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Keywords:

  • ascidian;
  • oocyte;
  • embryo;
  • maturation;
  • fertilization;
  • a-v/D-V/A-P axes;
  • polarity

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. OOGENESIS, MATURATION, AND POLARIZATION OF THE OOCYTE: SPECIFICIATION OF A PRIMARY ANIMAL–VEGETAL AXIS
  5. MULTIPLE CORTICAL AND CYTOPLASMIC REORGANIZATIONS BETWEEN FERTILIZATION AND FIRST CLEAVAGE
  6. DV AND AP AXES ARE SPECIFIED BEFORE FIRST CLEAVAGE BY CORTICAL AND CYTOPLASMIC REORGANIZATIONS
  7. EARLY CLEAVAGES: A SERIES OF ASYMMETRICAL DIVISIONS
  8. POLARIZATION OF OOCYTES AND EMBRYOS: ASCIDIANS COMPARED WITH OTHER ORGANISMS AND THE QUESTIONS THAT REMAIN
  9. Acknowledgements
  10. REFERENCES

The dorsoventral and anteroposterior axes of the ascidian embryo are defined before first cleavage by means of a series of reorganizations that reposition cytoplasmic and cortical domains established during oogenesis. These domains situated in the periphery of the oocyte contain developmental determinants and a population of maternal postplasmic/PEM RNAs. One of these RNAs (macho-1) is a determinant for the muscle cells of the tadpole embryo. Oocytes acquire a primary animal–vegetal (a-v) axis during meiotic maturation, when a subcortical mitochondria-rich domain (myoplasm) and a domain rich in cortical endoplasmic reticulum (cER) and maternal postplasmic/PEM RNAs (cER-mRNA domain) become polarized and asymmetrically enriched in the vegetal hemisphere. Fertilization at metaphase of meiosis I initiates a series of dramatic cytoplasmic and cortical reorganizations of the zygote, which occur in two major phases. The first major phase depends on sperm entry which triggers a calcium wave leading in turn to an actomyosin-driven contraction wave. The contraction concentrates the cER-mRNA domain and myoplasm in and around a vegetal/contraction pole. The precise localization of the vegetal/contraction pole depends on both the a-v axis and the location of sperm entry and prefigures the future site of gastrulation and dorsal side of the embryo. The second major phase of reorganization occurs between meiosis completion and first cleavage. Sperm aster microtubules and then cortical microfilaments cause the cER-mRNA domain and myoplasm to reposition toward the posterior of the zygote. The location of the posterior pole depends on the localization of the sperm centrosome/aster attained during the first major phase of reorganization. Both cER-mRNA and myoplasm domains localized in the posterior region are partitioned equally between the first two blastomeres and then asymmetrically over the next two cleavages. At the eight-cell stage the cER-mRNA domain compacts and gives rise to a macroscopic cortical structure called the Centrosome Attracting Body (CAB). The CAB is responsible for a series of unequal divisions in posterior–vegetal blastomeres, and the postplasmic/PEM RNAs it contains are involved in patterning the posterior region of the embryo. In this review, we discuss these multiple events and phases of reorganizations in detail and their relationship to physiological, cell cycle, and cytoskeletal events. We also examine the role of the reorganizations in localizing determinants, postplasmic/PEM RNAs, and PAR polarity proteins in the cortex. Finally, we summarize some of the remaining questions concerning polarization of the ascidian embryo and provide comparisons to a few other species. A large collection of films illustrating the reorganizations can be consulted by clicking on “Film archive: ascidian eggs and embryos” at http://biodev.obs-vlfr.fr/recherche/biomarcell/. Developmental Dynamics 236:1716–1731, 2007. © 2007 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. OOGENESIS, MATURATION, AND POLARIZATION OF THE OOCYTE: SPECIFICIATION OF A PRIMARY ANIMAL–VEGETAL AXIS
  5. MULTIPLE CORTICAL AND CYTOPLASMIC REORGANIZATIONS BETWEEN FERTILIZATION AND FIRST CLEAVAGE
  6. DV AND AP AXES ARE SPECIFIED BEFORE FIRST CLEAVAGE BY CORTICAL AND CYTOPLASMIC REORGANIZATIONS
  7. EARLY CLEAVAGES: A SERIES OF ASYMMETRICAL DIVISIONS
  8. POLARIZATION OF OOCYTES AND EMBRYOS: ASCIDIANS COMPARED WITH OTHER ORGANISMS AND THE QUESTIONS THAT REMAIN
  9. Acknowledgements
  10. REFERENCES

Ascidians have played an important role in our understanding of development since Chabry performed in 1887 the first experimental manipulation on blastomeres and Conklin suggested in 1905 that “organ forming substances” were located in distinct regions of the zygote (Chabry,1887; Conklin,1905a,b). Conklin gave specific names to peripheral domains (he called them “plasms”), which could be recognized in the zygote by their colors and granularity (ectoplasm, myoplasm, endoplasm). Conklin further observed that these domains relocalized in a stereotyped manner and that, as cleavages proceeded, the different “plasms” segregated into specific blastomeres (the myoplasm ending up in muscle cells).

The major relocalizations that take place between fertilization and first division became known as the first and second phases of “ooplasmic segregation,” a term mostly used in the ascidian field (Sardet et al.,1989; Jeffery,1995). We have proposed to use different terms such as “cytoplasmic and cortical reorganizations” and “segregation of cytoplasmic and cortical domains” to familiarize a larger community with the changes involved in axis specification in ascidians (Roegiers et al.,1999; Sardet et al.,2005).

Fifty years after Conklin's discoveries, Italian biologists led by the cleric Reverberi (Reverberi,1956) and more recently Japanese investigators working with N. Satoh and H. Nishida (Satoh,1994; Nishida,1997) have built upon the notion that specific cytoplasmic and cortical domains relocalize and segregate into blastomeres with different fates. They have performed key ablation and transplantation experiments and succeeded in identifying some of the molecular determinants involved. These experiments have shown that, in mature ascidian oocytes “determinants” for muscle (identified as the maternal mRNA macho-1), endodermal and epidermal cells of the tadpole embryo are already present in the oocyte and are distributed in a shallow gradient manner along the animal–vegetal (a-v) axis (Nishida,2002,2005; Satou et al.,2002; Sawada et al.,2005). After fertilization, these domains and the determinants they contain are concentrated and relocalized first along the future dorsoventral (D-V) and then along the anteroposterior (A-P) axes.

Our aim is to review what is known about the structure and composition of the cytoplasmic and cortical domains, what determinant macromolecules they contain, and how these domains form, relocalize, and segregate in different blastomeres. We also provide a large archive containing 30 FILMs illustrating the reorganizations (see “Film archive: ascidian eggs and embryos” at http://biodev.obs-vlfr.fr/recherche/biomarcell/). Other review articles in this special issue focus on the determinant macromolecules themselves and in particular the RNA determinants and their functions in development (see 2007 reviews by Prodon et al. and Kumano and Nishida in this special issue).

OOGENESIS, MATURATION, AND POLARIZATION OF THE OOCYTE: SPECIFICIATION OF A PRIMARY ANIMAL–VEGETAL AXIS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. OOGENESIS, MATURATION, AND POLARIZATION OF THE OOCYTE: SPECIFICIATION OF A PRIMARY ANIMAL–VEGETAL AXIS
  5. MULTIPLE CORTICAL AND CYTOPLASMIC REORGANIZATIONS BETWEEN FERTILIZATION AND FIRST CLEAVAGE
  6. DV AND AP AXES ARE SPECIFIED BEFORE FIRST CLEAVAGE BY CORTICAL AND CYTOPLASMIC REORGANIZATIONS
  7. EARLY CLEAVAGES: A SERIES OF ASYMMETRICAL DIVISIONS
  8. POLARIZATION OF OOCYTES AND EMBRYOS: ASCIDIANS COMPARED WITH OTHER ORGANISMS AND THE QUESTIONS THAT REMAIN
  9. Acknowledgements
  10. REFERENCES

Mature Oocyte Is Highly Polarized (Figs. 1E, 2f,g, and FILMS 5,6,7,8,9)

The fertilizable mature oocytes of the half a dozen ascidian species used for research (Satoh,1994) are polarized along a primary (a-v) axis defined with respect to the cortical position of a small meiotic apparatus arrested in the first metaphase of meiosis (see Fig. 1E, 2f,g, and FILMS 5–9). The positioning of the meiotic spindle under the surface is necessary for meiotic chromosome reduction and emission of polar bodies. How the spindle is anchored at the animal pole is not known, but in ascidians as in mouse oocytes, the surface of the egg adjacent to the meiotic spindle is different with respect to microfilament density. In Phallusia mammillata, a small actin microfilament-rich cap forms in the animal pole cortex above the spindle surrounded by a region depleted in microfilaments (Sardet et al.,1992). A similar depletion in microfilaments has been observed at the animal pole of Ciona eggs (Chiba et al.,1999; Prodon et al.,2006).

