Formation and Localization of Cytoplasmic Domains in Leech and Ascidian Zygotes

Inl. J. D<\. lIio!. ~!: 1075-1U8~ 119981

Formation and localization of cytoplasmic domains in leech and ascidian zygotes

JUAN FERNANDEZ", FABRICE ROEGIERS2, VIVIANA CANTILLANA' and CHRISTIAN SARDET2 rDeparramento de 8;010g(8, Facu/rad de Ciencias, Universidad de Chile, Santiago. Chile and 2Unire de 8iologie Cellula ire Marine URA 671 CNRS/Universite Pierre et Marie Curie, Observatoire Station Zoologique, Villefranche~sur-Mer, France

CONTENTS

Introduction u .." ...... 1075 Leech and ascidian oocytes. zygotes and embryos ...... 1076 Formationand localization of cytoplasmic domains. 1078 Fertilization and meiotic events Pronuclear movements and interphase events Mitotic events and the partitioning at cytoplasmic domains Mechanisms and perspectives . "...... 1080 Fertilization and early meiotic events Late meiotic events Interphase and mitotic events Summary ...... 1082

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Introduction coming from Drosophila embryos, has shown that determinants can be heterogeneously distributed macromolecules whose location Most eggs display an heterogeneous distribution ot organelles may be or may not be necessarily coincident with microscopically and cytoskeletal elements established during oogenesis and/or identifiable structures (Davidson. 1986: St Johnston and Nusslein- aher tertilization and subsequent cell cycles (see Capco, 1995). Vol hard. 1992: Nusslein-Volhard, 1996). The best example of a Heterogeneities are easily visualized in eggs that have microscopi- determinant associated with a structure is that of the oskar mRNA in cally discernible cytoplasmic domains due to HH::!accumulation of Drusopflila. As a result or cytoskeletal reoryalli.latiorls, oskar mRNA organelles and sometimes pigmented vesicles or granules is normally present in a distinct cytoplasmic domain known as "germ (Fernandez. 1980: Sardet et al" 1994). These distinct cytoplasmic plasm". Ectopic localization of this mRNA promotes the appearance domains were first perceived a century ago in eggs of protostomes of germ plasm and functional pole cells in ectopic regions of the and deuterostomes, including those of leeches, sea urchins, blastula (Ephrussi and Lehmann. 1992). Except for germ plasms. ascidians, molluscs and insects (Whitman, 1878: Boveri. 1895: which exhibit common structural and biochemical features in egg Conklin. 1897,1905: Wilson, 1925). The presence of naturally-marked cytoplasmic regions has al- --- lowed cell lineage analysis and the demonstration that cell fate ,\/Jl'r/,'iflli(J//J rw'ri ill rhjl 1m/wI: .\T. .mim,tl Ic'ltlpl.l,m: (;"\1\. rt'1111tl,nnw- ;HI,IClil1~ hod\-; <:H, dIlHloT1; <:R. Ctll1lranioll lil1~; (:Y. nt.I,lc'T: fR, depended on the presence or absence of particular domains. Early ('ndt'ptHlI1il" l'l'UCllhll1l; FJ.:. It'II1.llt" kan(ll1It..-e~: (;S, gr.n" 'lit II: 111' P:I, observations formed the basis of a theory of "cytoplasmic localiza- irHI'ilullrillhll'phau': K,\I, J..antHII('Tt"; \IH,lIwridillllal 1I.1I1I\'1If flllllr;lrtinl1: tion" (Wilson. 1925). It was gradually recognized that visible as well :'IIF. IIIOt1,I'[t"r fiber'\: \11. tir~1 lIu'I,ll'ha'>t" olllleio,i,= \In. mOIl.',I('r; \11', as invisible cytoplasmic regions might contain 'determinants' that 1II.lle prulIIl1lc"us: \IS, meiotiC' 'pilUllt'; PI\,lir'l pCII.\! hl,(h; PI',pt'linudt,'u" caused the cells thHt inherited these domains to follow different p].I'1II dom.till: PR. pu].lr riuj,{: ~C. 'penn ll'l1lru:'UlIIl': :0.:'\. SPt'rIt1lll1ch:u': differentiation programs. In the last 2 decades evidence, principally \"1". \'eK"lallt'lopla'll1: Z:\. I~~oll' Illlclt'll',

---- .Address for reprints: Departamento de Biologla. Facultad de Ciencias, Universidad de Chile, Casil1a 653, Santiago, Chile. FAX: 56.2 271 2983. e.mail: [email protected]

021 ~-6!82/98/S I0.00 ~ l'IH"'l'r~\\ f'rinlc.JmSp4in 1076 J. Femdnde: el al.

from a wide variety of species (electron dense RNA-rich granules acrosome and flagellum as well as the single sperm mitochondrion immersed in a "nuage" of mitochondria, see Rango and Lehman, are found nearby (Fernandez ef al., 1994). In rare cases polyspermy 1996), other recognizable cytoplasmic domains exhibit a widely occurs, and sperm nuclei are found at the periphery of the egg in both different structure, compositionand localization(Sardet et al., 1994; hemispheres. Capco, 1995). This is reflected in the various names given to them Study of the embryology of leeches goes back to Whitman (myoplasm, pole plasm, teloplasm). Yet common mechanisms must (1878,1887), who was the first investigator to attempt cell lineage be at work in the formation, positioning and maintenance of these analysis using glossiphoniid leech embryos. Embryonic develop- domains. Cytoskeletal elements (microtubules, microfilaments and ment in leeches was first considered to be essentially determinate possibly intermediate filaments) have been implicated in the progres- and to rely on invariant and independent cell lineages (Mori. 1932). sive formation, positioning and relocation of these domains with More recently it was shown that development of some cell lines respect to the embryonic axes (Sardet et al.. 1994; Fernandez and also depends on cell interactions (Blair and Weisblat, 1984; Olea, 1995; Etkin, 1997; Gard el aI., 1997). There is also increasing Shankland, 1987) and that indeterminacy and transtating take evidence that cytoskeleton-associated motors, and their membrane place under certain experimental conditions (Shankland and receptors, are a major factor in the transport, anchorage and possibly Weisblat, 1984; Weisblat and Blair, 1984). Like many protostome in the function of mRNAs and other determinants (Glatzer and embryos, the leech embryo undergoes spiral cleavage. A series of Ephrussi, 1996; Bassell and Singer, 1997; Gavis, 1997; Goodson el unequal stereotyped divisions results in blastomeres of different al., 1997). size, position, structure and fate. These give rise to precursor cells Leech and ascidian eggs were among the first cells in which whose descendants can be unambiguously identified (Fernandez formation of microscopically visible domains were associated with and Olea, 1982; Weisblat el.aI.., 1987). A particularfeature of leech cell fate determination (Whitman, 1878; Conklin, 1905). In this development is that the ectoderm and mesoderm are produced by review, we compare the nature of the cytoplasmic domains and the a discrete number of stem cells (telobiasts), located at the caudal mechanisms responsible fortheir localizations in leech and ascidian end of the embryo, that inherit particular cytoplasmic domains zygotes. We also consider their importance for development. (teloplasms) (Fernandez, 1980; Fernandez and Stent, 1980). Recent reviews, books and papers have appeared that deal with These domains, originally located at both the animal and vegetal these and other species (Sardet el. al., 1994; Capco, 1995; King. poles of the zygote, are produced through a series of cytoplasmic 1996; Pilon and Weisblat, 1997). rearrangements that will be described and discussed in this review. Another interesting feature of leech embryonic development deals Leech and ascidian oocytes, zygotes and embryos with segmentation. Leeches have a constant number of segments (32) with an additional non-segmented prostomium. The leech Leeches are protandric annelids that lay cocoons containing embryo is a particularly convenient model for the study of the from a few to several dozen eggs fertilized in the ovaries. Diameter cellular and molecular events involved in segment determination of laid zygotes ranges from about 100 ~m (Hirudinid leeches) to and organization (Fernandez and Stent, 1980; Weisblat el. al." several mm (Glossiphoniid leeches). Some species breed several 1988,1994; Shankland el al., 1991). times a year (Hel.obde/./a) while others (Theromyzon) breed only Ascidians are hermaphroditic marine invertebrates once in their life and then die. The hirudinid leech Hirudomedicinalis (urochordates. tunicates) with a small genome. Most species and the glossiphoniid leeches Hel.obde/./a robusta, Hel.obde/./a exhibit indirect embryonic development but a few species develop triserialis and Theromyzon rude have been used for developmen- directly (Satoh, 1994). Most ascidians develop rapidly into simple tal studies at cellular and molecular level. swimming tadpoles whose organization is typical of the chordate Atthe beginning of the breeding season, when leeches are males, embryo. Within days of hatching, tadpole embryos attach to the the animals copulate. The transmitted sperm is retained inside the substrate and metamorphose into a reproductive sessile adult. body for periods of weeks to months. The animals then become Ascidians are deuterostome organisms characterized by stere- female and produce mature oocytes that are fertilized internally otypic bilaterally symmetrical cleavage divisions that produce a before proceeding to first metaphase (MI) of meiosis. As soon as the blastula with a relatively small number of cells in a few hours. The zygotes are laid, development is initiated. In some leeches fate of these blastomeres, up to the tadpole and juvenile stages, (Theromyzon) development is direct while in others (Hirudo) it is has been partly or completely traced in Ciona intestinalis, Phallusia indirect, giving rise to a cryptolarva. Development to sexual maturity mammil.lata, Sl.yel.a partil.a and Hal.ocynthia roretzi (Zalokar and can take weeks or months (Fernandez et al., 1992). in the laid zygote Sardet, 1984; Venuti and Jeftery, 1989; Nishida, 1992,1996,1997). one can clearly distinguish an acellular chorion, a thin layer of In addition, study of closely related interhybridizing aseidian spe- peripheral cytoplasm deficient in yolk (ectoplasm) and a core of yolk ciesthat develop into tadpole larvae with orwithout tails has yielded platelet-rich cytoplasm (endoplasm) (see Panel A, Fig. 1). Except for novel information about differentiation of embryonic tissues (Swalla the animal pole region which contains a clear area occupied by the and Jeftery, 1990; Swalla et al.., 1993). meiotic spindle, and the presence of larger yolk platelets in the Ascidian oocytes can be fertilized internally or externally de. vegetal hemisphere, the newly-laid zygote lacks distinct identifiable pending on the species. The sperm undergoes an acrosomal cytoplasmic domains. A network of microfilaments is present across reaction as it goes through an acellular chorion lined with follicle the entire ectoplasm, whereas microtubules seem to be restricted to cells. This layer can be chemically removed to study fertilization the meiotic spindle (Fernandez et al.., 1987). When the zygote is laid, and embryonic development (Zalokar and Sardet, 1984). Oocytes, the nucleus derived from the fertilizing sperm lies at the periphery of ranging in diameter from about 150 ~m (Ciona, Pha/./usia, Styel.a) the animal hemisphere, often near the animal pole. The sperm- to 300 ~m (Halocynl.hia), can be obtained in large quantities in derived centrosome has not yet grown astral microtubules and the appropriate seasons or throughout the year. They are usually screw-shaped nucleus contains condensed chromatin. The naked arrested in MI of meiosis ready to be fertilized. Leech aud (/scidian :ygoles 1077

