miR-430和miR-92对鲆鲽胚胎发育早期克氏泡中胞液流的网络调控

Flatfish is famous of the asymmetric transformation during metamorphosis. There is high mortality rate during metamorphosis because of greatly change in larval body. It's an urgent question needed to be solved in flatfish breeding. The molecular mechanism of morphologic left/right asymmetry is, however, largely unknown. Monocilia have been proposed to establish the left/right (LR) body axis in vertebrate embryos by creating a directional fluid flow that triggers asymmetric gene expression. Furthermore, the miR-430 and miR-92 played an essential role in Nodal signal pathway which was responsible for asymmetry gene expression and Kupffer's vesicle formation. The roles of miR-430 and miR-92 is unknown still in cilia fluid flow and asymmetric transformation of flatfish. Here, we establish an available approach to investigate the roles of miR-430 and miR-92 in flatfish by injection of Target Protectors and LNA-antimiRNAs into embryos. After knockdown or overexpress miR-430 and miR-92 or perturb the Nodal fluid flow, some genes, which were relative to Nodal signaling pathway, marker genes in mesoderm and endoderm, endoderm progenitor and dorsal forerunner cell (DFC),will be detected by in situ hybridization and qRT-PCR; Cilia structure and the Nodal fluid flow in kupffer's vesicle will be observed by Laser Scanning Confocal Microscope.Those exploring of this research should improve our knowledge in the regulate mechanism of Nodal fluid flow and asymmetry left/right body of flatfish.

鲆鲽变态时由于体内剧烈变化导致仔鱼大量死亡，这是鲆鲽养殖亟待解决的问题，但对鲆鲽变态机理目前尚不清楚。研究表明胚胎发育早期克氏泡中纤毛摆动形成的胞液流调控着左右不对称基因的表达，同时，miR-430和miR-92参与了与左右不对称相关的Nodal信号途径的调节和克氏泡中纤毛的形成，但二者与胞液流之间的关系以及是否对鲆鲽变态起调控作用还未见报导。本项目以miR-430和miR-92为切入点，对鲆鲽克氏泡中胞液流形成的调控网络进行研究，通过比较靶基因保护者和锁核苷酸两种技术，建立鲆鲽miRNAs的功能研究体系；通过抑制和过量表达miR-430和miR-92以及调控胞液流，检测Nodal信号途径关键基因、中内胚层标记基因、克氏泡形成前体- - 背部前驱细胞标记基因的表达，以及激光共聚焦显微镜观察纤毛结构和克氏泡胞液流的变化。此课题在探求胞液流形成的调控网络基础上，为鲆鲽体态偏转机理研究提供理论基础。

牙鲆变态发育过程眼睛从左右对称转变到右眼偏转到左侧的变态机理还不甚清楚。本课题从仔鱼变态发育不同时期的转录组和miRNA表达为出发点，探讨了miR-430和miR-92早期胚胎发育及变态过程中的作用。结果显示：miR-430可能通过靶基因lefty和sqt影响克氏泡中纤毛发育，进而控制着胚胎左右轴的正常形成；miR-92可能是通过靶基因gata5调控内胚层，从而实现对牙鲆变态发育进程的促进作用；进一步结合变态发育时期转录组分析，表明miR-430和miR-92可能通过以上过程调节其靶基因参与糖酵解、钾离子通道、甲状腺激素和钙信号等通路，进而调控仔鱼的信号转导、能量代谢、细胞增殖和凋亡等过程来介导牙鲆的体态偏转。