In all species of ascidians examined, the a-v axis is characterized by the presence of a basket-like distribution of a subcortical domain rich in mitochondria (Sawada and Schatten,1989; Swalla et al.,1991; Jeffery,1995). This domain situated 1–3 microns from the surface is 7–20 microns thick, depending on the size of oocytes, which vary in diameter from 100 to 300 microns in different species. This mitochondria-rich domain has been named the myoplasm (Conklin,1905a), because the bulk of it is progressively segregated to the primary muscle cells of the ascidian tadpole tail. The myoplasm basket lines the vegetal hemisphere and equatorial region of the oocyte and is open in the animal pole region (see FILM 5). In addition to its very high density of mitochondria, the myoplasm contains vesicles (which in some species are conveniently pigmented) and is characterized by a lower density of microtubules than the rest of the cytoplasm. The myoplasm has a very low density of Endoplasmic Reticulum (ER) compared with the deeper cytoplasm. However, in places, the thick mat of mitochondria is traversed by cytoplasmic corridors densely populated with ER tubes and sheets linking the deeper cytoplasm to the cortex (Fig. 2h; Speksnijder et al.,1993; Prodon et al.,2005).

There are only a few reports of specific proteins localized to the myoplasm (Nishikata et al.,1987; Jeffery,1995). One antibody commonly used to label the myoplasm is NN18 or anti-p58, a monoclonal originally made against vertebrate neurofilaments that was thought to label an intermediate filament network in the myoplasm (Swalla et al.,1991) but that, in ascidian embryos, recognizes mitochondrial ATP synthase (Nishikata, personal communication; Prodon et al.,2005; Patalano et al.,2006). Two cytoskeletal proteins enriched in the myoplasm with possible structural roles are ankyrin and myoplasmin C1, which binds to p58 (Jeffery and Swalla,1993; Jeffery,1995; Chiba et al.,1999). In addition a variety of specific mRNAs localize to the myoplasm or mitochondria (Swalla and Jeffery,1996; Oka et al.,1999; Sasakura et al.,2000; Makabe et al,2001; Yamada et al.,2005), but the significance of this association has not been investigated.

Other domains are also distributed inhomogeneously along the a-v axis in Ciona intestinalis, Phallusia mammillata, and Halocynthia roretzi. A monolayer of ER tubes and sheets lines the plasma membrane, and the density of this network increases along the a-v axis, ER sheets becoming more prominent in the vegetal cortex (FILM 9; Sardet et al.,1992; Prodon et al.,2005). Although this thin layer of rough ER is in continuity with the rest of the ER network in the oocyte, it has particular attributes. It is characterized by its physical attachment to the plasma membrane by means of contact sites (see FILM 9) and it binds specific maternal postplasmic/PEM RNAs (at least PEM-1, macho-1, Hr-POPK-1, PEM-3; Sardet et al.,2003; Nakamura et al.,2005; Prodon et al.,2005) as well as specific proteins (A. Paix and C. Sardet, unpublished observations). We have called this distinct cortical ER layer the cER, and because of the mRNAs associated with it, we call this cortical domain the cER-mRNA domain. The thin (0.5- to 1-micron) cortical layer containing the plasma membrane and cER-mRNA domain can be easily isolated as “cortical fragments” in Phallusia, Ciona, and Halocynthia (Fig. 2i–k; Sardet et al.,1992,2003; Nakamura et al.,2005; Prodon et al.,2005). We have also noted that in isolated cortical fragments, the density of microfilaments lining the plasma membrane increases along the a-v axis (Sardet et al.,1992; Prodon et al.,2005), apparently reflecting the situation in the whole oocyte (Chiba et al.,1999). Finally, in Phallusia, vesicles within the oocyte (including large yolk granules) are distributed inhomogeneously along the a-v axis (Gualtieri and Sardet,1989) and of course in Styela, pigmented granules, which Conklin first observed are also distributed in a graded a-v manner (Conklin,1905b). One unresolved question is that of the existence and distribution of the germ plasm at this stage. Vasa (a germ plasm marker in many animals) like other postplasmic/PEM RNAs accumulates in the posterior pole of Phallusia and Ciona embryos (Fujimura and Takamura,2000; Takamura et al.,2002; Patalano et al.,2006). In oocytes and zygotes, we have observed that a high density of Vasa-positive granules exists at the interface between the cER-mRNA domain and the myoplasm, constituting a presumptive germ plasm domain (C. Sardet, C. Djediat, P. Dru, A. Paix, unpublished observations).

a-v Polarity Is Established During Meiotic Maturation (Figs. 1A–E, 2a–f, and FILMS 1–4)

Before Germinal Vesicle BreakDown (GVBD), a uniform peripheral layer of mitochondria is established as mitochondria move out from a perinuclear location during vitellogenesis (Prodon et al.,2006; Figs. 1A, 2a,b). The asymmetric polarization of this subcortical mat of mitochondria, which constitutes the bulk of the myoplasm, occurs during meiotic maturation and takes 2–3 hr in C. intestinalis (Fig. 1C–E, FILMs 2, 4; Prodon et al.,2006). Not much is known about what causes maturation in the natural situation, but it can be artificially induced in “Stage III” oocytes, which are those Prophase I-arrested oocytes that are fully grown, contain a GV, and are ready to mature (Fig. 1A; Sakairi and Shirai,1991; Prodon et al.,2006). Maturation can be triggered by simple exposure of Stage III oocytes to sea water in H. roretzi (Sakairi and Shirai,1991), C. intestinalis (Satoh,1994; Prodon et al.,2006), and P. mammillata (F. Prodon and A. McDougall, personal communication). In Ciona, breakdown of the germinal vesicle (GVBD) and the formation of a centrally located meiotic apparatus take approximately 30 min to 1 hr after dilution in sea water. In maturing oocytes of C. intestinalis, the first visible sign of polarization is the microfilament-dependant migration of the meiotic apparatus toward the oocyte's surface. After the localization of the meiotic apparatus under the animal pole, the myoplasm polarizes along the a-v axis approximately 1–2 hr later (Figs. 1C–E, 2a–d), and this polarization is blocked by microfilament inhibitors but not by microtubule inhibitors (Prodon et al.,2006). In Ciona, as in mouse oocytes (Verlhac et al.,2000; Maro and Verlhac,2002), it is presumably the migration of the meiotic apparatus toward an apparently random site of the oocyte's surface that constitutes the first symmetry-breaking event, although it cannot be excluded that a favored “animal pole site” pre-exists before maturation in the Ciona oocyte. In other ascidian species, there is evidence of asymmetry existing before GVBD. Indeed, in H. roretzi (a convenient species for studying maturation because large numbers of Stage III oocytes are stored in the gonad) and a few other ascidian species, the GV is not in the center of the oocyte and the meiotic apparatus apparently migrates toward the closest region of the surface (Sawada and Schatten,1988; Sakairi and Shirai,1991; see Prodon et al.,2006). In Molgula manhattensis, there is an even earlier indication of polarity related to the presence of basophilic domains situated on one side of the GV (Kessel,1983). We do not yet understand the mechanisms that preside over meiotic apparatus migration and the progressive movement of the subcortical myoplasm domain away from the animal pole region. Examination of time-lapse recordings of polarizing Ciona oocytes reveal suggestive surface contractions and cytoplasmic/cortical flows probably related to meiotic cell cycle progression, which may provide the force for meiotic spindle migration and/or polarization of organelles (FILM 3; Prodon et al.,2006).

Maternal RNAs Polarize in the Cortex Along the a-v Axis (Figs. 1D,E, 2e,f)

A remarkable feature of mature ascidian oocytes is the large population of cortical maternal mRNAs (more than 40 in C. intestinalis), which are distributed in a gradient of increasing density along the a-v axis (Figs. 1D,E, 2e,f; Yamada et al.,2005; Yamada,2006). These interesting and varied mRNAs include one identified developmental determinant (macho-1) involved in muscle and posterior cell fate (Nishida and Sawada,2001; reviewed in Nishida,2005). It has been suggested that these maternal cortical mRNAs be called postplasmic/PEM RNAs, because they concentrate and segregate into a small Posterior End Mark in the posterior region (called postplasm) of the zygote and embryo (Yoshida et al.,1996; Sasakura et al.,1998,2000). The postplasmic/PEM mRNAs are divided into two classes: the majority, which display a polarized distribution in the oocyte, are called Type I, whereas those that appear unlocalized until after fertilization are known as Type II (for review, see Sardet et al.,2005, and Prodon et al. and Kumano and Nishida in this issue).