Panel A: Leech. Figs. 1-12. Cytoplasmic reorganizations in the leech Theromyzon rude from oviposition to first cleavage. With the exception of Figures 7 and 9, each figure represents a mendional section of a zygote. In all figures rhe animal pole of the zygote is up. Figures 1-3 show D zygotes in the first meiotic cell cycle, figures 4 and 5 zygotes in the second meiotic cell cycle, figure 6 a zygote termrnatlng early first interphase. figures 7 and 8 zygotes in mid-interphase. figures 9and 10 zygotes m late first interphase and figures 11 and 12 cleaving zygotes, TImes of development at room temperature for these ddferent stages are mdlcated below. 1. Structure of the zygote at '" the time of laying, The layer of peripheral cytoplasm (ectoplasm) contams numerous microfilaments as well as organelles (represented here by mltochondnal. CH. chorion; MS. meiotic spindle; SC, sperm centrosome; SN. sperm nucleus, 2. Formation of the gray spot (about 0, 5 h of development), The developing spIndle has " , rotated about 90<>and its peripheral pole, tethered to the future / " ..... animal pole. forms the gray spot (GSi. 3. Contraction nng dunng " meiosIs I (about 15 h of development). The contraction ring (Cm, moving towards the animal pole (arrows), is composed of numer- ,., " ous actin filamenrs as well as organelle-nch ectoplasm. 4. Monaster -~-' t; formation and establishment of an animal pole cytoplasmic domain (about 2 h of development). The first polar body (PB) has been t.I 1:1 released and the sperm centrosome onglnated the nascent monaster (MO), that appears associated wIth the male pronucleus (MP). , The ectoplasm formed a cup-shaped cytoplasmic domain in the " animal pole region. 5, Monaster centratlon, formation of the pennu- '" clear plasm domam and release of the second polar body (about 2.5 h of development). The sperm centrosome and male pronucleus have reached the center of the zygote and the monaster microtubule bundles now e.\tend throughout the Internal cytoplasm (endoplasm). CY, cytasters: FK, female karyomeres; PP, pennuclear lID w plasm domain. 6, Formation of the amphidiaster, centratlon of the female karyomeres and thickening of the ecroplasmlc layer (about 3 h of development) The pennuclear plasm domain, that encloses

the amphidiaster, has grown Into a disk-shaped structure In the center of the zygote, The decondensmg female karyomeres. to- ChrO/l/(!",mlt"1 . .Hicwwl>ulf'l gether wirh organelle-nch cytoplasm. have migrated from the .\lrtl',"/r/>II,Jlia ,\IICl'llfililmnrr\ animal pole along a subset of monaster fibers (MF). Note that the thickened ectoplasm includes numerous organelles and cytasters. 7. Surface view of a zygote at the peak of furrowing (about 3.5 h of development). The zygote adopts a pumpkm-like appearance due to formation of annular furrows (polar rings. PR) and mendlonal bands of contractIOn (MB), This is the most common furrowing pattern detected dunng mid inrerphase. Organelle-nch ectoplasm. actm microfilaments and microtubules appear concentrated at the wallof the furrows. The discontinuous Ime marks the animal!vegetala'(is and corresponds to the plane of the mend,onal sectton displayed m Figure 8,8, Accumulation of ectoplasm at the wall of the polar nngs and mendional bands of contraction. This drawing illustrates the Internal structure of the zygote in Figure 7 viewed In a meridional section not passmg through the tipS of the furrows. NotIce that the female karyomeres have completed thelf centration and lie adjacent to the male pronucleus at the center of the pennuclear plasm domam. Meanwhile. the subset of microrubules assocIated with the migration of the female karyomeres have started shortemng towards the animal pole. PRo polar nngs 9 Surface view of a zygote showmg poleward displacement of the polar rings and "shorrenmg" of the meridional furrows (about 4 h of development). ThIs moddlcation of the furrowmg pattern pnor ro mitosis (arrows mdlcate the poleward displacement of the rings and bands of contraction) ISaccompanied by bipolar displacement of organelle-nch ectoplasm, actm microftlaments and mlcrotubules, 10. Complerlon of teloplasm formation (about 4,5 h of developmenr) Furrowing has ceased and cytoplasm enriched in organelles and cytoskeletal elements accumulate at the poles of the zygote to form the animal (A T) and vegetal (Vn teloplasms. The zygote nucleus (ZN) has formed at the center of the dumbbell-shaped perinuclear plasm domain. 11. First cleavage division (about 5 h of development!. The mitotic spindle has a large aster located in the CD sector and a small aster located in the AB sector of the zygote. Astral micro tubules penetrate mto the substance of the teloplasms and reach the zygote cortex, Karyomeres (KM) formed at the end of the first cleavage divIsion appear close to the poles of the cleavage spmdle. 12. The f"st two blasromeres (about 6 h of development). The cleavage furrow has divided the zygote unequally and the large CD blastomere has mheored most of the animal and vegetal teloplasms and the large aster The enlarged pennuclear plasm (compare wirh Fig. 5). however. has divIded symmetncally and thus the two blastomeres inherit similar amounts of this cytoplasmic domain.

