SULT4A1 in Zebrafish Development

 描述（由申请方提供）：本提案的总体目标是在遗传学上易处理的斑马鱼（Danio rerio）模型系统中建立发育期间和成体中磺基转移酶（SULT）4A 1的调节和功能。SULT 4A 1是一种孤儿酶，基于其结构同源性，已被纳入胞质SULT基因家族。SULT 4A 1 mRNA和蛋白质选择性地表达于所检查的脊椎动物的神经元中。SULT 4A 1在神经系统中的功能是令人感兴趣的，因为该蛋白质在哺乳动物中的氨基酸序列大于98.5%相同，并且在脊椎动物物种（包括斑马鱼）中几乎90%相同。在人类中，基于传递不平衡研究及其与精神病理学的关联，SULT 4A 1基因已被鉴定为精神分裂症发生的易感基因。SULT 4A 1基因缺失也与Phelps-McDermid综合征有关，这是一种广泛的认知和发育缺陷综合征。最近，我们的实验室报道了斑马鱼胚胎中SULT 4A 1表达的瞬时抑制与光转导中多个基因的显著失调有关。这是SULT 4A 1与生理功能的首次关联。迄今为止，没有令人信服的证据，其底物的选择性或活性已确定的酶在任何物种或以下细胞或细菌的表达和纯化的高度同源性的斑马鱼SULT 4A 1的酶在其他脊椎动物提供了一个有价值的模型系统，检查其发育和生理调节和功能。活性位点和敲除SULT 4A 1突变体的产生将用于研究其活性丧失对斑马鱼发育、行为、视觉和神经元特性的影响。TALEN已被用于产生5AA活性位点缺失和移码敲除SULT 4A 1突变体，其存活至成年并作为纯合子繁殖。SULT 4A 1突变体提供了一个框架，用于更好地了解斑马鱼中SULT 4A 1的功能和活性，具体目标如下所述。由于在脊椎动物进化中许多脑功能和特性的高度保守性，斑马鱼系统将为SULT 4A 1在人类神经生物学中的作用提供有价值的见解。1)研究干扰SULT 4A 1活性或表达对斑马鱼发育的影响。将在TALEN衍生的SULT 4A 1突变体中进行胚胎和成体的形态特征、基因表达和繁殖的分析。2)鉴定SULT 4A 1活性位点缺失和敲除的斑马鱼幼体和成体的行为表型。将进行视觉和行为研究，以深入了解SULT 4A 1功能障碍的神经影响。3)分析SULT 4A 1在斑马鱼眼内光信号转导中的作用。斑马鱼胚胎中SULT 4A 1表达的瞬时抑制导致参与光转导的多个基因的表达上调。将在SULT 4A 1突变体中检查视网膜功能和信号转导，以鉴定受影响的神经元通路。

 DESCRIPTION (provided by applicant): The overall goal of this proposal is to establish the regulation and function of sulfotransferase (SULT) 4A1 during development and in adults in the genetically tractable zebrafish (Danio rerio) model system. SULT4A1 is an orphan enzyme that has been included in the cytosolic SULT gene family based on its structural homology. SULT4A1 mRNA and protein are selectively expressed in neurons in the vertebrates examined. The function of SULT4A1 in the nervous system is of interest since the protein is greater than 98.5% identical in amino acid sequence in mammals and almost 90% identical in vertebrate species, including zebrafish. In humans, the SULT4A1 gene has been identified as a susceptibility gene for the occurrence of schizophrenia based on both transmission disequilibrium studies and its association with psychopathology. SULT4A1 gene deletion has also been linked to Phelps-McDermid syndrome, a generalized cognitive and developmental deficit syndrome. Recently our laboratory has reported that transient inhibition of SULT4A1 expression in zebrafish embryos is associated with the significant dysregulation of multiple genes in phototransduction. This is the first association of SULT4A1 with a physiological function. To date, no convincing evidence as to its substrate selectivity or activity has been identified for the enzyme in any species or following cell or bacterial expression and purification The high homology of zebrafish SULT4A1 to the enzyme in other vertebrates provides a valuable model system for the examination of its developmental and physiological regulation and function. Generation of both active site and knockout SULT4A1 mutants will be used to investigate the effects of its loss of activity on zebrafish development, behavior, vision and neuronal properties. TALENs have been utilized to generate both 5 AA active site deletion and frameshift knockout SULT4A1 mutants that survive to adulthood and breed as homozygotes. The SULT4A1 mutants provide a framework for developing a better understanding of the function and activity of SULT4A1 in zebrafish as elaborated by the following specific aims. Due to the high conservation many brain functions and properties in vertebrate evolution the zebrafish system will provide valuable insights into the role of SULT4A1 in human neurobiology. 1) To investigate the effects of disrupting SULT4A1 activity or expression on zebrafish development. Analysis of morphological characteristics of embryos and adults, gene expression, and reproduction will be carried out in the TALEN-derived SULT4A1 mutants. 2) To identify behavioral phenotypes in larval and adult SULT4A1 active site deletion and knockout zebrafish. Vision and behavioral studies will be performed to generate insights into the neural effects of SULT4A1 dysfunction. 3) To analyze the role of SULT4A1 in phototransduction in the zebrafish eye. Transient inhibition of SULT4A1 expression in zebrafish embryos resulted in the up-regulation of expression of multiple genes involved in phototransduction. Retinal function and signal transduction will be examined in the SULT4A1 mutants to identify affected neuronal pathways.