High resolution fluorescent in situ hybridization in mature (stage IV) oocytes shows that Type I postplasmic/PEM RNAs are in fact concentrated as a thin layer (1–2 microns thick) situated between the plasma membrane and the myoplasm (Fig. 2f). In situ localization of RNAs retained in isolated cortical fragments shows that, in Ciona, Phallusia, and Halocynthia, several postplasmic/PEM RNAs are bound to the cER monolayer distributed in a gradient of increasing density along the a-v axis, together constituting the cER-mRNA domain (Fig. 2i–k,n; Sardet et al.,2003,2005; Nakamura et al.,2005; Prodon et al.,2005). Before GVBD (Stage III oocyte), several postplasmic/PEM RNAs (PEM-1, PEM-3, macho-1) are already located beneath the surface as a thin unpolarized layer and evenly distributed in thicker patches (Fig. 2e; Prodon et al.,2006; A. Paix and C. Sardet, unpublished observations). After GVBD, these patches sandwiched between the plasma membrane and the myoplasm spread into a thin polarized layer in C. intestinalis (Figs. 1B,E, 2e,f). We do not know yet when or how the cER-mRNA domain is established, but it appears that, like mitochondria, postplasmic/PEM RNAs move out from a perinuclear location during vitellogenesis and then polarize along the a-v axis during maturation at approximately the same time as the myoplasm polarizes (i.e., 2–3 hr after GVBD in C. intestinalis; Fig. 2e,f; Prodon et al.,2006).

MULTIPLE CORTICAL AND CYTOPLASMIC REORGANIZATIONS BETWEEN FERTILIZATION AND FIRST CLEAVAGE

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. OOGENESIS, MATURATION, AND POLARIZATION OF THE OOCYTE: SPECIFICIATION OF A PRIMARY ANIMAL–VEGETAL AXIS
  5. MULTIPLE CORTICAL AND CYTOPLASMIC REORGANIZATIONS BETWEEN FERTILIZATION AND FIRST CLEAVAGE
  6. DV AND AP AXES ARE SPECIFIED BEFORE FIRST CLEAVAGE BY CORTICAL AND CYTOPLASMIC REORGANIZATIONS
  7. EARLY CLEAVAGES: A SERIES OF ASYMMETRICAL DIVISIONS
  8. POLARIZATION OF OOCYTES AND EMBRYOS: ASCIDIANS COMPARED WITH OTHER ORGANISMS AND THE QUESTIONS THAT REMAIN
  9. Acknowledgements
  10. REFERENCES

The fertilizing sperm activates the quiescent stage IV oocyte arrested in metaphase of meiosis I (Fig. 1F,G). In addition, the sperm brings spatial and temporal information to the oocyte, which before fertilization displays a radial symmetry along the a-v axis. Fertilization can occur anywhere on the oocyte's surface, but analysis of sperm entry in dechorionated oocytes of P. mammillata shows a bias in sperm fusion, being most frequent in the animal pole region and less frequent in the vegetal hemisphere (Speksnijder et al.,1989b; Roegiers et al.,1995).

Fertilization initiates a series of physiological, cell cycle, cytoskeletal, and developmental events that we have represented in Figure 1F–J using a time scale, which is that followed by Ciona and Phallusia zygotes at 18–19°C (first cleavage occurring approximately 1 hr after fertilization at that temperature).

Although there are clearly two major phases of reorganization (corresponding to the so-called first and second phase of “ooplasmic segregation”) between fertilization and first cleavage, careful examination using the transparent dechorionated zygote of P. mammillata shows that up to nine successive events and phases can be distinguished (Roegiers et al.,1999). In recent years, we have observed that most of these processes also occur in C. intestinalis (Prodon et al.,2005; F. Prodon, A. Paix, and C. Sardet, unpublished observations). Here, we outline the events and successive phases in chronological order from fertilization to cleavage.

First Major Phase of Reorganization: From Fertilization to Meiosis Completion

Fertilization/contraction phase: 0–5 min PF (Figs. 1F–G, 2l–n, and FILMS 10–15).

A first consequence of sperm fusion with the egg is that it causes a calcium wave, which starts from the site of fertilization (see pink arrow in Fig. 1F) to traverse the egg at a speed of 8–9 microns/sec (Speksnijder et al.,1989a,1990a; McDougall and Sardet,1995). This calcium wave triggered by a sperm factor has pleiotropic effects (Carroll et al.,2003; Whitaker,2006). Within 1–2 min of the onset of the fertilization/calcium wave a cortical contraction is initiated, which is probably mediated by Rho proteins (Yoshida et al.,2003). The calcium increase also stimulates mitochondrial respiration and causes the meiotic cell cycle to resume, leading to the emission of a first polar body (Nixon et al.,2000; Dumollard et al.,2003).

The large reorganization of the oocyte caused by the fertilization/contraction phase has traditionally been called “the first phase of ooplasmic segregation” (Sardet et al.,1989; Specksnijder et al.,1990a; Chiba et al.,1999). The rise in free calcium (from 0.5 to 7–10 micromolar) triggers an acto-myosin driven cortical contraction wave starting on the side of the egg where fertilization occurred (Specksnijder et al.,1990a; Roegiers et al.,1995). This microfilament-dependent cortical contraction spreads through the oocyte generally toward the vegetal hemisphere culminating in a protrusion or bulge called the “vegetal/contraction pole” (abbreviated CP in Figs. 1, 2; Sawada and Osanai,1985). The cortical contraction causes the myoplasm and cER-mRNA domains to concentrate around the vegetal/contraction pole (Figs. 1F,G, 2l–n, FILMs 10, 11, 12). The contraction also results in the formation of three new domains: a surface region rich in microvilli at the vegetal/contraction pole as well as domains rich in ER and yolk on the cytoplasmic face of the myoplasm (Roegiers et al.,1999; Dumollard and Sardet,2001). The roughly a-v directionality of the contraction is apparently due to the fact that, regardless of the site of sperm entry, the oocyte's acto-myosin cortical basket can only contract in a general animal to vegetal direction probably because the cortical microfilament network is less dense around the animal pole (Sardet et al.,1992; Chiba et al.,1999).

Another major consequence of sperm entry and fertilization/contraction phase is the introduction under the oocyte's surface of a paternal basal body and nucleus, which both undergo the same vegetally driven translocation along with maternal cortical and subcortical organelles, such that the basal body (becoming a male centrosome) and sperm nucleus (paternal chromosomes) will generally be relocated to the vegetal cortex (FILMs 13,14; McDougall and Sardet,1995; Roegiers et al.,1995; Dumollard and Sardet,2001). This rapid vegetal translocation of the sperm centrosome and nucleus (completed approximately 2–3 min after fertilization) had previously led to the mistaken notion that fertilization always occurred in the vegetal hemisphere. As soon as the sperm centrosome penetrates, it begins to nucleate microtubules and gather ER in the center of the nascent aster. The vegetally translocating sperm centrosome/ER accumulation is the initiation site of a series of smaller postfertilization (PF) calcium waves that traverse the egg after the main fertilization/calcium wave (Speksnijder et al.,1989a,1990a). We have given the name “pacemaker 1” to this moving calcium wave pacemaker situated at the male centrosome (Fig. 1F, pink arrow; Dumollard and Sardet,2001).

Oscillation and meiotic completion phase: 5–30 min PF (Fig. 1G–H, and inset, and FILMs 10,12,13,14,16,22).

The characteristic bulge formed in the vegetal/ contraction pole of zygotes of Phallusia and Ciona disappears soon after the first polar body is emitted (5–7 min PF; see inset between Fig. 1G and H). The smaller calcium waves that follow the initial fertilization wave cease when the first polar body is emitted and MPF activity drops to a low level. MPF activity starts rising again approximately 10–13 min after fertilization (McDougall and Levasseur,1998; Russo et al.,1998; Levasseur and McDougall,2000). Then a new series of periodic calcium waves starts, as MPF activity remains high for 10–15 min and then drops before the second polar body is emitted (approximately 30 min PF). MAPK activity reaches maximum value at 10–15 min after first polar body emission and drops before completion of meiosis, as does MPF activity.

