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Oocyte meiotic state, developmental plasticity, and independence of cytokinesis from karyokinesis during early development in the Cnidarian Nematostella vectensis
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OOCYTE MEIOTIC STATE, DEVELOPMENTAL PLASTICITY, AND
INDEPENDENCE OF CYTOKINESIS FROM KARYOKINESIS DURING EARLY
DEVELOPMENT IN THE CNIDARIAN NEMATOSTELLA VECTENSIS












By

JEFFREY A. WILCOX


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

UNIVERSITY OF FLORIDA


2001














ACKNOWLEDGEMENTS


This dissertation is dedicated to my wife, Sandra Ricardo-Wilcox. Her support,

her counsel, and her patience was unwavering, and made this all worthwhile.

I extend particular thanks to Dr. Wallis Clark, Jr. for introducing me to

Nematostella, and for his skillful mentoring. A finer model of professorial leadership is

unimaginable. He knew when to encourage me, when to discourage me, and when to

leave me to my own devices. He has been and, hopefully, will continue to be, the

greatest influence on my future as a scientist. I extend thanks to his wife, Beth, for her

many kindnesses, and for allowing Wally to spend as much time with me as was

necessary.

I greatly appreciate Dr. Frank Chapman for his support, for his guidance, and for

the endless hours of intellectual jousting he has invested in making me a better student

and scientist. We may not always have agreed, but we certainly did discuss the issues.

I give thanks to the faculty, staff, and students of the Department of Fisheries and

Aquatic Sciences for their support and encouragement.

I am deeply grateful for the friendship and assistance of Chulhong Park. I learned

a great deal about kindness, respect, proper student attitude, Korea, and Korean food,

during the years we lived and worked together in the Reproduction and Development

Laboratory. I am proud to be considered a member of his family, and I will strive to

deserve the honor this bears.














TABLE OF CONTENTS


ACKNOWLEDGEMENTS ii

ABSTRACT iv

CHAPTERS

I INTRODUCTION 1
Life History 3
Laboratory Culture 5
Statement of the Problem 6

2 MEIOTIC STATE OF THE OOCYTE AT FERTILIZATION 19
Methods 20
Results 22
Discussion 32

3 PLASTICITY OF EARLY DEVELOPMENT 37
Methods 41
Results 42
Discussion 53

4 THE INDEPENDENCE OF CYTOKINESIS AND 59
KARYOKINESIS DURING EARLY DEVELOPMENT
Methods 61
Results 65
Discussion 73

5 CONCLUSION 80
Nematostella vectensis Oocytes are Meiotically Complete 80
Nematostella vectensis Exhibited Three Patterns of Cleavage 82
Cytokinesis is Independent from Karyokinesis 85

REFERENCES 89

BIOGRAPHICAL SKETCH 94














Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

OOCYTE MEIOTIC STATE, DEVELOPMENTAL PLASTICITY, AND
INDEPENDENCE OF CYTOKINESIS FROM KARYOKINESIS DURING EARLY
DEVELOPMENT IN THE CNIDARIAN NEMATOSTELLA VECTENSIS

By

Jeffrey A. Wilcox

August 2001



Chair: Dr. Wallis H. Clark, Jr.
Major Department: Fisheries and Aquatic Sciences


The aims of this study were to determine the meiotic state of the egg at

fertilization, to characterize the multiple pathways used in early development, and to

experimentally manipulate cytokinesis and karyokinesis to determine whether they were

one sequential regulatory pathway or two intimately associated independent pathways.

Developing oocytes displayed a germinal vesicle, whereas spawned eggs displayed a

pronucleus prior to fertilization. Fertilized eggs did not eject polar bodies prior to

initiation of cleavage. Nematostella vectensis Stephenson 1935 can be validly added to

the short list of known Cnidarians which are meiotically complete at fertilization.

Early development in Nematostella showed indeterminant cleavage and two

syncitial cleavage pathways. Nematostella blastomeres which exhibited indeterminant

cleavage remained totipotent through the third cleavage. Nematostella exhibited syncitial








cleavage remained totipotent through the third cleavage. Nematostella exhibited syncitial

cleavage patterns in some embryos in most egg masses, either delayed-cytokinesis or re-

fusion into a syncitium between the 4-cell and 32-cell stage. The re-fusion form of

syncitial cleavage was previously reported in three other Anthozoans. This research also

confirmed previous reports of delayed-cytokinesis cleavage in Anthozoans. All three

patterns of early development resulted in successful embryogenesis.

This species provided a unique opportunity to evaluate the regulation of

cytokinesis and the dependency of cytokinesis on karyokinesis. Treatments with

1 ug/mL cytochalasin-D and 1 ig/mL aphidicolin were compared to control treatments to

determine whether cytokinesis was a karyokinesis-dependent function or an independent

function of the cytoplasm. Nematostella embryos arrested cytokinesis under

cytochalasin-D treatment, became multinucleate, and cleaved to the appropriate number

of blastomeres upon washing. This treatment mimicked the natural deferred-cleavage.

Nematostella continued to cleave post-aphidicolin treatment for more than six cell

cycles and produced equal-sized enucleate cells in the absence of (S-phase of)

karyokinesis. These results were similar to those reported in certain sea urchins by

Nagano et al. in 1981 and Hirai et al. in 1984. If cytokinesis required a preceding

karyokinesis, suppression of S-phase and karyokinesis would block cytokinesis; this did

not occur. Cytokinesis, therefore, was independent from karyokinesis, in early embryos

of Nematostella vectensis, and did not require karyokinesis as a predecessor event.














CHAPTER 1
INTRODUCTION


Nematostella vectensis Stephenson 1935 is a small, burrowing, estuarine sea

anemone (Cnidaria: Anthozoa: Hexacorallia: Actinaria: Edwardsiidae) with interesting

characteristics worthy of scientific exploration. It is a hardy, gregarious species found

intertidally and subtidally on both the East and West Coasts of North America, as well as

on the West Coast of Britain, where it is considered endangered. Nematostella vectensis

from Britain (Stephenson 1935), Nematostella pellucida from Woods Hole,

Massachusetts (Crowell 1946), and Nematostella sp. from Oregon and California, were

all determined to be one cosmopolitan species (Hand 1957). This diminutive

cosmopolitan species is the only species of sea anemone reported to release eggs in a

jelly mass (Crowell 1946). Capable of simultaneous sexual and asexual reproduction

throughout the year, Nematostella, at this time, is the only sea anemone whose life cycle

has been dependably closed in captivity (Hand and Uhlinger 1992)(Figure 1-1).

Cnidarians are the most primitive living Metazoans which undergo gastrulation to

form specialized (diploblastic) tissues. Anthozoa, including Nematostella and the Corals,

are considered the most primitive of the living Cnidarians, since they possess no medusa

stage. As such, Nematostella now provides an opportunity to examine developmental

processes and pathways in an animal representative of the most primitive living class of

the most primitive living phylum of Metazoan organisms. Nematostella possesses the

complete genetic and biochemical suites necessary to successfully perform gastrulation,







2
metamorphosis, separation of the sexes, nerve and sensor formation, muscle formation,

and body-part regeneration. Because of the conservative nature of biochemical pathways

among and beyond Metazoa, it is likely that many of the genes and gene cascades present

in Nematostella will be echoed throughout the more advanced Metazoan phyla.

Nematostella was shown to possess HOX homeobox genes and the even-skipped gene of

Drosophila sp. (Finnerty and Martindale 1997).

As the only member of the Subclass Hexacorallia with a closed lifecycle under

laboratory conditions, it is presently our best model for reproduction in the Corals.

Nematostella has been proposed as a model estuarine bioassay organism and already has

demonstrated a sensitivity to cadmium chloride, a heavy metal (Harter 1996).

Nematostella is herein proposed as a model for biomedical research due to its

indeterminant cleavage, initially documented as regulative to the four-cell stage by

Uhlinger (1997) and extended to fully totipotent at the eight-cell stage and, at least

partially, totipotent at the sixteen-cell stage in this report.

Nematostella eggs and embryos are large size and easily manipulated. Their

embryos progress rapidly through development to the planula stage at room temperature,

and will do so throughout a wide range of salinities. Their rate of development is easily

manipulated via temperature regulation. Metamorphosis to juvenile is direct with no

intermediary stages, and may occur rapidly or after prolonged delay. Adults are hardy

and easy to maintain. Establishment of clonal populations and full sibling pure-strain

populations is relatively easy to achieve. Development, growth, and maturation is rapid,

such that the capacity for both sexual and asexual reproduction can be achieved in as

little as 67 days post-fertilization (Hand and Uhlinger 1992).







3
During early development, zygotes within a single egg mass can exhibit up to

three different patterns for achieving the cell number necessary to perform blastula

formation. Most cleave mitotically directly to blastula formation. Others will mitotically

cleave to individual blastomeres (4, 8, 16, or 32), then fuse into a multinucleate mass,

thereafter reinitiating mitotic cleavage and proceeding to blastula formation. Still others

just wait, appearing to be infertile eggs, and then suddenly cleave to 8, 16, or 32 cells and

thereafter proceed mitotically to blastula formation. By exhibiting a unique lack of lock-

step synchrony between karyokinesis and cytokinesis, which is the normal characteristic

associated with mitotic cleavage, Nematostella provided an opportunity to explore the

presumed dependency of cytokinesis on karyokinesis.


Life History

Nematostella vectensis is an Edwardsiid sea anemone with a burrowing physa,

twelve or more tentacles on both their perfect mesenteries (macrocnemes), and imperfect

mesenteries (microcnemes). Only their macrocnemes bear gametes. Cnidae are most

concentrated on the tentacles, but are present over the entire body surface, both externally

and internally. Nematosomes (flagellated spherical bodies comprised of numerous

agglutinous basotrichus cnidae) are unique to this species and circulate throughout the

body and tentacles. Nematostella is reported to measure no more than 1.5 cm total length

when captured from the wild, and is reported to exist in unisex (presumably clonal)

populations (Hand and Uhlinger 1994). They are gregarious, have reported population

densities exceeding 80,000/m2 in the wild, and display no intra-species aggression or

cannibalism (Frank and Bleakney 1976). Under laboratory conditions, they grow to more

than 10 cm in length, populations can exceed 400,000 m2 in culture (Unpublished data,







4
Wilcox 1999) and a single individual can produce up to 96 clones in one season

(Uhlinger 1997).

Nematostella is found in low-energy estuarine habitats, typically intertidally or

subtidally on mudflats, the upper reaches of salt marshes, and in permanent tide pools.

They can tolerate and successfully reproduce in salinities from five parts per thousand

(ppt) to 44 ppt. Like all sea anemones they are carnivorous, and are reported to consume

ostracods, gastropod larvae (Kneib 1988), bivalve larvae, small crustaceans, and even

small fish. Nematostella is reported to be a prey organism for the Grass Shrimp,

Palaemonetes pugio (Kneib 1985).

Nematostella is gonochoristic, with no sexual dimorphism, no known

hermaphroditism, and no known switching of sex due either to genetics or environmental

cues. Females release tens to hundreds of large, isolethical eggs (170 Pm to 220 Jim) in

an adherent gelatinous mass and do so regularly, on an eight-day cycle, under culture.

Eggs are immediately invested in the egg-mass jelly, along with hundreds of

nematosomes, upon release to the lumen of the gut, and promptly extruded (Hand and

Uhlinger 1992). Nematostella sperm are small, with conical heads and a single

flagellum, similar to other known Anthozoa (Dewel and Clark 1974). Sperm easily

penetrate the fresh egg mass; consequently, embryos in a cohort cleave in close

synchrony. Males and females are easily sexually synchronized, even when cultured

independently, and females can release more than one egg mass during a spawning cycle

(Uhlinger 1997). The nematosomes embedded in the spawned egg mass have been

documented to deter predation by fishes.







5
Asexual reproduction is independent of sexual reproduction and is achieved by

transverse fission. Nematostella is only the fifth sea anemone reported to produce clones

by transverse fission. Pedal laceration, budding, fragmentation, and longitudinal fission

are the more common forms of asexual reproduction in the Anthozoa (Uhlinger 1997).

Fission produces a physal fragment with no siphonopharynx or tentacles and often no

visible gastro-mesentery, unlike the oral fragment. Growth of the tentacles and

siphonopharynx is rapid, and feeding can resume in a week. The presence of a food

bolus can prolong the time required to complete re-establishment of a complete feeding

organism (Uhlinger 1997). The frequency of asexual fission shows no correlation to

water quality parameters, food supply, sexual maturity, or spawning frequency (Hand and

Uhlinger 1994).


Laboratory Culture

Nematostella vectensis used for this research were hybrids from cultures CH2 and

CH6 (collected in Chesapeake Bay, Maryland) maintained by Dr. Cadet Hand, Bodega

Marine Laboratory. These strains have been under closed-lifecycle laboratory culture

since 1988. When maintained on a regulated photoperiod of 14 h on/10 h off, and fed

Artemia salina three times a week, Nematostella released eggs within 24 h of their

weekly water change. Up to 270 eggs have been reported embedded in a mass of jelly.

Upon fertilization, cleavage occurred every 20 min or so at room temperature, embryos

gastrulated in less than 12 h and developed to the early ciliated planula stage in 24 to

36 h. Metamorphosis to juvenile occurred in less than three days or was delayed up to

60 d (Hand and Uhlinger 1994).







6
Normal laboratory procedure was to maintain groups of Nematostella in 150 mL

crystallizing dishes filled with 1 OOmL of 11 to 15 ppt natural sea water (NSW) covered

with watch glasses to limit evaporation. When egg laying occurred, the identification of

known females was straightforward. Known females were thereafter segregated to

separate bowls. If spermiation was observed, segregation of known males followed;

otherwise putative males were exposed to fresh egg masses, or portions thereof, for

microscopic confirmation of spermiation and subsequent segregation. Planulae from

fertilized egg masses were cultured separately and fed by introducing one or two dying

Artemia sp. to the bowl to provide an infusion of zooplankton for metamorphosing

juveniles.

Feeding practices had included providing pieces (1 to 2 mm3) of clam ovary once

weekly to each animal (Uhlinger 1997), but Hand reported that this was not necessary for

prolonged culture and repetitive spawning, Artemia sp. alone being sufficient (Personal

Communication 1999). Mature Nematostella will readily take Bio-Diet pellets (750 Pim

or 1 mm), minced shrimp, or lobster, water-packed tuna, sardines, or even whole first-

born Gambusia sp., with no ill effect. Non-motile feed stuffs require dropping the food

directly onto the waiting tentacles, approximating the wafting of feed to the anemone by

tidal flow. Over-feeding, leading to water fouling, is the most common culture problem.



Statement of the Problem

This dissertation focuses on three questions in Nematostella vectensis: 1) What is

the meiotic state of the egg when it is fertilized? 2) What is the nature of the early

developmental plasticity observed? Is early cell fate patterning determinant (mosaic or







7
Type I: Invariant) or indeterminant regulativee or Type II: Variant)? What role does the

Type III: Syncitial pathway play in this species? 3) Does cytokinesis depend on a

preceding karyokinesis for establishment of cleavage planes or for licensure to occur?

Can cytokinesis be experimentally separated from karyokinesis in this species, as has

been achieved in certain Echinoderms?


Meiotic State at Fertilization

The meiotic state of the oocyte at fertilization has been used as a defining

characteristic of an organism since the beginnings of the science of embryology. An egg

by definition is an oocyte that is capable of being fertilized. For different species,

oocytes can become eggs at several meiotic states: before completion of Prophase I-

Germinal Vesicle stage (GV), during Metaphase I or II, during Anaphase II, or after the

completion of meiosis (Masui 1985). Meiotic arrest at the GV stage, with a greatly

enlarged 4N nucleus and one or more nucleoli, for accumulation of oocyte resources, is

ubiquitous among Metazoa. Among most Metazoan species meiotic arrest usually recurs

at a second point in the cycle, to await fertilization, and this point varies characteristically

among phyla, among classes of a phylum, and possibly among genera of the same family

(Masui 1985).

