Differences in cell responsiveness to an inductive signal contribute to the emergence of a variety of tissue types during animal development. In ascidian embryos, the Fibroblast Growth Factor (FGF) signal secreted from endoderm cells induces several different tissue types, such as notochord, mesenchyme and brain, at different positions in the embryo at the 32-cell stage. We show here in Halocynthia roretzi that FoxA and Zic are required for notochord formation in cells that receive the FGF signal. We also show that these transcription factors, only when both are supplied, are able to induce ectopic expression of the brachyury gene, a notochord-specific marker, in cells of all the three germ layers in an FGF-dependent manner. These results suggest that FoxA and Zic confer notochord-specific responsiveness to FGF signaling. Further analyses including knockdown and over-expression experiments showed that combinatorial inputs from maternally supplied and zigotically activated factors lead to overlapping expression of FoxA and Zic in the presumptive notochord cells, which eventually activate the expression of the brachyury gene in cooperation with FGF signaling. Our data illustrate how a complex gene network specifies the notochord at its specific position within the embryo.

Suppression of zygotic transcription in early embryonic germline cells is tightly linked to their separation from the somatic lineage. Many invertebrate embryos utilize localized maternal factors that are successively inherited by the germline cells for silencing the germline. Germline quiescence has also been associated with the underphosphorylation of Ser2 of the C-terminal domain (CTD-Ser2) of RNA polymerase II [1-3]. Here, using the ascidian Halocynthia roretzi, we identified a first deuterostome example of a maternally localized factor, posterior end mark (PEM), which globally represses germline transcription. PEM knockdown resulted in ectopic transcription and ectopic phosphorylation of CTD-Ser2 in the germline. Overexpression of PEM abolished all transcription and led to the underphosphorylation of CTD-Ser2 in the somatic cells. PEM protein was reiteratively detected in the nucleus of the germline cells and coimmunoprecipitated with CDK9, a component of posterior transcription elongation factor b (P-TEFb). These results suggest that nonhomologous proteins, PEM and Pgc of Drosophila [3-5] and PIE-1 of C. elegans [1, 6, 7], repress germline gene expression through analogous functions: by keeping CTD-Ser2 underphosphorylated through binding to the P-TEFb complex. The present study is an interesting example of evolutionary constraint on how a mechanism of germline silencing can evolve in diverse animals.

An important step in early embryonic development is the allocation and segregation of germ layer fates into distinct embryonic regions. However, the mechanism that segregates the mesendoderm into mesoderm and endoderm fates remains largely unknown in most animals. Here, using ascidians, a primitive chordate, we show that these fates are segregated by partitioning of asymmetrically localized Not mRNA from the mesendoderm cell to its mesodermal daughter. Migration of the mesendoderm cell nucleus to the future mesoderm-forming region, release of Not mRNA from the nucleus, Wnt5α-dependent local retention of the mRNA, and subsequent repositioning of the mitotic spindle to the center of the cell are each required for the asymmetric localization and partitioning of Not mRNA. Our results show that nuclear migration plays an unexpected role in asymmetric cell divisions that segregate germ layer fates in chordate embryos.

Extrinsic fibroblast growth factor (FGF) signal and intrinsic factors that determine the response of the signal-receiving blastomeres to FGF regulate mesoderm patterning in embryos of the ascidian Halocynthia roretzi. To investigate how cells integrate information from extrinsic and intrinsic inputs, we examined Brachyury (Hr-Bra) promoter activity in the early embryo. Hr-Bra, which encodes a key transcription factor for notochord development, is expressed exclusively in notochord precursors in a manner dependent on the FGF-MEK-MAPK-Ets signaling pathway and on the intrinsic factors Zic and FoxA. Reporter gene expression driven by the 900-bp upstream region of the Hr-Bra promoter was detected as early as the 110-cell stage in notochord precursors by in situ hybridization with a LacZ probe. Deletion analysis combined with MEK inhibitor treatment demonstrated that the -598/-499 region carries FGF-responsiveness. Electrophoretic mobility shift assay identified three Ets-binding sites in this region that were required for promoter activity. Further deletion analysis conducted by injecting eggs with reporter constructs at higher concentration suggested that the -398/-289 region also has enhancer activity, although ectopic reporter expression was detected in nerve cord and endoderm precursors. The -398/-289 region has a Zic-binding site that was also essential for the enhancer activity. These results indicate that Ets- and Zic-binding sites are critical for the initiation of Hr-Bra expression. In conclusion, information from both extrinsic and intrinsic factors is integrated at the level of enhancer of the target gene by direct binding of the transcription factors to the enhancer region.

According to the three-signal model of mesoderm patterning in Xenopus, all mesoderm, with the exception of the Spemann organizer, is originally specified as ventral type, such as lateral plate and primary blood islands. It is proposed that the blood islands become restricted to the ventralmost mesoderm because they are not exposed to the BMP-inhibiting activity of the Spemann organizer. We present evidence here that, contrary to predictions of this model, the blood islands remain ventrally restricted even in the absence of Spemann organizer signaling. We further observed that inhibition of FGF signaling with a dominant negative receptor resulted in the expansion of the blood island-forming territory with a concomitant loss of somite. The requirement for FGF signaling in specifying somite versus blood island territories was observed as early as midgastrulation. The nonoverlapping expression domains of Xnr-2 and Xbra in the gastrula marginal zone appear to mark presumptive blood island and somite, respectively. Inhibition of FGF signaling with dominant negative receptor leads to an expansion of Xnr-2 expression and to a corresponding reduction in Xbra expression. On the other hand, we found no evidence that manipulation of BMP signaling, either positively or negatively, altered the expression domains of Xnr-2 and Xbra. These results suggest that FGF signaling, rather than BMP-inhibiting activity, is essential for restriction of the ventral blood islands to ventral mesoderm.