During this period between first and second polar body emission, the repetitive calcium waves are generated with gradually increasing and then decreasing amplitudes (0.5 to 2 micromolar) and with frequencies of 1–3 min, depending on egg batches (Speksnijder et al.,1990b; Dumollard and Sardet,2001). All these meiotic calcium waves are emitted from the “pacemaker 2,” which is located at the position of the vegetal/contraction pole even after the protrusion subsides, and more precisely in the cER-mRNA domain, which has accumulated there (FILMs 13, 14; pink arrow in inset between Fig. 1G and H; McDougall and Sardet,1995; Carroll et al.,2003). As a consequence, for 20 to 30 min, the vegetal region is exposed to higher calcium concentrations than the rest of the zygote (Speksnijder et al.,1989a,1990b).

It is also clear that there is a certain amount of cross-talk between the cER domain and the tightly packed mitochondria in the myoplasm because repetitive calcium waves have an influence on the activation of mitochondrial metabolism (Dumollard et al.,2003). Mitochondrial function is necessary in turn to sustain the calcium wave pacemaker 2. Although there are periodic contractions of the zygote that correspond to the passage of each calcium wave, they do not appear to change the stratification of the domains around the vegetal/contraction pole (FILMs 12, 16, 22). The most conspicuous changes during this meiotic oscillation period are the displacement of the aster from the cortex to the subcortical area, the progressive growth of microtubules and accumulation of ER around the sperm aster, and the formation of ER-rich microdomains in the vegetal cytoplasm (FILM 22; Roegiers et al.,1999; Dumollard and Sardet,2001).

Interphase/Vegetal Button Stage: 30–40 min PF (Fig. 1H and FILMs 16,17,23)

Just after repetitive meiotic calcium waves and associated periodic contractions cease, a second polar body is extruded at the animal pole and nuclear membranes form around male and female chromosomes yielding pronuclei. The centrosome duplicates and microtubules grow to fill up the posterior region and extend into the animal pole region (Fig. 1H). In zygotes of Phallusia and Ciona at least, a second transient protrusion we have called the “vegetal button” (VB in Fig. 1H) appears at the site where the vegetal/contraction pole had previously formed 25 min earlier (Fig. 1H, FILM 16, 17, 23; Roegiers et al.,1999). Like the vegetal/contraction pole, the vegetal button is a microfilament-dependent structure and is characterized by a tuft of long microvilli filled with microfilaments. This protrusion lasts approximately 10 min and varies in size and shape from one batch of egg to another. It ranges from being a minuscule peduncle to a larger bulge or multiple tiny bulges. The vegetal button contains an electron dense granular area, which we have called the “vegetal body,” that is surrounded by a high density of cER (Roegiers et al.,1999). We have suggested that the vegetal body contains germ plasm-like granules, an idea reinforced by our observations that, in Phallusia, Vasa mRNA is concentrated in that region (A. Paix, P. Dru, and C. Sardet, unpublished observations). We have also pointed out that there may exist some similarities between the vegetal button and polar lobes, the transient vegetal protrusions characteristic of zygotes and early embryos of annelids and molluscs (Conrad et al.,1990).

Second Major Phase of Reorganization (and Its Multiple Subphases): From Meiosis Completion to First Mitotic Division

The completion of the meiotic cell cycle results in the formation of male and female pronuclei around the chromosomes and the growth of a large microtubular sperm aster in the future posterior pole (Fig. 1H, FILMs 10, 16). Interactions between microtubular structures and the cortex and changes in the contractile properties of the cortex are then driven by progression of the mitotic cell cycle as it passes through interphase (30–40 min), mitosis (45–55 min), and cytokinesis (55–60 min). The large aster structures the cytoplasm throughout this period, contacting male and female pronuclei and interacting with the cortex, in particular the cortical region nearest the aster (future posterior pole). The microtubule-dependent motions of the male pronucleus toward the center of the zygote and then of both pronuclei toward each other signal the onset of the second major phase of reorganization, traditionally called “the second phase of ooplasmic segregation” (Chiba et al.,1999; Roegiers et al.,1999). This second major phase of reorganization in fact comprises a series of distinct events and three subphases involving not only microtubules but also microfilaments: two successive microtubule-mediated translocation phases, one slow and one fast, and finally a microfilament-mediated vegetal relaxation phase preceding the first cleavage (Sardet et al.,1989; Roegiers et al.,1999; Prodon et al.,2005).

Migration of pronuclei: 30–40 min PF (Fig. 1H,I and FILMs 10,16,18,21,22).

In the period during which the vegetal button grows and resorbs, the female pronucleus (which is often multilobed) starts migrating along sperm aster microtubules toward the male centrosomal area, which is now disc shaped due to the duplication of the sperm-introduced centrosome (FILMs 18, 21, 22). Simultaneously the centrosome pair and the male pronucleus move toward the female pronucleus and egg center, causing the cER and myoplasm, which appear attached to the male pronucleus, to begin their translocation (FILM 18; Sardet et al.,1989; Speksnijder et al.,1993; Roegiers et al.,1999).

Slow posterior translocation phase: 30–45 min PF (Fig. 1H,I and FILMs 10,20–23).

The large sperm aster, which has developed at the end of meiosis completion, is clearly connected to the posterior cortex by means of numerous microtubules that course along the posterior cortex and also traverse the mitochondria-rich myoplasm domain by means of ER-rich corridors that extend all the way to the cortex (FILMs 19, 20; Roegiers et al.,1999; Chiba et al.,1999). That the posterior cortex has special interactions with microtubules is shown by observations on live and fixed zygotes and on cortical fragments isolated at the time the major reorganizations of the posterior region begin. The posterior cortex, which is very rich in microtubules underlying the plasma membrane, vibrates at a high frequency during this phase (FILMs 19, 20, 21; Roegiers et al.,1999). These rapid deformations of the surface are abolished in the presence of the microtubule inhibitor nocodazole. In addition, cortical fragments isolated from the posterior region retain many attached microtubules, whereas cortical fragments isolated from other regions of the zygote do not (Prodon et al.,2005; C. Sardet and H. Nishida, unpublished observations).

As the female pronucleus starts migrating along astral microtubules toward the duplicated sperm aster and male pronucleus, these latter structures move away from the cortex and toward the center of the egg (FILM 18). Time-lapse recordings show that the basket- or bowl-shaped myoplasm, which appears tethered to the paternal centrosomes and nucleus, undergoes jerking motions toward the posterior pole and the center of the zygote (FILMs 10, 16, 20). This phase, which we named the “slow posterior translocation phase,” lasts approximately 15 min (Sardet et al.,1989; Roegiers et al.,1999). The vibrations and translocations are clearly microtubule-driven and are indicative of tensions between the large aster moving with respect to the cortex and the bulky subcortical myoplasm and elements of the cER, which have some attachment to the cortex (FILM 20). This phase causes the posterior translocation of the cER-mRNA and myoplasm domain at an average speed of 5 microns/min (FILMs 10, 21, 22; Sardet et al.,1989).

Fast posterior translocation phase: 45–50 min PF (Figs. 1I,J, 2o,p, and FILMs 10,16,21,22,23).

The fast translocation phase is brief and is characterized by a smooth and rapid displacement of the duplicated male centrosome, the adjoining male and female pronuclei, the microtubule-rich/ER-rich aster, and myoplasm and cER-mRNA domains toward the posterior and center of the zygote at an average speed of 25 microns/min (Sardet et al.,1989; Roegiers et al.,1999). Time-lapse observations suggest that the resistance to motion of the aster with respect to the cortex is relieved. The bottom of the bowl-shaped mitochondria-rich myoplasm tears in the vegetal pole region such that the bulk of the myoplasm (but not all of it) accompanies the aster and relocates posteriorly while a smaller portion moves anteriorly (FILMs 10, 22). The rim of the myoplasm bowl remains intact, forming a girdle of subcortical myoplasm which encircles the entire equatorial region (Sardet et al.,1989; Roegiers et al.,1999). Time-lapse confocal microscopy observations of eggs injected with an oil droplet saturated with 1,1′, di-octadecyl-3,3,3′,3′,-tetramethylindo-carbocyanine perchlorate C16(3) (DiIC16(3)) to label the ER network show that the entire cER network moves with the bulk of the myoplasm to the posterior region of the zygote (Figs. 1I,J, 2o,p, FILMs 22, 24; Roegiers et al.,1999; Prodon et al.,2005).

Mitosis, the vegetal relaxation phase, and cleavage: 50–60 min PF (Fig. 1J and FILMs 10,22,23,24).