Experimental embryology of ascidians, dating back to the days reorganization (ooplasmic segregation), which are described and of Chabry (1887) and Conklin (1905), showed that precursor discussed in this review. In recent years these morphogenetic blastomeres of muscle cell lineages contained cytoplasmic do. movements. and the developmental potential of parts of zygotes mains. or plasms (myoplasm), inherited from particular regions of and embryos, have been analyzed rigorously (Bates and Jeffery, the zygote. These domains are established before or afterfertiliza- 1987; Nishida, 1994,1996,1997; Sardet et a/., 1994; Yamada and lion as a result of two main episodes of cytoplasmic and cortical Nishida, 1996). These studies, complemented with the analysis of 1078 J. Fl'I"lldl1ll1'~ 1'1al.

early expression of certain genes (including homologs of verte- concentration of a subcortical mitochondria-rich region (the brate genes), have revealed that the differentiation of some cell myoplasm) in the vegetal hemisphere. The myoplasm is a thin types (such as primary muscle cells) is highly determinate while subcortical layer that forms during oocyte maturation (Swalla el al., that of other cells (such as notochord cells) relies on cell interac- 1991). It is readily observed in living eggs of some species (Styela) tions (Satoh el al.. 1996; Corbo el al" 1997). The composition. because italso contains naturally pigmented (yellow/orange) vesi- cleavage pattern and lineage of all the cells in the early embryo are cles (Conklin, 1905; Jeffery and Meier. 1983) and is bestvisualized now known in detail, reinforcing the potential of the ascidian using fluorescent dyes that accumulate in mitochondria (Sardet et embryo as a modeltounderstandearly ontogeny of deuterostomes al., 1989). The cortical contraction moves the introduced sperm at a molecular, cellular and physiological level (Nishida, 1997). nucleus and centrosome vegetaily (Speksnijder elal., 1989).lIalso creates a new cytoplasmic domain sandwiched between the Formation and localization of cytoplasmic domains myoplasm and the microfilamentous cortex and microvilli-rich surtace of the contraction pole (Speksnijder et al.. 1993). This new Fertilization and meiotic events cytoplasmic domain consists of numerous sheets and tubes of In the leech, meiosis is initiatedinthe ovary and is completed endoplasmic reticulum (ER) (Panel S, Figs. 2 and 3). In both 150 to 180 min aher egg laying (Panel A, Figs. 1-5). Although the ascidian and leech zygotes the end result of these microfilament- sperm has already penetrated the egg when it is laid, the zygote driven cortical contractions is the redistribution of cell components stored in the leech body remains arrested in M I of meiosis. The such as mitochondria, cytoskeletal elements and probably many zygote only resumes meiosis once out of the body (Fernandez and associated organelles with respect to the future position of the Olea, 1982). The first detectable sign that meiosis has resumed is embryonic body axes. In leeches the contraction moves symmetri- the reorientation of the meiotic spindle, such that its long axis cally, with respect to the animal/vegetal axis, towards the animal becomes perpendicular to the surface of the animal pole. Mean- pole whereas in ascidians the contraction moves vegetally but may while, the peripheral pole of the meiotic spindle together with its be asymmetrical with respect to the preexisting animal-vegetal astral microtubules have approached the zygote surface where axis (Sawada and Osanai, 1985; Sardet el al" 1989; Jeffery. 1995; they form the gray spot (a lighter area that will constitute the animal Roegiers el al.. 1995). pole) (Panel A, Figs. 1 and 2). This phenomenon is blocked by When thefirst polar body is emiNed in the leech zygote (after about colchicine and hence depends on microtubules (Fernandez et al., 90 min of development), small cytaslers with short microtubules are 1990). The gray spot becomes detectable about 30 min after already detected in the ectoplasm. Cytaslers are small microtubule zygote laying (Fernandez, 1980). An hour later a cell constriction, organizing centers also found in the cytoplasm of zygotes from other the contraction ring, appears at the equator and progresses toward species (see Harris el al.., 1980; Maro el al" 1988; Fernandez el a/., the animal pole at a speed of about 30 ~m/min (Panel A. Fig. 3). 1994). Shortly aher emission of the first polar body, bundles of long Concomitantly, the animal pole surface begins to bulge up in microtubules (astral fibers) grow from around the centrosome pro- anticipation of first polar body formation. Formation and displace- vided by the fertilizing sperm (panel A. Fig. 4). As the astral fibers ment of the contraction ring is inhibited by cytochalasin B, indicat- grow longer, the centrosome together with the sperm nucleus, ing that these processes involve actin filaments. Examination of the acrosome, mitochondrion and flagellum move toward the center of contraction ring reveals that it contains not only a high concentra- the zygote (Panel A, Figs. 4 and 5). A large monaster develops while tion of actin filaments, but also microvilli, granular material, vesicles the spiral-shaped nucleus unwinds and its chromatin begins to and numerous mitochondria as well as other organelles, all of decondense (Fernandez et al., 1994). Aher 150-180 min of develop- which move towards the animal pole. Hence, poleward translation ment, a transient deformation of the vegetal pole accompanies the of the meiotic contraction ring is coupled to both emission of the first emission of the second polar body (Fernandez el al" 1987). This is polar body and segregation of plasmalemmal domains. the actin followed by formation of karyomeres, enclosing the female post- cytoskeleton and organelles. This is the first episode of ooplasmic meioticchromosomes, in the thickenedectoplasm underlying the segregation in the leech zygote (Fernandez el al., 1990). The animal pole (Panel A, Fig. 5) (Fernandez and Olea, 1995). cytoplasmic domain thus created appears as a distinct disk- By comparison, completion of meiosis in ascidians is character- shaped region beneath the animal pole (Panel A, Fig. 4). ized by periodic contraction-relaxations of the zygote surface in Ascidian eggs, which are also generally arrested in MI phase of synchrony with periodic calcium waves. These waves are initiated meiosis at the time of fertilization (Halocynthia, Styela, Ciona, in the vegetal pole area from the ER-rich domain of the zygote and Phalfusia), also undergo a characteristic cortical contraction which propagate cortically towards the animal pole (Speksnijder el al., is dependent on actin filaments (Jeffery and Meier, 1983.1984; 1990; McDougall and Sardet, 1995). The monaster generated Sawada and Osanai, 1985; Sardet el al., 1989; Roegiers et al., around the sperm-derived centrosome grows slowly in the zygote 1995). II is the fertilizing sperm, that preferentially penetrates the periphery between the first and second meiotic metaphase (Panel egg in the animal hemisphere, what triggers the cortical contraction S, Figs. 4,5 and 6). The zygote then rounds up and the second polar (Panel S, Figs.l and 2). This contraction starts close to the site of body is emitted. Depending on the ascidian species being consi- sperm entry and progresses towards the vegetal hemisphere at a dered, this process is completed after 20 to 40 min of development speed of about 80 Ilm/min, forming a characteristic microfilament (Sawada and SchaNen, 1988; Sardet el al., 1989). At the end of and microvilli-rich protrusion called the contraction pole (Panel B, meiosis, a large subcortical monaster is found in the vegetal Fig. 3). The progress of the corticalcontractionin ascidian zygotes hemisphere of the zygote where the sperm centrosome has been clearly depends on a calcium wave that traverses the cell at a dragged by the contraction wave (Sardet el al., 1989; Roegiers el speed of 5 Ilm/sec fromthe sperm entry site (Speksnijder et al., al., 1995). As the astral rays reach the animal pole, the female 1990; McDougall and Sardet, 1995; Roegiers el al., 1995). In chromosomes are enclosed in karyomeres and a nuclear envelope ascidians, this firstphase of ooplasmic segregation leads to the forms around the decondensing sperm chromatin. The female Leech and asddian ;ygoles 1079

