Wnt5a调控中华鳖背甲脊发育的信号转导通路及机理

The carapacial ridge is a key embryonic tissue in the forming progress of turtle shell. It is still not clear the molecular mechanism of carapacial ridge development and how it regulates the forming of turtle shell.The applicant’s previous studies found that Wnt5a may play an important role in regulating the development of carapacial ridge in turtles. However,it is still not known how Wnt5a regulates the development of carapacial ridge and its mechanism of action also needs to be clarified. Therefore,this research will use the embryos of Chinese soft-shell turtle as material. In order to reveal the role of Wnt5a gene in regulating the development process of carapacial ridge,we will silence and over-express Wnt5a in carapacial ridge tissue using RNA interference and gene over-expression methods.Then, we will analysis the effect of abnormal expression of Wnt5a on histological structure and specific marker gene expression of carapacial ridge. In addition, in order to illustrate the signal transduction pathway of Wnt5a regulates carapacial ridge development in Chinese soft-shell turtle,we check Wnt signal pathway members expression, such as Fz Daam JNK and β-catenin after Wnt5a gene was silenced and over-expressed in vivo. The above results will elucidate the mechanism and signal transduction pathway of Wnt5a regulates carapacial ridge development of Chinese soft-shell turtle. The research might provide a theoretical basis for revealing the forming mechanism of turtle shell and lay a foundation for guiding the production practice of Chinese soft-shell turtle.

背甲脊是鳖甲形成的关键胚胎组织，但对背甲脊发育的分子机制及其调控鳖甲形成的作用机理，仍不清楚。申请人发现，在已鉴定的候选基因中，Wnt5a可能是调控背甲脊发育的关键候选基因，有必要深入探究其作用机理。本项目以中华鳖胚胎为研究材料，利用RNA干扰和基因过表达技术，在背甲脊中沉默和过表达Wnt5a基因，分析异常表达Wnt5a对其表型、组织结构和标记基因表达的影响，揭示Wnt5a调控背甲脊发育的机制；通过体内试验，探究沉默/过表达Wnt5a对非经典Wnt信号通路成员Fz、Daam、JNK以及经典Wnt信号通路关键成员β-catenin等的影响，阐明“Wnt5a-Wnt信号通路-中华鳖背甲脊发育”的关系，揭示Wnt5a调控中华鳖背甲脊发育的信号转导通路。以上结果为揭示鳖甲形成机制提供理论依据，为指导生产实践奠定基础。

背甲脊是鳖甲形成的关键胚胎组织，本试验以中华鳖背甲脊为研究对象，运用多组学关联分析、RNA干扰技术和基因过表达技术等，研究Wnt5a调控背甲脊发育的作用机理及信号转导通路。利用多组学关联分析及验证试验揭示Wnt信号通路是调控中华鳖背甲脊发育的关键信号通路，Wnt5a在中华鳖背甲脊发育过程中起重要作用。通过Wnt通路图（ko04310）转录和翻译基因热图绘制显示Wnt5a/PCP信号通路在背甲脊发育过程中激活。利用RNA干扰技术在背甲脊原代细胞中沉默Wnt5a基因，结果显示沉默组背甲脊原代细胞中Dvl1、Dvl3、Ror2、Mapk8、Mapk9和Damm1表达量显著低于对照组，而Dvl2的表达量无显著变化。且，在背甲脊组织中沉默Wnt5a基因，结果也显示沉默组Dvl1、Ror2、Mapk8、Mapk9和Damm1表达量显著低于对照组。此外，在背甲脊原代细胞中转染pcDNA3.1-Wnt5a质粒高表达Wnt5a基因，结果显示Dvl1、Ror2、Mapk8、Mapk9、Damm1、Fzd4的表达量显著高于对照组，而Lef-1、Ctnnb1的表达量与对照组无显著差异。综上所述，Wnt5a通过Wnt5a/JNK信号通路调控中华鳖背甲脊发育。另外，利用RNA干扰技术在背甲脊原代细胞中沉默β-catenin基因，沉默组CCND1和Lef1表达量显著低于对照组。利用流式细胞仪检测沉默组背甲脊原代细胞增殖和凋亡情况，显示沉默组凋亡细胞数量高于对照组，增殖细胞数量低于对照组。此外，背甲脊在硬壳龟背甲形成过程中也起关键作用，本试验通过对中华草龟不同时期背甲脊组织进行转录组测序，获得了中华草龟背甲脊发育时期的全基因表达谱。再利用差异表达基因分析和验证试验揭示了Fgf、Bmp、Sox、Wnt基因是中华草龟背甲脊发育的关键候选基因。通过差异表达基因KEGG通路富集分析及基因表达趋势分析显示Wnt信号通路在中华草龟背甲脊发育早期表达量高，Hedgehog信号通路在晚期表达量高。另外，Wnt蛋白互作调控网络分析揭示Wnt信号通路与Fgf，Bmp和Shh相互作用构成一个复杂的调控网络。总之，Wnt信号通路是调控龟鳖背甲脊发育的关键信号通路，Wnt5a通过Wnt5a/JNK通路调控中华鳖背甲脊发育，本研究为进一步揭示龟鳖背甲形态发生机制提供了新视角，具有重要意义。