Mesoderm in ascidians is induced in the equatorial region (marginal zone of the vegetal hemisphere) mainly by a signal from the vegetal cells (Fig. 6A; Nishida,2005), as has been shown in vertebrates (Kimelman,2006). Whereas members of the transforming growth factor-β superfamily induce mesoderm in vertebrates (Feldman et al.,1998; Kofron et al,1999; Agius et al.,2000; Takahashi et al.,2000), FGF signaling represented by FGF9/16/20 plays a major role in mesoderm induction in ascidian embryos (Imai et al.,2002a,2003; Kumano et al.,2006). The FGF9/16/20 signal emanating from endoderm cells induces the notochord and mesenchyme in the anterior and posterior regions of the cleaving embryo, respectively (Fig. 6A; Imai et al.,2002a,2003; Kumano et al.,2006). As observed in the case of the notochord and mesenchyme induction, mesoderm in many organisms is induced in different cell types by the same signal (Cui et al.,1996; Kim et al.,2000; Kumano et al.,2001; Kobayashi et al.,2003). This finding is because regionalization within the mesoderm-arising territory takes place before the induction and provides different competence to respond to the inducing signal. In Halocynthia, maternal Macho-1 is responsible for this preinduction regionalization (Fig. 6C). This is another function of this protein, after its function as a muscle determinant as mentioned above. Maternal transcripts for Macho-1 become localized to the future posterior pole by means of ooplasmic movements after fertilization (Fig. 2G–I, see the article by Prodon et al. in this issue) and are inherited by the posterior blastomeres, and the protein confers posterior-specific responsiveness to the FGF9/16/20 signal at the 32-cell stage or later, when induction occurs (Fig. 6C; Kobayashi et al.,2003). A combination of Macho-1 and FGF9/16/20 signaling appears to lead to the expression of the twist-like-1 gene in the presumptive mesenchyme cells, which regulates all the mesenchymal genes known to date that are expressed at later stages (Imai et al.,2003). In the anterior region, on the other hand, competence to respond to the FGF9/16/20 signal appears to arise because of the absence of Macho-1 (Fig. 6C; Kobayashi et al.,2003). Zygotically expressed FoxA and Zic have recently been identified as intrinsic competence factors for notochord induction (Wada and Saiga,2002; Kumano et al.,2006). Maternally derived Macho-1, therefore, might suppress the function of FoxA and Zic in the posterior region. The FGF9/16/20 signal also induces the secondary muscle and the secondary notochord by means of activation of nodal signaling in cells (a pair of b6.5 blastomeres) in the animal hemisphere (Hudson and Yasuo,2005,2006).
Figure 6. Fate specification in the vegetal hemisphere of the ascidian embryo proposed in Halocynthia. A: A diagram of a 32-cell stage embryo. Vegetal view with anterior to the top. Notochord and mesenchyme are induced in the anterior four blastomeres (white at the top) and posterior four blastomeres (red hatched at the bottom), respectively, by FGF signaling (light-blue arrows) from endoderm cells (yellow). The hatched blastomeres contain Macho-1 as an intrinsic factor. At this stage, both anterior and posterior blastomeres do not have their fates restricted yet. B: A diagram of a 64-cell stage embryo. At the division to the 64-cell stage, the induced blastomeres divide to produce daughters with induced fates (notochord in pink and mesenchyme in green) at the positions closest to the endoderm, and daughters with default fates (nerve cord in purple and muscle in red) away from the source of the inducer. Each of the sister cells has its fate restricted to a single kind. The sister cells are connected with blue bars. C: (a–d) A model for binary specification of notochord vs. nerve cord and mesenchyme vs. muscle fates. (a,b) Simplified drawing of notochord and mesenchyme induction shown in A and B. Default fates (nerve cord anterior and muscle posterior) arise in both daughters when induction is blocked (c), whereas induced fates (notochord anterior and mesenchyme posterior) are assumed by both daughters when FGF signaling is applied over the entire surface of the mother cells (d). (a,b,e,f) A model for regionalization of the mesoderm by maternal Macho-1. Maternal Macho-1 is responsible for the difference in competence between the anterior and posterior regions to respond to FGF signaling. (e) The anterior-derived notochord/nerve cord arises in a mirror image in both anterior and posterior regions of the embryo without Macho-1. (f) The posterior tissues are duplicated in the anterior region when Macho-1 is ectopically expressed there. NC, nerve cord; Not, notochord; En, endoderm; Mes, mesenchyme; Mus, muscle. Colors are used as in Figure 4.
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Molecular cascades that lead to the expression of the notochord-specific brachyury gene (Yasuo,1993,1994) at the 110-cell stage have been extensively studied. Maternal β-catenin activates both intrinsic and extrinsic factors that are required for brachyury expression. FoxD and FoxA, both activated by β-catenin as early as the 16-cell stage (Imai et al.,2002b,2006; Kumano et al.,2006), are required for the production of Zic (Imai et al.,2002c,2006; Kumano et al.,2006) in the primary lineage, which together with FoxA confers notochord-specific responsiveness to FGF signaling (Wada and Saiga,2002; Kumano et al.,2006). The expression domains of FoxA and Zic overlap only in the presumptive notochord cells (Shimauchi et al.,1997; Wada and Saiga,2002; Imai et al.,2002c) in the anterior half of the embryo. The expression of the notochord inducer FGF9/16/20 is also activated by β-catenin (Imai et al.,2002a; Kumano et al.,2006). The secondary notochord is induced by Notch/Delta signaling in Ciona (Corbo et al.,1998; Imai et al.,2002b,2006; Hudson and Yasuo,2006), although this may not be the case in Halocynthia (Darras and Nishida,2001a; Akanuma et al.,2002). A recent study shows that the nodal signal from b6.5, both directly and indirectly by means of activation of Delta2, regulates brachyury expression in the secondary lineage (Hudson and Yasuo,2006).