Once the male pronucleus and female pronucleus are in the center of the zygote and the bulk of myoplasm and cER domains have moved posteriorly by means of microtubule generated forces, nuclear membranes disperse and a small mitotic apparatus surrounded by very large asters forms in the center of the zygote (Fig. 1J, FILM 10). It is between this time and the onset of first cleavage that a general surface movement (visualized with attached surface particles) is initiated from the vegetal pole region and progresses toward the equator at a speed of 0.15 microns/second (FILM 23; Roegiers et al.,1999). This “surface relaxation wave,” which depends on actin microfilaments, completes the translocation of the myoplasm and cER domain toward the posterior/ equatorial region. This movement is considered a “relaxation,” because surface particles spread out farther from one another over the egg as opposed to concentrating closer together as during the earlier “contraction” triggered by fertilization. We believe that the precleavage relaxation wave is mediated by cell cycle factors, by analogy with surface contraction/relaxation waves described in precleaving zygotes of Xenopus and the ctenophore Beroe (Beckhelling et al.,2000). At the end of this final readjustment of domain positioning, cleavage is then initiated in the vegetal region (FILMs 23, 24). Because the duplicated centrosomes generally align perpendicular to the a-v axis and equidistant from the posterior domains, the first cleavage plane forms along the a-v (or D-V) axis, bisecting the myoplasm and cER-mRNA domains and distributing them equally into the first two blastomeres (Fig. 1K, FILM 24).

DV AND AP AXES ARE SPECIFIED BEFORE FIRST CLEAVAGE BY CORTICAL AND CYTOPLASMIC REORGANIZATIONS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. OOGENESIS, MATURATION, AND POLARIZATION OF THE OOCYTE: SPECIFICIATION OF A PRIMARY ANIMAL–VEGETAL AXIS
  5. MULTIPLE CORTICAL AND CYTOPLASMIC REORGANIZATIONS BETWEEN FERTILIZATION AND FIRST CLEAVAGE
  6. DV AND AP AXES ARE SPECIFIED BEFORE FIRST CLEAVAGE BY CORTICAL AND CYTOPLASMIC REORGANIZATIONS
  7. EARLY CLEAVAGES: A SERIES OF ASYMMETRICAL DIVISIONS
  8. POLARIZATION OF OOCYTES AND EMBRYOS: ASCIDIANS COMPARED WITH OTHER ORGANISMS AND THE QUESTIONS THAT REMAIN
  9. Acknowledgements
  10. REFERENCES

Site of Sperm Entry Influences the Position of the Dorsal Pole

It has been generally believed that, in ascidians, the contraction pole forms at the vegetal pole, which is defined as the antipode of the location of the meiotic apparatus/polar bodies (Bates and Jeffery,1988). Although this appears to be true in eggs observed at low resolution, high resolution observations of dechorionated P. mammillata eggs show that the fertilization/contraction wave, which depends on sperm entry site, is often asymmetric with respect to the a-v axis and results in a contraction pole angled away from the vegetal pole toward the side opposite the site of sperm entry (Fig. 1G, FILM 15; Speksnijder et al.,1990a; Roegiers et al.,1999). Depending on the position of the sperm entry site the location of the contraction pole can be shifted by up to 45–60 degrees with respect to the location of the vegetal pole. The shift is maximal when sperm entry occurs in the equatorial or vegetal region. Experiments using particles to mark the vegetal/contraction pole show that its position (which is also the position of the vegetal button) will be the future site of gastrulation (Roegiers et al.,1995; Nishida,1997). After morphogenetic movements, the site of gastrulation corresponds to the dorsal side of the embryo, and the polar bodies (animal pole) are located on the ventral side (see the representation of embryonic axes in Fig. 2 in Nishida,2005). Complementary observations on dechorionated eggs of C. intestinalis indicate that the scenario elaborated for Phallusia probably holds true for Ciona, but in earnest we do not know if this situation reflects what happens within the intact chorion or in other species such as H. roretzi, where the opacity of eggs make such detailed observations of spatial coordinates of sperm entry and consequent reorganizations difficult.

Position of the Sperm Centrosome After Translocation Defines the Posterior Pole

The sperm-derived centrosome and pronucleus specify the localization of the future posterior pole of the embryo, because they position the myoplasm and cER-mRNA domains that confer posterior position and fates on the cells that inherit them (see Prodon et al. and Kumano and Nishida in this special issue). The final location of the sperm centrosome is a result of the three successive subphases of reorganizations described above depending on microfilaments, then on microtubules and again on microfilaments (Fig. 1F–J). First the acto-myosin driven fertilization/contraction wave relocalizes the centrosome and nucleus introduced by the fertilizing sperm vegetally, thus defining a position on a meridian that will correspond to the posterior region. Second, the growth of sperm aster microtubules and their interaction with the cortex drive the translocation of the myoplasm and cER-mRNA domain toward the sperm entry side of the zygote (Sardet et al.,1989; Speksnijder et al.,1990a; Roegiers et al.,1999). Finally, we have pointed out that, in Phallusia, a microfilament-mediated relaxation wave completes the relocalization of myoplasm and cER-mRNA domain toward a more equatorial position. This final relocalization event specifies the exact position of the future posterior pole.

Cortical and Cytoplasmic Reorganizations Relocalize Developmental Determinants

An important consequence of the reorganizations of the first cell cycle is to position maternal determinants. These developmental determinants must be properly localized to ensure their distribution into the appropriate lineage to pattern the embryo (Nishida,1997,2005). From ablation and electrofusion of different peripheral regions (containing both cortex and subcortex) of oocytes and zygotes, the localizations of three tissue determinants (muscle, endoderm, ectoderm) and two morphogenetic determinants (unequal cleavage and gastrulation) have been mapped (reviewed in Nishida,2002,2005, and Kumano and Nishida in this issue). After the first major phase of reorganization (Fig. 1F,G), all determinants except for ectoderm are concentrated in the vegetal region, two of which (determinants for muscle and unequal cleavage) move to the posterior region after meiosis completion. The posterior region rich in postplasmic/PEM RNAs, cER, and the putative germ plasm formed during the second major phase of reorganization (Fig. 1I,J) has been named the PVC (Posterior Vegetal Cytoplasm) by Nishida and his colleagues (Nishida,1997). Using the large zygote of the Japanese ascidian H. roretzi, they have been able to excise this PVC region and electro-fuse the resulting piece with other regions of operated or nonoperated zygotes and have thus changed the anterior into a posterior pole (Nishikata et al.,1999; Nishida,2002). These remarkable experiments have shown that the PVC not only contains the muscle and posterior patterning determinant (now identified as macho-1) but also contains the morphogenetic determinant that causes asymmetric divisions in the posterior pole by means of a macroscopic cortical structure called the Centrosome Attracting Body (CAB: see next paragraphs and article by Kumano and Nishida in this special issue).

Some of these maternal determinants are associated with identifiable domains. We have observed that the fertilization/contraction phase of reorganization results in the stratification of at least five cytoplasmic and cortical domains around the vegetal/contraction pole (Roegiers et al.,1999). The acto-myosin–driven contraction concentrates two pre-existing domains (cER-mRNA and adjacent myoplasm) and causes new domains to appear (a domain rich in surface microvilli, and cytoplasmic domains respectively rich in ER and rich in yolk vesicles located on the cytoplasmic side of the myoplasm). The muscle determinant (i.e., macho1) and unequal cleavage determinant (i.e., the precursor of the CAB, see below) clearly locate at the level of the cER-mRNA domain and follow its movements, first to the vegetal/contraction pole and then to the posterior pole.

Less is known about the specific location or identity of the other determinants. Experiments based on either ultraviolet irradiation or micromanipulation of regions of the egg and zygote show that determinants for gastrulation are situated close to the surface in the vegetal/contraction pole and that they do not move posteriorly after meiosis completion but rather disperse throughout the vegetal cortex and subcortex (Nishida,1996; Bates and Jeffery,1987; Bates,2004; Jeffery,1990; reviewed in Jeffery,2001). The location and movement of the gastrulation determinant resembles the relaxation spreading of the domain rich in surface microvilli, which becomes uncoupled from the posterior translocation of the myoplasm and cER-mRNA domains. The endoderm determinant shows a similar vegetal concentration followed by an equatorial spreading (Nishida,1993) and thus may also correspond to the microvilli-rich domain or possibly the yolk-rich domain. The animal ectoderm determinant may be situated in the “clear ectoplasm” described by Conklin (Conklin,1905a), but we have detected no conspicuous organelles or molecular markers that could define this domain.

