in association with oxycephaly.
The present study indicated that cleft palate in rat embryos
in response to maternal hypervitaminosis A was not due to the failure
of shelf movement to occur. When the orientation of palatine shelves
was observed during early developmental stages it became evident that
in a major percentage of the embryos from vitamin A treated mothers,
shelf movement was initiated on time and that cleft palate developed
in these embryos in spite of the fact that the barrier of the tongue
had been crossed and the palatine shelves were horizontal (Kochhar,
'64).
Larsson's (’62) findings related the production of cleft palate
in embryos from cortisone treated mice to inhibition of acid-mucopoly¬
saccharide synthesis within the palatine shelves which, therefore, failed
to develop enough force within themselves to enable them to move from
the vertical to the horizontal position. This conclusion was based on
35
radioautographic studies which revealed that incorporation of S
into palatine shelves and other areas of cleft palate embryos was
depressed. Larsson suggested that other agents like hypervitaminosis
A which also possibly reduced the amount of acid-mucopolysaccharides
in tissues might be teratogenic due to their possession of this
biochemical property. In the present investigation palatine shelves
and other tissues of embryos from hypervitaminotic A rats did not
35
show reduction in the incorporation of S .
Many morphological factors were encountered in embryos from
hypervitaminotic A rats which were suggestive of participation in the
development of cleft palate. In the first place it had been seen that

93
cleft palate resulted when insufficient mesenchymal tissue outfolded
from the maxillary process into palatine shelf, resulting in the latter
either being very small or absent. In most cases, however, the reduc¬
tion in size of palatine shelves occurred only posteriorly while the
size of palatine shelves in anterior regions was comparable to that
in normal embryos. In still other embryos cleft palate resulted in
spite of the apparent presence of sufficient palatal tissue, both
anteriorly and posteriorly, to bridge the gap across the roof of the
oral cavity.
In all treated embryos the development of the maxillary bone
primordium was defective while it was at the preosteoblastic stage of
organization. Some of the preosteoblastic cells underwent heterotopic
transformation into chondroblasts and chondrocytes which were surrounded
by a typically metachromatic matrix. In subsequent development this
heterotopic cartilage progressively replaced the maxillary bone until
in 17 day old embryos the maxilla was represented by scattered bone
trabeculae at the periphery of the heterotopic cartilage. This
maxillary cartilage was instrumental in producing massive maxillomandi¬
bular ankylosis and through its Intervention the reduced maxilla came
to communicate with mandible. Abnormal infoldings of oral epithelium
adjacent to the maxillary cartilage entrapped some of the palatal
mesenchymal tissue. Finally, ectopic dental laminae were also associated
with this heterotopic maxillary cartilage.
The reduction in size of the maxillary bone due to the presence
of maxillary cartilage appeared to participate in the production of
cleft palate in at least two ways. Firstly the development of dental
laminae for upper molars was abhortive and did not encounter the

94
maxillary bone and the two limbs of the lamina diverged and thus
separated the palatal tissue from the maxilla. Secondly, due to the
absence of the maxillary bone in its usual position with respect to
the palate, the likelihood exists that normal pressure and stress of
the developing bone to aid the progression of the horizontal palatine
shelves towards the midline could be partially or completely absent in
the treated embryo. Both these factors explained the persistent presence
of widely separated but horizontal palatine shelves at the comers
between the nasal septum and the tongue.
Another abnormality seen in the treated embryos was the more
medial origin of the maxillary dental laminae. This contributed to
reduce the amount of mesenchymal tissue available for formation of
secondary palate by limiting the lateral extent of the palatine shelves.
Embryonic S-^ Incorporation Following Maternal Hypervitaminosis A
Ample evidence exists to demonstrate that after the parenteral
administration of radioactive sulfate into pregnant rat, sulfate becomes
transferred across the placenta to be utilized for tissue formation by
the embryo (Layton et al., '50; Dziewiatkowski, '53; Friberg and Eingertz,
>56). Dziewiatkowski ("53) also demonstrated that maternal injections
35
of 5 sulfate resulted in incorporation into embryonic cartilage 30
times greater than in the maternal cartilage and 15 times greater in
embryonic muscle than in the mother's muscle. Consistent with previous
findings (Friberg and Eingertz, '56), the highest uptake of occurred
in mesenchymal areas or its derivatives. Cartilage was the most active
in this regard in 15, 16, and 17 day old control embryos. The

