SULT4A1 in Zebrafish Development

 描述(由申请人提供)：这项提案的总体目标是在遗传易驯化斑马鱼(Danio Rerio)模型系统中建立磺基转移酶(Sult)4A1在发育和成鱼中的调节和功能。SULT4A1是一种孤儿酶，根据其结构同源性已被归入胞质sult基因家族。SULT4A1的mRNA和蛋白在被研究的脊椎动物的神经元中选择性地表达。SULT4A1在神经系统中的功能令人感兴趣，因为该蛋白在哺乳动物中的氨基酸序列同源性超过98.5%，在包括斑马鱼在内的脊椎动物中的同源性几乎为90%。在人类中，SULT4A1基因已被确定为精神分裂症发生的易感基因，这是基于传递不平衡研究及其与精神病理学的关联。SULT4A1基因缺失也与菲尔普斯-麦克德米德综合征有关，菲尔普斯-麦克德米德综合征是一种广泛性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SULT4A1表达的瞬时抑制与多个基因在光传导中的显著失调有关。这是首次将SULT4A1与生理功能联系起来。到目前为止，还没有令人信服的证据表明该酶在任何物种或随后的细胞或细菌表达和纯化中具有底物选择性或活性。斑马鱼SULT4A1与其他脊椎动物中该酶的高度同源性为研究其发育和生理调节及其功能提供了一个有价值的模型系统。活性位点和敲除SULT4A1突变体的产生将被用来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已经被用来产生5个AA活性位点缺失和移码敲除SULT4A1突变体，这些突变体存活到成年并以纯合子的形式繁殖。SULT4A1突变体为更好地理解SULT4A1在斑马鱼中的功能和活性提供了一个框架，具体目的如下。由于SULT4A1在脊椎动物进化中的高度保守性，许多大脑功能和特性，斑马鱼系统将为SULT4A1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SULT4A1活性或表达对斑马鱼发育的影响。在TALEN来源的SULT4A1突变体中，将对胚胎和成体的形态特征、基因表达和繁殖进行分析。2)鉴定幼体和成体SULT4A1活性位点缺失和敲除斑马鱼的行为表型。将进行视觉和行为研究，以深入了解SULT4A1功能障碍的神经影响。3)分析SULT4A1在斑马鱼眼睛光信号转导中的作用。瞬时抑制SULT4A1在斑马鱼胚胎中的表达导致与光信号转导相关的多个基因表达上调。将在SULT4A1突变体中检查视网膜功能和信号转导，以确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.