EARLY CLEAVAGES: A SERIES OF ASYMMETRICAL DIVISIONS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. OOGENESIS, MATURATION, AND POLARIZATION OF THE OOCYTE: SPECIFICIATION OF A PRIMARY ANIMAL–VEGETAL AXIS
  5. MULTIPLE CORTICAL AND CYTOPLASMIC REORGANIZATIONS BETWEEN FERTILIZATION AND FIRST CLEAVAGE
  6. DV AND AP AXES ARE SPECIFIED BEFORE FIRST CLEAVAGE BY CORTICAL AND CYTOPLASMIC REORGANIZATIONS
  7. EARLY CLEAVAGES: A SERIES OF ASYMMETRICAL DIVISIONS
  8. POLARIZATION OF OOCYTES AND EMBRYOS: ASCIDIANS COMPARED WITH OTHER ORGANISMS AND THE QUESTIONS THAT REMAIN
  9. Acknowledgements
  10. REFERENCES

First Cleavage Partitions cER-mRNA and Myoplasm Domain Equally: 60 min PF (Fig. 1K and FILM 24)

As a result of the reorganizations described above, at the end of the first cell cycle, the bulk of the crescent-shaped myoplasm is situated on the posterior side of the zygote, with the smaller cER-mRNA domain (containing postplasmic/PEM and embedded putative germ plasm) nestled between the myoplasm and the plasma membrane (Nakamura et al.,2005; Prodon et al.,2005; Patalano et al.,2006). From this position, these two domains will be divided in their middle during first cytokinesis (Fig. 1K, FILM 24) and then partitioned into the posterior blastomeres of the embryo as a result of the alternating orientations of subsequent cleavage planes.

The first cleavage, which follows the general direction of the D-V axis, bisects the myoplasm and cER-mRNA domain. We have noticed that invagination of this first cleavage plane often starts in the vegetal region just after the surface relaxation wave (FILMs 23, 24). This cleavage yields two daughter cells equal in size, which will give rise to the left and right sides of the bilaterally symmetrical embryo (Nakauchi and Takeshita,1983; Morokuma et al.,2002). When separated at the two-cell stage, each blastomere will form a half-size tadpole, which has either left or right characteristics showing that they are already slightly different in developmental potential. Although the first two blastomeres are identical in detectable organelle content and domain distribution, they have been reported to be different with respect to channel permeability and calcium signaling (Albrieux and Villaz,2000). One possible explanation for such a difference between the first two blastomeres could be that the small piece of membrane situated in the vegetal/contraction pole is inherited unequally, with resulting differences in the segregation of membrane channels (Arnoult et al.,1996).

Second and Third Cleavages Partition the cER-mRNA and Myoplasm Domains Into Posterior Vegetal Blastomeres: 90 min PF (Fig. 1L,M)

The second cleavage is perpendicular to the first and separates the two posterior blastomeres containing the myoplasm and cER-mRNA domains from the two anterior blastomeres (Fig. 1L). When raised separately, these anterior and posterior blastomeres that are equal in size but not in content have different developmental potential (Nishikata and Satoh,1991; Ohtsuka et al.,2001). The third cleavage is equatorial, separating animal from vegetal halves with the cER domain and the bulk of the myoplasm inherited by the posterior-vegetal B4.1 blastomeres, and a small amount of myoplasm partitioned into b4.1 animal posterior blastomeres (Fig. 1M; Roegiers et al.,1999). The distribution and layering of the myoplasm and cER-mRNA domains at the eight-cell stage resemble that at the two-cell stage, but a relaxation occurs during the four-cell stage such that both domains transiently lose their compacted appearance. Observations of live, fixed, or extracted embryos indicate that cortical differentiations occur at the posterior pole during the two- and four-cell stages with respect to microvilli, actin microfilaments, and aPKC protein (C. Sardet, J. Chenevert, and A. Paix, unpublished observations) and accumulations of refractile granules (Iseto and Nishida,1999).

The segregation of posterior domains and associated developmental determinants results from the alternating cleavage planes, which are dictated by the orientation of the mitotic spindle. Experiments that change the position of the cleavage planes or delocalize the myoplasm (and probably cER-mRNA domain) change the developmental potential of the resulting blastomeres (Whittaker,1982). What controls the orientation of these early cleavage planes? Possibly the myoplasm itself or the cER-mRNA domain (giving rise to the CAB, see below) influences spindle position because the posterior vegetal B4.1 blastomeres which contain the bulk of the myoplasm and cER-mRNA domains at the eight-cell stage are bigger than the other blastomeres and protrude posteriorly. Recent observations indicate that the precursor structure of the CAB may influence the orientation of mitotic spindles before the eight-cell stage (T. Negishi, H. Nishida; C. Sardet, unpublished observations).

Fourth, Fifth, and Sixth Cleavages Separate the Myoplasm From the cER-mRNA Domain by Means of Asymmetric Divisions: 2–3 hr PF (Figs. 1M,N, 2q–y, and FILMs 25–29)

Starting at the eight-cell stage, the cER-mRNA domain containing germ plasm granules forms the bulk of the CAB, a macroscopic disc-shaped structure nestled between the plasma membrane and the myoplasm. The CAB directs a series of three asymmetric divisions resulting in blastomeres that differ in size and content (FILMs 25–26). In the B4.1 pair of blastomeres the CAB “attracts” a centrosome toward the posterior cortex so that the spindle positions excentrically and cleavage is unequal, generating one smaller cell containing the CAB (B5.2) and a larger cell (B5.1) containing most of the myoplasm (Fig. 1M,N). This asymmetric division is repeated twice more such that at the 64-cell stage the cER-mRNA domain (including the putative germ plasm) is partitioned into two small posterior cells (B7.6) and the bulk of the myoplasm is segregated into six primary muscle cell precursors (B7.4, B7.5, B7.8 pairs).

CAB: A Specialized Structure Implicated in Asymmetric Cleavages and mRNA Localization and Translation

The role of the CAB in unequal cleavage has been demonstrated by micromanipulation experiments: when posterior fragments of the zygote (PVC) are removed no CAB is formed and the B4.1 cells cleave equally, whereas fusion of posterior fragments to an anterior position causes extra CAB formation and unequal cleavage in an ectopic site (Nishikata et al.,1999). The mechanism of centrosome attraction is not yet understood, but it is thought that some component of the CAB facilitates capture of plus ends of microtubules and a pulling action on one centrosome (Nishikata et al.,1999). Observations of living embryos show a migration of the duplicated centrosome and interphase nucleus toward the posterior cortex during which the surface at the position of the CAB ripples and forms a transient protrusion (Fig. 2r, FILM 25; Patalano et al.,2006). In fixed specimens, microtubules extend from the centrosome to the cortex and a conspicuous bundle connecting the nucleus to the CAB can sometimes be detected (Nishikata et al.,1999; Patalano et al.,2006). The observation of a kinesin-like protein in the CAB region (Nishikata et al.,1999) and the recent demonstration that the polarity proteins aPKC, PAR-6, and PAR-3 accumulate on the cortical face of the CAB (Patalano et al.,2006) provide clues as to possible molecular mechanisms by which this cortical domain may influence microtubule dynamics or anchoring (Figs. 1K–N, 2v–y, FILMs 25–29).

The CAB appears to be a conserved feature of ascidian embryos. Interestingly, this peripheral structure had been observed over a century ago in the original reports of ascidian embryonic development. Chabry (1887) described “une petite saillie en forme de mamelon” (literally “small nipple-shaped protrusion”) along the surface of posterior-vegetal blastomeres of the eight-cell embryo of Ascidia Aspersa. Both Castle and Conklin made remarkable drawings delimiting the “regions of clear protoplasm” in the posterior-most blastomeres of 8- to 64-cell embryos of C. intestinalis (Castle,1894, 1896) or Styela/Cynthia (Conklin,1905a, c). This important structure was then neglected until being rediscovered as a posterior disc in extracted Halocynthia embryos (Hibino et al.,1998; Nishikata et al.,1999; Iseto and Nishida,1999). Since then, the CAB has been documented in embryos of Ciona, Halocynthia, Phallusia, and Molgula, and now the colonial ascidian Botrylloides using in situ localization of mRNAs and proteins, labeling of ER, DIC optics, or electron microscopy (Brown and Swalla,2007; Hibino et al.,1998; Iseto and Nishida,1999; Nishikata et al.,1999; Sardet et al.,2003; Patalano et al.,2006; Gyoja,2006). The CAB when it is disc-shaped attains dimensions of 20 × 10 × 5 microns in Ciona and Phallusia and more than double that size in the large embryos of Halocynthia.