95
35
distribution of S -labelled material coincided with the occurrence of
35
toluidine blue stainable metachromatic component. Fixation of 5 by
tissues of embryos from hypervitaminotic A rats, however, needs special
mention. In 15 and 16 day old treated embryos, whose mothers were
35
treated with vitamin A on 9, 10, and 11 days of gestation and S -
sulfate injected 48 hours before autopsy, the mesenchymal tissue in
the abnormally shaped palatine shelves and in other areas revealed a
35
very much higher S incorporation than soen in comparable areas of
control embryo. The same occurrence was true of nasal capsule carti¬
lages, Meckel's cartilage and cartilage models in the limb. Though
maxillary and mandibular osteoblastic tissue of control and treated
embryos had comparable S J incorporation, the presence in the treated
embryos of heterotopic cartilages, which actively fixed S-^, made
maxilla of treated embryos much more radioactive than the controls.
35
Experiments investigating the nature of S -labelled component
in tissue sections seem to indicate that after histological processing
35
the only left in the tissue is that present in a bound form
(Dziewiatkowski, '58); it occurs in a close association with substances
which are metachromatic and stain with Alcian blue and colloidal iron
methods (Curran and Kennedy, '55). In this light it would be safe to
assume that S-^5 detected in tissues in the current study is not
present free as inorganic sulfate but has been incorporated into a
newly synthesized material in the intercellular ground substance or
matrix.
The present report is the only study which examines the in vivo
35
effect of maternal vitamin A treatment on fixation of S by the
embryo. In tissue culture very different results were obtained by

96
Foil, Kellanby and Pele (’56) who reported that embryonic cartilage
in an excess vitamin A medium suffers a dissolution of its matrix as
35
evidenced by removal of previously incorporated S . These results
were substantiated by other studies on the effects of excess vitamin A
on adult cartilage and bone, both in vivo and in vitro (Thomas et al.,
*60 and Fell and Thomas, '60). Besides dissolving the cartilage matrix,
hypervitaminosis A in intact animals was also reported to impair the
ability of chondrocytes to synthesize cartilage matrix in tissue culture
(McElligott, '62). Such effects were not evident in 15 and 16 day old
embryos after maternal hypervitaminosis A. On the other hand more
35
3 activity was detected in these embryos than in embryos from control
mothers.
The concentration of vitamin A in fetal tissues following maternal
hypervitaminosis A was not investigated here. Giroud, Gounelle and
Martinet ('56 and *57, referred to in Kalter and Warkany, '59) reported
a slight increase in vitamin A concentration in rat fetuses after
treating their mothers with large doses of vitamin A. In case such
an increase in vitamin A concentration is also present in embryonic
tissues in the present study, it has resulted in increased incorporation
of into the embryo. Increased rate of S^5 uptake following vitamin
A administration was also observed by Dziewiatkowski ('54) in the
skeleton and skin of rats which were previously on vitamin A deficient
35
diet. He also demonstrated that this incorporation of S had occurred
into sulfo-mucopolysaccharides as their sulfate groups. Wolf, Varandani
and Johnson (16l) have shown that in the presence of vitamin A the
incorporation of -radioactivity into mucopolysaccharides by colon
homogenate of vitamin A deficient rats is increased.