Most metazoans ovulate meiotically-arrested eggs that must complete meiosis and

formation of a pronucleus after activation by sperm, and before fusion of their pronuclei

with the already-formed spermatozoan pronuclei, in order to complete re-establishment

of the diploid state in the zygote. Egg activation and pronuclear fusion comprise the two-

step process termed fertilization. Yolky oocytes typically complete nuclear DNA

duplication (S phase of meiosis) and arrest at the GV stage of meiosis to undergo







8
vitellogenesis (Masui 1985). At this stage, the chromosomes remain decondensed, and

the nucleolus is (or nucleoli are) rapidly transcribing rRNA precursor transcripts and

mRNA precursors for rRNA-associated proteins. Other mRNA precursors are

presumably being generated away from the nucleolus. During this period much maternal

mRNA is typically incorporated into the maturing oocyte, although species exist that

apparently derive no maternal mRNA. Sufficient materials must be sequestered to

sustain subdivision of the fertilized egg into the proper number of cells required by a

particular species to undergo blastulation and gastrulation.

Once the oocyte has reached the diameter characteristic of that species, oocyte

growth terminates, and the egg re-enters meiosis (except for those species that fertilize at

the GV stage). Re-entry into meiosis invokes nuclear envelope breakdown (temporarily

creating one diffusable plasm of the normally membrane segregated cytoplasm and

nucleoplasm), chromosomal condensation, centrosomal movement toward opposite poles,

and spindle formation. Once the condensed 4N chromosomes are aligned on the

equatorial plane of the spindle, Metaphase I is achieved. Either arrest occurs here or a 2N

polar body is ejected (at the animal pole) and arrest occurs during the Metaphase II

equatorial alignment of the remaining sister chromatids. In most species, balanced

physical strains on the meiotic spindle apparently favor arrest at Metaphase I or II,

although arrest (rarely) occurs at Anaphase II. This second meiotic arrest can endure for

minutes or decades (e.g., Mammals), and stereotypically requires egg activation after

ovulation by a fertilizing sperm to release the arrest (Masui 1985). Some organisms can

release meiotic arrest after ovulation, but before sperm penetration, by utilizing

environmental (e.g., ionic) induction of egg activation, albeit in the presence of sperm,







9
such as occurs via Mg2+ in Penaeoidian shrimp (Pillai and Clark 1987). Experimentally,

egg activation can be induced via mechanical pricking of the oolemma (Briggs et al.

1954) or using calcium ionophore A23187.

The meiotic state of both ovulated eggs and pre-ovulatory oocytes can be

determined in Nematostella vectensis by visually inspecting unfertilized eggs and

histological sections of gonadal cysts. Nucleoli are sites of intense nuclear transcription

for rRNA (Gilbert 1996). Therefore, nucleoli are de facto indicators of chromosomal

decondensation. Visible nucleoli indicate that meiotic maturation has not progressed

beyond mid-Prophase I. Following release of primary meiotic arrest at GV, nuclear

envelope breakdown occurs (the nucleoplasm is no longer distinguishable from the

cytoplasm) and chromosomal condensation occurs. Absence of nucleoli and the presence

of condensed chromosomes would allow determination of the meiotic state based on the

arrangement of chromosomes and presence or absence of polar bodies. If the

chromosomes were aligned on the metaphase plate and no polar bodies were evident, the

egg would be arrested at Metaphase I. If the chromosomes were aligned on the

metaphase plate and one polar body was evident, the egg would be arrested at Metaphase

II. If chromosomes were separated and one polar body was present, the egg would be

arrested at Anaphase II. If both polar bodies were evident, the nucleus was greatly

reduced in size, no nucleoli and no chromosomes were present, the egg would be

meiotically complete. Polar bodies are generated during telophase I and II, and contain

condensed chromosomes whose DNA content matches that remaining in the egg. Use of

a DNA-quantifying fluorescent stain would produce equal intensity signals from the polar

body and the egg.







10
IfNematostella were ovulating meiotically complete eggs, those eggs would

display a distinct small pronucleus nucleoplasmm clearly separated from the cytoplasm

implies that the nuclear envelope has reformed), and no condensed chromosomes (which

de-condense following telophase II). If polar bodies remained in the gonadal cyst, the

spawned egg mass would contain none. Upon fertilization, no chromosomal

condensation nor polar body ejection(s) would occur prior to amphymixus and pronuclear

fusion. The embryo would only display chromosomal condensation and nuclear envelope

breakdown immediately prior to the first cleavage. If Nematostella were ovulating

meiotically-arrested eggs which were environmentally activated, the spawned egg mass

would retain the polar bodies and their fluorescent signal would either be double in

intensity (1N:2N for the haploid egg and first polar body) or equal in intensity (IN: 1N for

the haploid egg and second polar body). This author presumed that the rate of meiotic

maturation (two segregations and two cytokinetic polar body ejections) would be quicker

than two cleavage cycles of the fertilized embryo due to the absence of S-phase DNA

synthesis and time for chromosomal condensation, thus meiotic maturation was predicted

to require less than 35 min.

Nemahelminthes, most Platyhelminthes, some Annelids, many Molluscs, and

many Crustaceans release eggs which have not proceeded past the GV stage of meiosis.

Enteroprocts, some Arthropods (Insects), some Annelids, some Molluscs, and some

Platyhelminthes (Planarians), all release eggs which are arrested at the Metaphase I of

meiosis. Virtually all of Chordata (except two reported and still contested) arrest the egg

at Metaphase II, as do some Crustaceans. Certain Echinoderms (sea urchins and sea







11
lilies), and some Ascidians are among the few other taxa reported, along with the known

Cnidarians, to release meiotically complete eggs (Ginzburg 1971, Masui 1985).

Two Anthozoans: Metridium marginatum (=M senile) (McMurrich 1897) and

Renilla koellikeri (Wilson 1889), were reported to release meiotically mature ova nearly a

century ago. Spaulding (1974) confirmed pronuclear eggs in Peachia quinquecapitata.

The Cnidarians known to release meiotically complete oocytes now include one member

of the Scyphozoa: Nausithoe aurea (Morandini 1999). Little recent literature otherwise

disputes or substantiates these observations. While The Atlas of Invertebrate

Reproduction and Development states that "all Cnidarians release meiotically complete

oocytes," only four species of the more than 10,000 species of Cnidarians have actually

been documented. This dissertation serves to validly add Nematostella vectensis as the

fifth member to this list.


Plasticity Of Early Development

From fertilized egg, how does Nematostella vectensis proceed to subdivide to

produce the cellularity necessary to sustain blastula formation? What level of a priori

commitment to function do individual blastomeres display? Is Nematostella more similar

to the determinant Caenorhabditis elegans, to indeterminant Homo sapiens, or to

syncitial Drosophila melanogaster in their pattern of early development?

Nematostella demonstrates a high degree of developmental plasticity. During

early development embryos from the same spawn simultaneously display normal mitotic

cleavage resulting in morula formation, while other embryos display one of two syncitial

cleavage patterns before morula formation. The first of these syncitial patterns is

deferred-cleavage generating multiple nuclei in a single cell before initiating cleavage.






12
The occurrence of this form of syncitial cleavage in Anthozoa was first described by

Gemmill in 1920, and is confirmed in this present report. The second pattern of syncitial

development is refusion, wherein embryos undergo apparently normal mitotic cleavage,

thereafter temporarily refusing into a multi-nucleate single cytoplasm, as documented in

earlier studies of Cnidarians by Wilson (1883), McMurrich (1891), Gemmill (1920), and

Wietrzykowski (1910) and later reported in Nematostella by Uhlinger (1997). All three

early patterns of development result in successful embryogenesis.

An organism's mode of early development has been used as a defining

characteristic since the inception of the science of embryology. The dogma is that all

embryos within a species display a singular, characteristic pattern of development or fail

to develop properly, if at all.

Certain phyla display radial cleavage, whereas other phyla display spiral,

rotational, discoidal, or syncitial cleavage. Some juveniles are small replicas of the

parents (direct development), others are dramatically different and must metamorphose to

become adults (indirect development). Numerous categorizations can and have been

established. Subgroups within some phyla have evolved patterns distinct from the parent

group; for example, Penaeid shrimp undergo radial cleavage (Hertzler and Clark 1992),

whereas most Crustacea utilize spiral cleavage. Documentation of the characteristic

patterns) displayed by Nematostella during early development is a requisite part of the

life-history of this model species.

A key pattern of early development in Metazoa falls into three broad categories:

1) determinant or mosaic, wherein the commitment and differentiation of any given

blastomere is dominated by asymmetrically segregated intracellular cytoplasmic







13
determinants, primarily mRNAs, their promoters, and their inhibitors; 2) indeterminant

or regulative, wherein inter-cellular cues dominate the commitment and differentiation of

any given blastomere; or 3) syncitial, wherein the commitment and differentiation of

prospective blastomeres is determined by trans-acting morphogenic substances and

differential local response thresholds prior to the initial cytoplasmic subdivision, which

occurs all at once, after multiple karyokinesis, and normal mitotic regulative cleavage

proceeds thereafter (Slack 1996). These categories were re-specified as Type I for

invariant (determinant/mosaic) cleavage, Type II for variant (indeterminant/regulative)

cleavage, and Type III for syncitial cleavage (Davidson 1990); Davidson speculated upon

the gray areas between and beyond these three categories, and he speculated regarding

how differing patterns of transcriptional regulation can account for these differences

(Davidson 1988, 1989, 1990).

Determinant (Type I) embryos with blastomeres removed or destroyed in place

ablatedd) during early cleavage either fail to develop, or develop abnormally: that is,

without the parts that would have been produced by the missing blastomeres; mosaic

blastomeres relocated to a novel location within an embryo will still generate the part

they were initially committed (determined) to become, not the part appropriate to the new

location. Failure of the blastomeres of two-cell embryos to adhere together results in the

production of two varieties of dysfunctional embryos, or the lethal termination of

development. The nematode, Caenorhabditis elegans, is presently the dominant model

organism for mosaic development, although many Metazoans follow this pattern for

some portion of their development.







14
Indeterminant or Regulative (Type II) embryos with blastomeres removed, or

ablated during early cleavage will regulate the "morphogenic field" to replace the missing

blastomeres, that is they alter the pattern of cell fate specification of the remaining

blastomeres via an interaction of cis-acting and trans-acting signals, thereby regenerating

a complete suite of cell lineages, and thereafter developing normally and with all parts

(albeit derived juveniles are proportionally smaller than juveniles from the full ova).

Regulative embryos with blastomeres removed or ablated during early cleavage will still

develop the parts that would have been produced by the missing blastomeres; regulative

blastomeres relocated to a novel location within an embryo generate the part appropriate

to the new location. Identical twins can be produced easily by this developmental

pattern; failure of the first blastomeres to adhere together, merely results in

volumetrically smaller, otherwise normal juveniles. Mammals are a prime example of

regulative development, but many invertebrates are regulative as well.

Syncitial (Type III) embryos undergo repeated karyokinesis without cytokinesis

to generate multiple nuclei before to establishing numerous one-nucleus blastomeres in

the embryo in one simultaneous cleavage event. After cleavage to multicellularity,

normal mitotic cleavage ensues, without further syncitial karyokinesis. Commitment to

differentiate into specific tissues is regulated by the interaction of multiple soluble

morphogenic substances in overlapping fields of influence with morphogenic substances

localized to specific regions of the syncitium (Slack 1996). The simplest model for this

concept is the local-source, diffused sink model, which involves a single concentration

gradient for a morphogenic substance and differential responses of cytoplasmic regions

based on activation thresholds. This system can work by providing a single confluent







15
biochemical soup unimpeded by the intricacies of signal cascades associated with cell

and nuclear membrane receptors or the complications of osmosis and diffusion through

such membranes. In Drosophila sp., the dominant syncitial development model system,

subdivision of a huge egg into many thousands of committed cells occurs all at once.

E. B. Wilson reported deferred-cleavage and refusion of blastomeres in Renilla

koellikeri, a soft coral, in 1883. He considered this early syncitium to be a normal

component of this species' development. McMurrich reported spontaneous refusion of

blastomeres in sea anemones in 1897. While observing cleavage in Metridium

marginatum (= M senile), he described the "well-marked" formation of a secondary

syncitium, which he termed "refusion." He did not elect to determine whether this

refusion phenomenon was normal in Metridium sp. development, or not (McMurrich

1897). "Refusionnement" was subsequently reported for Edwardsia beautempsi

(Wietrzykowski 1914). Wietrzykowski observed this phenomenon so frequently that he

concluded it was normal for this species. Nematostella is in the same family as

Edwardsia beautempsi, and they share this refusion trait. Gemill (1920) reported similar

refusion, as well as deferred-cytokinesis, in Metridium dianthus (=M. senile). Spaulding

reported deferred-cleavage syncitial development in Peachia quinquecapitata in 1974.

The literature is otherwise nearly silent on refusion and delayed cleavage; Adiyodi and

Adiyodi (1983), Spaulding (1974), or Hand and Uhlinger (1990, 1992, 1994) are the

only identifiable sources to cite the previous authors' reports.

Nematostella displayed this peculiar refusion (coalescence) of blastomeres in

-15% of the embryos, as well as delayed cytokinesis (syncitial development) in -10% of

the egg mass embryos, in the culture used in this research (hybrids of CH2 x CH6).







16
Individual egg masses occasionally exhibited much higher rates of either refusion or

deferred-cleavage, up to 67% of a cohort. Uhlinger (1997) reported that 29% of the

embryos of the strains of Nematostella he used for his doctoral research (CH2 & CH6

pure strains) typically underwent a blastomere refusion event before initiation of the fifth

cleavage. In other strains, the refusion frequency within an egg mass ranged from 2% to

60%. Formation of a temporary syncitium between all or some of the blastomeres is

reported to have little impact on the interval period between mitotic cleavage events. For

example, from the 4-cell to 8-cell stage was roughly the same time interval whether

refusion and resegmentation occurred or not.

In order to determine whether the developmental pathway of the majority cohort

(the group with direct cleavage to blastula) of a Nematostella egg mass was Type I or

Type II, certain questions need to be answered. Do isolated first, second, or third

cleavage blastomeres (2-, 4-, 8-cell stage) successfully develop to planula and do they

result in normal or abnormal juveniles? If they produce abnormal juveniles, which

blastomeres' absence generates which defect? Do embryos with blastomeres ablated in

situ regulate to replace missing parts or are they abnormal? Which blastomere ablation

generates which defect? Can the embryos with excised or ablated blastomeres survive

metamorphosis from planula to juvenile? Abnormal planula or developmental failure

resulting from experimental manipulation constitutes verification of Type I/Indeterminant

development. Normal planula resulting from the blastomere isolation and from

blastomere ablation experiments constitutes verification of Type II/Determinant early

development.