The formation of the CAB appears to occur progressively in phases. First, the cER-mRNA domain is put in place in the posterior pole as a result of the multiple reorganizations of the first cell cycle described above (Sardet et al.,2003; Nakamura et al.,2005; Prodon et al.,2005). At this stage, this posterior domain can be considered the precursor of the CAB (we propose the name “preCAB”). At first cleavage, the preCAB marks the posterior location where the CAB will form but does not yet attract the centrosome. Then during the two- and four-cell stages, other components gradually concentrate at the preCAB site: these components include in particular the complex of polarity proteins PAR3/PAR6/aPKC (Patalano et al.,2006), as well as extraction-resistant surface granules (Iseto and Nishida,1999) and a tuft of microfilament-rich microvilli (A. Paix and C. Sardet, unpublished observations). Morpholino knockdown experiments indicate that Hr-POPK-1, a localized postplasmic/PEM RNA encoding a kinase, plays a key role in concentration and positioning of the cER and other components of the CAB, such as putative germ plasm granules during this period (Nakamura et al.,2005). By the eight-cell stage, the CAB is fully mature, has acquired a multilayered structure and is able to attract durably the centrosome and position the spindle in an excentric position for asymmetric cleavage (Prodon et al.,2005; Patalano et al.,2006). All these observations suggest that the preexisting PVC posterior domain (including the cER-mRNA domain and putative germ plasm) emits signals to recruit polarity proteins and a specific dynamic patch of microfilaments and microvilli at the level of the posterior pole. The basic multilayered structure of the CAB is retained during the 8-, 16-, and 32-cell stages, but it undergoes dynamic relaxation/contraction movements with each cell cycle, being widespread and thinnest in interphase and most compact and thickest during mitosis (Prodon et al.,2005; Patalano et al.,2006).

In addition to its role in unequal cleavage, the CAB is also proposed to be important for localizing translation and for segregation of the germ line. Analysis of the Ybox protein CiYB1, a major component of RNPs that concentrates in the CAB and associates with postplasmic/PEM RNAs, suggests that this protein contributes to the translational control of localized mRNAs in eggs and embryos (Tanaka et al.,2004). Although there is little known about the proteins encoded by postplasmic/PEM mRNAs, data obtained with an antibody to PEM-3 is consistent with the idea that postplasmic/PEM RNAs are translated in the CAB at or before the eight-cell stage (Satou,1999). The CAB-containing blastomeres are thought to be primordial germ cells, because the CAB is rich in electron-dense granules resembling germ plasm (Hibino et al.,1998; Iseto and Nishida,1999; Prodon et al.,2005) and the germ cell-specific marker Vasa (Fujimura and Takamura,2000; Takamura et al.,2002; Patalano et al.,2006). It has also been suggested that zygotic transcription and cell division are inactivated in the pair of cells that inherit the CAB (Tomioka et al.,2002). In fact, a recent study shows that the small posterior B7.6 cells containing the CAB resume cleavage at the time of gastrulation and form two distinct daughter cells, one B8.11 inheriting the CAB and the other B8.12 giving rise to the germ line (Shirae-Kurabayashi et al.,2006, and Prodon et al., this issue).

It should also be emphasized that many other blastomeres in the early embryo undergo asymmetric division to yield daughter cells of distinct fates and/or unequal sizes, including the larger sisters of the CAB-containing cells (Tassy et al.,2006) as well as blastomeres that segregate notochord from other cell fates (Darras and Nishida,2001; Minokawa et al.,2001; Yasuo and Hudson,2007), although in these other cases no CAB-like structure has been detected.

POLARIZATION OF OOCYTES AND EMBRYOS: ASCIDIANS COMPARED WITH OTHER ORGANISMS AND THE QUESTIONS THAT REMAIN

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. OOGENESIS, MATURATION, AND POLARIZATION OF THE OOCYTE: SPECIFICIATION OF A PRIMARY ANIMAL–VEGETAL AXIS
  5. MULTIPLE CORTICAL AND CYTOPLASMIC REORGANIZATIONS BETWEEN FERTILIZATION AND FIRST CLEAVAGE
  6. DV AND AP AXES ARE SPECIFIED BEFORE FIRST CLEAVAGE BY CORTICAL AND CYTOPLASMIC REORGANIZATIONS
  7. EARLY CLEAVAGES: A SERIES OF ASYMMETRICAL DIVISIONS
  8. POLARIZATION OF OOCYTES AND EMBRYOS: ASCIDIANS COMPARED WITH OTHER ORGANISMS AND THE QUESTIONS THAT REMAIN
  9. Acknowledgements
  10. REFERENCES

We can consider that the past decade has provided a reliable description of the events, structures, and key macromolecules essential for the polarization of the ascidian oocyte and the reorganizations that occur after fertilization. It is also clear that localized macromolecules and particularly some cortical mRNAs specify the embryonic axes and define the identity of embryonic cell types. In this sense, the ascidian embryo joins the small club of model organisms (Drosophila melanogaster, Caenorhabditis elegans, Xenopus laevis) where localized mRNAs and/or PAR proteins play key roles in embryonic polarization (Pellettieri and Seydoux,2002). It is now important to understand in detail the mechanisms at work and to compare them with those used by other organisms to specify axes and cell types. We have previously reviewed this question as applied to a handful of model organisms, including ascidians (Prodon et al.,2004; Sardet et al.,2004; and charts of comparative axis formation at: http://biodev.obs-vlfr.fr/recherche/biomarcell/ and click on “Embryo Comparisons Poster”). Such a comparative analysis reveals the wide range of strategies used in the animal kingdom for the establishment of embryonic axes. In a small number of cases such as the embryo of the fly D. melanogaster, developmental axes are established well before fertilization while in other organisms such as the nematode worm C. elegans these axes are set up only after fertilization. Ascidians represent an intermediate scenario similar to that in the frog X. laevis, in which a primary a-v axis acquired during oogenesis is remodeled by sperm entry to yield the embryonic axes.

In this review, we provide a time line for the physiological, cell cycle, cytoskeletal, structural, and macromolecular processes that polarize the oocyte and reorganize the zygote. Some of the causative relationships between these events still elude us, in part because they often belong to distinct “fields of study.” It is reasonable to assume that a common repertoire of physiological and cell cycle events are the master regulators of cytoskeletal transformations and of changes in the cytoplasm and cortex. In turn, these cortical and cytoplasmic changes drive the translocation and positioning of organelles and macromolecules. Finally the spatial and temporal control of protein synthesis or protein activation depends on physiological and cell cycle factors as well as on factors localized during oogenesis, maturation, or after fertilization (Sardet at al.,2004, Wilhelm and Smibert,2005).

Polarization of Oocytes

There are still many unanswered questions concerning the polarization of the ascidian oocyte along the a-v axis, including—How do oocytes originate, and does their history impart some spatial polarity information? —When and how do mitochondria and postplasmic/PEM mRNAs reach the oocyte's periphery? What roles do microtubules, microfilaments, and the cER network play in the translocation?—How are mRNAs and organelles anchored in the cortex and subcortex? When and how does the cER become a distinctive domain? How does the meiotic apparatus position itself under the animal pole surface? How is this area cleared of RNAs and organelles during maturation?

In many organisms, oocytes acquire spatial coordinates (a-v and/or A-P, D-V) in the gonad (Prodon et al.,2004). In ascidians, we do not know if oocytes inherit a polarity landmark with respect to their position in the gonad or site of previous cell division. The prerequisite identification of oocyte stages, as defined by their growth, differentiation, and relationship to surrounding follicle cells in different ascidian species, has begun (Okada and Yamamoto,1999; Yamamoto and Okada,1999; Chiba et al.,2004; Prodon et al.,2006). These descriptions should now include a thorough analysis of the location of different classes of postplasmic/PEM RNAs (Type I and Type II: see Prodon et al., this issue) during oogenesis and maturation and after fertilization and in particular of those RNAs that are bound to a cER network in the mature oocyte (macho-1, PEM-1, PEM-3, POPK-1; Sardet et al.,2003; Nakamura et al.,2005; Prodon et al.,2005). It is also essential to understand the information that lies in the noncoding regions of postplasmic/PEM RNAs that can lead to their cortical localization and association with cER or other cellular structures (Sasakura and Makabe,2002, and see Prodon et al., this issue).

