Molecular identification of muscle cell determinants in ascidian eggs.

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The Halocynthia roretzi tadpole larva has 42 striated muscle cells.Cell lineage studies show that 28 of these cells are derived from B-line (B4.1 cell-pair) , 4 cells from A-line (A4.1 cell-pair) , and 10 cells from b-line (b4.2 cell-pair) blastomeres. The B-line muscle cells differentiate autonomously mediated by cytoplasmic determinants. The muscle determinants are localized in the myoplasm. The aim of this study is a molecular identification of muscle determinants.1. Ciona savignyi eggs are separated into nucleated red fragments and enucleated brown, clear and black fragments by centrifugation. Fertilized red fragments generate partial embryos containing epidermis but no muscle cells. However, muscle development can be promoted in red fragments by fusion with black fragments. The other fragments show much less potential to promote muscle differentiation, suggesting that the determinants are concentrated in black fragments. The capacity of black fragments to promote muscle differentiation is diminished by uv irradiation and partially restored by injection of black-fragment poly (A) +RNA into uv-irradiated black fragments. Isolation and characterization of cDNA clones for black fragment mRNAs is being carried out to identify candidates for muscle determinants.2. We characterized an ascidian homolog (AMD1) of MyoD.The AMD1 gene begins to be expressed in the primary muscle lineage during the 32-cell stage, at the same time as a muscle actin gene. Because AMD1 transcripts are not detected in eggs, it is unlikely that AMD1 is the muscle determinant.3. In HAlocynthia five muscle actin genes form a cluster within a 30 kb region of the genome. A 103 bp sequence in the 5' flanking region of each gene is necessary and sufficient to drive muscle actin expression in larval muscle cells.