Molecular identification of muscle cell determinants in ascidian eggs.

罗氏盐蛙蝌蚪有42个横纹肌细胞，细胞谱系研究表明，其中28个细胞来自B系（B4.1细胞对），4个细胞来自A系（A4.1细胞对），10个细胞来自b系（b4.2细胞对）卵裂球。B系肌细胞的分化由细胞质决定簇介导。肌肉决定因子位于肌浆中。本研究的目的是对肌肉决定因子进行分子鉴定.通过离心将赛维氏玻璃海鞘卵分离成有核的红色碎片和去核的棕色、透明和黑色碎片。受精的红色碎片产生部分胚胎，含有表皮，但没有肌肉细胞。然而，通过与黑色碎片融合，可以促进红色碎片中的肌肉发育。其他片段显示出更少的潜力，以促进肌肉分化，这表明决定因素集中在黑色片段。黑色片段的能力，以促进肌肉分化的紫外线照射减少和部分恢复注射黑色片段聚（A）+RNA到紫外线照射的黑色片段。分离和鉴定黑色片段mRNA的cDNA克隆，以鉴定肌肉决定子的候选者。我们的特点是海鞘同源物（AMD1）的MyoD。AMD1基因开始表达的主要肌肉谱系在32细胞阶段，在同一时间作为肌肉肌动蛋白基因。由于AMD1转录本在鸡蛋中未检测到，因此AMD1不太可能是肌肉决定因子。在HALocynthia五肌肉肌动蛋白基因形成一个簇内的30 kb的基因组区域。在每个基因的5'侧翼区中的103bp序列是驱动肌肌动蛋白在幼虫肌细胞中表达所必需的并且是足够的。

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.