17
Dependency of Cytokinesis on a Preceding Karyokinesis

It was long believed that cytokinesis only followed karyokinesis, which laid the

template for the cleavage plane, and acted as a licensing event for cytokinesis. In mitosis,

and cleavage, the normal course of events requires duplication of the decondensed

nuclear DNA from 2N to 4N, segregation of the centrosomes toward opposite poles,

formation of the spindle fibers and condensation of the DNA into chromosomes,

attachment of the chromosomes to the spindle fibers and equatorial alignment,

segregation of the chromosomes toward opposite poles, cytokinesis, and duplication of

the centrosomes. During syncitial development, cytokinesis can be temporarily excluded

from this sequence to generate a multi-nucleate cytosol. When cytokinesis is finally

invoked, cell divisions occur among all chromosome sets resulting in multiple one-

nucleus-per-cell blastomeres in one single stroke. Only in Meiosis II, where the specific

goal is to reduce the cellular DNA to IN, do any Metazoan cells undergo cytokinesis

without a preceding S-phase, and even here karyokinesis precedes cytokinesis. Even

Metazoan cells which perform their functions without nuclei (e.g., Mammalian red blood

cells) are generated via normal mitosis, complete with nuclei, and thereafter eject them;

rather than being cleaved off of a precursor cell via cytokinesis without a preceding

karyokinesis. Therefore, it was presumed that cytokinesis was dependent on

karyokinesis.

Evidence has accumulated, however, that the concept of the dependency of

cytokinesis on a preceding karyokinesis may be incorrect. Research demonstrated that

many of the controls for cytokinesis and karyokinesis lie within the cytoplasm,

particularly with the centrosome. It was demonstrated that cytokinesis occurs between







18
supernumerary spindle poles (which contain centrosomes), and not just between those

spindles bearing chromosomes; but that cytokinesis does not occur between spindle-less

chromosome pairs (Sluder and Reider 1985a). Research already demonstrated that

certain echinoderm embryos can cleave multiple times after blockage of S-phase DNA

duplication, which effectively blocked karyokinesis, with aphidicolin (Hirai et al. 1984).

These results are consistent with research that confirmed that centrosomal precursor

reserves would support multiple cycles of centrosomal replications (Hinchcliffe et al.

1999), only if the centrosomes of the cytoplasm are responsible for establishment of the

cleavage plane, not the karyokinesis of chromosomes. Licensure of cytokinesis similarly

must not be a function of S-Phase or karyokinesis, or at most, one cycle of cytokinesis

should have occurred. These lines of evidence strongly implied that a preceding

karyokinesis may not be required for cytokinesis.

Nematostella vectensis provides a unique opportunity to evaluate the regulation of

karyokinesis & cytokinesis. The early developmental plasticity of this species prompted

us to examine the effects of interfering with the natural course of karyokinesis and

cytokinesis. In other words, if we blocked cytokinesis for multiple cell cycles, producing

an artificial syncitium, when we released the arrest, would subsequent cleavage and

normal development occur? The converse of this experiment was to block karyokinesis

to determine whether cytokinesis would proceed and, if so, to what extent?














CHAPTER 2
MEIOTIC STATE OF THE OOCYTE AT FERTILIZATION


The meiotic state of the oocyte at fertilization has been used as a defining

characteristic of an organism since the beginnings of the science of embryology. Meiotic

arrest at the germinal vesicle (GV) stage ofprophase I for oocyte growth is ubiquitous

among Metazoa, and secondary arrest occurs during meiosis in representatives of most

phyla (Masui 1985). Meiotic arrest to await fertilization is documented to occur in

different species at different stages of meiosis, and this secondary arrest can endure for

hours or decades (as in Mammals). Sperm induced egg activation releases the meiotic

arrest and induces meiotic maturation and pronuclear formation in nearly all of these

species (Masui 1985).

Cnidarian oocytes which have been confirmed as having completed meiosis prior

to fertilization include three representatives of the Anthozoa: Renilla koellikeri (Wilson

1889), Metridium senile (McMurrich 1897, Gemmill 1920), Peachia quinquecapitata

(Spaulding 1974), and one representative of the Scyphozoa: Nausithoe aurea (Morandini

1999). Although The Atlas of Invertebrate Reproduction and Development claimed that

"all Cnidarians" release meiotically complete eggs (Conn 1996), considering that there

are over 10,000 living species in this phylum, and that few have been examined, this

seemed to be a remarkably presumptive statement. Not all Cnidarian eggs are equal; the

Cnidaria contains species whose early development is Type I: determinant or mosaic, and

species which are Type II: indeterminant or regulative. It would not be surprising should

19







20
future research determine that Cnidarian species exist which do not release meiotically

complete ova. In this chapter we provide data that confirm Nematostella vectensis can be

validly added to the short list of known Cnidarians which release meiotically complete

eggs.



Methods

Nematostella used in this research were hybrids from cultures CH2 and CH6

(collected from Chesapeake Bay, Maryland) maintained by Dr. Cadet Hand, University

of California, Davis, Bodega Marine Laboratory. Animals were held at room

temperature (20 to 25 C) in 100 mL 15 ppt natural sea water (NSW) in 150 mL

crystallizing dishes covered with watch glasses, on a regulated photoperiod of 14 h light/

10 h dark, and fed Artemia salina three times weekly. Nematostella shed egg masses

approximately 24 h after weekly water changes.

Spawned eggs were stained with Hoechst 33342 (bis-benzimide, H-1399, Sigma)

at 1 tg/mL and immediately inspected for condensed DNA using epifluorescent

microscopy or stained and preserved in 5% Formalin, 100 mmol PIPES for 2 h and stored

in 100 mmol PIPES overnight for subsequent inspection.

Mature non-ovulating females were imbedded in paraffin and sectioned.

Specimens were vitally stained with Hoechst 33342 at 1 gtg/mL for 12 h, lethally

anesthetized with 10% MS-222 (tricaine methanesulfonate, Aquatic Eco-Systems), and

fixed in 10% formalin overnight. Samples were dehydrated in an ethanol series

(50/70/85/95/100/100%) for 30 min each. The final 100% ethanol was held for 1 h.

Samples were then run through a xylene/ethanol replacement series (50/75/100/100%) for







21
30 min each, except the final 100% xylene which was held for 1 h. Samples were then

run through a xylene/paraffin replacement series (50/75/100%) for 1 h each. Paraffin

embedded samples were sectioned at 10 gtm on a Reichert-Jung microtome with a

disposable microtome razor, and a subset of samples secondarily stained with Delafield's

hematoxylin and counterstained with eosin (H/E) (Humason 1979).

Using an alternative technique, ovulating females were imbedded in medium-

density epoxy (Spurr 1969, Electron Microscopy Services) and sectioned. Samples were

fixed in 4% glutaraldehyde for 2 h, and oxidized with 1% osmium tetraoxide for 15 to 30

min. Samples were run through an ethanol dehydration series (50/70/85/95/100%) for 30

min each. The final 100% ethanol was held for 1 h. Samples were then run through an

acetone/ethanol replacement series (50/75/100/100%) for 30 min each, except the final

100% acetone which was held for 1 h. Samples were then run through an epoxy/acetone

replacement series (50/75/100/100%) for 1 h each, except the final 100% epoxy which

was overnight. Samples were transferred to fresh epoxy in a labeled aluminum boat and

incubated at 70C for 16 h. Cured samples were cut from the epoxy, mounted on

aluminum ultramicrotomy plugs (Ted Pella), and trimmed in preparation for sectioning.

Thin sections (< 0.25 pm) were cut with a Sorvall MT-2 ultramicrotome using a glass or

diamond knife, mounted, stained with 1% toluidine blue-0.1% sodium borate solution

(Humason 1979) and observed using bright field microscopy.

Samples were observed on an Olympus BH2 upright microscope mounted with

Nomarski differential interference contrast (DIC) optics and mercury-arc ultra-violet

(UV) illumination, 10X and 20X apochromatic UV objectives, and UG-1 UV filter block.

Images were recorded via Olympus PA-10 automated 35 mm film camera, Olympus







22
DP-11 digital camera, by videocamera via videotape recorder, or Image-1 Software

(Universal Imaging) on a desktop computer.

Due to the large diameter of Nematostella eggs, imaging was improved with

gentle compression. Whole egg imaging slides were prepared by gluing small

rectangular sections (-3 mm x 30 mm) of #1 coverslips on either side of the slide

specimen mounting area with iso-cyanoacrylate (e.g., SuperGlue) to support a 40 mm x

30 mm #1 coverslip, allowing some compression without rupture of the sample.


Results

Spawned eggs were released embedded in an adherent jelly mass containing

spherical bodies termed nematosomes. Eggs in an egg mass numbered from 10 to 270.

Eggs were relatively uniform in size within each egg mass, with almost all eggs

measuring between 170 pm and 220 gm (Figure 2-1). Sperm easily penetrated this egg

mass jelly, and embryos often cleaved in close synchrony as a result (Figure 2-2). The

nematosomes were flagellated, multicellular aggregates of basotrichous cnidae (Figure

2-3). The nematosome was distinctly larger than the pronucleus of an egg (Figure 2-4),

and was predicted to be similarly larger than a polar body, which should approximate the

diameter of the pronucleus.

Oocytes developed in outpockets along the macrocnemes. In immature females,

sagittal sections revealed a thin uniform layer of cells delimiting oocytes in various stages

of vitellogenesis from the lumen of the gut. In mature females, the outer layer of cells

had thickened, and now possessed numerous dark-staining secretary cells. These

secretary, goblet-shaped cells were reported to correspond to Frank and Bleakney's Type

C secretary cells (Uhlinger 1997) (Figure 2-5).








































Figure 2-1. Nematostella vectensis Eggs and Nematosomes. Eggs were relatively
uniform in size within an egg mass.
















*0 ,
s


1


3 *
* 200pam


Figure 2-2. Nematostella vectensis Cleavage. Freshly fertilized egg mass, note that the
embryos cleaved holoblastically to equal blastomeres.


V

S."


t


Ok d







































Figure 2-3. Nematosome. Nematosomes are flagellated multi-cellular spherical bodies
comprised ofbasotrichous cnidae. Nematosomes are found only in
Nematostella vectensis, they circulate throughout the coelenteron and are
embedded in the egg mass jelly.








































Figure 2-4. Nematostome and Pronucleus of Nematostella vectensis. The nematosome is
distinctly larger than the pronucleus and therefore predicted to be distinctly
larger than a polar body.






27
Oocytes were surrounded only by their oolemma, without extracellular matrices,

and developed without follicle cells or nurse cells. Oocytes were contiguous to one

another without intervening cells or cytoplasmic bridges. No interdigitation of

membranes occurred between the cells of the gonadal cyst and the surface of the oocyte,

nor between adjacent oocytes. Cysts with oocytes appeared to have a greater density of

dark staining (goblet shaped) cells than non-gonadal gastromesentery tissues and the

majority of these cells were larger at the end directed toward the lumen of the gut rather

than toward the interior of the gonadal cyst.

In mature non-ovulating females, and in actively ovulating females, immature

cysts contained from one to seven oocytes oocytes that ranged from 25 itm to 220 tm

diameter, retained an enlarged GV, one or more nucleoli, no condensed chromosomes,

and maintained a clear distinction between the cytoplasm and the nucleoplasm, which

implied a complete nuclear envelope. Oocyte nuclear volume comprised from -5% to

-3% of the total cellular volume at the GV stage (decreasing in percentage as the size of

the oocyte increased) (Figure 2-5, Table 2-1). No gonadal cysts, therefore no oocytes,

were identified in the oral or physal regions of the gastromesentery.

Oocytes in mature ovulating cysts contained no GV, no nucleoli, and no clear

distinction between cytoplasm and nucleoplasm in stained sections, under either H/E or

TB staining (Figure 2-6, Table 2-1). No cysts containing oocytes which lacked a GV

contained more than one oocyte.

Spawned eggs displayed a distinct pronucleus under DIC imaging (Figure 2-7,

Table 2-1). The pronucleus comprised -0.1% of the cell volume in ovulated oocytes and

spawned eggs. The pronucleus was located at or near the outer perimeter in most of the
































Prophase Nematostella vectensis Oocytes In Cyst. The GV nucleus and
nucleoli were visible in Prophase-I-arrested oocyte. Note that as many as
seven oocytes occurred in one cyst. The GV was ~5% of the total cell
volume in this image. The dark staining cells corresponded to Frank and
Bleakney's type C secretary cells. H/E stain.


Figure 2-5.


Loeb, It


MOPPP7 ^Thitf
a- .,& L .". 04,i-11, r







































Nematostella vectensis Oocyte Completing Meiosis. The oocyte had
initiated meiotic maturation and had not been released from the cyst. Note
that the GV and nucleoli were no longer present. This was a representative
<0.25 pm thin-section of several hundred serial sections taken through this
epoxy-embedded specimen. Toluidine blue stain.


Figure 2-6.















$ .....


Figure 2-7. Pronuclear Nematostella vectensis Egg. Ovulated unfertilized egg with
distinct pronucleus (Nomarski image).









Table 2-1. Nematostella vectensis Oocyte Characteristics


Cystic Oocyte


25-220 pm
Oocyte


GV nucleus


Nuclear
membrane


Nucleolus or
Nucleoli


Polar bodies
ejected


Pronucleus
Formed


Yes


Yes



Yes



No



No


-220 gm Oocyte
Pre-ovulatory Ovulatory


Yes


Yes



Yes



No


Not until
Pronucleus


No



Presumably


Yes


Released Egg


Egg



No


Yes



No



No



Yes


Zygote



No


Yes



No



No


Yes







32
eggs, possessed no condensed chromosomes, and no nucleoli. Eggs were relatively

uniform in size within a single egg mass, and ranged from 170 gtm to 220 urm. No eggs

smaller than 170 ulm occurred in spawned egg masses, and eggs ejected no polar bodies

between fertilization and first cleavage (Table 2-1).


Discussion

Nematostella vectensis oocytes showed meiotic arrest only once, at the GV stage of

prophase I. Oocytes developed surrounded only their oolemma and without follicle cells,

or nurse cells (e.g., gastrodermally-derived trophonema cells, or trophocytes). Oocytes

were contiguous to one another without intervening cells, membranes, or cytoplasmic

bridges. No interdigitation of membranes occurred between the cells of the gonadal cyst

and the surface of the oocyte, nor between adjacent oocytes. Many Cnidarians display

complex interdigitations of oocyte membrane with the surrounding gastromesentery cells.

Cnidarians utilize many different schemes to advance oocyte development, ranging from

strictly autosynthetic generation to phagocytosis of up to 10,000 heterosynthetic nurse

cells (Grassi et al. 1995). Only one Cnidarian, a Scyphozoan, Haliclystus octoradiatus,

has been reported to have follicle cells; Scyphozoans typically increase in oocyte

diameter via phagocytosis of reserves from apoptotic trophocytes, and often exhibit

complex rugation of the oolemma to provide increased surface area for endocytosis

(Eckelbarger 1992). The Hydrozoa exhibits extracellular oocyte nutrition via

phagocytosis either of nurse cells (Grassi et al. 1995) or adjacent oocytes (Aizenshtadt

1980). Other Anthozoans, such as Actinia equina utilize phagocytosis of exuded

trophocyte cell products to increase oocyte reserves and exhibit complex rugation of the

oolemma to facilitate heterosynthetic nutrition (Eckelbarger 1988). Cysts with oocytes







33
appeared to have a greater density of secretary cells than nearby non-gonadal

gastromesentery tissues and the majority of these cells were larger at the end directed

toward the lumen of the gut rather than toward the interior of the gonadal cyst. What role

these secretary cells may play in Nematostella oocyte nutrition was not determined.

While Nematostella does not appear to receive heterosynthetic oocyte nutrients from

nurse cells, follicle cells, or other accessory cells, when observed using light microscopy,

this possibility is not precluded. Electron microscopy or autoradiography may be able to

address this issue in the future.

Unruptured gonadal cysts, in both mature non-ovulating females and in actively

shedding females, possessed oocytes which displayed an enlarged GV, one or more

nucleoli, no condensed chromosomes, and a clear distinction between the cytoplasm and

the nucleoplasm, which implied an intact nuclear envelope. As many as seven oocytes of

different sizes often occurred within one cyst, and they ranged from 25 Pm to 220 utm

diameter. Oocyte nuclear volume comprised from ~5% to -3% of the total cellular

volume at the GV stage (decreasing in percentage as the size of the oocyte increased).

