Roles of Mouse Sad-1 in Presynaptic Differentiation

描述（由申请人提供）：突触由两个隔室组成：突触前（轴突末端）和突触后（靶膜）。在突触形成过程中，突触前区室和突触后区室都有分化。关于突触后分化，我们已经了解了很多，但突触前分化的分子机制仍然很大程度上是未知的。确定对突触前发育很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。在秀丽隐杆线虫中，突触前分化需要丝氨酸/苏氨酸激酶Sad-1: Sad-1突变体的突触囊泡聚集减少，Sad-1的过表达导致囊泡错定位。本研究旨在阐明小鼠Sad-1同源物在突触前分化中的功能。在小鼠中，Sad-1有两个同源物，Sad-1A和b。我们实验室最近培育的双敲除小鼠，在出生时瘫痪并死亡。第一个目的是通过抗体染色和原位杂交来观察Sad-1组织和细胞分布。这将提供有关Sad-1在何处运行的信息。第二个目的是表征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敲除小鼠的组织或分离神经元中进行检查。第三个目标是产生CRE介导的条件性Sad-1敲除。这将有助于动物在出生后存活，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