鸡卵巢SWH和SLIT/ROBO近分泌信号协同调控卵泡发育的双模态分子机制

鸡的产蛋性状决定于卵泡募集和等级化建立的发育过程，SWH和SLIT/ROBO信号通路在此过程中发挥着重要作用。本课题组前期研究表明两通路组分互作对颗粒细胞增殖产生共抑制效应，故推测SWH和SLIT/ROBO通路可能以“共激活——促抑制”和“信号互作——竞争促抑”双模态作用调控卵泡发育，但机制不清楚。本项目拟对这两个通路近分泌信号（SLIT2/3）及所介导的调节因子（LAST1/2、YAP1和CDC42等）协同调控卵泡发育的机制开展研究，通过采取免疫共沉淀、双荧光报告基因检测、RNAi和DuolinkPQ原位定量等方法揭示配体SLIT2/3可共激活SWH和SLIT/ROBO两途径、诱发胞内信号分子或核心调节因子间互作和活性状态转变、引起信号转导时空调节变化、产生竞争性促抑控制，阐明SWH和SLIT/ROBO近分泌信号协同调控卵泡发育的双模态机制，为鸡遗传资源评价和分子育种研究提供科学依据。

Egg production performance is one of the most important economic traits for hens, which are significantly divergent between higher-yield layers and lower-yield layers during their reproductive phases. The remarkable differentiations between them depend upon the cyclic process of ovarian prehierarchal follicle recruitment and hierarchy estiblishment. More recent studies have demonstrated that SWH and SLIT/ROBO signal pathways play a pivotal role in regulating follicular development. However, the molecular mechanism of the two pathways founction on the process remain poorly understood now. This work is aimed to reveal the biological roles and synergic regulative mechanisms of SWH and SLIT/ROBO signal pathways in hen ovarian follicle development mediated by the candidate key regulators (including LAST1/2, YAP1 and CDC42) under the stimulation of ligand SLIT2/3 as a juxtacrine regulatory molecule. Co-immunoprecipitation and dual luciferase reporter assay are conducted to investigate the reciprocal reactions between the regulators of LAST1/2 and CDC42, and YAP1 and CDC42, following the SLIT2/3 being detected to simultaneously trigger the regulations of the pathways by binding to its receptors ROBOs and NF2 that acts as a membrane stabilizing protein. Then, the methods of siRNA interference and gene overexpression are designed to prove the functions of the target genes (LAST1/2, YAP1 and CDC42)to govern the hen ovarian follicular development in vitro; And Duolink PQ method is utilized to study the activities and effects of the regulators on the development of the differentiate follicles after and before phosphorylation of the target proteins. Meanwhile, using the in vitro test, the biological effects of the SWH and SLIT/ROBO signals on hen prehierarchal follicle development was explored by knock-down or overexpression of the ROBOs and/or NF2 genes in the incubated ovarian granulose cells, respectively. The functional variations caused by the changes of spatio-temporal expression patterns of the mRNA and protein of the candidate genes examined are mainly to further confirm the synergic regulative mechnism of the SWH and SLIT/ROBO signal transduction pathways in the target follicle development. Taken collectively, the findings of these studies are very helpful in elucidating the role and dual modality molecular mechanism of the pathways in prehierarchal follicles development and dominant follicle selection in hen. This work is expected to provide a novel train of thought and useful data for chicken genetic resource evaluation and chicken molecular breeding to improve egg laying performance. It has a very important scientific significance and application value to carry out this study on the molecular regulatory mechanism for the involvement of the SWH and SLIT/ROBO pathways in the hen ovarian follicular development in this area.