Oocytes from mature ovulating cysts contained no GV, no nucleoli, and no clear

distinction between cytoplasm and nucleoplasm in stained sections, which implied that

nuclear envelope breakdown had occurred and meiosis was proceeding prior to ovulatory

release. No ovulating cysts contained more than one oocyte. Egg masses were

comprised of eggs of a uniform size range (170 utm to 220 gim) while unovulated,

unruptured cysts contained oocytes of various sizes (45 gm to 220 gm), rather than

oocytes of uniform sizes. Where did the smaller oocytes disappear to?







34
No phagocytotic vesicles were evident in thin sections, no multiple DNA signals

were evident under fluorescent microscopy. Speculation would lead toward the

possibility that, like some of the Hydrozoa, e.g., Hydra oligactis and Hydra attenuata

(Aizenschtadt 1980), Nematostella may accumulate yolk materials by fusion of the

remaining oocytes prior to becoming competent to be ovulated. If this did occur, fusion

of the oolemmas and digestion of the incoming nuclear DNA to nucleotide reserves

would be all that would be required. Evidence for this was never observed. Whether this

is the case or not remains an interesting question.

No secondary arrest occurred after oocyte growth was completed and meiosis was

resumed. Once primary arrest was released, meiosis and ovulation proceeded without

further arrest, until the egg had generated the female pronucleus and been released to the

lumen. Released oocytes rapidly become encased in a jelly mass produced by secretary

cells in the gastromesentery tissues (Uhlinger 1997). This egg jelly mass has

incorporated nematosomes comprised of clusters of agglutinous cnidae (Uhlinger 1997).

Eggs in a spawned jelly mass possessed distinct, small pronuclei when viewed using

Nomarski DIC optics. Upon fertilization, no loss of distinction of the pronucleus

occurred and often a second, presumably the sperm pronucleus, could subsequently be

identified. No polar bodies were ever witnessed being ejected adjacent to the female

pronucleus of a spawned egg. Only immediately prior to the initiation of cleavage would

this pronucleus disappear.

Hoechst 33342 fluoresces 20X brighter when bound to DNA than when unbound

to DNA (Haugland 1996), therefore unbound background fluorescence was

inconsequential. Oocytes did possess a general glow, however, presumably due to the







35
fluorochrome binding to mtDNA. Due to the small size, huge number, and random

distribution ofmitochondria in the mature oocyte, this was not a point-source; therefore

this did not seriously interfere with identifying polar bodies or chromosomes, which were

point-sources of fluorescence. Hoechst 33342 has been previously documented to

fluoresce proportionately to the quantity of DNA it binds (Dresser et al. 1993). This

fluorescent DNA stain, therefore, would illuminate polar body I chromosomes (2N) twice

as brightly polar body II chromosomes (IN). Nematostella possess very small mitotic

figures and their polar bodies would be similarly very small, but their DNA fluoresces

effectively. Hoechst 33342 also stained the DNA of the nematosomes and the

community of protozoa present in the spawned egg mass. Due to the large size and

multicellularity of the nematosomes, and the small size of the mitotic figures, it was not

possible to misidentify polar bodies as nematosomes or vice versa. Due to the ease with

which condensed chromosomes were identified in rapidly cleaving embryos using this

DNA fluorochrome, identification of chromosomes in polar bodies ejected into the egg

mass should not have been problematic.

Ovulated Nematostella eggs were rapidly encased in egg jelly by the secretary

cells of the gastromesentery (Uhlinger 1997). If meiotic maturation occurred after

ovulation, it was presumed that the polar bodies ejected would either be entrained

adjoining the egg at the animal pole or would be entrained in the jelly immediately

adjacent to the egg. IfGV or Metaphase I eggs were released, and eggs activated to

complete meiosis by ionic induction, chromosomes would have been evident and polar

bodies I and II would have been ejected. If Metaphase II eggs were released, equatorial

chromosomes would have been evident, and polar body II would have been ejected.







36
Only if meiosis were completed prior to release of eggs would no condensed

chromosomes be evident or no ejected polar bodies evident. No condensed chromosomes

nor ejected polar bodies were ever recorded in the eggs embedded in hundred of

unfertilized egg masses. No condensation of chromosomes preceding ejection of polar

bodies was ever recorded in fertilized egg masses. Chromosomal condensation was only

recorded immediately priorito the onset of cleavage.

In the early studies of meiotic state at fertilization, the absence of chromosomal

condensation and/or polar body ejection following fertilization was considered one

empirical diagnosis for ovulation ofmeiotically complete eggs. The eggs of most species

analyzed contained condensed Metaphase chromosomes and so were readily visualized

using available stains and light microscopy. Polar body ejection was witnessed in many

of the species observed, following fertilization, which facilitated determination of

whether the Metaphase chromosomes were at MI or MIl. Renilla koellikeri (Wilson

1883), and Metridium senile (McMurrich 1897) were reported to be meiotically complete

at ovulation. In Nausithoe aurea, the one documented Scyphozoan, the released

(unfertilized) oocytes were enclosed by the egg envelope, and usually contained 2 polar

bodies, although in some cases 3 or 4 polar bodies were reported (Morandini 1999).

Nematostella vectensis was confirmed to release eggs that were meiotically

complete before fertilization. This was consistent with the other known Cnidaria;

Cnidaria, however, is a very diverse Phyla, with four classes and over 10,000 species.

Only a few species in two classes have been rigorously analyzed, therefore it remains

scientifically premature to assume that "all" Cnidaria, or even "all" Anthozoans and

Scyphozoans, generate meiotically complete, pronuclear eggs.














CHAPTER 3
PLASTICITY OF EARLY DEVELOPMENT

Nematostella vectensis Stephenson 1935 is a small, cosmopolitan, burrowing,

estuarine sea anemone (Cnidaria: Anthozoa: Hexacorallia: Actinaria: Edwardsiidae). An

early documentation of the sexual nature of Nematostella reproduction was the result of

studies in Nova Scotia (Frank and Bleakney 1976). Descriptions of their taxonomy

(Hand 1957), culture and modes of reproduction (Hand and Uhlinger 1992),

biogeography (Hand and Uhlinger 1994), and some aspects of their early development

(Hand and Uhlinger 1994, Uhlinger 1997) have been produced at the Bodega Bay Marine

Laboratory over the last forty-five years. This chapter documents details of the pathways

followed for cell line specification during early cleavage and development. What level of

a priori commitment to cell lineage specification (Davidson 1990a) do individual early

blastomeres display? Is Nematostella more similar to mollusks (Type I, determinant or

mosaic), to insects (Type III, syncitial), or to mammals (Type II, indeterminant or

regulative) in their pattern of early development?

An organism's mode of early development has been used as a defining

characteristic since the inception of the science of embryology. It is generally presumed

that all embryos within a species display a singular, characteristic pattern of development

(Wilson 1925). Due to the distinct clustering of this patterning across the animal

kingdom, scientists have developed vocabulary to describe, differentiate, and associate

the various patterns. A suite of patterns will serve to specify and distinguish one phylum







38
from another, one class from another, occasionally, even one species from another.

Certain phyla display radial cleavage, whereas other phyla display spiral, rotational,

discoidal, or syncitial cleavage. Some juveniles are small replicas of the parents (direct

development), others are dramatically different and must metamorphose to become adults

(indirect development). Numerous categorizations can and have been established

(Wilson 1925). Subgroups within some phyla have evolved patterns distinct from the

parent group; for example, Penaeid shrimp undergo radial cleavage (Hertzler and Clark

1992), whereas most Crustacea utilize spiral cleavage (Wilson 1925). Several

Anthozoans have been reported to display more than one pattern of early development.

Edwardsia beautempsii (Wietrzykowski 1910, 1914), Renilla koellikeri (Wilson 1925),

and Metridium senile (McMurrich 1898, Gemmill 1920), each exhibit both normal

mitotic cleavage and refusion, with Metridium senile reported to show deferred-

cytokinesis as well (Gemmill 1920). This research provides detailed documentation of

the multiple characteristic patterns displayed by the Cnidarian Nematostella.

Key patterns of early development in Metazoa fall into three broad categories,

classically termed: determinant or mosaic: wherein the commitment and differentiation of

any given blastomere is dominated by intra-cellular cues: asymmetrically distributed

cytoplasmic determinants (primarily mRNAs); indeterminant or regulative, wherein inter-

cellular cues dominate the commitment and differentiation of any given blastomere; or

syncitial, wherein the commitment and differentiation of prospective blastomeres is

determined by fields of multiple morphogenic substances prior to the initial cytoplasmic

subdivision which occurs late and all at once, after multiple karyokineses, and normal

mitotic regulative cleavage proceeds thereafter (Slack 1996).







39
These classical categories have been rewritten as Type I for invariant

(determinant or mosaic) cleavage, Type II for variant (indeterminate or regulative)

cleavage, and Type III for syncitial cleavage (Davidson 1990). Some organisms do not

fall easily into just one category. Examples of difficult species to categorize into such a

theoretical construct would include the four cited Anthozoans, that display syncitial

refusion during otherwise regulative cleavage, or the sea urchin embryo, that appears

regulative only through the first cleavage, separated 2-cell blastomeres will each develop

normally (Driesch 1900), but that is mosaic in development thereafter.

Determinant (Type I) embryos with blastomeres removed or ablated (i.e.,

destroyed in place) during early cleavage either fail to develop or develop without the

parts that would have been produced by the missing blastomeres. Type I embryos with

blastomeres ablated will develop missing the part the targeted blastomere was committed

to become; the remaining blastomeres cannot generate the missing cell lineage (Conklin

1905a). The nematode, Caenorhabditis elegans, is presently the dominant model

organism for Type I development, although many Metazoans, including some Cnidarians

such as the Hydrozoan Aglantha sp. (Freeman 1983), follow this pattern during their

early development.

Indeterminant (Type II) embryos with blastomeres removed or ablated during

early cleavage will "regulate the morphogenic field" (Slack 1996) to replace the missing

blastomeres and develop into normal embryos (Spemann 1903). The mouse, Mus

norvegicus albus, is the dominant developmental model of regulative development, but

many invertebrates, including Cnidarians, are regulative as well (Wilson 1898, Wilson

1925, Freeman 1983).







40
Syncitial (Type III) embryos undergo karyokinesis without cytokinesis to

generate multiple nuclei prior to establishing numerous one-nucleus blastomeres in one

simultaneous cleavage event. Certain Arthropods (primarily Insects) commonly utilize

the deferred-cytokinesis form of Type III development. Commitment to differentiate into

specific cell lineages is regulated by the interaction of multiple soluble morphogenic

substances in overlapping fields of influence interacting with morphogenic substances

localized to specific regions of the syncitium (Slack 1996). In the fruit fly, Drosophila

melanogaster, the dominant syncitial development model system, subdivision of a large

egg into more than 10,000 committed cells occurs all at once (Raff and Glover 1988).

Determining which paths Nematostella follows in becoming a multicellular whole

is requisite to understanding the subsequent developmental processes in this species. The

focus of this research is the early developmental plasticity exhibited by Nematostella

prior to the blastula stage. Within a single egg mass embryos appear to follow three

distinct early paths in generating the cell number needed to sustain blastula formation,

that is: they demonstrate Type II cleavage, and the previously reported forms of Type III

cleavage: refusion and deferred-cytokinesis. This paper confirms the previous reports of

refusion and deferred-cytokinesis occurring among the Anthozoa of Cnidaria. Metridium

marginatum (= senile) was the only Anthozoan previously reported to undergo both

refusion and deferred-cytokinesis (Gemmill 1920). Nematostella vectensis becomes the

second Anthozoan species reported to exhibit Type II and both Type III characteristics

simultaneously.







41
Methods

As stated in Chapter 2, Nematostella vectensis were hybrids obtained from

cultures CH2 and CH6 (Chesapeake Bay, Delaware) maintained at the Bodega Marine

Laboratory by Dr. Cadet Hand. Animals were held at room temperature (20-25C) in 100

mL 15 ppt natural sea water (NSW) in 150 mL crystallizing dishes covered with watch

glasses, fed Artemia sp. 3 times weekly, and maintained on a regulated photoperiod of 14

h light/I 0 h dark. Dishes were cleaned and water changed weekly. Nematostella

maintained on this regimen shed egg masses approximately 24 h after weekly water

changes. Unfertilized egg masses (or portions thereof) were presented to known males

for fertilization. All trials were conducted at room temperature with room temperature

solutions.

De-jellying of spawned egg masses was accomplished by use of a 10 mmol final

concentration of dithiothreitol (DTT, D-9163, Sigma) in 15 ppt NSW. A 5X (50 mmol)

stock solution was prepared by dissolving 0.34 g DTT in 50 mL of 15 ppt NSW. This

stock was diluted 5:1, volume for volume (V/V) with egg masses in 15 ppt NSW (e.g.

250 pl of DTT 5X stock to 1 mL 15 ppt NSW with eggs for a final volume of 1.25 mL

yielding a 10 mmol DTT final concentration), and incubated for 3 to 5 min with gentle

agitation. DTT was removed by three washes of 3 min each.

De-jellied blastomeres were separated manually with pulled glass dissecting

needles. De-jellied blastomeres were separated chemically by extended immersion in 10

utmol DTT, and washed for 2 min in 15 ppt NSW three times. Blastomere ablations were

performed with pulled glass dissecting needles. Random clusters of blastomeres were

generated by manual separation, chemical separation, and ablation.







42
Selected embryos were stained with the DNA fluorochrome, Hoechst 33342,

utilized at 1 pg/mL final concentration. A 1 000X stock solution of 1 mg/mL of dye was

prepared by dissolving 10 mg dye in 10 mL ddH-20, vortexed for three to five min,

wrapped in aluminum foil to block light degradation, and stored at 4C. An aliquot of

this stock solution was diluted 500:1 immediately prior to use with 15 ppt NSW to make

a 2X (2 .g/mL) working solution. This 2X working solution was applied 1:1 V/V, to the

15 ppt NSW holding the treatment and control embryos.

Embryos were manipulated on an Olympus SZH dissecting microscope and

observed on an Olympus BH2 upright microscope with Nomarski differential

interference contrast optics, mercury-arc UV illumination, 10X, 20X UV objectives, and

UG-1 UV filter block mounted. Images were recorded via Olympus PA-10 automated

35 mm film camera, Olympus DP-I 1 digital camera, by videocamera via videotape

recorder, or Image-1 Software (Universal Imaging) on a desktop computer.


Results

All isolated blastomeres from 2-cell and 4-cell embryos developed normally to

planulae and metamorphosed juveniles. Results from trials conducted with eggs in jelly

and dejellied eggs were the same. Manual separation required more trials to obtain

survival of 30 complete sets of planulae from individual embryos than did chemical

separation (Table 3-1, Figure 3-1). Isolated blastomeres from 8-cell embryos often failed

to develop to the planula stage, such that most replicates of eight blastomeres isolated

from a single embryo yielded from seven to no surviving planulae or juveniles. A

complete set of eight juveniles, all isolated as individual blastomeres from a single

embryo, did survive in three trials out of more than 42 replicates (Table 3-2).