97
Dissolution of cartilage matrix observed by Thomas et al.
(160) seems to result only when the animals are dosed with very large
amounts of vitamin A. Thomas et al. (*60) and McSlligott ('62) admin¬
istered to the rabbits 1 million i.u. of vitamin A daily for 5 to 7
days to bring about dissolution of cartilage matrix. Dosage of
vitamin A employed for teratogenesis in the present study was not
sufficiently high to dissolve the matrix of either maternal cartilage
or the cartilage of 15 and 16 day embryos. On the other hand it was
noticed that in the epiphyseal cartilage of adult rats receiving
teratogenic doses of vitamin A (60,000 i.u. per day for 3 days) the
matrix showed more intense metachromasia with toluidine blue than in
rats receiving only cottonseed oil. Depletion in cartilage matrix of
its motachromatically stainable component was, however, observed when
the dosage of vitamin A was increased to 100,000 i.u. or 200,000 i.u.
daily for three days.
Dissolution of cartilage matrix may be responsible for the
35
detection of decreased incorporation observed in the cartilage of
17 day old treated embryos. Palatal mesenchymal tissue of such
35
embryos also incorporated less S than comparable areas of the control
embryo. This could possibly be due to decreased growth encountered in
the unfused palatine shelves (Larsson, '62).
In conclusion it can be said that cleft palate in rat embryos
after maternal hypervitaminosis A was not associated with any specific
reduction in the amount of intercellular ground substance during the
period of palatal closure as far as it could be determined on the
basis of toluidine blue metachromasia and incorporation.

9S
Interaction Between Hypervitaminosis A and Cortisone
The present study supports the conclusion of Cohlan and Stone
('61) that cortisone injections do not potentiate the teratogenic
manifestations of hypervitaminosis A in the rat embryos. There may be
a slight protection afforded by cortisone against the production of eye
defects by hypervitaminosis A. This protection may be only apparent
rather than real, since different degrees of microphthalmia may
escape detection with the method of examination employed.
It is of interest to note that though vitamin A and cortisone
administered singly to mothers reduce the mean weight of the fetuses,
but if administered together the fetal weight was higher and approached
the normal level. This may be explained on the basis of previous
observations that hydrocortisone inhibited the action of vitamin A on
the cartilage of embryonic bone explants (Fell and Thomas, '6l) and
on the cartilage of intact animals (Thomas et al., '63). Since there
was a possibility that the dissolution of cartilage matrix as an effect
of excess vitamin A was mediated through the release of a protease
from lysosomes of chondrocytes (Lucy, Dingle and Fell, '61 and Dingle,
’61), and since it was separately shown that less lysosomal enzymes
were released under the influence of hydrocortisone, both in vivo
(Weissmann and Dingle, ’61) and in vitro (De Duve, Wattiaux and Wibo,
’6l), it might be indicated that hydrocortisone stabilizes the lysosomal
membrane against the disruptive effects of excess vitamin A. The
validity of this explanation or the extent to which it applies to the
present situation, however, cannot be verified here.

SUMMARY
Pregnant rats were given high doses of vitamin A, cortisone or
papain during the second week of gestation in an effort to produce
cleft palate in their embryos. Only vitamin A, administered in doses
of 60,000 i.u. each day by oral intubation on days 9 to 11 or 10 to
12 of pregnancy, resulted in cleft palate in more than 80$ of embryos
from such mothers. Cortisone and papain produced no teratogenic
effects. Cortisone, however, when injected into the pregnant animals
already receiving teratogenic doses of vitamin A afforded some pro¬
tection against the weight loss occurring in embryos from mothers
treated with vitamin A alonej such treatment with cortisone did not
modify the incidence of cleft palate induced by hypervitaminosis A.
Early morphogenesis of secondary palate was studied in rat
embryos from normal and hypervitaminotic A mothers by means of histo-
chemical and radioautographic techniques. In the control embryo before
the movement of palatine shelves from a vertical to a horizontal posi¬
tion took place it was noticed that the shelves contained as one of
their histological components an extension of preosteoblastic tissue
from the maxilla. This tissue was one of the first components of the
palatine shelf to extend medially into an outfolding which started the
process of shelf movement.
Morphological changes which resulted in cleft palate in embryos
after maternal hypervitaminosis A differed from those described by
99

