at a colloquium entitled " Biology of Developmental

The B-line presumptive muscle cells of ascidian embryos have extensive potential for self-differentiation dependent on determinants prelocalized in the myoplasm of fertilized eggs. Ascidian larval muscle cells therefore provide an experimental system with which to explore an intrinsic genetic program for autonomous specification of embryonic cells. Experiments with egg fragments suggested that maternal mRNAs are one of the components of muscle determinants. Expression of larval muscle actin genes begins as early as the 32-cell stage, prior to the developmental fate restriction of the cells. The timing of initiation of the actin gene expression proceeds the expression of an ascidian homologue of vertebrate MyoD by a few hours. Mutations in the proximal E-box of the 5' flanking region of the actin genes did not alter the promoter activity for muscle-specific expression of reporter gene. These results, together with results of deletion constructs of fusion genes, suggest that muscle determinants regulate directly, or indirectly via regulatory factors other than MyoD, the transcription of muscle-specific structural genes leading to the terminal differentiation. During early embryogenesis in animals, the developmental fate of embryonic cells is specified either autonomously or conditionally (1-4). In the case of autonomous specification, particular maternal information or morphogenetic determinant is prelocalized in a certain region of the egg cytoplasm and is segregated during cleavage to a certain lineage. This information serves to regulate, either directly or indirectly, the transcription of genes that are required for specific functions of cells (1, 5, 6). One of the examples of autonomous embryonic cell specification is the lineage that gives rise to larval tail muscle cells in the ascidian embryo (6, 7). During embryogenesis of the ascidian Halocynthia roretzi, 42 unicellular and striated muscle cells are formed in the larval tail, which are associated with active locomotion of the larva. Lineage analysis has shown that the B4.1 cell pair of the bilaterally symmetrical 8-cell embryo gives rise to 28 muscle cells in the anterior and middle part of the tail, A4.1 pair gives rise to 4 muscle cells in the posterior part of the tail, and the b4.2 pair gives rise to 10 muscle cells at the tip of the tail (Fig. 1; ref. 8). Presumptive muscle cells of the B-line (primary lineage) have extensive potential for self-differentiation or autonomous development, while those of the Aand b-lines (secondary lineage) are unable to differentiate autonomously (9). Even if B4.1 cells are isolated from the 8-cell embryo and division of the isolated cells is arrested with cytochalasin B immediately after isolation, the cells eventually develop a marker of muscle differentiation (10). Therefore, the B-line presumptive cell is a self-sustained system in regard to muscle differentiation. From the turn of the century, the existence of muscle determinants in so-called myoplasm of ascidian eggs has been suggested by several observations and experiments (11, 12). Recently, convincing evidence for the presence of muscle determinants has been offered by Nishida (13). B4.1 cells isolated manually from Halocynthia 8-cell embryos were divided into fragments that were with and without myoplasm. The enucleated myoplasm was fused with nonmuscle lineage a4.2 cells and the fusion products were allowed to develop into partial embryos. Nearly all of the partial embryos produced markers of muscle differentiation, while none of those from the fusion of a4.2 cells with enucleated B4.1 fragments without myoplasm produced such markers. Therefore, ascidian larval muscle cells provide an experimental system with which to explore an intrinsic genetic program for autonomous specification of embryonic cells. In this system, as shown in Fig. 1A, muscle determinants, presumably via zygotic transcription factors, eventually activate muscle-specific structural genes. We describe and discuss here results of recent studies. Muscle Determinants: Their Molecular Nature and Segregation Mechanisms Molecular Nature of Muscle Determinants. As mentioned above, recent experiments clearly showed the presence of muscle determinants in the myoplasm of ascidian eggs. Several studies have already been undertaken to elucidate the molecular nature of muscle determinants (6). Jeffery (14) isolated the yellow myoplasm from Styela plicata eggs and found that the myoplasm fraction contains at least 15 polypeptides that are undetectable in the other cytoplasmic fraction. However, there were no detectable prevalent mRNAs specific to the myoplasm, as determined by extraction and translation of myoplasmic poly(A)+ RNA in an in vitro system (14). Nishikata et al. (15) produced monoclonal antibodies that specifically recognize components of the myoplasm of Ciona intestinalis eggs. One of the antigens, named myoplasmin-Cl, is a single 40-kDa polypeptide of the cortex of the myoplasm. The myoplasmin-Cl is implicated in muscle differentiation, because injection of its antibody into fertilized eggs partially Abbreviations: DFL, deep filamentous lattice; PML, plasma membrane lamina; bHLH, basic helix-loop-helix; RT-PCR, reverse transcription-PCR. *To whom reprint requests should be addressed. e-mail: satoh@ ascidian.zool.kyoto-u.ac.jp. tPresent address: IDAC, Tohoku University, Aobaku, Sendai 980-77, Japan. 9315 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 9316 Colloquium Paper: Satoh et al.