Table 3-1. Manual Disassociation of Nematostella vectensis Blastomeres:
Blastomeres from Embryos Survive to Planula Stage


2-cell embryo T= 30 N= 54 S= 90
4-cell embryo T= 30 N= 103 S= 86
8-cell embryo T= 12 N= 71 S= 74

T= Number of embryos manually teased apart
N= Number of blastomeres surviving to planula stage
S= Survival percentage



Table 3-2. Totipotency of Nematostella vectensis Blastomeres:
All Blastomeres from One Embryo Survive to Planula Stage


All 2 blastomeres of 2-cell embryo Yes T= 30 N= 27%
All 4 blastomeres of 4-cell embryo Yes T= 30 N= 24%
All 8 blastomeres of 8-cell embryo Yes T= 43 N= 3%
All 16 blastomeres of 16-cell embryo No T= 45 N= 0%

T= Number of trials
N= Number of complete surviving sets
S= Survival percentage

Note: Many of the 8-cell blastomeres survived chemical disassociation through planula
formation and metamorphosis to juvenile (see Fig. 2), but most sets were
incomplete, with 7, 6, or 5 surviving cohort members. Additionally, while some of
the 16-cell blastomeres survived chemical disassociation through planula formation,
all sets were incomplete. If even one complete set of blastomeres survived the
trials, totipotency and Type II: Indeterminant development was supported at that
stage.
































60


50


40


30


20


10



eN 00 CC CC- 00 CC CC 00so oc
0 C O 0 0 0 0 0 0 0
fet eN rs .Tt 0^o r


Figure 3-1. Percent Survival: Nematostella vectensis Blastomere Isolations. Blastomeres
were manually and chemically separated. Isolated blastomeres developed
normally, but smaller blastomeres had higher rates of mortality. Chemically
isolated blastomeres had a higher rate of survival than manually isolated
blastomeres at the 8-cell and 16-cell stage. Planula and juveniles which
developed from fractional embryos were smaller than planula and juveniles
derived from whole embryos.







45
Isolated blastomeres chemically generated from 16-cell embryos progressed to the

beginning ofgastrulation but failed to develop to planula stage in unfiltered 15 ppt NSW.

Random clusters of blastomeres from 4-cell (2/2, 3/1), 8-cell (4/4, 5/3, 6/2) and

16-cell (8/8, 9/7, 10/6, 11/5, 12/4) embryos developed normally to planulae and

metamorphosed juveniles (Table 3-3, Figure 3-2). Partially ablated embryos from 2-cell

(1 remaining blastomere), 4-cell (3, 2, 1 remaining blastomeres), 8-cell (7, 6, 5,4, 3, 2

remaining blastomeres), and 16-cell (15, 14, 13, 12, 11, 10,9,8, 7,6, 5,4 remaining

blastomeres) embryos developed normally to planulae and metamorphosed juveniles

(Table 3-4, Figure 3-3). Planulae and juveniles derived from blastomere separations and

blastomere ablations were proportionately smaller than planulae and juveniles deriving

from whole embryos.

In both the natural egg mass, and among dejellied embryos, a variable fraction of

embryos, which displayed refusion Type III syncitial development characteristics, would

initiate normal cleavage (Figure 3-4) only to refuse into a temporary syncitium at the 2-,

4-, 8-, 16- (or 32-cell) stage, thereafter cleaving to the next stage, as previously described

(Uhlinger 1997). Confirmation that refusing embryos contained the appropriate nuclei

number at the initiation of refusion was achieved with DNA fluorochrome and

epifluorescent microscopy (Figure 3-5). Refusion was displayed by <15% of a typical

egg mass, but occasionally was displayed by >60% of a mass cohort.

Embryos which displayed deferred-cytokinesis Type III syncitial development

characteristics would delay initiation of cleavage, generating 4, 8, 16, or 32 nuclei prior

to cleaving to the appropriate cell number. The presence of supernumerary nuclei was

confirmed with DNA fluorochrome and epifluorescent microscopy (Figure 3-6).









Table 3-3. Chemical Disassociation of Nematostella vectensis Blastomeres


Blastomere Isolations
2 cell embryo T= 30 N= 57 S= 95%
4 cell embryo T=30 N=114 S=95%
8 cell embryo T= 30 N= 185 S= 77%
16 cell embryo T= 30 N= 63 S= 13%

Blastomere Clusters
2 of 4 cell embryo T= 30 N= 28 S= 93%
3 of 4 cell embryo T= 30 N= 30 S= 100%

2 of 8 cell embryo T= 30 N= 23 S= 77%
3 of 8 cell embryo T= 30 N= 23 S= 77%
4 of 8 cell embryo T= 30 N= 28 S= 93%

2 of 16 cell embryo T=30 N=4 S= 13%
3 of 16 cell embryo T=30 N=6 S=20%
4 of 16 cell embryo T= 30 N= 22 S= 73%
5 of 16 cell embryo T=30 N=19 S=63%
6 of 16 cell embryo T= 30 N= 20 S= 67%
7 of 16 cell embryo T= 30 N= 22 S= 73%
8 of 16 cell embryo T= 30 N= 26 S= 87%

T= Number of embryos chemically disassociated
N= Number of blastomeres surviving to planula stage or
Number of blastomere clusters surviving to planula stage
S= Survival percentage

Note: Few isolated 16-cell blastomeres, and no complete cohort sets survived to the
planula stage, while many 2-cell clusters from the 16-cell embryos survived to the
planula stage. Individual blastomeres from 16-cell embryos may be at the lower
limit of resources required to support cleavage to the number of cells necessary to
successfully form a gastrula despite retained totipotentency or differentiation of
some lineages may have initiated between the 8-cell and 16-cell stage.








Table 3-4. Random Manual Ablation ofNematostella vectensis Blastomeres:
Remaining Embryos Survive to Planula Stage


2-cell embryo 1 ablated T= 37 N= 30 S= 81%

4-cell embryo 1 ablated T= 35 N= 30 S= 86%
4-cell embryo 2 ablated T= 38 N= 30 S= 79%
4-cell embryo 3 ablated T= 43 N= 30 S= 70%

8-cell embryo 1 ablated T= 30 N= 24 S= 80%
8-cell embryo 2 ablated T= 30 N= 26 S= 87%
8-cell embryo 3 ablated T= 30 N= 21 S= 70%
8-cell embryo 4 ablated T= 30 N= 21 S= 70%
8-cell embryo 5 ablated T= 30 N= 17 S=57%
8-cell embryo 6 ablated T= 30 N= 7 S= 23%
8-cell embryo 7 ablated T= 30 N= 4 S= 13%

T= Number of trials
N= Number of surviving clusters
S= Survival percentage

Note: Ablation of blastomeres allowed cell-membrane from the destroyed cell to remain
adhered to the surviving cell(s) so trans-membrane signal-receptor loci were not
disrupted. Development of normal embryos indicated that blastomeres were
regulative to the absence of the missing living cell(s), not to missing trans-
membrane signals. Ablation negatively impacted survivability by increasing the
nutrient levels of the culture medium, which stimulated protozoan population
explosions. That any 8-cell embryos with 7 blastomeres ablated survived, was
testimony to the hardiness of these embryos; that they developed into small normal
planula, was proof that they were regulative.

































-3 0 0 0 0 '


FIGURE 3-2:


Percent Survival: Nematostella vectensis Embryos With Random Clusters
Of Blastomeres. Random clusters of blastomeres were generated from
embryos by treatment with DTT at 10 gg/mL final concentration for
prolonged duration and agitation. Individual blastomeres were not
included in this trial. Due to the random nature of chemical disassociation,
clusters were not composed of the same blastomere lineages in each
instance, that is, in clusters, some were the A & B blastomeres, others
were A & C, or B & C, or A, B, & D, or A, C, & D, etc. Survival to
juvenile of members of virtually all trials indicated that no specific
blastomere were required for normal development.

Chemical disassociation of: 4 cell embryos yielded isolated blastomeres
and, 2 and 3 blastomere clusters; 8 cell embryos yielded isolated
blastomeres and, 2 through 7 blastomere clusters; 16 cell embryos yielded
isolated blastomeres and 2 through 15 blastomere clusters.

Since the data had already documented that one half an embryo could
develop normally, the 5 through 7 blastomere clusters of the 8-cell
embryos, and the 9 through 15 blastomere clusters of the 16-cell embryos
were not plotted.








49




90

80

70

C* 60

Z 50

40
(-
30

20

10


e4 CC 00 0 CC C CC C
o 0 0 0 0 0 0 0 0 0 0
-. (el (^- e'a (** T 'ri r-~



Figure 3-3: Percent Survival: Manual Nematostella vectensis Blastomere Ablations.
Ablation trials were conducted to determine whether the remaining cells were
utilizing membrane contact or inter-cellular cues to perform regulation.
Failure of embryos to replace the cell lineages of the ablated blastomeres
would have indicated that membrane contact was the cue utilized in
regulation.

2 cell embryos had one cell ablated.
4 cell embryos had one, two, or three blastomeres ablated.
8 cell embryos had one to seven blastomeres ablated.

With increasing numbers of blastomeres ablated, embryo mortality increased,
and survival to juvenile decreased. It was not determined whether this was a
function of increased rate of disease, increased rate of predation by protozoa,
decreased viability of the remaining embryo to sustain cleavage to the cell
numbers needed to gastrulate and planulate, or some combination of the
three.


















* ...,:* : ,- -










I... -
: .,"-. : ^ir^ Vf



' :,. *..: ^



%.^:


Normal Mitotic Cleavage in Nematostella vectensis. The blastomeres were
distinct, equal, and individual, and displayed classic radial holoblastic
cleavage. Note that karyokinesis had been completed, distinct paired nuclei
were evident, and that cleavage to the eight cell stage was imminent
(Nomarski image).


Figure 3-4:
























I ; Ii y .;. . . . .


50 Wun


Figure 3-5. Nematostella vectensisType III Refusion After Initiation Of Cleavage: 4 Cell.
The formerly individual blastomeres had begun to refuse into a single
temporary syncitium. The location of the former intervening cell membranes
were still visible. Treatment with the DNA fluorochrome Hoechst 33342
demonstrated the continued presence of 4 nuclei despite the dissolution of the
individual blastomeres (Nomarski and UV).

















































Figure 3-6: Nematostella vectensisType III Deferred-Cleavage. This embryo had
undergone karyokinesis without initiating cytokinesis. Multiple nuclei were
distinguishable. Gentle compression of the embryo accounted for the
irregular outline and afforded better visualization of the multiple nuclei.
Treatment with Hoechst 33342, verified the presence of DNA at the sites of
the presumptive nuclei (Nomarski image and UV).







53
This deferred cleavage usually comprised <5% of an egg mass cohort, but occasionally

was displayed by >60% of a cohort.

No embryos were observed which displayed both Type III patterns. Of the few

deferred-cytokinesis Type III embryos from which 4-cell blastomeres were isolated, or 8-

cell blastomere clusters were generated, no difference in survival from the Type II

embryos was noted. No experimentally manipulated embryos displayed the refuision

syncitial pattern after treatment.


Discussion

In Nemalostella vectensis, in both the natural egg mass and among de-jellied

embryos, a variable fraction of embryos displayed refusion Type III syncitial

development characteristics, and would initiate normal cleavage only to refuse into a

temporary syncitium at the 2, 4, 8, 16 or 32-cell stage, thereafter cleaving to the next

stage. Confirmation that refusing embryos contained the appropriate nuclei number at

the initiation and termination of refusion was achieved with DNA fluorochrome and

epifluorescent microscopy. Refusion was displayed by <15% of a typical egg mass, but

occasionally was displayed by >60% of a mass cohort. Uhlinger (1997) reports that 29%

of the embryos of the strains of Nematostella he used for his doctoral research (CH2 and

CH6) typically underwent a blastomere refusion event prior to initiation of the fifth

cleavage. In other strains, the refusion frequency within an egg mass ranged from 2% to

60%. Formation of a temporary syncitium between all or some of the blastomeres is

reported to have little impact on the interval period between mitotic cleavage events. For

example, from the four cell to eight cell stage was roughly the same time interval whether

refusion and resegmentation occurred or not. In his 1997 dissertation, Uhlinger reports







54
that four-cell refusion embryos contain four nuclei, and this is confirmed by our

epifluorescent data. Our data did not confirm whether this variability was exclusively a

function of strain variation, as proposed by Uhlinger (1997), or whether it was effected

by seasonality, stocking density, cross strain fertilization, or the effect of individual

variation. E.B. Wilson reported refusion of blastomeres in Renilla koellikeri, a soft coral,

in 1883. He considered this early syncitium to be a normal component of this species'

development. Reported observation of re-fusion cleavage in Metridium marginatum

(now M senile), the second Cnidarian to have described a "well-marked formation of a

secondary syncitium", did not determine whether this phenomenon was either normal or

abnormal for Metridium sp. development (McMurrich 1897). "Refusionnement" was

subsequently reported for Edwardsia beautempsi (Wietrzykowski 1914). Wietrzykowski

observed this phenomenon so frequently that he concluded it was normal for this species.

Nematostella is in the same family as Edwardsia beautempsi, and they share this refusion

trait. Gemill reported refusion in Metridium dianthus (now also M senile) in 1920. The

literature is otherwise nearly silent on refusion; Adiyodi and Adiyodi (1983) and Hand

(Unpublished data 1990) or Hand and Uhlinger (1992, 1994) being the only identifiable

sources which cite the previous authors reports. Nematostella became the fourth

Actinarian species reported to display this unusual behavior.

In this present study, Nematostella were observed to exhibit deferred-cytokinesis

Type III syncitial development characteristics. Spaulding (1974) reviewed development

in sea anemones, adding Peachia quinquecapitata to the four previously reported

anemones to exhibit this developmental pattern. Among these anemones, embryos would

delay initiation of cleavage, generating 4, 8, or 16 nuclei prior to cleaving to the







55
appropriate cell number. With Nematostella, the presence of supernumerary nuclei was

confirmed with a DNA fluorochrome and epifluorescent microscopy. This deferred

cleavage usually comprised <5% of an egg mass cohort, but occasionally was displayed

by >60% of a cohort. This research confirms the previous reports of deferred-cytokinesis

Type III cleavage in Cnidaria.

Normal development in Nematostella proceeds as radial, holoblastic, equal,

cleavage (Uhlinger 1997). Within a single egg mass, the largest subset of embryos,

usually greater than 80% of an egg mass, will cleave in this manner, displaying the

sequential cycling of karyokinesis and cytokinesis typical of embryonic cleavage. Within

this same egg mass, however, a variable fraction of embryos can revoke cytokinesis

temporarily and thereafter re-establish synchronous cleavage cycling, and another smaller

variable fraction of the same egg mass can defer cytokinesis for several karyogamy

cycles prior to establishing synchronous cleavage cycling, with all pathways producing

normal planula and juveniles.

Nematostella's re-fusion of cleaving blastomeres among only a subset of embryos

in an egg mass remains an interesting conundrum. Speculation regarding possible

alternative reasons would include redistribution of mis-distributed cytoplasmic

determinants, realignment of misarranged spindle axes, equalization of mechanical

stresses between blastomeres, and, possibly, that no purpose is served at all. The

experimental data generated allowed us to immediately discount the redistribution of

cytoplasmic determinants. Nematostella eggs must be relatively uniform in internal

distribution of cytoplasmic materials. The first and second cleavage are meridianal and

would subdivide laterally skewed distributions of cytoplasmic materials (such as that







56
found in the germ plasm of Penaeid shrimp oocytes), but would not subdivide vertical

asymmetries of materials along the animal/vegetal (A/V) axis (such as that found in frog

eggs and sea urchin eggs). The third cleavage is equatorial and would subdivide such a

skewed A/V distribution. Neither possible version of asymmetrical cytoplasmic

determinant distribution is supported by the survival of small, but normal appearing,

juveniles from all blastomeres of an 8-cell embryo isolation, nor by survival of juveniles

from embryos with randomly ablated blastomeres, nor by the survival of random clusters

of blastomeres from 16-cell embryos. If localized asymmetries are not present in the egg,

it would not be plausible to arrest cytokinesis in a small fraction of eggs in a spawned

mass, to redistribute evenly distributed materials. It is possible to conceive of

experiments to test the other speculative motives for refusion. It may be possible for

molecular biochemists to illuminate which translational pathway is being activated or not

being activated to induce the phenomena of refusion.