Panel B: Ascidian. Figs. 1-12. Cytoplasmic reorganizations between fertilization and first cleavage division in the zygote of the ascidian Phal/usia mammillata. All figures are meridional sections of the egg along theammal/v'egetal axis except for Figure 11 (equatorial section of an egg in mitosis) and Figure 12 (oblique surface vlewof a cleaving egg seen from the postenor pole). Only cortical and subcortical cytoplasmic domains and large cytoplasmic structures (asters. spmdles, nuclei) are represented. 1. fgg at the time of fertilization. A cortical basket of actin mlcrofilaments, sheets and tubes of fR is sandwiched between the plasma membrane and a mitochondria-rich ,., . . domain called the myoplasm (this domam also contams vesicles and a low density of fR which are not indicated). The meiotic spindle (MSJ of the egg arrested in metaphase of the first meJOtlccell cycle, lies at the penphery and parallel ro the egg surface. The basket of E.J 111 actin microfilaments opens towards the animal pole 2. Sperm penetraTion. The sperm penetrates preferentially m the animal hemisphere, introducing Its centrosome and nucleus (5). The entering sperm triggers a calcium wave which causes the cortical mlcrof,lament basket to contract. The contraction nng (CA) propa- gates vegetally (arrows) draggmg with It the mitochondria-rich . . myoplasm 1M) as well as the sperm nucleus (SJ and adjomlng centrosome. 3. Formation of the contraction pole (5 mm after fertilization). A contraction pole (CP) forms in the vegetal hemisphere. It is characterized by numerous microvilli, actm ID 1:1 mlcrofilaments and abundant sheets and tubes of ER. The sperm chromosomes and centrosome have moved vegeta/ly with the contraction wave. 4. Completion of the first meiotic cell cycle (10 mm after fertilization). The first polar body (PB) has been emitted The contraction pole has resorbed but an accumulation of cortical fR perSists m the vegetal hemisphere. A monaster (MO) develops around the introduced sperm centrosome. 5. Oscillation period dunng meiOSIS (10-20 min after fertilization). The zygote exhibits periodical contractions and relaxations as 10 to 20 calcium waves w If.! propagate rhythmically from the site of the vegetal/y-Iocalized contraction pole. Monaster microtubules grow in length. 6. Com- pletioll of the second meiotic cell cycle (20-25 min after fertilization). Completion of meiosis is charactenzed by emisSion of the second polar body and formation of the female karyomeres (FK). The Chronw.wmu . monaster begms to grow rapidly throughout the mtenor of the . .\'Ur:l~l4J ,\"c"ifil

karyomeres then move along the astral rays towards the centro- microtubules in the centripetal accumulation of organelles around some and sperm pronucleus (Panel S, Figs. 6 and 7). the leech centrosome to form this domain. Interestingly, active In both leech and ascidian zygotes a large number of organelles gathering and proliferation of mitochondria are both involved in the gather around the sperm centrosome and derived monaster. In growth of the perinuclear plasm (Fernandez et a/., 1994). In leeches, this accumulation constitutes the so-called 'perinuclear ascidians the sperm monaster gathers ER and a wedge-shaped plasm" domain (Panel A, Fig. 5). Drug studies have implicated domain of the mitochondria-rich myoplasm (see Panel S, Fig. 6). 1080 1. Fern,;nde: et (/1.