100
Walker and Fraser (’57) in embryos from cortisone treated mice. No
delay in the movement of palatine shelves from vertical to horizontal
position occurred in vitamin A treated embryos. Larsson (’62) had
suggested that the teratogenic action of cortisone in the production
of cleft palate in mouse embryos could be correlated with the presence
of reduced amounts of acid-mucopolysaccharides in the affected palate.
Assuming that the amount of intercellular ground substance present in
connective tissue was indicative of its acid-mucopolysaccharide content,
no reduction in the former (determined by S-^ radioautography coupled
with toluidine blue staining) was revealed in the defective palate or
other areas of vitamin A treated embryos. Dosage of vitamin A employed
was not sufficiently high to dissolve the matrix of either maternal
cartilage or the cartilage of 15 and 16 day embryos. In fact, under
35
such conditions, not only was S incorporation into the embryonic
tissues increased during the period of palatal closure, but an augmenta¬
tion in the intensity of metachromatic staining in the matrix of mater¬
nal epiphyseal cartilage was also observed.
Many morphological factors were encountered which possibly were
responsible for cleft palate in such embryos. These were: (1) Less
than normal amount of mesenchymal tissue outfolded from maxillary process
resulting in overly narrow palatine shelves. (2) Size of the maxillary
bone was reduced due primarily to replacement with heterotopic carti¬
lage (perhaps the horizontally positioned palatine shelves failed to
advance towards the midline because of lack of directive influence of
the maxillary bone). (3) The two limbs of dental laminae for upper
molars, which were not lodged in maxillary bone because of defects in
the latter, diverged and thus separated the palatal tissue from the

101
maxilla. (4) Frequently the dental laminae for the upper molars arose
from oral epithelium more medially than in controls and thus limited
laterally the amount of mesenchymal tissue which could participate in
the formation of secondary palate and alveolar process. (5) Abnormal
infoldings of oral epithelium appeared to entrap some of the palatal
mesenchymal tissue which should have been included in the formation of
secondary palate.

LITERATURE CUED
Asling, C.W., M.M. Nelson, II.L. Dougherty, K.V. Wright and H.M. Evans
I960 The development of cleft palate resulting from maternal
pteroylglutamic acid deficiency during the latter half of gesta¬
tion in rats. Surg. Gynec. Obstet., 111:19.
Baxter, H. and F.C. Fraser 1950 Production of congenital defects in
offspring of female mice treated vdth cortisone. McGill M.J.,
12:245.
Blandau, R.J., J.L. Boling and W.C. Young 1941 The length of heat in
the albino rat as determined by the copulatory response. Anat.
Rec., 72:453.
Bostrom, K. 1952 On the metabolism of the sulfate group of chondroit-
insulfuric acid. J. Biol. Chera., 196:477.
1953 Chemical and autoradiographic studies on the sulfate
exchange in sulpho-mucopolysaccharides. Ark. Kemi, 6:43.
Boyd, G.A. 1955 Autoradiography in biology and medicine. New York,
Academic Press, Inc.
Brachet, J. 1942 La localisation des acides pentosenucleiques dans les
tissus animaux et les oeufs d'Amphibiens en voie de development.
Arch. Biol. Paris, 53:207.
Burston, W.R. 1959 The development of cleft lip and palate. Ann. Roy.
Coll. Surg. Engl., 25:225.
Callas, G. and B.E. Walker 1963 Palate morphogenesis in mouse embryos
after X-irradiation. Anat. Rec., 145:6l.
Cohlan, S.Q. 1953 Excessive intake of vitamin A as a cause of congenital
anomalies in the rat. Science, 117:535.
Cohlan, S.Q. and S.M. Stone 1961 Observations on the effect of experi¬
mental endocrine procedures on the teratogenic action of hyper-
vitaminosis A in the rat. Biol. Neonat., ¿:330.
Corner, G.W. I960 Congenital Malformations: The Problem and tne Task.
In "First International Conference on Congenital Malformations"
p. 7, J.B. Lippincott, Philadelphia.
102