Experimental manipulations of embryos consisted of culturing individual

blastomeres from embryos, to determine whether all blastomeres from an individual

embryo were capable of generating complete, albeit smaller than normal, planulae and

juveniles. Data revealed that Nematostella embryos possessed this capacity at the 2-cell,

4-cell, and 8-cell stage, with a dramatic reduction of the percent survival at the 8-cell

stage. Further experimental manipulations involved tracking the fate of clusters of

blastomeres, and embryos with ablated (missing) blastomeres. Clusters of blastomeres,

comprised of 4-cell trials (2/2 or 3/1), 8-cell (4/4, 5/3, 6/2) and 16-cell (8/8, 9/7, 10/6,

11/5, 12/4) embryos and embryos with one to many blastomeres ablated, comprised of 2-

cell (1 ablated), 4-cell (1-3 ablated), 8-cell (1-7 ablated), or 16-cell stage (1-13 ablated),







57
were cultured for observation of development to planulae and juveniles. All partial

embryos demonstrated the capacity to survive to the juvenile stage, with decreasing rate

of survival with decreasing size of experimental fragment. This blastomere-cluster

survival data confirms that embryos with missing blastomeres are capable of regulating

their "morphogenic field", that is, they can alter cell fate determination to compensate for

blastomere loss (Slack 1996), and thus confirms that Nematostella embryos do not

become determinant after the first equatorial cleavage as Sea Urchin embryos do.

The juveniles resulting from the blastomere ablations and from the blastomere

isolations (both being categories of partial embryos) were proportionately smaller at

metamorphosis than those juveniles which developed from an entire embryo. These

smaller embryos took longer to grow to two centimeters expanded length than did

unmanipulated embryos, and had a higher mortality. Speculation regarding the reasons

for this slow compensatory growth would include the increased difficulty of obtaining

food, and the comparatively larger size of competitors and predators relative to the

diminutive juveniles. Feeding success may be reduced by increased difficulty to capture

normal prey or may be reduced by a shift to smaller or less nutritious prey.

Nematostella clearly demonstrated Type II indeterminate cleavage. A tremendous

resiliency of cell fate specification was found. Nematostella is an excellent model of

indeterminate development. Individual blastomeres from embryos with two, four, and

eight cells were totipotent. Through the third cleavage Nematostella blastomeres remain

uncommitted and fully capable of producing a functional metamorphosed juvenile.

Nematostella embryos are also capable of exhibiting two distinct forms of Type III

cleavage: both deferred cytokinesis and refusion; why they do this remains a mystery.







58
Nematostella joins the select group of organisms, currently comprised of four Actinarians

and the Sea Urchin (but not other echinoderms), which fail to fall easily under either the

classical or the modem theoretical construct for categorizing early development. If the

rubric, "The exceptions make the rule" holds, then clearly further work needs to be done

on the modem theoretical construct for specification of cell fate in early development.















CHAPTER 4
THE INDEPENDENCE OF CYTOKINESIS AND KARYOKINESIS
DURING EARLY DEVELOPMENT


Nematostella vectensis Stephenson 1935, an estuarine sea anemone (Cnidaria:

Anthozoa) demonstrates a high degree of developmental plasticity. During early

development embryos from the same spawned egg mass simultaneously display normal

mitotic cleavage resulting in morula formation, while other embryos display one of two

syncitial cleavage patterns prior to morula formation. The first of these syncitial patterns

exhibits deferred-cleavage generating multiple nuclei in a single cell. The occurrence of

this form of syncitial cleavage in Nematostella is first described in this present report,

although this pattern has been reported to occur in other sea anemones "with large yolky

eggs" (Spaulding 1974). The other pattern of syncitial development is performed by

embryos which undergo, apparently normal mitotic cleavage, thereafter exhibiting

refusion. This was documented in early studies of Cnidarians by Wilson (1883),

McMurrich (1891), Gemmill (1920), Wietrzykowski (1910, 1914), and later reported in

Nematostella by Uhlinger (1997). All three early patterns of development result in

successful embryogenesis. This species thus provides a unique opportunity to evaluate the

variable regulation of karyokinesis and cytokinesis.

The plasticity of this species prompted us to examine the effects of interfering with

the natural course of karyokinesis and cytokinesis. In other words, if we blocked







60
arrest, would subsequent cleavage and normal development occur? The converse of this

experiment was to block karyokinesis to determine if cytokinesis would proceed and, if so,

to what extent?

It had long been believed that, in animals, cell division was a singular process.

Cytokinesis only followed karyokinesis, which laid the template for the plane of the

division of the cell (Wolfe 1972), and acted as a licensing event for cytokinesis

(Satterwhite et al. 1992, Wheatley et al. 1997). Evidence has continued to accumulate,

however, that the concept of the dependency of cytokinesis on a preceding karyokinesis

may be incorrect. Early work with enucleate eggs of the amphibian Triton (Fankhauser

1934) and sea urchins (Harvey 1936, 1940) demonstrated cleavage without karyokinesis.

More recent research has demonstrated that the controls for cytokinesis lie within the

cytoplasm (Briggs et al. 1951, Nagano et al. 1981, Hirai et al. 1984), particularly with the

activities of the centrosome and spindle (Hinchcliffe et al. 1998, 1999, Sluder et al. 1985a,

1985b, 1986, 1987, 1989, 1990, 1995, 1999). It has been demonstrated that cytokinesis

occurs between supernumerary spindle poles, and not just between those spindles bearing

chromosomes; but that cytokinesis does not occur between spindle-less chromosome pairs

(Sluder and Rieder 1985). Research has already demonstrated that echinoderms can

cleave a few times after blockage of karyokinesis with aphidicolin (Hirai et al. 1984),

which is consistent with research that confirmed that centrosomal precursor reserves

would support multiple cycles of centrosomal replications (Hinchcliffe et al. 1998).

Research has demonstrated linkages between oscillations in cyclin protein concentrations,

the cyclical formation of Mitosis Promoting Factor, and the regulation of karyokinesis and







61
cytokinesis. These lines of evidence imply that a dependent preceding karyokinesis may

not be required for cytokinesis.

In this chapter we have demonstrated that cytokinesis can be blocked and released,

resulting in normal development, mimicking embryos which follow natural deferred-

cleavage syncitial development. Secondly, we have demonstrated that blocking S-phase of

karyokinesis resulted in continued cytokinesis up to blastula formation which produces

relatively normal appearing embryos composed predominantly of enucleate cells.


Methods

As reported in Chapter 2, Nematostella vectensis were hybrids obtained from

cultures CH2 and CH6 (Chesapeake Bay, Delaware) maintained at the Bodega Marine

Laboratory, University of California, Davis, by Dr. Cadet Hand. Animals were held at

room temperature (20 to 25C) in 100 mL 15 ppt natural sea water (NSW) in 150 mL

crystallizing dishes covered with watch glasses, liberally fed Artemia salina 3 times

weekly, and maintained on a regulated photoperiod of 14 h light/ 10 h dark. Dishes were

cleaned and water changed weekly. Nematostella maintained on this regimen shed egg

masses approximately 24 h after weekly water changes. Unfertilized egg masses (or

portions thereof) were presented to known males for fertilization. All trials were

conducted at room temperature with room temperature solutions.

De-jellying of spawned egg masses was accomplished by use of a 10 mmol final

concentration of dithiothreitol (DTT, Sigma) in 15 ppt NSW. A 5X (50 mmol) stock

solution was prepared by dissolving 0.34 g DTT in 50 mL of 15 ppt NSW. This stock was

diluted 5:1, volume for volume (V/V) with egg masses in 15 ppt NSW (e.g. 250 pL of







62
DTT 5X stock to 1 mL 15 ppt NSW with eggs for a final volume of 1.25 mL yielding a 10

mmol DTT final concentration), and incubated for 3 to 5 min with gentle agitation. DTT

was removed by 3 washes of 3 min each.

Control embryos were monitored for cleavage cycle timing, and rate of

refusion/deferred cleavage within a spawn cohort. Control treatments received 15 ppt

NSW 1:1 V/V concurrently to experimental cohorts receiving treatments at 1:1 V/V.

In order to verify that the carrier solvent, dimethylsulfoxide (DMSO, D-8779,

Sigma), was not the active agent in observable responses to experimental treatment, a

control subset was treated with a final concentration of 0.1% DMSO without any treatment

solute, for comparison to NSW control embryos and to treatment embryos. A 10X (1%)

stock solution of 1 mL of DMSO in 100 mL of 15 ppt NSW was diluted 5:1 for a working

solution of 2X (0.2%) DMSO. 2X working solution was diluted 1:1 V/V for a final

effective concentration of 0.1% DMSO for the carrier-solvent control.

Cytochalasin-D (C-8273, Sigma) was utilized to reversibly arrest cytokinesis at 1

ug/mL final concentration. A 20X (20 utg/mL) stock solution was prepared by dissolving

1 mg cytochalasin-D in 1 mL DMSO, this diluted 50:1 with 15 ppt NSW, divided into

aliquots and frozen at -20C. Aliquots were thawed immediately prior to use and diluted

10:1 with 15 ppt NSW for a 2X (2 ug/mL) working stock concentration. Treatment was

2X working solution 1:1 V/V, dilution in 15 ppt NSW with embryos for an effective

concentration of 1 tg/mL cytochalasin-D in 0.1% DMSO in 15 ppt NSW. Fertilized

embryos were treated with cytochalasin-D and arrested for three cleavage cycles. Embryos

were stained with Hoechst 33342 or DAPI (4,6 Diamidino-2-phenylindole

dihydrochloride, D-9542, Sigma) and a subset preserved, while the other subset was







63
washed 3 times, 5 min each, in 15 ppt NSW to release cytokinetic arrest, and observed for

reinitiation of cleavage and determination of the number of nuclei per blastomere.

Aphidicolin (A-0781, Sigma) was utilized at 1 ug/mL final concentration to arrest

karyokinesis. A 20X (20 ug/mL) stock solution was prepared by dissolving 1 mg

aphidicolin in 1 mL DMSO, this diluted 50:1 with 15 ppt NSW, divided into aliquots and

refrigerated at 4C. Aliquots were diluted 10:1 with 15 ppt NSW for a 2X (2 ig/mL)

working concentration. Treatment was 2X working solution 1:1 V/V, dilution in 15 ppt

NSW with embryos for an effective concentration of 1 ug/mL aphidicolin in 0.1% DMSO

in 15 ppt NSW.

Both DNA fluorochromes, Hoechst 33342 and DAPI, were utilized at 1 uIg/mL

final concentration. A 1000X stock solution of 1 mg/mL of dye was prepared by

dissolving 10 mg dye in 10 mL ddH20O, vortexed for 3 to 5 min, divided into aliquoits,

wrapped in aluminum foil to block light degradation, and stored at 4C. An aliquot of this

stock solution was diluted 500:1 immediately prior to use with 15 ppt NSW to make a 2X

(2 ug/mL) working solution. This 2X working solution was applied 1:1 V/V, to the 15 ppt

NSW holding the treatment and control embryos.

Samples were observed immediately or preserved in 3% Formalin, 100 mmol

PIPES in 15 ppt NSW for 2 h, then transferred to 100 mmol PIPES in 15 ppt NSW.

Controls were preserved concurrently to experimental treatments.

Samples to be prepared for epoxy embedding (Spurr 1969) were preserved in 4%

glutaraldehyde for 2 h, and oxidized with 1% osmium tetraoxide for 15 to 30 min.

Samples were run through an ethanol dehydration series (50/70/85/95/100%) for 30 min

each. The final 100% ethanol was held for 1 h. Samples were then run through an







64

acetone/ethanol replacement series (50/75/100/100%) for 30 min each, except the final

100% acetone which was held for 1 h. Samples were then run through an epoxy/acetone

replacement series (50/75/100/100%) for 1 h each, except the final 100% epoxy which was

overnight. Samples were transferred to fresh epoxy in a labeled aluminum boat and

incubated at 70C for 16 h. Cured samples were cut from the epoxy, mounted on

aluminum ultramicrotomy plugs (Pella), and trimmed in preparation for sectioning. Thin

sections (< 0.25 gim) were cut with a Sorvall MT-2 ultramicrotome using a diamond or

glass knife, mounted, stained with 1% toluidine blue-0.1% sodium borate solution

(Humason 1979) and observed under bright field microscopy.

Samples were observed on an Olympus BH2 upright microscope with Nomarski

DIC optics and mercury-arc UV illumination, 10X, 20X UV objectives, and UG-1 UV

filter block mounted. Images were recorded via Olympus PA-10 automated 35 mm film

camera, Olympus DP-11 digital camera, by videocamera via videotape recorder, or Image-

1 Software (Universal Imaging Corporation) on a desktop computer.

Due to the large diameter of Nematostella embryos, imaging is improved with

gentle compression. Live imaging slides were prepared by gluing small rectangular (-3

mm x 30 mm sections) of#1 coverslips on either side of the slide specimen mounting area

with iso-cyanoacrylate (e.g., SuperGlue() to support a 40 mm x 30 mm #1 coverslip,

allowing some compression without rupture of the sample.







65
Results

Untreated Nematostella vectensis embryos, either in egg mass jelly or post-DTT

jelly removal, cleave mitotically for most (>80%) of the typical spawn cohort

(see Chapter 2, Page 29, Figure 2-la). The pattern of deferred-cleavage, wherein a

fertilized embryo does not initiate cleavage until 4, 8, 16, or 32 nuclei are present in < 5%

of a typical cohort; however, the occasional spawn occurred wherein the majority of the

embryos (>60%) displayed this pattern. The pattern of refusion, wherein an embryo

initiates mitotic cleavage, and sometime before the fifth cleavage coalesces into a

syncitium, thereafter resuming mitotic cleavage, was exhibited by <15% of a typical

cohort; here again, the occasional spawn would display refusion among the majority of

embryos (> 60%). All untreated embryos displayed one nucleus per cell after initiation or

resumption of normal mitotic cleavage. There was broad variation of the occurrence of

both types of syncitial cleavage between different egg masses, under identical regimens,

often on the same night. This variation was previously reported in the literature

(Uhlinger 1997), who ascribed the differences to variation between CH2 and CH6 culture

strains. Our data indicate that these differences exist between members of the same strain,

the hybrids of CH2 x CH6 being our only strain at this point (Figure 4-1).

Treatment with 0.1% DMSO solution alone was conducted to determine the effects

attributable to DMSO, due to its use as a carrier solvent for the treatment solutes used in

the following trials. DMSO at 0.1% concentration did not induce the effects attributable

to treatment solutes in the other trials. DMSO treated embryos cleaved in near synchrony

with and displayed similar percentages of cleavage patterning to paired NSW controls. All

DMSO treated embryos displayed one nucleus per cell, whether







66
viewed whole using Nomarski DIC or fluorochrome imaging, or viewed with brightfield

microscopy for epoxy embedded thin sections.

In an attempt to mimic Type III deferred-cleavage, fertilized eggs were treated with

cytochalasin-D. These trials were conducted on one-half of an egg mass, while the other

half of the egg mass acted as an indicator of the natural rate of deferred cleavage

(Table 4-1, Figure 4-2). This treatment blocked cytokinesis in Nematostella while leaving

karyokinesis apparently unaffected in both capacity and timing. Treatment of uncleaved

embryos for two cleavage cycles resulted in single cells bearing four nuclei, and four

cleavage cycles resulted in single cells with sixteen nuclei (Figure 4-3).


