Characterization of a Novel Myoplasmic Protein in Ascidian Embryos

本实验旨在研究一种新的58kd蛋白（p58）在海鞘幼虫尾肌分化中的作用。海鞘胚胎被用作研究细胞自主分化的模型系统，因为胚胎细胞继承了卵的局部区域肌浆，将在分离时成为幼虫肌细胞。即将进行的实验将验证p58参与幼虫尾部肌肉决定的假设。P58在卵发生早期合成，卵黄发生时集中在卵肌质中，后期在幼虫尾肌细胞中大量存在。我已经对一个克隆进行了测序，该克隆是通过筛选含有NN18的表达文库分离出来的，NN18是一种在western blots上识别p58的抗体。该克隆编码一种与转导蛋白相关的新蛋白，果蝇分裂E（spl）信息增强子m9/10和人类核蛋白。E（spl）是Notch基因组的一员，突变分析表明，它通过与Notch（一种类似egf的跨膜蛋白）相互作用，在细胞命运决定中起作用。我建议用海鞘胚胎作为模型系统来研究这些独特基因的作用方式。PI将首先通过northern杂交和原位杂交检测p58基因在胚胎发生过程中的时空表达。p58与卵细胞骨架成分可能的相互作用将通过双免疫荧光标记和共聚焦显微镜以及超微结构免疫定位进行研究。p58在胚胎发生中的作用将通过功能丧失和功能获得实验来检验。胚胎中的P58功能将通过向卵中微注射抗体来抑制，并记录对发育和幼虫尾肌形成的影响。在通常不表达该基因的细胞中表达该基因的效果将通过向胚胎中微量注射cDNA克隆或纯化蛋白来分析。在大多数海鞘物种中，卵发育成蝌蚪幼虫，具有明显的头部和含有条纹肌肉细胞的尾巴，但在无尾的无年生胚胎中，在“幼虫”阶段没有肌肉细胞。无尾海鞘在卵肌浆和幼虫尾肌细胞中都缺乏p58的定位，因此我们将在无尾海鞘胚胎中检测p58基因的表达，以探索这些物种的卵细胞骨架如何组织不同。这些实验将测试p58可能在海鞘自主肌细胞决定中发挥作用的可能性。＊＊＊

9304958 Swalla The proposed experiments investigate the role of a novel 58 kD protein (p58) in ascidian larval tail muscle differentiation. Ascidian embryos are used as a model system to study cell autonomous differentiation because embryonic cells which inherit the myoplasm, a localized region of the egg, will become larval muscle cells in isolation. The experiments to be performed will test the hypothesis that p58 is involved in larval tail muscle determination. P58 is synthesized early in oogenesis, then is concentrated in the egg myoplasm during vitellogenesis, and later is abundant in larval tail muscle cells. I have sequenced a clone isolated by screening an expression library with NN18, an antibody which recognizes p58 on western blots. This clone encodes a novel protein related to transducin, the Drosophila Enhancer of split E(spl) message m9/10, and human nuclear proteins. E(spl) is a member of the Notch group of genes, which have been shown by mutational analysis to function in cell fate decisions through interactions with Notch, an EGF-like transmembrane protein. I propose to use the ascidian embryo as a model system to study the mode of action of these unique genes. The PI will first examine the temporal and spatial expression of the p58 gene during embryogenesis by northern and in situ hybridization. Possible interaction(s) of p58 with egg cytoskeletal components will be investigated by double immunofluorescent labelling followed by confocal microscopy, and also by ultrastructural immunolocalization. The role of p58 during embryogenesis will be examined by loss-of-function and gain-of-function experiments. P58 function in the embryo will be inhibited by microinjecting antibodies into the egg and documenting the effect on development and larval tail muscle formation. The effect of expressing the gene in cells that do not normally express it will be analyzed by microinjection of cDNA clones or purified protein into the embryo. In most ascidian species, the egg develops into a tadpole larva with a distinct head and a tail containing bands of striated muscle cells, but in anural (tailless) embryos, there are no muscle cells in the "larval" stage. Anural (tailless) ascidians lack both localization of p58 in their egg myoplasm and larval tail muscle cells, so that expression of the p58 gene will be examined in anural embryos to explore how the egg cytoskeletal may be organized differently in these species. These experiments will test the possibility that p58 may have a role in autonomous muscle cell determination in ascidians. ***