ZNF378在胚胎视网膜发育中的作用及机制研究

The retina is the primary function of the visual system structure. During retina development, the factors which determine the differentiation of retinal precursor cells are not only the key to study the mechanisms of retina development, but also the foundation for gene therapy to retinal degenerative diseases. ZNF378 highly expressed at the eraly stage of retina development. In our project, we used reverse genetics method to repress the expression of NF378，and found that knockdown of ZNF378 caused a microphthalmia phenotype, and also affected the stratification and maturation of retina. More importantly, knockdown of ZNF378 changed conserved genetic regulatory network. Based on the preliminary results, we firstly design our project to investigate the effect of ZNF378 downregulation on retina development by loss-of-function method. Secondly, we explore the role of ZNF378 in chromatin remodeling and histone modifications by FISH and ChIP methods. Finally, we use ABE and WB techniques to examine the PAT activities of ZNF378 during retina devlopment in zebrafish. Our study will provide a good basis to reveal the mechanisms of the choice of cell fate during retinogenesis. More importantly, it will provide new clues to detect the mechanism and clinical treatment of Retinal degeneration.

视网膜是视觉系统的第一级功能结构。视网膜发育过程中，有哪些因素影响视网膜前体细胞的分化方向，不仅是在分子水平上研究视网膜发育的关键，也是视网膜变性疾病基因治疗的基础。ZNF378在斑马鱼胚胎视网膜发育早期有着独特的表达模式。抑制ZNF378表达，斑马鱼胚胎将出现明显的小眼畸形，视网膜的分层与成熟受到抑制。同时，保守的遗传调控机制也发生改变。本项目拟在前期研究的基础上，利用loss-of-function等方法，探讨ZNF378基因缺失对视网膜细胞发育的影响；使用荧光原位杂交、染色质免疫共沉淀等技术，研究染色质重塑、组蛋白共价修饰和相关修饰酶的表达活性；并利用酰基-生物素交换法、Western blot等技术，明确ZNF378在视网膜发育过程中的棕榈酰转移酶活性及调控机制。为进一步研究在视网膜发育过程中细胞种类选择的调控机制奠定基础，同时为视网膜变性疾病的基因治疗提供理论基础和实验数据。

视网膜发育过程中，有哪些因素影响视网膜前体细胞的分化方向，不仅是在分子水平上研究视网膜发育的关键，也是视网膜变性疾病基因治疗的基础。ZNF378在斑马鱼胚胎视网膜发育早期有着独特的表达模式。抑制ZNF378表达，斑马鱼胚胎将出现明显的小眼畸形，视网膜的分层与成熟受到抑制。进一步证实ZNF378棕榈酰化Noggin1，促进Noggin1胞外分泌及调控视网膜前体细胞命运决定因子Islet1、NeuroD4、Crx等表达，进而有助于斑马鱼视网膜的发育。这些结果揭示了转录因子、信号通路分子及蛋白翻译后修饰在视网膜前体细胞命运决定阶段的相互作用网络，为视网膜发育的基础研究提供大量线索，同时为视网膜变性疾病的基因治疗提供理论基础和实验数据。