Table 4-1. Control Versus Cytochalasin-D Treatment Cleavage Patterns in
Nematostella vectensis

Mitotic Deferred Refusion Infertile

Control: / egg mass 74% 8% 13% 5%

Treatment: /2 egg mass 0 % 93% 0% 7%


Release of cytokinetic arrest resulted in direct cleavage to blastomeres containing one
nucleus, and exhibited normal development thereafter. None of the treated embryos
displayed refusion after release of cytokinetic arrest.

















100 .




70 .




40 --

30-

10
0

IM Mtotic 0 Deferred U Refusion U No Cleavage



Figure 4-1. Nematostella vectensis CH2 x CH6 Hybrids: Cleavage Variation. Cleavage
followed three distinct patterns: normal mitotic cleavage Type II
indeterminant, deferred-cleavage Type III syncitial, and re-fusion Type III
syncitial, with all patterns resulting in normal development. Normal cleavage
of the embryos in an egg mass produced a wide range of results. On average
80% would undergo mitotic cleavage, 5% would delay cleavage for several
cycles and thereafter cleave to the appropriate number of one nucleus
blastomeres, and 15% would initiate mitotic cleavage only to re-fuse into a
temporary syncitium. The occasional egg mass was skewed in favor of either
re-fusion or deferred cleavage, this accounts for the large error bars.

























A4O


Cytochlasin


1-I
Aphidicoin


SNMtotic Deferred Refision No deavage


Figure 4-2.


Cleavage Patterns by Treatment in Nematostella vectensis. Egg masses were
divided in half, one half was untreated to act as control indicator of the natural
rate of deferred and refusion cleavages. Treatments with cytochalasin-D and
aphidicolin at 1 gg/mL were conducted on the other halves. This graphic
represents pooled data showing that treatments skewed the cleavages almost
exclusively toward either deferred cleavage for cytochalasin-D (cytokinesis
was suppressed, but karyokinesis proceeded) or mitotic cleavage for
aphidicolin (karyokinesis was suppressed, but cytokinesis proceeded). No
refusion was observed in treated embryos, and the rate of no-development was
higher.


MoVB











Since cytokinesis and karyokinesis are naturally disjunct for a time in early

Nematostella development, the reciprocal experiment was to attempt to block

karyokinesis, rather than cytokinesis. Therefore, aphidicolin was applied to fertilized eggs

and cleaving embryo. This treatment blocked S-phase DNA synthesis, but not cytokinesis,

in Nematostella. Embryos treated at the 2-, 4-, or 8-cell stage, all responded by continuing

cytokinesis in the absence of karyokinesis. Aphidicolin treated embryos cleaved a

minimum of five times post-treatment. Treatment with aphidicolin resulted in embryos

with multiple chromosome-free blastomeres, as evidenced by a lack of DNA-fluorochrome

signal (Figure 4-4). Not only were chromosomes missing, but distinct nuclei were not

apparent in these blastomeres. Epoxy embedded thin sections of treated embryos revealed

blastomeres with no visible nuclei, no chromosomes, nor separation of nucleoplasm from

cytoplasm. Nomarski DIC imaging revealed no distinct nuclei, and fluorochrome imaging

revealed no distinct nuclei, thus many blastomeres were demonstrably enucleate (Fig 4-3).

The intensity of the UV-stimulated, DNA-bound fluorochrome signal did not visibly

diminish during continued cytokinesis among cells retaining DNA under aphidicolin

treatment. Observation using both Normarski DIC and DNA fluorochrome confirmed that

the blastomeres cleaved following aphidicolin treatment were of equal size, whether

enucleate or containing nuclear DNA (Figure 4-5).

















































Cytochalasin-D Treatment Results in Nematostella vectensis. Treatment of
fertilized eggs with cytochalasin-D resulted in multi-nucleate embryos, thus
mimicking the natural deferred-cleavage exhibited by Nematostella (see
Figure 3-6). Arrows point to nuclei. Treatment with Hoechst 33342 provides
confirmation of DNA at the presumptive nuclear loci. Embryo is not round
due to gentle compression to improve imaging (Nomarski and UV).


Figure 4-3.



















































Aphidicolin Treatment Results in Nematostella vectensis. Untreated embryos
stained with Hoechst 33342 displayed distinct nuclei in each blastomere
during cleavage. Even cells which were above or below the plane of focus
glowed brightly enough to identify the presence of nuclei. Aphidicolin-treated
embryos produced enucleate blastomeres, as evidenced by their lack of DNA-
fluorochrome signal (UV). Images are same scale.


Figure 4-4.






































Figure 4-5. Further Aphidicolin Results in Nematostella vectensis. Embryo was treated at
the 4-cell stage. Treatment with aphidicolin resulted in blastomeres of
relatively equal size, rather than resulting in four sequentially half-sized
blastomere classes.







73
Discussion

Normal cleavage, whether determinant or indeterminant is comprised almost

exclusively of S-phase and M-phase without G1 or G2 (Wolfe 1972). Cytokinesis occurs

only during the telophase of M-phase, under normal conditions, thus cytokinesis follows

karyokinesis in a sequential manner during non-syncitial cleavage. Syncitial cleavage

temporarily bypasses cytokinesis and thereby doubles cellular DNA content and

centrosome number for each vacated cycle of cytokinesis (Raff and Glover 1988).

Centrosomal duplication occurs concurrently with S-phase, therefore doubling in

synchrony with DNA replication (Sluder and Lewis 1987, Sluder et al. 1999). The

capacity to subdivide one syncitial cell into many, all at once, is consistent with the

research regarding cytokinesis occurring between spindle pairs (Sluder and Rieder 1985,

Sluder et al. 1999). No attempt was made to determine if the blockage of cytokinesis

induced by cytochalasin-D parallels the intracellular method of suppressing cytokinesis,

although this remains an interesting question. De-jellying of the egg mass induced no

increase or decrease in the percent of embryos displaying either form of syncitial cleavage,

and no visible effect on normal development.

Syncitial cleavage, specifically deferred-cytokinesis, is common in Arthropod

Insects, e.g., Drosophila sp. (Raft'and Glover 1988), occurs in some Crustaceans,

e.g., Macrobrachium rosenbergii (Lynn 1981), and in some Cnidarians (Gemmill 1920,

Spaulding 1974), e.g., Nematostella vectensis. This form of cleavage is generally invoked

only in large isolethical eggs as a means of rapidly subdividing a large egg volume without

the timing delay required for repeated cytokinesis (Slack 1996). Refusion, the formation

of a temporary syncitium from cleaving blastomeres, was reported in Renilla koellikeri by







74
E.B. Wilson in 1883, in Metridium marginatum (= M senile) by McMurrich in 1891 and

Gemmill in 1920, and in Edwardsia beautempsii by Wietrzykowski 1910 & 1914.

Refusion in Nematostella was reported by Uhlinger in 1997. The refusion form of

syncitial cleavage has been reported only in the Subclass Hexacorallia (Cnidaria) and may

be restricted to these organisms. It is interesting to note that of the few observations of the

existence of deferred-cleavage in Cnidaria, the most recent was by Spaulding (1974), and

to note that no research has been undertaken.

The display of refusion-syncitial development by only a subset of embryos in

Nematostella, implied that not all of the embryos are equal in all aspects, and possibly

implied that these refusing Nematostella were performing some necessary internal

reorganization of materials or structures during the period of refusion. Potential

candidates for invoking refusion in only a subset of embryos included redistribution of

cytoplasmic determinants or reserves, equalization of mechanical stresses or blastomere

volumes, reorganization of the mitotic apparatus, or adjustment of internal chemistry.

Chapter 3 has documented that Nematostella blastomeres are totipotent through the third

(equatorial) cleavage, this removed redistribution of cytoplasmic determinants or reserves,

and equalization of stresses or blastomere volumes, as likely candidates. Analysis of

spindle orientation between neighboring blastomeres during the early refusion phase is the

next target for evaluation. While Nematostella spindles were very small compared to the

size of the egg, introduction of fluorescently labeled tubulin should reveal whether all

spindles were similarly oriented in non-refusing embryos and whether those entering

refusion had spindles in different orientations in different blastomeres.







75
Cytochalasin-D treatment arrested cytokinesis, but not karyokinesis, and thereby

produced multinucleate embryos indistinguishable from natural syncitial embryos.

Release of cytokinetic arrest resulted in cleavage to single nucleus blastomeres and normal

development thereafter, thereby mimicking natural (deferred-cleavage) syncitial

development. However, we cannot claim that the actin depolymerization resulting from

the cytochalasin-D treatment is the same process utilized by the embryo to naturally arrest

cytokinesis. Research is being conducted to determine how many cycles of cytokinesis

can be vacated prior to release while the embryo retains the capacity for cleaving to single

nucleus blastomeres and proceeding through normal development. DMSO treatment alone

produced no significant effect on cleavage patterns, timing, or survival of embryos.

Embryos treated with aphidicolin continued cytokinesis in the absence of S-phase

DNA synthesis. Similar phenomenon was first reported in sea urchin embryos (Nagano et

al. 1981, Hirai et al. 1984) in apparent contradiction to prior work which reported sea

urchin embryos arresting cleavage in the presence of aphidicolin treatment (Ikegami et al.

1979). The contradiction was resolved when it was determined both research groups were

correct, and that different species of sea urchin embryos responded to aphidicolin

treatment differently. If karyokinesis of the 2N chromosomes present in pre-treatment

blastomeres occurred, haploid cells (IN) would be produced by the first cleavage, and

fractionally aneuploid cells would be produced in subsequent cytokinetic cycles (V N, /4

N, etc.) If karyokinesis of the 2N chromosomes present in pre-treatment blastomeres did

not occur, two possibilities existed. The 2N blastomere could cleave to an enucleate cell

which was incapable of further cytokinesis and a 2N cell for each cycle of cytokinesis,

thereby producing sequentially smaller enucleate blastomeres. Alternately, the 2N







76
blastomere could cleave to an enucleate cell which was capable of further cytokinesis and

a 2N cell for each cycle of cytokinesis, thereby producing all equal sized blastomeres. The

results of each pattern would be clearly discernable by epifluorescent microscopy. In the

first instance, the DNA-bound fluorochrome signal would get progressively smaller and

weaker as cytokinesis proceeded and the originally 2N chromosomes were repeatedly

subdivided. In the second instance, the number of enucleate (DNA-bound- fluorochrome-

free) cells would directly reflect the number of cytokinetic events; e.g. three cycles of

cytokinesis of an embryo treated as a zygote would produce three enucleate cells of

different size (half egg, quarter egg, eighth egg) and one (eighth egg) 2N cell. Only if

enucleate cells underwent cytokinesis would three cleavage cycles produce eight equal

cells, one with 2N DNA and seven without. Similarly, treatment of a 4 cell embryo would

only produce a morula with four visible DNA signals, if enucleate cells were repeatedly

undergoing cytokinesis in the absence of DNA replication (Figure 4-6).

No visible diminution of DNA-bound fluorochrome signal was detected, therefore,

subdivision of 2N chromosomes did not occur. Intensity of DNA-bound fluorochrome

signal has been determined to be an effective quantification of DNA present (Dresser

1993). These results confirm that karyokinesis does not occur in the absence of S-phase in

Nematostella. Equal blastomeres were generated in the appropriate number for the

number of cleavage cycles observed. Therefore, not only blastomeres possessing 2N

chromosome sets were capable of cytokinesis, but chromosome-free, enucleate

blastomeres underwent repeated cytokinesis.

















0 0oineIN
Karyokinesis proceeds


DNA signal diminishes


Cytokinesis requires
karyokinesis, DNA
signal = 1/8th of 2N


002
No Karyokinesis


Only Blastomere with
DNA Divides


Cytokinesis requires
chromosomes, blasto-
meres unequal, DNA
signal = 0 or 2N


0No Karyokinesis
No Karyokinesis


Enucleate Blastomeres Divide


Cytokinesis not dependent
on Karyokinesis, blasto-
meres equal, DNA signal =
0 or 2N


Possible Aphidicolin Treatment Results. Aphidicolin blocks DNA
replication. Repeated karyokinesis and cytokinesis will produce fractional
chromosome sets (left). Cytokinesis without karyokinesis could occur only
in nucleated blastomeres, yielding sequentially smaller blastomeres
(middle) or could occur in enuculeate blastomeres, as well, yielding equal
blastomeres (right).


Figure 4-6.







78
Thin sections of aphidicolin treated embryos showed no identifiable nucleus, and

no separation ofnucleoplasm from cytoplasm, in most blastomeres of an embryo. The

number of identifiable condensed DNA sets was consistent with the blastomere number at

treatment (e.g., treatment at the four cell stage, for three cleavage cycles prior to

preservation, would reveal either four or eight sets of condensed DNA in thirty two cells,

depending upon whether S-phase was completed prior to aphidicolin treatment). The

present evidence revealed that these blastomeres were not just achromosomal, but

enucleate. This research thus documented that achromosomal, apparently enucleate,

Nematostella blastomeres continued cytokinesis in the absence of S-phase of karyokinesis

for multiple cleavage cycles.

The continued cytokinesis of the enucleate blastomeres of Nematostella under

aphidicolin treatment was not entirely unexpected, but it was exciting. Most of the

preceding research had been conducted in Echinoderms, exclusively with sea urchins. In

as much as sea urchins were determinant following the first cleavage, Nematostella

became the first indeterminant model to display this useful trait. Prior research utilizing

aphidicolin treatments of indeterminant cells had generally resulted in cytokinetic arrest,

rather than continued cell division; this was apparently a result of using growing cell

cultures rather than cleaving embryos (Sluder and Lewis 1987). Just as with the

Echinoderm species which continued cytokinesis in the presence of aphidicolin,

Nematostella embryos arrested immediately prior to blastula formation, and the embryo

subsequently lysed. This implied that some nuclear function is requisite to blastula

formation.







79
Cytokinesis had long been considered to be a function dependent on karyokinesis

as a prior activating or authorizing event. Results of this research demonstrated that

cytokinesis can function independently of karyokinesis, and that passage through S-phase

and karyokinesis was not required for induction of or effective progress through

cytokinesis. Evidence now confirmed that cytokinesis was not a dependent pathway,

licensed by passage through an S-phase, or a checkpoint, nor was it oriented by the

metaphase alignment of chromosomes. It was an independent, albeit intimately

associated, function of the cytoplasm and its organelles. Research has confirmed that cells

undergo cytokinesis between spindles, not chromosomes, and irrespective of the presence

or absence of chromosomes (Sluder and Rieder 1985). These results imply that

centrosomal replication is occurring repeatedly post-treatment in vivo, which is consistent

with in vitro experimentation of centrosomal replication capacity (Hinchcliffe et al. 1999).

This indeterminant species unique response to aphidicolin treatment will allow

Nematostella vectensis to serve as a new and useful model for analysis of cytoplasmic

regulation of karyokinesis and cytokinesis.














CONCLUSION


Nematostella vectensis Oocytes are Meiotically Complete at Ovulation

Nematostella exhibited meiotic arrest only once, at the GV stage of prophase I.

Unlike most other Metazoans, no secondary arrest occurred when oocyte growth was

complete and meiosis was resumed. Released pronuclear oocytes rapidly became encased

in a jelly mass produced by secretary cells in the gastromesentery tissues (Uhlinger 1997).

This egg jelly mass contained many nematosomes, comprised of clusters of agglutinous

cnidae (Uhlinger 1997), presumably for the protection of the eggs.

Oocytes were present in outpockets of the macrocnemes. In mature females the

oocytes were enclosed in small discontinuous pockets herein referred to as gonadal cysts.