103
Curran, R.C. and J.S. Kennedy 1955 The distribution of the sulphated
mucopolysaccharides in the mouse. J. Pathol. Bacteriol., 70:
449.
EfeDuve, C., R. Wattiaux and K. Wibo 1961 Effects of fat-soluble com¬
pounds on lysosomes in vitro. (Abstract) Biochem. Pharmacol.,
8:30.
Deuschle, F.M., J.?. Geiger and J. Warkany 1959 Analysis of an anomalous
oculodentofacial pattern in newborn rats produced by maternal hyper-
vitaminosis A. J. Dent. Res., 38:149.
Dingle, J.T. 1961 Studies on the mode of action of excess of vitamin
A. 3. Release of a bound protease by the action of vitamin A.
Biochem. J., 79:509.
Dziewiatkowski, D.D. 1951 Isolation of chondroitin sulfate-S-^ from
articular cartilage of rats. J. Biol. Chem., 189:187.
1953 Sulfate-sulfur metabolism in the rat fetus as indicated
by sulfur-35. J. Exp. Med., 98:119.
1954 Vitamin A and endochondral ossification in the rat as
indicated by the use of sulfur35 and phosohorus32. J. Exp. Med.,
100:11.
1958 Autoradiographic studies with s35-Sulfate. Int. Rev.
Cytol., 7:159.
Evans, H.J. and G. Clingen 1953 Effects of cortisone acetate on rat
feti. Anat. Rec., 117:624.
Eainstat, T. 1954 Cortisone-induced congenital cleft palate in rabbits.
Endocrinology, 55:502.
Fell, H.3. and E. Mellanby 1952 The effect of hypervitaminosis A on
embryonic limb bones cultivated in vitro. J. Physiol., 116:320.
1953 Metaplasia produced in cultures of chick ectoderm by
high vitamin A. J. Physiol., 119:470.
Fell, H.B., E. Mellanby and S.R. Pele 1956 Influence of excess
vitamin A on the sulfate metabolism of bone rudiments grown in
vitro. J. Physiol., 134:179.
Fell, H.3. and L. Thomas I960 Comparison of the effects of papain
and vitamin A on cartilage. II. The effects on organ cultures
of embryonic skeleton tissue. J. Exp. Med., 111:719.
1961 The influence of hydrocortisone on the action of excess
vitamin A on limb bone rudiments in culture. J. Exp. Med.,
114:343.

104
Feulgen, R. and H. Rossenbeck 1924 Mikroskopisch-chemischer Nachweis
einer Nukleinsaure vom Typus der Thymonukleinsaure und die
da.ra.uf beruhende elektive Farbung von Zellkernen in mikroskopischen
Praparaten. Z. Phys. Chen., 135:203.
Fitch, N. 1957 An cmbryological analysis of two mutants in the house
mouse, both producing cleft palate. J. Exp. Zool., 136:329.
1961 Development of cleft palate in mice homozygous for the
shorthead mutation. J. Morph., 109:151.
Frape, D.L., R.S. Allen, V.C. Speer, Y.W. Hays and D.V. Carton 1959
Relationship of vitamin A to S-^7 metabolism in the baby pig. J.
Nutrition, 68:189.
Fraser, F.C. 1955 Thoughts on the etiology of clefts of the palate and
lip. Acta Genet., ¿058.
I960 Some experimental and clinical studies on the causes of
congenital clefts of the caíate and the lio. Arch. Pediat.,
77:151.
Fraser, F.C. and T.D. Fainstat 1951 Production of congenital defects
in the offspring of pregnant mice treated \jith cortisone.
Pediatrics, 8:527.
Fraser, F.C., H. Kalter, 3.S. Walker and T.D. Fainstat 1954 The experi¬
mental production of cleft palate with cortisone and other
hormones. J. Cell. Comp. Physiol., 43:237.
Friberg, U. and N.R. Ringertz 1956 An autoradiographic stud;/ on the
uptake of radiosulfate in the rat embryo. J. Embryol. exp.
Morph., 4:313.
Gebauer, E. 1954 Zur A-Eypervitaminose und Schwangerschaft. Die
Pharmazie, 2*684.
Giroud, A., E. Gounelle and M. Martinet 1956 Concentration de la
vitamine A chez la mere et le foetus au cours de la teratogenese
par hypervitaminose A. Compt. rend. soc. biol., 150:2064.
1957 Donnees quantitatives sur le taux de la vitamine A chez
le rat lors d'experiences de teratogenese par hypervitaminose A.
Bull. soc. chim. biol., 39:331.
Giroud, A. and M. Martinet 1954 Fentes du palais chez I'erabryon de
rat par hyperviatminose A. Compt. rend. soc. biol., 148:1742.
1955 Malformations diverses foetus de Rat suivant les stades
d1administration de vitamine A en exces. Compt. rend. soc.
biol., 149:1088.