Because young previtellogenic oocytes do not continue oogenesis outside of the gonad, they can only be studied in situ, so it is difficult to imagine how mechanisms involved in translocation of postplasmic/PEM RNAs and myoplasm can be approached. In contrast later stages in the establishment of a-v polarity can be tackled in C. intestinalis using spontaneously maturing oocytes (Prodon et al.,2006). Maturation and polarization should in principle be easier to investigate in Halocynthia where large quantities of stage III immature oocytes can be readily obtained (Sakairi and Shirai,1991). Micromanipulations performed to displace the GV or remove or transfer the meiotic spindle should tell us whether specific cortical sites that attract the GV or the spindle exist, as in the case in some echinoderms and annelids (Lutz et al.,1988; Miyazaki et al.,2000,2005). In ascidians, the migration of the meiotic spindle toward the surface resembles what has been described for mouse oocytes in that microfilaments play a major role (Maro and Verlhac,2002) but where and how acto-myosin creates force to move the spindle remains unknown. Specifically, we need to understand whether the surface movements and cortical flows observed during meiotic maturation are a cause or consequence of movements of the deeper cytoplasm. We believe that this contractile activity of the cortex is linked to the progression of the meiotic cell cycle and may reflect “surface contraction waves” triggered by the uneven distribution of factors that regulate the cell cycle (Beckhelling et al.,2000). Surface contraction waves triggered by mitotic cell cycle factors have already been characterized in the large eggs of X. laevis where they propagate from the animal pole (where the mitotic spindle is located) toward the vegetal pole with the effect of concentrating small germ plasm islands into larger aggregates that move vegetally and eventually segregate into primary germ cells (Perez-Mongiovi et al.,1998). This and other questions make it essential to study the temporal and spatial variations in cell cycle factors and related activities (MAPK, Mos, and so on) during maturation of ascidian oocytes, as has been done for meiosis completion after fertilization (Russo et al.,1998; Nixon et al.,2000).

Fertilization and the Reorganizations That Follow

There are many unresolved questions concerning the numerous changes triggered by fertilization. The following are a few examples of such questions and speculative answers based on what is known in other models.

Why does sperm preferentially enter the animal hemisphere? This finding is a property shared with some amphibians, which has not received much attention. Based on older observations made in sea urchins (Longo et al.,1986), we suggest that this tendency may be related to the ability to stabilize sperm fusion, which likely depends on calcium signaling differences. This in turn could be related to the a-v gradient distribution of cER (Sardet et al.,1992).

How does the sperm-triggered calcium wave generate the microfilament-driven contraction of the egg? The chain of events including the roles of G proteins (Rac, Rho, CDC42) and kinases (MLCK) could probably be worked out considering that synchronous fertilization of a large number of eggs is easily achieved and that cortical fragments can be prepared to analyze all intermediate stages of the contraction (Prodon et al.,2005). In vivo imaging approaches pioneered in X. laevis and C. elegans zygotes may be used to investigate these questions (Munro et al.,2004; Ma et al.,2006).

How are postplasmic/PEM mRNAs relocalized after fertilization? We have provided a partial answer to this question by showing that some postplasmic/PEM RNAs are bound to the cER network and move with it (Sardet et al.,2003; Prodon et al.,2005), and this may also apply to some vegetally localized RNAs in X. laevis (Chang et al.,2004). This mRNA attachment to the ER raises in turn many other questions: how many of the postplasmic/PEM RNAs are bound to the cER, how are they attached? Is binding to ER a way to regulate mRNA translation, as recent findings in D. melanogaster and C. elegans suggest (Decker and Parker,2006, and see review by Prodon et al. in this issue)?

How does the sperm-derived aster rotate against the cortex and relocalize the cER-mRNA domain and myoplasm posteriorly? We think it is significant that, in ascidians, in X. laevis and in C. elegans large translocations of organelles and macromolecules occur in interphase just after meiosis completion when the female pronucleus migrates along microtubules to meet the male pronucleus (Houliston et al.,1994; Sardet et al.,2004; Prodon et al.,2004). This finding suggests that cell cycle-driven transformations of astral microtubules and the cortex promote a sort of “cortical rotation” as seen in Xenopus or ascidian zygotes or “cortical flow” as described in C. elegans (Houliston et al.,1994). Undoubtedly, we need to know what microtubular motors are at work, how they are regulated by cell cycle factors, and where they interact with cortical structures to provide the force for movement (Labbe et al.,2004; Marrari et al.,2003,2004).

Early Cleavages and Embryonic Polarity

At the end of the first mitotic cell cycle, the posterior pole of the ascidian embryo contains many localized postplasmic/PEM RNAs, several of which play a role in proper patterning of the posterior of the embryo (reviewed by Nishida,2005, and by Prodon et al. and Nishida et al. in this issue). It is now essential to understand when these localized maternal mRNAs are translated and what functions are performed by the proteins they encode. Given the composition of the CAB, which is rich in ER and ribosomes, it is tempting to speculate that one purpose of localizing maternal mRNAs to this posterior structure is to regulate their translation. We know very little about translation of endogenous postplasmic/PEM RNAs, except for an early report of the localized production of PEM-3 protein in the posterior pole (Satou,1999) and the possible role of the RNA binding protein CiYB1 in regulating translation (Tanaka et al.,2004). That injected antisense and/or morpholino oligonucleotides (MO) produce early phenotypes (Nishida and Sawada,2001; Satou et al.,2002; Kondoh et al.,2003; Nakamura et al.,2006) suggests that some of the postplasmic/PEM RNAs are translated soon after fertilization. It is expected that important proteins such as macho1 and POPK-1 will be specifically synthesized in the posterior pole. Future studies with specific antibodies are now needed to understand the translational patterns of postplasmic/PEM RNAs.

The localization of the polarity proteins aPKC, PAR3, and PAR6 at the CAB probably does not depend on localized translation, because their mRNAs are homogeneously distributed (Patalano et al.,2006). Rather it is hypothesized that polarity proteins are recruited to the posterior pole by some signal provided by the preexisting cER-mRNA preCAB structure. One possibility is that the accumulation of the PAR complex may be activated by a small G protein as is the case in vertebrate cells (Etienne-Manneville,2004), because two of the localized postplasmic/PEM RNAs encode regulators of G proteins (CDC42 GEF and rhoGAP; Satou and Satoh,1997; Philips et al.,2003; Yamada et al.,2005). Testing these ideas will require a better understanding of the pattern of translation of postplasmic/PEM RNAs as well as a detailed functional analysis of these maternal factors (see Nakamura et al.,2006, and review by Prodon et al. in this issue).

Another main unresolved issue is the mode of action of the CAB. A dissection of the centrosome attraction mechanism will require time-lapse observations of centrosome motion and microtubule dynamics, as well as analysis of the forces involved and the roles of microtubular motors and of polarity proteins using inhibitors and dominant-negative constructions (Grill et al.,2001; McCarthy and Goldstein,2006). One complementary approach is to try to reactivate microtubule sliding on isolated cortical fragments that retain the CAB and attached microtubules following the strategies used on cortical fragments of Xenopus zygotes (Marrari et al.,2003,2004). The unequal cleavages directed by the ascidian CAB call to mind the generation of smaller cells in early embryos of other organisms (Gonczy,2002; Cowan and Hyman,2004). Interestingly, in many of these cases, including sea urchin micromeres, C. elegans posterior blastomeres, Drosophila pole cells, and ascidian posterior blastomeres, the smaller daughter cells generated by unequal cleavage are proposed to give rise to the germ line (Takamura et al.,2002; Tomioka et al.,2002; Fujii et al.,2006). The ability to visualize, isolate, and manipulate the CAB structure is a distinct advantage, and future studies of the ascidian model will help to establish what aspects of the mechanism of unequal cell division are conserved among different organisms.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. OOGENESIS, MATURATION, AND POLARIZATION OF THE OOCYTE: SPECIFICIATION OF A PRIMARY ANIMAL–VEGETAL AXIS
  5. MULTIPLE CORTICAL AND CYTOPLASMIC REORGANIZATIONS BETWEEN FERTILIZATION AND FIRST CLEAVAGE
  6. DV AND AP AXES ARE SPECIFIED BEFORE FIRST CLEAVAGE BY CORTICAL AND CYTOPLASMIC REORGANIZATIONS
  7. EARLY CLEAVAGES: A SERIES OF ASYMMETRICAL DIVISIONS
  8. POLARIZATION OF OOCYTES AND EMBRYOS: ASCIDIANS COMPARED WITH OTHER ORGANISMS AND THE QUESTIONS THAT REMAIN
  9. Acknowledgements
  10. REFERENCES