Itshould be noted that in the ascidians PhalJusia and Cionaa small h of development) (Panel A. Figs. 7 and B) (Fernandez et al., 19B7). microfilament-and-ER-rich protrusion, the "vegetal button", forms When meridional furrows form, organelles move in the plane of the transiently at this time at the site where the contraction pole first ectoplasm and accumulate within the walls of the rings and bands of formed. The vegetal button appears at the time the second polar contraction. Actin filaments have been implicated in these processes body forms and subsides about 10 min later (Panel B, Fig. 6) (Fernandez et aI., 199B). The poleward movement of the contraction (Sardet et al., 19B9: Roegiers et ai, 1995). rings and meridional bands of contraction, during late first interphase (about 4 h of development), is accompanied by a massive bipolar Pronuclear movements and interphase events translocation of organelles and cytoskeletal elements (Panel A, Fig. After the completion of meiosis in the leech, the female 9). This leads to the accumulation of organelles, microtubules and karyomeres coalesce andtogetherwith the surrounding organelle- actin filaments at both zygote poles and the establishment of the new rich cytoplasm start moving centripetally toward the zygote center cytoplasmic domains called teloplasms (Panel A, Fig. 10) (Fernandez (Panel A, Fig. 6). This movement occurs along a subset of and Olea, 1995). Drug treatment indicates that these movements monaster microtubules that link the animal pole to the central towards the poles depend on both microtubules and actin filaments. perinuclear plasm domain. These animalpalata center microtubules Teloplasm formation, like the previous expansion of the perinuclear are more stable than the other astral microtubules and persist for plasm domain, involves both recruitment and proliferation of mito- a longer time. As the karyomeres move along these microtubules chondria (Fernandez et al.. 199B). The teloplasms are cytoplasmic towards the zygote center, they start fusing and their chromatin domains destined to be inherited by large caudally-situated embry- decondenses. The fusing karyomeres enter the enlarged perinu- onic stem cells called teloblasts. The teloplasm of these cells is clear plasm domain and reach the male pronucleus (Panel A, Fig. utilized in the manufacture of the ectodermal and mesodermal 8). Following completion of chromatin decondensation the female founder cells that will give rise to the paired germinal bands of the pronucleus forms besides the male pronucleus (Fernandez and embryo (Fernandez, 19BO:Fernandez and Stent. 19BO). Olea, 1995). In ascidians, the female karyomeres also migrate toward the Mitotic events and the partitioning of cytoplasmic domains center of the sperm monaster which in this case is situated in an In ascidians the first cleavage spindle is small and situated at the asymmetric position. Awedge of the mitochondria-rich myoplasmic center of the zygote. Each spindlepole is associated with a large domain occupies about a quarter of the monaster near the zygote aster whose microtubules often reach the zygote surface. The cortex and is closely apposed to the envelope of the male pronu- cleavage plane bisects the zygote generating 2 identical blastomeres cleus (Panel B, Fig. 7). In both leech and ascidian zygotes, the in which the cytoplasmic domains exhibit mirror image distributions paternally-derived centrosome duplicates at the time the female (Panel B, Figs. 11 and 12). During the subsequent cleavage karyomeres start moving along the astral rays (early during the first divisions, the domains are progressively allocated to different interphase). In this manner, a disk-shaped perinuclear plasm blastomeres with different cell fates (Venuti and Jeffery, 19B9: domain houses the amphidiaster in the leech zygote (Panel A, Nishida, t 992, 1997; Satoh, 1994 : Satoh et al., 1996). Figs. 6 and B). In ascidians, the amphidiaster with its disc-shaped In leeches the first cleavage spindle is huge and asymmetric, centrosomes together with the male and female pronuclei, and the with one spindle pole (corresponding to the CD sector of the cytoplasmic domains embedded in the aster, migrate to the zygote zygote) much larger than the other (corresponding to the AB sector center (Panel B, Figs. 7 and B). The translocation of the mitochon- of the zygofe). The asymmetric positioning of the spindle is such dria-rich myoplasm and adjoining ER-rich domain are coordinated. that the cleavage furrow divides the zygote unequally (Panel A, They move in 2 characteristic phases, first in a slow phase (6 ~lm/ Figs. 1t and 12). As a consequence, the larger CD blastomere min) and then in a fast and smoother phase (25 ~lm/min) (Sardet et frequently inherits the entire animal and vegetal teloplasms al., 1989). These movements depend mostly on microtubules. (Fernandez et al... 19B7). They result in that the cup-shaped myoplasm first buckles and then ruptures (Panel B, Figs. 7 and B). The bulk of the mitochondria-rich Mechanisms and perspectives myoplasmic domain and the vegetally situated ER-rich domain move along the cortex towards the equatorial and central region of Fertilization and early meiotic events the zygote (Panel B, Fig. 9). They form the so-called "yellow In ascidians it is clear that a first phase of cytoplasmic reorgani- crescent" in naturally pigmented species (Conklin, 1905: Jeffery zation, lasting about 5 min, is triggered by a cortical contraction. and Meier, 19B3). The ultimate position of this crescent depends on The zygote cortical microfilament basket. with its opening at the the location of the sperm aster at the end of meiosis and corres- animal pole, contracts in response to the intracellular release of ponds to the future posterior pole of the ascidian embryo (Sardet calcium caused by the fertilizing sperm. et al.. 19B9: Speksnijder et al., t 993: Jeffery, 1995). The sequence of events leading to this first phase of cytoplasmic In the leech zygote the ectoplasm thickens considerably during reorganization is likely to be the following: 1) transmission of an early first interphase (Panel A, Fig. 6). This process relies on the activation signal to the egg by a sperm factor and/or via activation centrifugal recruitment of organelles, from the neighboring endo- of a G protein (Whitaker and Swann, 1993: Sette et aI., 1997), 2) plasm, as well as on the proliferation of mitochondria in the ectoplasm release of intracellular calcium at the site of sperm entry. This event itself. Drug treatment shows that translocation of organelles to the is probably mediated by the secondary messenger InsP3 periphery of the egg is a microtubule-based process (Fernandez et (McDougall and Sardet, 1995: Albrieux et al. 1997); 3) initiation of al., 199B). Once the ectoplasm has thickened, intense furrowing a microfilament-based cortical contraction which drags surface activity leads to the formation of 2 contraction rings (polar rings) lying proteins, cortical and subcortical organelles, as well as the nucleus towards the poles of the zygote. A dozen meridional furrows linking and centrosome introduced by the sperm. If the sperm penetrates the rings then appear as the zygote enters midinterphase(about3 near the animal pole (the most common situation), the calcium Li'ccll awl (I.\"citlian :.ygOfi'S IOS I wave reaches simultaneously all sides of the opening of the observations).Localizedregulationof contractilecomponents in microfilament basket and the cortical contraction proceeds sym- the cortex by cell-cycle dependent phosphorylation may be in- metrically in the vegetal direction, forming a contraction pole volved in generating these contractions. A similar bout of exactly opposite the animal pole. IIthe sperm penetrates near the microfilament-driven contractions has been reported in the zy- equator the calcium wave reaches one side of the microfilament gotes of the phylogenetically related oligochaete TuMex at the basket first and this side contracts first. This side of the basket conclusion of the first meiotic division (Shimizu, 1982a). In this corresponds to the position of the sperm entry site. One conse- case, several meridionally-oriented equatorial grooves. rather quence of this situation is the asymmetrical contraction of the than one contraction ring. are formed. Rearrangements of the microfilament basket. In such cases, the resulting contraction pole preexisting actin filaments network have been documented (re- can be situated as much as 45 degrees away from the vegetal pole viewed in Shimizu. 1995). (Speksnijder et al.. 1990; Roegiers et al.. 1995). These events result in a large reorganization of the ascidian zygote cortex and Late meiotic events cytoplasm into distinguishable domains (mitochondria-rich Once the early microfilament-driven reorganizations have taken myoplasm/subcortical. ER-rich cytoplasm/microfilaments and mi- place the next phase of cytoplasmic reorganization in leech and crovilli-rich contraction pole region). These domains are radially ascidian zygotes is linked to the growth of an aster around the organized around a new developmental axis that predicts the centrosome introduced by the sperm (establishment of the first future site of gastrulation and dorso-ventral axis of the embryo. monaster). This microtubular structure is essential in the recruit- This axis is exactly coincident (ifthe contraction is symmetrical) or ment and relocalization of organelles and the establishment of new not coincident (if the contraction is asymmetrical) with the original cytoplasmic domains. animallvegetal axis of the zygote (Speksnijder et al.. 1990; Roegiers In ascidians. the aster grows slowly and at first symmetrically, et al.. 1995). beneath the cortex in a period of 20 min following emission of the The mechanism by which the initial calcium signal provokes the first polar body. Aher the second polar body is formed. the monas- contraction of the actin microfilament network remains to be ter grows in an asymmetric manner with its longest fibers reaching established. We need to know if there is assembly of new actin the animal region, where the female karyomeres have formed. The filaments and how organelles are connected to the contracting karyomeres then move toward the center of the monaster contain. microfilament network and concentrated in the various cytoplasmic ing the male pronucleus, presumably using microtubule motors domains. Other cytoskeletal elements (intermediate filaments) (see Houliston et al.. 1995; Vernos and Karsenti. 1996). may also be involved.Finally.we wouldliketo knowhowthese In leech zygotes the process is quite similar. The monaster is large reorganizations influence the distribution and functions of initially situated beneath the cortex and grows asymmetrically over membrane components. a period of about 60 min. As it grows the monaster glides in a One clear consequence of the creation of a contraction pole vegetal direction towards the center of the zygote following an arc- region highly enriched in microvilli, micro filaments and sheets and like path. 80th microfilaments and microtubules are involved in tubes of ER is the establishment of a calcium wave "pacemaker" monastercentration in the leech egg (Fernandez et al.. 1994). The capable of generating from 10 to 20 calcium waves during meiosis microtubules linking the centrosome to the animal pole region, (see McDougall and Sardet. 1995). As in mammalian eggs. oscil- where the newly-formed female karyomeres lie. are stabilized latory calcium signals in ascidian eggs are involved in the regula- allowing movement of the karyomeres toward the egg center. tion of the meiotic cell cycle (Speksnijder et al.. 1990; McDougall OrganeHes recruited from the adjacent endoplasm form a perinu- and Sardet. 1995; Russo et al.. 1996). These signals may also be clear plasm domain that also encloses the sperm-derived centro- necessary for proper embryonic development since ablation of a some and the developing male pronucleus. sizeable fraction of this region prevents a normal cleavage pattern In both ascidians and leeches growth of the sperm centrosome- and gastrulation (Bates and JeHery. 1987; Nishida. 1996). It derived monaster is clearly related to progression from MI to the MIl remains to be seen what domains and molecular and cellular stage of meiosis. This process is likely to depend on cell cycle functions are affected in such experiments. regulation of microtubule dynamics and motor activity (Vernos and Eggs of many other organisms also display cortical contractions Karsenti, 1996). In both ascidians and leeches these microtubule- triggered by the sperm as a consequence of transient elevations of driven events during meiosis II are accompanied by transient intracellular calcium. The best documented case is that of the microfilament-based deformation of the vegetal cortex. amphibian eggs in which the calcium wave initiated by the fertilizing Microfilament-driven deformation of the vegetal hemisphere also sperm is followed by a symmetrical contraction (along the animal/ takes place during meiosis of other invertebrate zygotes. In mol- vegetal axis). which drags subcortical organelles and the incorpo- lusc and annelid zygotes such deformations result in polar lobe rated sperm nucleus towards the animal pole (Cheer et al.. 1987). formation (Dohmen, 1983). Meridional furrows also form at the This poleward motion brings the male and female pronuclei closer equator of the Tubifex zygote during emission of the second polar to each other (Elinson. 1983) but is not thought to have major body (Shimizu. 1982a). Recent experiments by Shimizu (1997) consequences on the establishment of a developmental axis. indicate that in Tubifexthe reorganization of cortical actin network In leeches, the microfilament-driven meiotic contraction is clearly requires activation of Protein kinase C and an increase in intracel- delayed with respect to fertilization and zygote laying (about 90 lular calcium. min). The poleward progression of the contraction ringterminates withthe emission ofthe firstpolar body and the accumulation at the Interphase and mitotic events animal pole region of plasmalemma. organelles and actin fila- In ascidians. duplication of the sperm-derived centrosome dur- ments. At present we have not been able to detect calcium signals ing first interphase modifies the shape of the centrosomal region, at the time of contraction (Fernandez and Sardet. unpublished from a sphere to a disc. This starts a relocalization process that 1082 J. Femal/de: el al.