105
1956 Teratogenese par hautes doses de vitamine A en foncticn
des stades du development. Arch. anat. micr., 45:77.
Glueksohn-Waelsh, S. 1954 Some genetic aspects of development. Cold
Spring Harbor Symp. Quant. Biol., 19:41.
Gregg, N. 1941 Congenital cataract following German measles in the
mother. Trans. Ophth. Society of Australia, ¿:35.
1945 Rubella during pregnancy of the mother, with its
seauelae of congenital defects in the child. Med. J. Australia,
1:313.
Hale, F. 1933 Pigs born without eyeballs. J. Heredity, 24:105.
1935 The relation of vitamin A to anophthalmos in pigs. Am.
J. Ophthalmology, 18:1087.
1937 The relation of maternal vitamin A deficiency to micro¬
phthalmia in pigs. Texas State J. Med., 33:228.
Hulth, A. and 0. Westerborn 1959 The effect of crude papain on the
epiphyseal cartilage of laboratory animals. J. Bone & Joint Surg.,
â– 413:836.
Jost, A. 1956 The age factor in some prenatal endocrine events. Ciba
Foundation Colloq. on Ageing, 2:18.
Kalter, H. 1954 Inheritance of susceptibility to the teratogenic action
of cortisone in mice. Genetics, 39:185.
I960 The teratogenic effects of hypervitarainosis A upon the
face and mouth of inbred mice. Ann. N.Y. Acad. Sci., 85:42.
halter, H. and J. Warkany 1959 Experimental production of congenital
malformations in mammals by metabolic procedure. Physiol. Rev.,
¿2:69.
1961 Experimental production of congenital malformations in
strains of inbred mice by maternal treatment with hypervitarainosis
A. Amer. J. Path., 38:1.
K&mei, T. 1962 Embryological and histochemical studies on the arti¬
ficially induced cleft oalate in mice. Acta Anat. Kiooonica,
¿2*140.
Kochhar, D.M. 1964 The influence of hypervitarainosis A on the formation
of secondary palate in rat embryos. Anat. Rec., 148:302.
Kramer, H. and G.K. Windrum 1955 The metachromatic staining reaction.
J. Histochem. Cytochem., ¿:227.

106
Larsson, K.S. I960 Studies on the closure of the secondary palate.
II. Occurrence of sulpho-mucopolysaccharides in the palatine
processes of the normal mouse embryo. Exp. Cell Res., 21:498.
1962 Studies on the closure of the secondary palate. III.
Autoradiographic and histochemical studies in the normal mouse
embryo. Acta Morphol. Neerl. Scand., /¿;349.
1962 Studies on the closure of the secondary palate. IV.
Autoradiographic and histochemical studies of mouse embryos
from cortisone-treated mothers. Acta Morphol. Neerl. Scand.,
¿:369.
1962 Closure of the secondary palate and its relation to
sulpho-mucopolysaccharides. Acta Odontológica Scand., 20:1.
Larsson, K.S., H. Bostrom and S. Carlsoo 1959 Studies on the closure
of the secondary palate. I. Autoradiographic study in the normal
mouse embryo. Exp. Cell Res., 16:379.
layton, L.L.
in the
1951 Cortisone inhibition of mucopolysaccharide
intact rat. Arch. Biochem., 32:224.
synthesis
1951 Effect of cortisone upon chondroitin sulfate synthesis by
animal tissues. ?roc. Soc. Exp. Biol., 76:596.
Layton,
L.L., D.R. Frankel and S. Scapa 1950 Maternal sulfate utilized
by mammalian embryos and suckling young. Arch. Biochem.,
28:142.
Lazzaro, C. 1940 Sul meccanisno di chiusura del palato secondario.
Monit. Zool. Ital., 51:249.
Lucy, J.A., J.T. Dingle and H.B. Fell
action of excess of vitamin A.
proteases in the degradation of
72:500.
McCluskey, R.T. and L. Thomas 1958 The removal of cartilage matrix
in vivo by papain. Identification of crystalline papain protease
as the cause of the phenomenon. J. Exp. Med., 1C8:371.
1959 Removal of cartilage matrix in vivo by papain. Pre¬
vention of recovery with cortisone, hydrocortisone and prednisolone
by a direct action on cartilage. Amer. J. Path., 35:S19.
McElligott, T.F. 1962 Decreased fixation of sulfate by chondrocytes in
hypervitaminosis A. J. Path. Bacteriol., 83:347.
McManus, J.F.A. 1948 Histological and histochemical uses of periodic
acid. Stain Tech., 23:99.
1961 Studies on the mode of
2. A possible role of intracellular
cartilage matrix. Biochem. J.,