Some other types of mesoderm are induced by signals from nonendodermal cells. In Xenopus, the ventral blood islands, where blood cells originate, are induced by bone morphogenetic protein (BMP) signaling from ectoderm cells (Maeno et al.,1994; Kumano et al.,1999). In ascidians, the TLCs, precursors of blood cells, are induced at the 16-cell stage by a signal from ectoderm (Kawaminami and Nishida,1997). Recent studies suggest that this signal may be represented by nodal, which emanates from b6.5 (Imai et al.,2006; Hudson and Yasuo,2006). In addition to the signal, the TLC also requires several other factors, including FoxD and NoTrlc (Imai et al.,2003,2006), whose function is probably intrinsic. The TVCs, precursors of heart in ascidians, are also induced. Heart precursors are induced by FGF signaling in two cells (a pair of B8.9 blastomeres) of the four competent cells (daughters of the B7.5 blastomeres, B8.9 and B8.10) that constitute the heart-forming field and are characterized by expression of the bHLH gene Mesp (Davidson and Levin,2003; Satou et al.,2004; Davidson et al.,2005,2006). Without the induction, the presumptive heart becomes larval tail muscle (Nishida,1992; Davidson et al.,2005,2006). Details about the formation of blood and heart, including the events that happen at later stages, are reviewed in other articles in this issue.
Asymmetric cell divisions are observed during ascidian embryogenesis, as mentioned in the previous section “2-3 Cleavage pattern.” In organisms that are known for their invariant embryogenesis with a relatively small number of embryonic cells, such as C. elegans and ascidians, cell fates are often induced and determined in a cell cycle before the fates are restricted (Nishida,1996). In ascidian notochord or mesenchyme induction, cells are fated to become both notochord and nerve cord, or mesenchyme and muscle, respectively, at the time they are induced at the 32-cell stage (Fig. 4A). It is the next division to the 44-cell stage when the cells asymmetrically divide to provide one daughter that gives rise to notochord or mesenchyme as induced fates and the other that becomes nerve cord or muscle as default fates (Fig. 6B). The notochord or mesenchyme cells arise closer to the endoderm (the source of the FGF9/16/20 signal), while the nerve cord or muscle cells arise away from it (Fig. 6B). This asymmetric division depends largely on external signals (Nakatani et al.,1996; Kim and Nishida,1999; Minokawa et al.,2001). Both daughter cells could adopt induced fates (notochord and mesenchyme) when the entire surface of the mother cells is exposed to FGF signaling, but take on default fates (nerve cord and muscle) in the absence of the signal (Fig. 6C). No intrinsic factors have been identified so far as being localized on one side of the mother cells before the division. It is, therefore, likely that the position from which FGF9/16/20 signaling is presented determines the asymmetry. In C. elegans, Wnt signaling can function as positional cues in establishing the polarity in the EMS cell (Goldstein et al.,2006), in which endoderm is induced before its fate is restricted in the E cell after the next division (Goldstein,1992). It is interesting that a common strategy to induce tissues is used in two distant species, ascidians and C. elegans. Whether this is also the case in other organisms remains to be seen.
Cell-autonomous formation of primary muscle.
In contrast to the mesoderm tissues described above, the formation of the primary muscle in ascidians does not require cell–cell communication and is executed cell-autonomously. Maternal mRNAs for Macho-1 is present in a localized region of the egg and, at the eight-cell stage, are inherited by the B4.1 blastomeres, which give rise to the primary muscle and other tissues (Fig. 4A; Nishida and Sawada,2001). Macho-1 encodes a transcription factor and is necessary and sufficient for the expression of several genes such as tbx6 and myoD that are important for muscle formation (Nishida and Sawada,2001; Yagi et al.,2004,2005; Sawada et al.,2005). Mesoderm formation by maternally localized factors may be a common strategy in vertebrates. Maternal β-catenin restricted to the prospective mesoderm region is involved in early mesoderm induction in Xenopus embryos (Schohl and Fagotto,2003). This region is marked by Spemann organizer-independent early expression of myoD and is fated to become muscle (Kumano and Smith,2002). As mentioned above, the function of Macho-1 as a muscle determinant must be inhibited in other cells than the primary muscle cells. The Macho-1 protein is supposed to be present in every descendant of B4.1 (Kondoh et al.,2003). Among the cell types that are derived from B4.1, posterior endoderm, mesenchyme, and secondary notochord cells require FGF signaling to undo the function of Macho-1 as a muscle determinant (Kim et al.,2000; Kim and Nishida,2001; Kondoh et al.,2003). Macho-1, however, is required in the presumptive mesenchyme cells as an intrinsic competence factor for mesenchyme induction (Kobayashi et al.,2003). It is, therefore, likely that the function of Macho-1 as a transcriptional activator (Sawada et al.,2005) per se is retained at least in the presumptive mesenchyme cells and that only the activation of muscle-specific genes is inhibited by FGF signaling.