In ascidian embryos, a fibroblast growth factor (FGF) signal induces notochord, mesenchyme, and brain formation. Although a conserved Ras/MAPK pathway is known to be involved in this signaling, the target transcription factor of this signaling cascade has remained unknown. We have isolated HrEts, an ascidian homolog of vertebrate Ets1 and Ets2, to elucidate the transcription factor involved in the FGF signaling pathway in embryos of the ascidian Halocynthia roretzi. Maternal mRNA of HrEts was detected throughout the entire egg cytoplasm and early embryos. Its zygotic expression started in several tissues, including the notochord and neural plate. Overexpression of HrEts mRNA did not affect the general organization of the tadpoles, but resulted in formation of excess sensory pigment cells. In contrast, suppression of HrEts function by morpholino antisense oligonucleotide resulted in severe abnormalities, similar to those of embryos in which the FGF signaling pathway was inhibited. Notochord-specific Brachyury expression at cleavage stage and notochord differentiation at the tailbud stage were abrogated. Formation of mesenchyme cells was also suppressed, and the mesenchyme precursors assumed muscle fate. In addition, expression of Otx in brain-lineage blastomeres was specifically suppressed. These results suggest that an Ets transcription factor, HrEts, is involved in signal transduction of FGF commonly in notochord, mesenchyme, and brain induction in ascidian embryos.
The ascidian tadpole larva is thought to be the prototype for the ancestral chordate. Although ascidians show a highly determinate mode of development, recent studies suggest significant roles of cell-cell interaction during embryogenesis. To elucidate the signaling molecules responsible for the cellular interaction, we investigated an ascidian homologue of the transforming growth factor beta (TGF-beta) superfamily. HrBMPa is an ascidian member of the 60A subclass of the BMP subfamily. Molecular phylogenetic analysis suggested that HrBMPa branched prior to further divergence of vertebrate BMPs-5-8. The zygotic expression of HrBMPa was initiated around gastrulation. HrBMPa transcripts were first evident in precursor cells of the spinal cord, notochord, epidermis and nervous system, although signals in the first two regions quickly disappeared. In neurulae and early tailbud embryos, transcripts were evident in the adhesive organ, midline of the anterior dorsal neuroectoderm and midline of both ventral and dorsal ectoderm, suggesting that HrBMPa plays a major role in neuroectodermal cell differentiation during embryogenesis. This HrBMPa expression profile resembled that of Xenopus BMP-7, implying a primordial function of BMP-7 among vertebrate BMPs-5-8.
The Sox family is a large group of transcription factors that are characterized by the presence of a DNA-binding HMG domain. We isolated HrSoxB1, an ascidian homolog of the Sox gene that belongs to the B1 subclass of the Sox family, from Halocynthia roretzi. Expression was initiated as early as the 8-cell stage. During cleavage stages, HrSoxB1 was expressed in three quarters of embryonic blastomeres but not in posterior-vegetal (B-line) blastomeres. Misexpression of mRNAs of HrPEM but not of macho-1, whose maternal mRNAs are localized to the posterior-vegetal cytoplasm of eggs and early embryos, repressed the anterior-vegetal expression of HrSoxB1. This result suggests that the zygotic expression of HrSoxB1 is controlled by the localized maternal mRNA. When HrSoxB1 was overexpressed in early embryos, ectopic expression of HrBra, a gene for a transcription factor expressed in notochord blastomeres, occurred in the most posterior blastomeres (B7.5), although these blastomeres did not eventually differentiate into notochord but developed into muscle, as they do in normal embryogenesis. In later embryogenesis, HrSoxB1 was specifically expressed in neural plate cells. However, overexpression of HrSoxB1 did not affect the expression of a neural plate marker gene, HrETR-1.
The ADP/ATP translocase is the most abundant integral protein of the inner mitochondrial membrane and it is encoded by the nuclear DNA. Because mitochondria in the ascidian egg appear to be segregated into blastomeres of muscle lineage, we examined the expression of a gene for ADP/ATP translocase during embryogenesis of the ascidian Halocynthia roretzi. Sequence analysis of a cDNA clone for the ascidian ADP/ATP translocase indicated that it contains a single open reading frame that encodes a polypeptide of 304 amino acids. The polypeptide showed extensive similarity to mammalian ADP/ATP translocases, with as much as 74% identity. The genome of H. roretzi contains a single gene, or two genes at most, for the protein. A large amount of maternal mRNA for ADP/ATP translocase was found in unfertilized eggs and early embryos. The amount of this mRNA decreased after the 64-cell stage, and the mRNA became barely detectable in tailbud embryos, although zygotic transcript for the protein was evident in adult tissues. Both in situ hybridization and Northern blot analyses demonstrated that the mRNA is distributed in the entire cytoplasm of unfertilized eggs. The mRNA is segregated during embryogenesis not only into blastomeres of muscle lineage but also into those of non-muscle lineage.
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