Characterization of a Novel Myoplasmic Protein in Ascidian Embryos

9304958斯瓦拉：拟议的实验研究了一种新的58kD蛋白(P58)在腹水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，因为继承肌浆的胚胎细胞将在隔离中成为幼虫肌肉细胞。肌浆是卵子的一个局部区域。将要进行的实验将检验p58参与幼虫尾部肌肉决定的假设。P58在卵子发生早期合成，在卵黄发生过程中集中在卵肌质中，后期在幼虫尾部肌细胞中大量合成。我已经用NN18筛选了一个表达文库，对一个克隆进行了测序。NN18是一种识别Western blotts上p58的抗体。该克隆编码一种与转导蛋白相关的新蛋白--果蝇Split E(SPL)信息增强子M9/10和人类核蛋白。E(SPL)是Notch基因家族中的一员，突变分析表明，这些基因通过与EGF样跨膜蛋白Notch的相互作用而在细胞命运决定中发挥作用。我建议使用海鞘胚胎作为一个模型系统来研究这些独特基因的作用模式。PI将首先通过Northern和原位杂交检测p58基因在胚胎发育过程中的时空表达。P58与卵细胞骨架成分的可能相互作用(S)将通过免疫荧光双标记法和共聚焦显微镜以及超微结构免疫定位来研究。P58在胚胎发育过程中的作用将通过功能丧失和功能获得实验来检验。通过将抗体微量注射到卵子中并记录对发育和幼虫尾部肌肉形成的影响，胚胎中的p58功能将被抑制。在不正常表达该基因的细胞中表达该基因的效果将通过向胚胎中显微注射cdna克隆或纯化蛋白来分析。在大多数海鞘物种中，卵发育成具有明显头部和尾巴的蝌蚪幼虫，其中含有条纹肌肉细胞，但在无尾(无尾)胚胎中，没有处于“幼虫”阶段的肌肉细胞。无尾(无尾)海鞘缺乏p58在其卵肌浆和幼虫尾部肌肉细胞中的定位，因此将在肛门胚胎中检测p58基因的表达，以探索这些物种的卵细胞骨架可能是如何不同地组织起来的。这些实验将测试p58可能在腹水自主肌肉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. ***