Oocytes developed surrounded only by their oolemmas and without follicle cells or nurse

(accessory) cells. Oocytes were contiguous to one another without intervening cells or

cytoplasmic bridges. No interdigitation of surfaces occurred between the cells of the

gonadal cyst and the surface of the oocyte, nor between adjacent oocytes. Cysts with

oocytes appeared to have a greater density of secretary cells than nearby non-gonadal

gastromesentery tissues and the majority of these cells were larger at the end directed

toward the lumen of the gut rather than toward the interior of the gonadal cyst. While no

visible phagocytotic vesicles were evident at the light microscopy level, endocytosis of

heterosynthetic nutrients was not precluded.







81
Immature oocytes displayed an enlarged GV, one or more nucleoli, no condensed

chromosomes, and an implied nuclear envelope, due to the clear distinction between the

cytoplasm and the nucleoplasm. Up to seven oocytes, that ranged between 25 gm and 220

uim diameter, occurred within one cyst. Oocyte nuclear volume comprised from ~5% to

-3% of the total cellular volume at the GV stage (decreasing in percentage as the size of

the oocyte increased). Oocytes from ovulating cysts contained no GV, no nucleoli, and no

clear distinction between cytoplasm and nucleoplasm in stained sections. No ovulating

cysts containing oocytes that lacked a GV contained more than one oocyte. Egg masses

are comprised of eggs of a uniform size range (170 u.m to 220 gm) while unovulated,

unruptured cysts contain oocytes of various sizes, rather than oocytes of uniform sizes.

Speculation would lead toward the supposition that, like some Hydrozoan Cnidarians

(Aizenschtadt 1980), Nematostella may accumulate yolk materials by fusing multiple

oocytes before becoming competent to be ovulated. Evidence for this speculation was not

observed. Whether this is the case or not remains an interesting question.

If meiotic maturation occurred after ovulation, either due to ionic activation of the

egg or by fertilization, chromosome condensation upon fertilization would provide a

dramatic fluorescent signal that the spawned egg was not pronuclear, similarly the polar

bodies ejected would be entrapped in the jelly immediately adjacent to the egg. This has

never been observed, despite hundreds of fertilization events being witnessed.

Nomarski DIC microscopy similarly provided evidence that the ovulated egg was

meiotically complete. Gentle compression of spawned eggs resulted in a plainly visible,

small, eccentrically located nucleus of-20 gm in diameter. Further evidence that the

spawned eggs were pronuclear was that, upon fertilization, no loss of distinction between







82
the small nucleus and the cytoplasm occurred (thus implying no nuclear envelope

breakdown) before the initiation of cleavage.

Nematostella was confirmed to yield meiotically complete eggs before

fertilization. This was consistent with the other known Cnidaria; Cnidaria, however, is a

very diverse Phyla, with four classes and over 10,000 species. Only a few species in two

classes have been rigorously analyzed, therefore it remains premature to assume that "all"

Cnidaria, or even "all" Anthozoans and Scyphozoans, generate fertilizable eggs in this

manner.



Nematostella vectensis Exhibited Three Patterns of Cleavage during Early Development

Normal development in Nematostella was documented as radial, holoblastic, equal

cleavage (Uhlinger 1997). Within a single egg mass, the largest subset of embryos,

usually greater than 80% of an egg mass, cleaved in this manner, and displayed the

sequential cycling of karyokinesis and cytokinesis typical of embryonic cleavage. In

Nematostella, in both the natural egg masses and among dejellied embryos, subsets of

embryos exhibited deferred-cleavage or refusion syncitial cleavage patterns. Some

embryos, therefore, deferred cytokinesis for several karyokinetic cycles before

establishing sequential karyokinesis-then-cytokinesis cycling, or revoked cytokinesis

temporarily and thereafter re-established sequential cycling; yet all pathways produced

normal planula and juveniles. In the occasional egg mass, either deferred-cleavage or

refusion syncitial cleavage patterns dominated, but never to the complete exclusion of

normal mitotic cleavage. De-jellying of the egg mass induced no increase or decrease in







83
the percent of embryos displaying either form of syncitial cleavage, and had no visible

effect on normal development.

Refusion embryos initiated normal cleavage only to coalesce into a temporary

syncitium at the 4, 8, 16 or 32-cell stage, and thereafter cleaved to the next stage, in

synchrony with those embryos that did not refuse. Refusion was displayed by <15% of a

typical egg mass, but occasionally was displayed by >60% of a mass cohort. This was

consistent with previous reports of refusion among Nematostella, which ranged from 2%

to 60% (Uhlinger 1997). Formation of a temporary syncitium between all or some of the

blastomeres had little impact on the interval period between mitotic cleavage events,

which was consistent with the prior report (Uhlinger 1997). Our data show variability that

was actually the effect of within strain variation, rather than between strain variation, this

did not confirm that cleavage variability was exclusively a function of strain variation, as

proposed by Uhlinger (1997). It remained undetermined whether cleavage variability was

effected by maturity, seasonality, stocking density, feed composition and nutrient value, or

water quality.

E. B. Wilson reported refusion of blastomeres in Renilla koellikeri, a soft coral, in

1883. He considered this early syncitium to be a normal component of this species'

development. Metridium senile (McMurrich 1897, Gemill 1920), and Edwardsia

beautempsi (Wietrzykowski 1914) are the only other species reported to display this

unusual behavior. The function served by re-fusion of cleaving Nematostella blastomeres

among only a subset of embryos in an egg mass remains an interesting conundrum.

Deferred-cleavage syncitial phase development was first reported in Metridium

senile eighty-one years ago (Gemmill 1920) and predicted in other yolky-egged sea







84
anemones twenty-six years ago (Spaulding 1974). In this present study, Nematostella are

reported to exhibit deferred-cytokinesis syncitial development (Type III) characteristics.

These embryos delayed initiation of cleavage, generating 4, 8, 16, or 32 nuclei before

cleavage to the appropriate cell number. Under a dissecting microscope, non-cleaving,

seemingly unfertilized, eggs, segmented into blastomeres and exhibited normal mitotic

cleavage thereafter. The presence of supernumerary nuclei was confirmed with a DNA

fluorochrome and epifluorescent microscopy. This deferred cleavage usually comprised

<5% of an egg mass cohort, but occasionally was displayed by >60% of a cohort. This

paper confirmed the previous reports of deferred-cytokinesis Type III cleavage in

Anthozoa, and added Nematostella vectensis to the four other species reported to do so.

Nematostella and Metridium senile remain the only two Anthozoans known to display all

three patterns of early development.

Experimental manipulations of embryos consisted of culturing individual

blastomeres from embryos, to determine whether all blastomeres from an individual

embryo were totipotent and capable of generating complete, albeit smaller than normal,

planulae and juveniles. Data revealed that Nematostella embryos indeed possessed this

capacity at the 2-cell, 4-cell, and 8-cell stage, with a dramatic reduction of the percent

survival at the 8-cell stage. Further experimental manipulations involved tracking the fate

of random clusters of blastomeres, and embryos with ablated blastomeres. This procedure

served to confirm the interactive regulation of cell fate specification among multicellular,

but incomplete, embryos. While all partial embryos demonstrated some capacity to

survive to the juvenile stage, the smaller the fragment, the higher the rate of mortality.

This blastomere-cluster survival data confirmed that Nematostella embryos exhibited no







85
Determinant/Type I development, and that they did not become determinant after the first

equatorial cleavage as Sea Urchin embryos did.

Nematostella clearly demonstrated Type II indeterminate cleavage. A tremendous

resiliency of cell fate specification was found. Through the third cleavage Nematostella

blastomeres remained uncommitted and fully capable of producing a functional

metamorphosed juvenile. Nematostella embryos were also capable of exhibiting two

distinct forms of Type III cleavage: both deferred cytokinesis and refusion; why they did

this remains a mystery. Nematostella vectensis joined the select group of organisms,

currently comprised of four Actinarians and the Sea Urchin (but not other Echinoderms),

that fail to fall easily under either the classical or the modem theoretical construct for

categorizing early development. If the rubric, "The exceptions make the rule" holds, then

clearly further work needs to be done on the modem theoretical construct for specification

of cell fate in early development.



Cytokinesis is Independent from Karyokinesis during Early
Development in Nematostella vectensis

In this dissertation, we demonstrated that cytokinesis can be blocked, creating a

syncitium, and thereafter released, which resulted in normal development; this mimicked

embryos that followed natural syncitial (deferred-cleavage or refusion) development.

Secondly, we demonstrated that blocking S-phase of karyokinesis resulted in continued

cytokinesis which produced relatively normal appearing embryos composed

predominantly of enucleate cells. Thus, in the early development of Nematostella,

cytokinesis functioned independently of S-Phase and karyokinesis. Due to the corollary

evidence provided in the literature, it was predicted that the centrosomes and/or the







86
centrosomal axis are solely responsible for determining the plane of cell division, and that

there were no critical checkpoints for cytokinesis during S-phase or karyokinesis.

Normal cleavage is comprised almost exclusively of S-phase and M-phase without Gi or

G2 (Wolfe 1972). Cytokinesis normally occurs only during the telophase of M-phase, thus

cytokinesis usually follows karyokinesis in a sequential manner. Syncitial cleavage

temporarily bypasses cytokinesis and thereby doubles cellular DNA content and

centrosome number for each vacated cycle of cytokinesis (Raff and Glover 1988).

Centrosomal duplication occurs concurrently with S-phase, therefore doubling in

synchrony with DNA replication (Sluder and Lewis 1987), but can be achieved in vitro in

the absence of S-phase (Hinchcliffe 1999).

Cytochalasin-D treatment arrested cytokinesis, but not karyokinesis, and thereby

produced multinucleate embryos indistinguishable from natural syncitial embryos.

Release of cytokinetic arrest resulted in cleavage to single nucleus blastomeres and normal

development thereafter, thereby mimicking natural (deferred-cleavage) syncitial

development. DMSO treatment (0.1% in 15 ppt NSW) alone produced no significant

effect on cleavage patterns, timing, or survival of embryos.

Embryos treated with aphidicolin continued cytokinesis in the absence of S-phase

DNA synthesis. A similar phenomenon was first reported in sea urchin embryos (Nagano

et al. 1981, Hirai et al. 1984) in apparent contradiction to prior work that reported sea

urchin embryos arresting cleavage in the presence of aphidicolin treatment (Ikegami et al.

1979). The contradiction was resolved when it was determined that different species of

sea urchin embryos responded to aphidicolin treatment differently. Treatment of fertilized

Nematostella eggs, resulted in cleavage to equal sized blastomeres, with only one







87
blastomere having a DNA fluorochrome signal, treatment of a 4 cell embryo produced a

64-cell morula with four visible DNA signals. Only if enucleate cells underwent

cytokinesis would three cleavage cycles produce eight equal cells, one with 2N DNA and

seven without. These results confirm that karyokinesis did not occur in the absence of S-

phase in Nematostella. Equal blastomeres were generated in the appropriate number for

the number of cleavage cycles observed. Thin sections of aphidicolin treated embryos

showed no identifiable nucleus, and no separation of nucleoplasm from cytoplasm, in

most blastomeres of an embryo. Therefore, not only blastomeres possessing 2N

chromosome sets were capable of cytokinesis, but chromosome-free, optically enucleate

blastomeres underwent repeated cytokinesis in the absence of S-phase and karyokinesis

for multiple cleavage cycles.

The research that first reported achromosomal cleavage as a result of aphidicolin

treatment was conducted exclusively with sea urchins (Nagano 1981, Hirai 1984). In as

much as sea urchins were determinant following the first cleavage, Nematostella became

the first indeterminant model to display this trait. Prior research utilizing aphidicolin

treatments of indeterminant cells has generally resulted in cytokinetic arrest, rather than

continued cell division; this was apparently a result of using cell cultures rather than

cleaving embryos (Sluder and Lewis 1987). Just as with the Echinoderm species which

continue cytokinesis in the presence of aphidicolin, Nematostella embryos arrested

immediately before blastula formation, and the embryo subsequently lysed. This implied

that some nuclear function is requisite to blastula formation.

Cytokinesis has long been considered to be a function dependent on karyokinesis

as a prior activating or authorizing event. Results of this research demonstrated that







88
cytokinesis can function independently of karyokinesis, and that passage through S-phase

and karyokinesis was not required for induction of or effective progress through

cytokinesis. Evidence now confirms that cytokinesis is not a dependent pathway, licensed

by passage through an S-phase, or a checkpoint, nor is it oriented by the metaphase

alignment of chromosomes. It is an independent, albeit intimately associated, function of

the cytoplasm and its organelles. Research has confirmed that cells undergo cytokinesis

between spindles, not chromosomes, and irrespective of the presence or absence of

chromosomes (Sluder and Rieder 1985). These current results implied that centrosomal

replication was occurring repeatedly post-treatment in vivo, which was consistent with in

vitro experimentation of centrosomal replication capacity (Hinchcliffe et al. 1999). This

indeterminant species' unique response to aphidicolin treatment will allow Nematostella

vectensis to serve as a new and useful model for analysis of cytoplasmic regulation of

karyokinesis and cytokinesis.














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BIOGRAPHICAL SKETCH


I have loved the ocean and all things biological since my first fishing trip when I

was four years old. I passed my youth happily mucking about in Liverpool, on the

Hudson River, and then on Tampa Bay. I received my Bachelor of Science degree in

Biology in 1971, from Florida State University, and received my Master of Education

degree in Science Education in 1977, from Florida Agricultural & Mechanical

University.

I designed and constructed one of the first high school fish farms in Florida, the

"Aqua-Garden" at the School for Applied Individualized Learning in Tallahassee in

1982. As a result of our successes, and the accompanying publicity about "fish-farming

teens with spiked-hair and nose-rings," I was honored as Leon County Teacher of the

Year in 1984.

I started Fossil Fish Farm in 1982, and marketed Grass Carp throughout North

Florida. In 1983, I was selected for the Governor's Interim Aquaculture Coordinating

Committee, whose industry group formed the Florida Aquaculture Association. In 1985,

I was elected as Secretary on the Board of Directors of the Florida Aquaculture

Association, and held this post with one hiatus until 1996.

In 1988, I was employed as an Assistant Professor to be the Florida A&M

University Aquaculture Extension Specialist. I designed and operated FAMU's

aquaculture facility, conducting research on polyculture of baitfish with foodfish as a







95
means of increasing small farm profitability. I designed a low-cost air-lift-driven self-

contained recirculation system and received funding from the University of Florida's

Center for Cooperative Agricultural Programs, to install three of these systems on small

farms and a larger mock-up at the FAMU REC in Quincy. One of these "hog-wire and a

liner" tanks has been operating non-stop for five years now, producing ornamentals, and

the larger FAMU unit is still producing catfish effortlessly.

Late in life, decided that what I really wanted to do was fundamental research

related to aquaculture. I was accepted by Dr. Clark at the Department of Fisheries and

Aquatic Sciences in 1997. My plan was to focus on sturgeon, but Dr. Clark introduced

me to the sea anemone, Nematostella vectensis, and encouraged me to use this animal as

a model to explore the fundamental functions of cell division. I am forever in his debt.

This nondescript little animal is fascinating, and has much to teach us regarding cell

biology. Nematostella is also a close relative of the Corals, and future studies may

eventually lead to induced spawning of Corals for reef restoration and production of

"Live Rock" for the aquarium trade. They may prove useful as a bioassay organism for

estuarine environments soon, as well.

It should be evident that I intend to continue to work with this animal for many

years to come. I am now trained both as a developmental biologist and as an aquaculture

extension professional. I will endeavor to apply my new expertise in useful and profound

ways. I will strive to make the University of Florida and the Department of Fisheries and

Aquatic Sciences proud.