DHHC16对胚胎端脑神经干细胞调控作用的研究

The DHHC domain protein is a group of protein associated with palmitoylation referred as one of post-translational lipid modification. To date, many proteins which played an important role in neural development have been found to be palmitoylated modification. Thus, the study of such proteins becomes a hot topic for researchers and attacts more and more attention..DHHC16, one of the DHHC domain protein family, was discovered from the cDNA library of zebrafish by our group. DHHC16 specifically expressed in the nervous system, and highly at the neural plate formation stage. In our project, we used reverse genetics method to repress the expression of DHHC16，and found that knockdown of DHHC16 caused a severe defect in the embryonic telencephalon and also affected the apoptosis and proliferation of the neural stem cell(NSC). Based on the preliminary results, we design our project to investigate the role and mechanisms of DHHC16 in NSCs' generation and fate decision by molecular and morphology techniques included IF, ISH, WB, IP and ABE methods. We also explore the function of the key domain features of DHHC16 and the role of DHHC16 PAT activity in signaling transduction during NSC development by analyzing gain-of-function phenotypes injected wild type or mutant DHHC16 mRNA..Our study will provide a good basis to reveal the function of other members in the DHHC protein family. More importantly, it will provide new clues to detect the mechanism and clinical treatment of neural tube malformations, especially in telencephalon malformations.

DHHC蛋白家族是一类介导蛋白棕榈酰化修饰的重要蛋白。最新研究表明，很多在神经发育过程中发挥重要作用的蛋白能被棕榈酰化修饰调节，因而此类蛋白的研究成为热点，该家族在神经发育中的研究引起越来越多的关注。DHHC16是本课题组率先从斑马鱼中克隆出的该家族成员,在神经系统特异性表达，且在神经板形成的关键时期高表达。利用反向遗传学手段,注射Morpolino抑制斑马鱼DHHC16表达，胚胎神经发育缺陷表现为端脑减小甚至缺失,端脑神经干细胞凋亡增加。本研究拟在前期基础上，通过原位杂交，WB，IP等方法，研究该基因对胚胎端脑神经干细胞生成和命运决定的作用及机制，通过显微注射基因野生型及突变型mRNA, 在体内研究其关键结构域的功能，并着重研究其棕榈酰化作用对神经干细胞发育信号分子的调控。本研究为揭示这类蛋白的功能提供了重要基础和新的切入点，也为神经管发育畸形特别是端脑缺陷的研究和临床治疗提供新线索。

蛋白质的翻译后修饰对于蛋白活性和功能的发挥起到关键作用，在神经发育过程中多种功能重要的蛋白能被棕榈酰化修饰调节。DHHC蛋白家族是一类介导蛋白棕榈酰化修饰的重要蛋白，在神经发育中的研究越来越受到重视。DHHC16是本课题组率先从斑马鱼中克隆出来的DHHC家族成员，在神经系统发育中特别是神经版形成的关键时期高表达。我们利用Morpolino技术特异性抑制DHHC16的表达，斑马鱼胚胎出现了端脑减小甚至缺失的表型，同时端脑神经干细胞增殖受损，凋亡增加。本课题充分利用原位杂交、IP、WB等技术研究了DHHC16对端脑神经干细胞生成及命运决定的作用和作用机制，构建缺失突变体，确定了关键结构域。并利用棕榈酰化水平检测方法，研究了棕榈酰化对神经干细胞发育的信号通路的调控。本研究为揭示此类蛋白在神经系统发育中的功能提供了重要的研究基础。