involves the coordinated translocation of the aster and the pronu- zygote of other leeches, it seems to be microtubule-dependent clei towards the center of the zygote and that of adjoining cytoplas- (Astrow et a/., 1989), whereas in Tubifex it appears to involve mic domains towards the future posterior pole of the embryo. This mainly microfilaments (Shimizu, 1982b, 1995). Although the large process is mainly dependent on the integrity of microtubules and zygote of glossiphoniid leeches and oligochaetes are favorable may be analogous to the "cortical rotation". In amphibian eggs, models to study the formation of cytoplasmic domains, much since microtubule-driven displacement of cytoplasmic regions remains to be done to disclose the respective roles of different likewise takes place in relation to the zygote cortex, it will be cytoskeletal elements and motors in vectorial translocation of informative to examine the organization and polarity of cortical and organelles and cytoskeletal elements. subcortical microtubules during these movements and to assess In the leech, the last and decisive event for the allocation of the importance of microtubule polymerization and displacement, cytoplasmic domains to one of the first two blastomeres is the with respect to other microtubules and organelles, as well as the generation and positioning of an asymmetric mitotic apparatus involvement of microtubule based motormoleculesinthisprocess (Panel A, Figs. 11 and 12). This allows unequal division of the (Houliston, 1994). The slow and fast phases of translocation that zygote such that the larger cell contains all or most of the animal we have described during this second phase of ooplasmic segre- and vegetal teloplasms. At present we have no clue as to what gation in ascidians probably correspond to the building up of determines the asymmetry of the mitotic spindle. It is possible that tensions brought about by the sliding of microtubules against the the teloplasms and the adjoining cortical regions, in which astral cortex and then to the release of tensions when the cup-shaped microtubules are embedded, affect the dynamic equilibrium of myoplasm tears. In ascidians, these movements ultimately result spindle pole microtubules in the CD sector of the egg (Fernandez in the relocalization of the bulk of cytoplasmic domains (mitochon- el a/., unpublished observations). In Tubilex, formation of an dria-rich and ER-rich domains). Because of the stereotyped clea- asymmetric cleavage spindle in the one cell embryo does not vage pattern of the ascidian embryo these domains will be inherited apparently depend on cortical mechanisms but rather on the by the 2 ventro-posterior blastomeres of the 8-cell embryo. These inheritance of a single maternal centrosome during mitosis (Ishii blastomeres will give rise autonomously to the primary muscle cells and Shimizu, 1997). In contrast, formation of an asymmetric of the ascidian tadpole and even develop muscle cells in isolation cleavage spindle in the two cell embryo of Tubifex relies on cortical (Satoh, 1994; Nishida, 1997). It is interesting to note that a differences dependent on cell contacts (Takahashi and Shimizu, particular cortical structure, called the CAB (Centrosome-Attract- 1997). ing Body), plays a key role in the unequal cleavage pattern of We have stressed similarities between leech and ascidian posterior blastomeres in 16-64 cell embryos (Hibino et al., 1998). zygote reorganization during the mitotic and meiotic cell cycles. It We may hypothesize that the CAB, or its precursor, is formed in the is evident, however, that there are also important differences. For posterior cortex during translocation and cent ration of the aster and example, in ascidians it is clear that the point of sperm entry and accompanying cytoplasmic domains. consequent localization of the sperm aster play an essential role in Centrosome duplication in the leech zygote also takes place in defining the localization of cytoplasmic domains and thus the the interphase period that follows completion of meiosis. The anterior/posterior axis of the embryo. In the leech zygote localiza- duplication, in this case, causes the perinuclear plasm domain to tion of cytoplasmic domains is dictated by the distribution of change its shape from spherical to ellipsoidal and finally into a cytoskeletal elements along the animal/vegetal axis that is deter- dumbbell-shaped body oriented perpendicularly to the animall mined during oogenesis. Other axes in the leech, such as that vegetal axis. In such a large zygote, restriction of mitotic activity running along cells AB and CD, do not appear to be strictly (meiosis) to the cytoplasm around female karyomeres, located in determined during oogenesis or by the site of sperm entry (Pilon the animal pole region of the zygote, might contribute to the stability and Weisblat, 1997).lt is also worth noting that unequal partitioning of some interphase microtubules, a situation that also occurs in the of cytoplasmic domains (teloplasms) is rather precocious in leeches even larger eggs of Beroe and Xenopus (Houliston et a/., 1995; taking place during the first mitotic cycles, whereas in ascidians, Perez-Mongiovi ef al., 1998). unequal partitioning of myoplasm and ER rich domains takes place By mid interphase distinct circular and meridional furrows, much later (at the 16-32 cell stage) when smaller posterior whose formation is dependent on microfilaments, appear at the blastomeres are formed (Nishida, 1997). leech zygote surface (Fernandez and Olea, 1995; Fernandez etal., 1998). It is not known what directs the formation of these furrows Summary and whether furrowing relies on the recruitment and contraction of preexisting microfilaments and/or on the polymerization of new Leech and ascidian embryos are well suited for the study of actin filaments. Microtubules and organelles accumulate at the certain developmental processes. Although leeches and ascidians walls of rings and bands of contraction. In this manner, organelles, belong to different bilateralia groups (protostomes and microtubules and microfilaments are brought together to concen- deuterostomes, respectively) they share important developmental trate in regions of thickened ectoplasm. The origin of ectoplasmic features and, in particular, the determinate character of their microtubules has not yet been fully determined but they may derive embryogenesis. In both types of embryos this property is related to from the numerous cytasters present in the ectoplasm during the presence of specific cytoplasmic domains that are selectively meiosis (see Panel A, Figs. 5 and 6). The bipolar displacement of allocated to different blastomeres during cleavage. In this review organelles, microfilaments and microtubules associated with the leech and ascidian eggs and zygotes are compared in terms of the poleward motion of the polar rings and the shortening of meridional structure of these cytoplasmic domains and of the cellular mecha- furrows results in a large accumulation of ectoplasm at both zygote nisms involved in their formation and localization. During meiosis poles. In the zygote of the leech T. rude this process involves both the zygote of leeches and ascidians undergo stereotypic actin- microtubules and microfilaments (Fernandez el al., 1998). In the dependent contraction movements related to both the emission of Let'ch lWei {lscielicm ::.ygote.\' 1083

the polar bodies and the formation and relocalization of cytoplas- EUNSON. A.P. (19831. Cytoplasmic phases 1Mfirst cell cycle olthe activated Irog egg mic domains. After completion of meiosis, during first interphase, Dev BioI. 100: 400-451. monaster microtubules nucleated from the sperm-derived centro- EPHRUSSI. A. and LEHMANN. R. (1992). Induclion 01 germ ceillormation by oskar. some playa key role in pronuclear migration, In addition, these Nature 358 387-392. astral microtubules direct the relocalization of cytoplasmic do- ETKIN. L D. (1997). A new lace for Ihe endoplasmic reticulum RNA localization Sc,ence 276 1092.1093. mains and the translocation and accumulation of organelles in the interior of the zygote. Microtubules and microfilaments, on the FERNANDEZ. J. (1980). Embryonic developmenl 01 the glosslphonlld leech Theromyzon rude: Characterization 01 developmental stages. Dev. B!O/. 76: 245- other hand, are involved in cortical reorganizations and organelle 262. trans locations in both zygote species during interphase and clea- FERNANDEZ. J. and OLEA. N. (1982). Embryonic development 01 glosslphonlld vage divisions. In the case of leech zygotes, this process leads to teeches. In Developmental Biology of Freshwater Invertebrates (EdS F.W. formation of characteristic polar cytoplasmic domains called Harrison and A.R. Cowden). A. R. Liss. New York.pp. 317-366. teloplasms. These domains are selectively inherited by teloblasts, FERNANDEZ. J. and OLEA. N. (1995). Formation of the temale pronucleus and precursor stem cells of ectodermal and mesodermal tissues in the reorganization and disassembly of the IIrst interphase cytoskeleton In the egg 01 leech embryo. In the ascidian zygote, the cytoplasmic movements the leech Theromyzon rude. Dev. BioI 71: 541.553 observed during interphase and mitosis lead to relocalization of the FERNANDEZ. J. and STENT, G.S. (1980). Embryonic development olthe glosslphoniJd leech Theromyzon rude:: Structure and development of the germinal bands. Dev. bulk of a mitochondria-rich domain, called the myoplasm. along Blo/. 78 407-434. with an endoplasmic reticulum-rich domain towards the future FERNANDEZ. J.. OLEA. N. and MATTE. C. (1987). Structure and development 01the posterior pole of the embryo. The myoplasm is inherited by a egg oltha glossiphonild leech Thsramyzon rude. Charactenzatlon 01 developmen- subset of posterior blastomeres committed to become the primary tal stages and structure of the early uncleaved egg. Developmem 100.211-225 muscle cells of the ascidian tadpole. FERNANDEZ. J.. OLEA. N. and TELLEZ. V. (19941. Formation 01 the male pronu. cleus. organization of the Ilrst Interphase monaster and establishmenl of the Acknowledgments perinUClear plasm domain In the egg of the glosslphonild leech Theromyzon rude. We thank Andrea Vbilla. Victor Guzman and Christian Rouvifne for Dev. BIOI. J64: 111.122. technical assistance, Sylvie Chemla torherhelp with the figures and Evelyn FERNANDEZ. J.. OLEA. N, TELLEZ. V. and MATTE. C. (1990). Siructure and Houliston, Janet Chenevert and Danielle Carre for their suggestions on the development 01the egg of the glosslphonlld leech TheromyzOI1 rude Reorganlza. manuscflpt. ThiS work was supported by grant 1950527 from Fondecyt and tlon 01 the renllized egg dUring completion of the first meiotIc diviSion. Dev. Bioi by ARC and AFM. 137: 142-154. FERNANDEZ. J.. OLEA, N.. UBILLA. A. and CANTILLANA. V. (1998). Formation 01 References polar cytoplasmic domains tleloplasmsl In the leech egg IS a three-step segrega. tlon process. In! J Dev. Bioi. 42. 149.162.