107
Messier, B. and CJP. Leblond 1957 Preparation of coated radioautographs
by dipoing sections in fluid emulsion. Proc. Soc. Exo. Biol. Med.,
£6:7.
Mtfrch, E.T. 1941 Chondrodystrophic dwarfs in Denmark. Copenhagen, E.
Munksgaard.
Nelson, M.M., C.W. Asling and H.M. Evans 1952 Production of multiple
congenital abnormalities in young by maternal pteroylglutamic
acid deficiency during gestation. J. Nutrition, 48:61.
Pearse, A.G.E. I960 Histochemistry, theoretical and applied. Boston,
Little, Brown and Co.
Pele, S.R. and H.B. Fell I960 The effects of excess vitamin A on the
uptake of labelled compounds by embryonic skin in organ culture.
Exp. Cell Res., 19:99.
Peter, K. 1924 Die Entwicklung des Saugetiergaumens. Ergebn. Anat.
Entwickl.-Gesch., 25:448.
Pitt, D.B. 1962 Congenital malformations: a review. Med. J. Australia,
Jan. 20:82.
Plotz, C.M., E.L. Howes, K. Meyer, J.W. Blunt and C. Ragan 1950 Action
of cortisone on mesenchymal tissues. Arch. Derm. Syph., 61:919.
Ragan, C., E.L. Howes, C.M. Plotz, K. Meyer and J.W. Blunt 1950 The
effect of ACTK and cortisone on connective tissue. Bull. N.Y.
Acad. Med., 26:251.
Schiller, S. and A. Dorfman 1957 The metabolism of mucopolysaccharides
in animals: The effect of cortisone and hydrocortisone on rat
skin. Endocrinology, 60:376.
Silagi, S. > 1962 A genetical and embryological study of partial comple¬
mentation between lethal alleles at the T locus of the house
mouse. Devel. Biol., j>:35.
Stark, R.B. and N.A. Ehrmann 1958 The development of the center of
the face with particular reference to surgical correction of bilateral
cleft lip. Plast. Reconstr. Surg., 21:177.
Stiles, K.A. and L.S. Pickard 1943 Hereditary malformations of the
hands and feet. J. Hered., 34:341.
Thomas, L. 1956 Reversible collapse of rabbit ears after intravenous
papain, and prevention of recovery by cortisone. J. Exp. Med.,
104:245.
Thomas, L., R.T. McCluskey, J. Li and G. Weissmann 1963 Prevention
by cortisone of the changes in cartilage induced by an excess of
vitamin A in rabbits. Amer. J. Path., 42:271.

