Characterization of a Novel Myoplasmic Protein in Ascidian Embryos

小行星9304958 拟议的实验调查的作用，一种新的58 kD蛋白（p58）在海鞘幼虫尾肌分化。 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质，将在隔离中成为幼虫肌细胞。 将要进行的实验将检验p58参与幼虫尾肌决定的假设。 P58在卵子发生早期合成，然后在卵黄发生期间集中在卵肌质中，随后在幼虫尾肌细胞中大量合成。 我对通过用NN 18（一种在蛋白质印迹上识别p58的抗体）筛选表达文库而分离的克隆进行了测序。 该克隆编码一种新的与转导素相关的蛋白质，即果蝇分裂增强子 E（spl）信息m9/10和人核蛋白。 E（spl）是Notch基因组的成员，其已经通过突变分析显示通过与Notch（EGF样跨膜蛋白）的相互作用在细胞命运决定中起作用。 我建议使用海鞘胚胎作为模型系统来研究这些独特基因的作用模式。 PI将首先通过北方和原位杂交检查胚胎发生过程中p58基因的时空表达。 将通过双重免疫荧光标记、共聚焦显微镜以及超微结构免疫定位来研究p58与卵细胞骨架成分可能的相互作用。 p58在胚胎发生过程中的作用将通过功能丧失和功能获得实验来检查。 通过将抗体显微注射到卵中并记录对发育和幼虫尾肌形成的影响，可以抑制胚胎中的P58功能。 通过将cDNA克隆或纯化的蛋白质显微注射到胚胎中来分析在通常不表达该基因的细胞中表达该基因的效果。 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细胞带的尾部，但在无尾（无尾）胚胎中，“幼虫”阶段没有肌细胞。 无尾（无尾）海鞘缺乏本地化的p58在他们的卵肌浆和幼虫尾肌细胞，使p58基因的表达将在无尾胚胎中进行检查，以探索如何卵细胞骨架可能在这些物种中的不同组织。 这些实验将测试p58可能在海鞘的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. ***