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

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.

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。

脊椎动物胚胎发生中，视黄酸（RA）作为形态发生素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信号图式后脑形成尚未见系统研究。在本研究中，我们围绕确认原肠早期RA效应基因是否为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、以及是否通过调控哪些靶基因的表达影响后脑的形成等开展工作，研究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。.通过实施本项目，我们发现敲除insm1b、lartb、meis2b、和znf703等原肠早期RA效应基因不影响斑马鱼胚胎的正常发育与生长。其中除znf703纯合突变胚胎的后脑发育有异常外，敲除lartb和meis2b的斑马鱼胚胎后脑发育正常，与这些RA效应基因不是斑马鱼发育与生长所必须一致。幸运的是，在众多的原肠早期RA效应基因中，我们证实了insm1b、zfnl1s和foxc1a是RA直接的效应基因。虽然insm1b不是斑马鱼生长与发育所必须，但缺少zfnl1s或foxc1a均可导致斑马鱼胚胎致死且后部后脑的前后轴发生异常。对zfnl1s在胚胎后脑发生中的作用与机制进行的深入研究结果显示敲降zfnl1s导致胚胎内RA信号水平下降破坏了RA信号的稳态，下调了pou5f3、sall4以及hoxb1b在原肠胚中的表达。进一步的研究证实zfnl1s通过作用于pou5f3和sall4的上游调控hoxb1b在后部神经外胚层的表达进而影响胚胎后部后脑的发生。我们的这些研究结果支持在斑马鱼原肠早期存在RA信号直接靶基因通过介导RA信号影响后部后脑前后轴的图式形成，但却未能确认在原肠早期是否存在这样的效应基因能够通过介导RA信号调控后脑菱脑身份标识基因在体节期的表达进而决定后脑分节特化。