ALBRIEUX. M.. SARDET.C. and VILLAZ, M. (1997). The two intracellular calcium FERNANDEZ, J., TELLEZ, V, and OLEA. N. (1992). Hirudinea. In Microscopic Anatomy Invertebrales. Vol. 7 (Eds FW. Harrison and S L. Gardiner). Wlley- channels. ryanodine receptor and inOSllol 1.4.5-tnsphosphate receptor play 0' different roles dunng fenllization In ascidlan. Dev. 8101. 189.174-185. L155. Ncw York. pp. 323 394. ASTROW. S.H.. HOLTON. B. and WEISBLAT, D.A (1989). Telopfasm formation in GARD. DL. CHA. B.J. and KING. E (1997). The organization and animal-vegetal a leech. He/obelia triserialls. is a microtubule-dependent process. Dev. BioI. 135 asymmetry of Cy10keralin filaments in stage VI Xenopus oocy1es is dependent 306-319 upon F-aclln and microtubules. Dev. Bioi. 184.95-114. BASSELL. G. and SINGER. R.H, (1997), mRNA and Cy1oskeletalldaments.ln Current GAVIS. E.R. (19971. Ellpedltlon to the pole: RNA localization in Xenopus and Opinion In Cell BIOlogy. (Ed. B. Geiger and E. Karsentl). Current BIOlogy Lid Drosophila Trends Cell BIoi. 7.485-492. London. Vol. 9. pp.l09-115. GLOTZER. J. B. and EPHRUSSI. A. (1996). mRNA localization and the cy1oskeleton. BATES. W.R. and JEFFERY. W.R. (1987). Localization of alll<11 dctcrminants in thc In $pmlnc1rs in Developmental Ellology. Vol. 7 (Ed. E. Karsentl). AcademiC Press vegetal pole region 01 ascldlan eggs. Dev. Blof. 124 65-76. Inc. pp. 357-365. BLAIR, S.S. and WEISBLA T, DA (1984). Cell interactions In the developing eplder. GOODSON. H.v.. VALETTI. C. and KREIS, T.E. (1997). Motors and membrane mis of the leech He/oMelia trisenalls Dev. BIOI. 101: 318-325 traffic. Curro Opln. Cell Bioi. 9: 18-28. BOVER!. T. (1895). Ueber die Betruchlungs-und Entwicklungsfahlgkeit kernloser HARRIS. p.. OSBORN. N. and WEBER, K. (1980). Distribution oltubulln-conlainlng Seelgel-eler Arch. fur Entwicklungsmechantk 2 3-92 slructures in the egg of the sea urchin Strongllocentrotus purpuralus from fertilization through first cleavage. J. Cell Bioi. 84 668.679 CAPCO. D. (1995). Cy10skelelal mechanisms during animal development. In CUffent Tnrir.s In npvelQrmentill R'OIQgy, Vol 31 (Ed D G. CC\pco) Academic Press. Inc. HIBINO. T.. NISHIKAT A. T. and NISHIDA. H. (1998). Centrosome-attracting body: a Orlando. pp. 1-501. novel struclure closely related to the unequal cleavages in Ihe ascldian embryo. Dev. Grol't'/h Ditter. 40: 85.95 CHABRY. L. (1887). Contribution a I"embryologle narmale et teratologlque des ascldles SImples. J. Anal. PhyStOI. (Pans) 23 167.319. HOULISTON. E. (1994). MIcrotubule translocation and polymensalion during cortical rotation in Xenopus eggs, Development 120: 1213.1220 CHEER. L.. VINCENT, J.P., NUCCITELLI, R. and OSTER, G. (1987). Conical activity in Verlebrate eggs I: The activation waves. J. Theor. Bioi. 124: 377-404. HOULISTON. E.. CARRE. D.. CHANG. P. and SARDET, C. (1995). Cytoskeleton and ctenophore development. CUrr Top. Dev. Blo/. 31: 41-63. CONKLIN. E.G. (1897). The embryology of Crep,dula J. Morphol. J3 98-140. ISHII. A. and SHIMIZU. T. (1997). Equalization 01 unequal Ilrst cleavage In the TubdeJl CONKLIN, E.G. (1905). The organization Bnd cell lineage of the 8SCldl8n egg. J. Aced egg by Introduction of an addlhonal centrosome: Implications for the absence 01 Nat/.Sci. (PhI/adelphia) 13. 1-119. cortical mechanisms for mitotic spindle asymmetry. Dev. BIoI. 189: 49-56 CORBO. J.C.. ERIVES. A.. GREGORIO. AD.. CHANG. A. and LEVINE. M. (1997) JEFFERY, W.R. (1995) Development and evolutIOn 01 an egg cytoskeletal domain In Dorsoventral patterning of !tie vertebrate neural tube is consel'\led Ina protochordate. ascldlans Curro Top. Dev Bioi. 31: 243-276. Development 124. 2335-2344. JEFFERY. W.R. and MEIER, S. (1983). A yellow crescent cy10skeletal domain In DAVIDSON, E, (1986) Gene activity in early development. Academic Press. San ascidlan eggs and Its role In early development. Dev. Bioi 96: 125-143. Diego JEFFERY. WA. and MEIER. S. (1984). Ooplasmic segregation of the myoplasmlC DOHMEN. M.R. (1983). The poJarpobe In 9ggs ofmofJuscs and annelids: structure. actin network in slratified ascidlan eggs. Roux's Arch Dev Bioi. 193: 257.262 composition and function In Time. Space and Pattern 1MEmbryontc Development (Eds. W. A. JeHery and R.A. Rail). A.R. Liss Inc. New York, pp. 197-220 KING, ML (1996). Molecular basis for cytoplasmic localizatIon. Dav. Genet. 19 183.189 1084 1. Ferndnde:. ef al.

