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).
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.