Roles of Mouse Sad-1 in Presynaptic Differentiation

描述(申请人提供)：突触由两部分组成：突触前(轴突终末)和突触后(靶膜)。在突触的形成过程中，突触前和突触后的隔室都会发生分化。有关突触后分化的研究已经很多，但突触前分化的分子机制仍然很大程度上是未知的。识别对突触前发育至关重要的基因将有助于填补这一空白，并促进对大脑功能的理解。在线虫中，突触前分化需要丝氨酸/苏氨酸激酶SAD-1：突触小泡聚集在SAD-1突变体中减少，过度表达SAD-1导致小泡错误定位。本研究旨在阐明小鼠SAD-1同源基因在突触前分化中的作用。在小鼠中有两个SAD-1的同源基因，SAD-1A和B。我们实验室最近培育的双基因敲除小鼠会瘫痪并在出生时死亡。第一个目标是通过抗体染色和原位杂交观察SAD-1的组织和细胞分布。这将提供有关SAD-1在哪里运行的信息。第二个目的是确定SAD-1基因敲除小鼠的表型。突触的形成以及神经发育的其他方面将在组织中或从基因敲除小鼠分离的神经元中进行检测。第三个目标是产生Cre介导的条件性SAD-1基因敲除。这将帮助动物活到出生后，并允许观察出生后的突触发生。

DESCRIPTION (provided by applicant): A synapse is comprised of two compartments: presynaptic (the axon terminal) and postsynaptic (the target membrane). During the formation of a synapse, the pre- and postsynaptic compartments both differentiate. Much has been learned about postsynaptic differentiation, but the molecular machinery of presynaptic differentiation remains largely unknown. Identification of the genes that are important for presynaptic development will help fill this gap and facilitate the understanding of brain functions. In C. elegans, presynaptic differentiation requires the serine/threonine kinase Sad-1: synaptic vesicle clustering is decreased in the Sad-1 mutant and overexpression of Sad-1 causes vesicles to mislocalize. This proposed study is aimed at elucidating the functions of the murine orthologues of Sad-1 in presynaptic differentiation. There are two orthologues of Sad-1 in mice, Sad-1A and B. Double knockout mice, recently generated in our lab, are paralyzed and die at birth. The first aim is to look at Sad-1 tissue and cellular distribution by antibody staining and in situ hybridization. This will provide information about where Sad-1 is functioning. The second aim is to characterize the phenotype of the Sad-1 knockout mice. The formation of synapses as well as other aspects of neurodevelopment will be examined in tissues or isolated neurons from knockout mice. The third aim is to generate CRE mediated conditional Sad-1 knockout. This will help the animals live past birth and allow observation of postnatal synaptogenesis