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Molecular identification of muscle cell determinants in ascidian eggs.

海鞘卵中肌细胞决定因素的分子鉴定。

基本信息

批准号：
05454652
负责人：
SATOH Noriyuki
金额：
$ 4.48万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05454652/
关键词：
cell differentiation determinants ascidian embryos muscle cells muscle actin genes MyoD gene transcriptional factors cytoplasmic information 分子的本質ホヤ

项目摘要

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.

Halocynthia roretzi 蝌蚪幼虫有 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 系肌细胞由细胞质决定簇自主分化。肌肉决定因素位于肌质中。本研究的目的是对肌肉决定因素进行分子鉴定。1．海鞘卵通过离心分离成有核红色碎片和无核棕色、透明和黑色碎片。受精的红色碎片产生含有表皮但不含肌肉细胞的部分胚胎。然而，红色碎片可以通过与黑色碎片融合来促进肌肉发育。其他片段显示出促进肌肉分化的潜力要小得多，这表明决定因素集中在黑色片段中。黑色片段促进肌肉分化的能力因紫外线照射而减弱，但通过将黑色片段poly(A)+RNA注射到经紫外线照射的黑色片段中而部分恢复。黑色片段 mRNA 的 cDNA 克隆的分离和表征正在进行中，以鉴定肌肉决定因素的候选者。2．我们鉴定了 MyoD 的海鞘同源物 (AMD1)。AMD1 基因在 32 细胞阶段开始在初级肌肉谱系中表达，同时作为肌肉肌动蛋白基因表达。由于在鸡蛋中未检测到 AMD1 转录本，因此 AMD1 不太可能是肌肉决定因素。3。在 HAlocynthia 中，五个肌肉肌动蛋白基因在基因组的 30 kb 区域内形成一个簇。每个基因 5' 侧翼区域中的 103 bp 序列对于驱动幼虫肌肉细胞中的肌肉肌动蛋白表达是必要且充分的。