原肠早期视黄酸效应基因在斑马鱼后脑发生中的作用及机制研究

项目名称： 原肠早期视黄酸效应基因在斑马鱼后脑发生中的作用及机制研究

项目编号： No.31471355

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 生物科学

项目作者： 赵庆顺

作者单位： 南京大学

项目金额： 88万元

中文摘要： 脊椎动物胚胎发生中，视黄酸（RA）在原肠胚背部预定后脑命运区域呈线性浓度梯度分布指导后脑图式形成。但是原肠期哪些基因最早介导了RA形态发生素信号图式后脑形成尚未见系统研究。在我们的前期工作中，通过基因表达芯片对斑马鱼原肠早期的表达基因进行高通量筛查，发现11个在体节期后脑中也有表达的RA效应基因。本研究中，我们将通过对以CRISPR/Cas9技术创建的上述基因敲除突变体胚胎的分析，研究这些原肠早期RA效应基因是否为斑马鱼胚胎后脑正常发育所必需、是否以直接靶基因的方式介导了RA信号图式后脑形成、是否通过影响胚胎内RA作为形态发生素信号的鲁棒性决定后脑的发生、以及是通过调控哪些靶基因的表达影响后脑的形成，从而验证在斑马鱼原肠早期存在RA信号效应基因通过介导RA信号图式后脑形成、进而调控菱脑身份标识基因在体节期的表达，决定后脑分节特化的科学假设。研究结果有利于深入揭示RA图式后脑形成的分子机制。

中文关键词： 斑马鱼；后脑发生；视黄酸效应基因；原肠胚；形态发生素

英文摘要： Existing in the presumptive hindbrain region of gastrula with a linear concentration gradient, retinoic acid (RA) plays essential roles in instructing hindbrain pattern formation during vertebrate embryogenesis. However, the identities of genes that initiate to pattern hindbrain development by directly mediating RA morphogen at gastrulation stage remains elusive. To identify the genes, we performed pioneer experiments by mining the gene expression microarray data of the 6 hpf zebrafish embryos treated with 1 uM RA from 0 through 6 hpf and then confirming the expression patterns of the genes that are responsive to RA treatment by whole mount in situ hybridization. Our preliminary results showed that there are 11 RA responsive genes that are expressed not only in the early gastrula (6hpf) but also in the developing hindbrain at somitogenesis stage. To explore the roles of the 11 RA responsive genes at early gastrula in patterning hindbrain development and the molecular mechanisms underlying their roles, we will generate knockout mutants of the 11 genes using CRISPR/Cas9 technology. Analyzing the knockout embryos, we will ask whether each of the RA responsive genes at early gastrula is essential to hindbrain development, whether the genes are the direct target ones of RA morphogen that patterning hindbrain development at gastrulation stage, whether the genes play their roles in patterning hindbrain development by maintaining the homeostasis of RA signal to increase its robustness, and what target genes of the RA responsive gens at early gastrula are responsible for the patterning hindbrain development. Our results will prove the hypothesis that the RA responsive genes at early gastrula of zebrafish regulate its hindbrain development by directly mediating the RA morphogen to pattern hindbrain development at gastrulation stage, following by activating the expressions of the identity marker genes in their corresponding rhombomeres. The results will reveal the molecular mechanisms underlying the roles of RA in hindbrain development.

英文关键词： zebrafish;hindbrain development;retinoic acid responsive genes;gastrula;morphogen