MARG, HQUUSTON. E. and PAINTRANO, M. (1988). Purification of meiotic B" SHIMIZU, T. (1995). Role of the cytoskeleton in the generation of spatial patterns in spindles and cytoplasmic asters Irom mouse oocytes. Dev. Bioi. 129: 275-282. Tubifex eggs. In Current Topics in Developmental BIology. Vol. 31. (Ed, D.G. MCDOUGALL, A. and SARDET, C. (1995). Function and characteristiCS of repetitive Capco). Academic Press, Inc. Orlando, pp. 197-235. calcium waves associated with meiosis. Curro BIoi. 5: 318-328. SHIMIZU, T. (1997). Reorganization of the cortical actin cytoskeleton during matura. MORI, Y. (1932). Entwicklung isolierter Blastomeren und abgetOteter alterer Keirne tiondlvision inthe Tubdexegg: possible involvement of protein kinase C. Dev. Bioi. van Clepsinesexoculata, Z. WISS. Zool. 141: 399-431. 188: 110-121. NISHIDA, H. (1992). Determination of developmental fates of blastomeres in ascidian SPEKSNIJDER. J.E.. JAFFE, L.F. and SARDET, C. (1989). Polarity of sperm entry embryos. Dev. Growth Olffer. 34: 253-262. in the ascidian egg. Dev. BiOi, 133: 180-184 NISHIDA. H. (1994). localization of deiermlnants for lormatlon of the anterior- SPEKSNfJDER, J.E., SARDET, C. and JAFFE, L.F. (1990). The activation wave of posterior aXIs in eggs of the ascidian Halocynthia roretzi. Development 120. 3093" calcium in the ascidlan egg and its role in ooplasmic segregation. J. Cell BioI. 1 to: 3104. 1589-1598. NISHIDA, H. (1996). Vegetal egg cytoplasm promotes gastrulation and is responSible SPEKSNIJDER,J.E., TERASAKI. M., HAGE. W..JAFFE, L. and SARDET. C. (1993). for speCification of vegetal blastomeres in embryos of the ascidian Halocynthla Polarity and dynamics of the endoplasmic reticulum during fertilization and roretzi. Development 122: 1271-1279. ooplasmic segregation In the ascidian egg. J. Cell BIOI. 120. 1337-1346. NISHIDA. H. (1997). Cell fate specification by localized cytoplasmic determinants and ST. JOHNSTON, D, and NOSSLEIN.VOLHARD, C. (1992). The origin of pattern and cell Interactions In ascidian embryos. Int, Rev. Gytol. 176: 245-306. polarity in the DrosophIla embryo. Ce1/58: 201-219. NUSSLEIN-VOLHARD. C. (1996). Gradients that organize embryo development. Sci. SWALLA, B,J. and JEFFERY. W.R. (1990). Interspecllic hybridization between an Am. 275: 38-43. anural and urodele ascidian: differential expression of urodele features suggests multiple mechanisms control anural development. Dev. Bioi. 142: 319-334. PEREZ-MONGIOVI. D.. CHANG. P. and HOULISTON. E. (1998). A propagated wave of MPF activation accompanies surface contraction waves at first mitosis in SWALLA, B.J.. BADGETT. M.A. and JEFFERY, W.R. (1991). Identification of a Xenopus. J. Gell SCI, 111: 385-393 cytoskeletal protein localized in the myoplasm of ascidian eggs: localization is modified during anural development. Development 1 t 1: 425-436 PILON. M. and WEISBLAT. DA (1997). Early events leading to fa Ie decisions during leech embryogenesis, Semlfl. Gell Dev. Bioi. 8 351.358 SWALLA, B.J., MAKABE, K.W., SATOH, N, and JEFFERY, W.R. (1993). Novel genes expressed differentially in ascidians with alternate modes of development. Devel- ROEGIERS. F., MCDOUGALL, A. and SAADET. C. (1995). The sperm entry paint opment. 119. 307-318. defines the orientation of the calcium-induced contraction wave that directs the first phase of cytoplasmic reorganization in the ascidian egg. Development 121: TAKAHASHI. H. and SHIMIZU, T. (1997). Role of the intercellular contacts in 3457-3466. generating an asymmetric mitotic apparatus in the Tublfex embryo. Dev. Growth Differ. 39. 351-362. RONGO. C, and LEHMANN, R. (1996). Regulated synthesis. transport and assembly 01 the Drosophila germ plasm, Trends Genet 12: 102-109 VENUTI, J. M. and JEFFERY, W.R. (1989), Cell lineage and determination of cell fate in ascidian embryos. IfIt J. Dev. Bioi. 33: 197-212. RUSSO. G,L.. KYOZUKA. K., ANTONAZZO, L., TOSTI. E. and DALE, B. (1996). Maturation promoting factor in ascidian oocytes is regulated by different intracel- VERNOS, I. and KARSENTI, E, (1996). Motors involved in spindle assembly and lular signals at meiosis I and II. Development 122. 1995-2003. chromosome segregation. Curro Qpin. Cell Bioi. I: 4.9. SARDET, C., MCDOUGALl. A. and HOULISTON, E. (1994). Cytoplasmic domains WEISBLAT, D.A, and BLAIR, S.S. (1984). Developmental indeterminacy in embryos in eggs. Trends Cell Bioi, 4: 166.172. 01 the leech Helabdelta tflserlalis, Dev. Bioi. t01: 326-335 SARDET, C., SPEKSNIJDER, J., INOUE, S. and JAFFE, L. (1989). Fertilization and WEJSBLAT, D.A., ASTROW, S.H.. BISSEN, ST, HO, RX, HOLTON. B. and ooplasmic movement in the ascidian egg. Development 105: 237-249. SETTLE, SA (1987). Earty events associated with determination of cell late in leech embryos. In Genetic Regulation ofDevelopment(Ed. W.F. Loomis). Alan R. SATOH. N. (1994). Developmentalbiologyof ascldlans. Cambridge University Press, Liss. Inc., New York. pp. Cambridge. 265-286. WEISBLAT, D.A., WEDEN. C.J. and KOSTRIKEN. R.C. (1994). Evolution 01 devel- SATOH. N., MAKABE. S.andSAIGA, H, (1996). The K.w" KATSUYAMA, Y., WADA, opmental mechanisms: Spatial and temporal modes of rostrocaudal patterning. In ascidian embryo: An experimental system for studying genetic circuitry for Current Topics embryonic cell specification and morphogenesis. Dey Growth Differ. 38: 325-340. of Developmental Biology, Vol. 29 (Ed. R. A. Pedersen). pp.101- 134. SAWADA. T. and OSANAI. K. (1985), Distribution 01 actin filaments in fertilized egg WEISBLAT. DA, PRICE. D.J. and WEDEN, C.J. (1988). Segmentation in leech of the ascidlan Clonia mtestinatls. Dev, BIOI. 111: 260-265. development. Development (Suppl.) 104: 161-168. SAWADA. T. and SCHATTEN. G. (1988). Microtubules in ascidian eggs during WHITAKER, M. and SWANN. K. (1993). lighting the fuse at fertilization. Development meiosis, lertilizatlon and mitosIS. Cell Motl/. Cytoskel. 9: 219-230. 117: 1-12. SETTE, C., BEVILACQUA. A., BIANCHINI, A., MANGIA, F., GEREMIA. R. and WHITMAN, C.O. (1 878). The embryology of Clepsine. O.J. Microsc. Sci. 18: 215-315 ROSSI, P. (1997). Parthenogenetic activation of mouse eggs by microinjectlon of a truncatedc-kittyrosine kinasepresentln spermatozoa. Development 124: 2267- WHITMAN, C.O, (1887). A contribution to the history of the germ-layers in Clepsine. 2274 J. Morphol. 1: 105-182. SHANKLAND, M. (1987). Differentiation of the 0 and P cell lines in the embryo of the WILSON, E.B, (1925). The Cell in Development and Heredity. 3rd edition. MacMillan. leech. II. Genealogical relationship of descendant pattern elements in alternative New York. developmental pathways. Dev. Bioi. 123: 97-107. YAMADA, A. and NISHIDA, H. (1996). Distribution of cytoplasmic determinants in SHANKLAND. M. and WEISBLAT, DA (1984). Stepwise commitment of blast cell unfertilized eggs of the ascidlan Halocynthia roretzi. Dev. Genes Evol. 206: 297- fates during the positional specification of the 0 and P cell lines in the leech 304. embryo. Dev. BIOI. 106: 326-342. ZALOKAR, M. and SARDET. C. (1984)_ Tracing of cell lineage in embryonic develop- SHANKLAND, M., MARTINDALE. M.a., NARDELLI-HAEFLIGER, D., BAXTER, E. ment of Phallusia mammillata(Ascidia) by vital staining of mitochondria. Dev. Bioi. and PRICE. D. J. (1991). Origin of segmental identity in the development of the 102: 95-205. leech nervous system. Development (Suppl.) 2: 29-38. SHIMIZU, T. (1982a). Development of the freshwater oligochaete Tubifex . In Developmental BIology of Freshwater Invertebrates (Ed. F.W. Harrison and R.A. Cowden). A.R. Liss. New York, pp. 283-316. SHIMIZU, T. (1 982b). Ooplasmic segregation in the Tubifexegg: Mode 01 pOle plasm accumulation and possible involvement of mlcrofilaments. Roux'sArch. Dev. Bioi Rflf'iT'f'd: A.pn11998 191: 246-256. ,\((t'/Jlflljol/lljbfjrflliOlJ:.1lj~\ 191)8