108
Thomas, L., R.T. McCluskey, J.L. Potter and G. Weissmarm I960 Com¬
parison of the effects of papain and vitamin A on cartilage. I.
The effects in rabbits. J. Exp. Med., 111:705.
Trasler, D.G., B.E. Walker and F.C. Fraser 1956 Congenital malforma¬
tions produced by amniotic-sac puncture. Science, 124:439.
Walker, 3.E. I960 A special component of embryonic mesenchyme.
Anat. Rec., 136:298.
1961 The association of mucopolysaccharides with morphogenesis
of the palate and other structures in mouse embryos. J. Embryo!,
exp. Morph., ^.i22.
Walker, B.E. and 3. Crain I960 Effects of hypervitaminosis A on palate
development in two strains of mice. Am. J. Anat., 107:49.
1961 Abnormal palate morphogenesis in mouse embryos induced
by riboflavin deficiency. Proc. Soc. Exp. Biol. Med., 107:404.
Walker, 3.E. and F.C. Fraser 1956 Closure of the secondary palate in
three strains of mice. J. Embryol. exp. Morph., ¿:176.
1957 The embryology of cortisone-induced cleft palate. J.
Embryol. exp. Morph., ¿:201.
Warkany, J. and R.C. Nelson 1940 Appearance of skeletal abnormalities
in the offsoring of rats reared on a deficient diet. Science,
92:383.
1941 Skeletal abnormalities in the offspring of rats reared on
deficient diets. Anat. Rec., 79:83.
Weissmann, G. and J.T. Dingle 1961 Release of lysosomal protease by
ultraviolet irradiation and inhibition by hydrocortisone.
Exp. Cell Res., 2£:207.
Wilson, J.G. 1959 Experimental studies on congenital malformations.
J. Chronic Diseases, 10:111.
Wolbach, S.B. 1947 Vitamin A deficiency and excess in relation to
skeletal growth. J. Bone and Joint Surg., 29:171.
Wolf, G. and P.T. Varandani I960 Studies on the function of vitamin A
in mucopolysaccharide biosynthesis. Biochim. Biophys. Acta,
¿2*501.
Wolf, G., P.T. Varandani and B.C. Johnson 1961 Vitamin A and mucopoly¬
saccharide synthesizing enzymes. Biochim. Biophys. Acta, 46:59.
Woollam, D.K.M. and J.W. Millen 1957 Effect of cortisone on the inci¬
dence of cleft palate induced by experimental hypervitaminosis A.
Brit. M.J., 2:1*97.

109
2willing, E. 1956 Genei:ic mechanism in limb development.
Harbor Symp. Quant. Biol., 21:349.
Cold Spring

APPENDICES

APPENDIX I
Analysis of Rockland Stock Diet for Rats
Protein
Fat
Fiber
Carbohydrate
Ash
Minerals
All vitamins present including
610-680 U.S.P. Units of
vitamin A per 100 grams
of diet.
per cent
24.27
4.15
4.86
56.23
7.78
5.50
111

APPENDIX II
Composition of Bouin,s Fluid
Saturated aqueous picric acid .... 75 parts
Formalin (40# formaldehyde) 25 parts
Glacial acetic acid 5 parts
Urea crystals 1 part
112

VITA
Devendrá Mohan Kochhar was born on March 10, 1938 in Sialkot,
Panjab, India. He received his primary and secondary education in
various states of Northern India. He attended Panjab University
College in Hoshiarpur, Panjab from September 1955 to June 1958 where he
majored in Zoology and graduated with honors with a Bachelor of Science
degree. He joined Panjab University, Chandigarh, Panjab in 1958 and
served as a teaching assistant in the Department of Zoology, and gradu¬
ated in September 1959 with a Master of Science degree with a major in
Zoology. His studies continued in the same department under a Research
Fellowship from the Council of Scientific and Industrial Research,
Government of India, New Delhi until July I960. From September I960
until the present time he has studied towards the degree of Doctor of
Philosophy in the Department of Anatomy at the University of Florida
College of Medicine, first under a University Graduate Assistantship and
then under a Predoctoral Training Fellowship from the National Institute
of Health.
Devendrá Mohan Kochhar was married in September 1962 to the
former Omila Sagar and has one child. He is a member of Sigma Xi, The
American Association for the Advancement of Science, The Southern
Society of Anatomists and The Panjab University Zoological Society.
He has accepted the position of Research Fellow at the rank of
Instructor in the Department of Anatomy, University of Florida College
of Medicine, Gainesville, Florida.

This dissertation was prepared under the direction of the chair¬
man of the candidate's supervisory committee and has been approved by
all members of that committee. It was submitted to the Dean of the
College of Medicine and to the Graduate Council, and was approved as
partial fulfillment of the requirements for the degree of Doctor of
Philosophy.
August, 1964
Dean, College of Medicine
Dean, Graduate School
Supervisory Committee: