miR-2954在鸡早期性腺发育中的调控作用及其分子机制研究

The revised model of sex determination based on cell-autonomous sex identify indicated that there are several primary parallel-acting factors regulating the sex differentiation of downstream gonads and somatic cells, while little was known about these factors. In our previous study, miR-2954 located on Z sex chromosome was identified as a male-biased expressed miRNA in chicken gonad during the period of sexual differentiation, which regulates some genes related gonad development like DMRT1. We speculated miR-2954 is probably a primary sex determination factor involved in early development of chicken gonads, and intend to further study its function and molecular mechanism in this project. We will detect the expression of miR-2954 in tissues and cells of chicken embryos and gonads during the onset of gonad differentiation via WISH and FISH, and investigate the changes of the histomorphology of gonads and the expression of marker genes related to gonad development after overexpression and knock-down of miR-2954 on chicken embryos. Then, the alterant downstream genes on chicken embryos and gonads of functional tested groups are identified by using RNA-seq technology, and target genes of miR-2954 are identified by CLIP-seq technology and validated some important targets by Dual-Luciferase reporter system on cell level; and based on these factors we will build the downstream signal pathways of miR-2954 affecting the development of embryonic gonad in chicken. Results in this project will contribute to the clarification of the regulatory functions and its mechanisms of miR-2954 as an upstream factor on chicken early sex development, complete the regulatory mechanism of chicken sex determination and sex differentiation, and provide experimental evidence for the new concept of sex determination.

基于细胞自主性别决定的修正模型认为鸡性染色体上存在多个初级性别决定因子并行指导下游性腺及体细胞性分化；但对这些因子的研究甚少。前期研究中我们鉴定了性分化前后鸡雄性性腺及体组织中高表达的Z染色体miR-2954，它可调控DMRT1等性腺发育基因，推测其可能是一个参与鸡早期性腺发育的初级性别决定因子；本项目即进一步研究其作用与分子机制。项目拟采用原位杂交技术研究miR-2954在性分化前后鸡胚及性腺中的组织和细胞表达；建立过表达和敲低miR-2954的鸡胚模型检测性腺组织形态及性腺发育标记基因表达的变化；利用RNA-seq技术鉴定过表达及敲低后性腺中改变的下游基因，结合CLIP-seq技术鉴定并验证的靶基因，构建miR-2954影响鸡胚早期性腺发育的信号通路。本研究将有助于阐明miR-2954在鸡早期性腺发育中的调控作用及其机制、完善鸡性决定与性分化调控机制，并为新的性别决定学说提供试验证据。

鸡的上游性别决定因子和性别决定机制至今尚未明确。基于鸡细胞自主性别决定的修正模型和本课题前期研究结果，我们认为位于Z染色体上的miR-2954可能是参与鸡早期性腺发育的一个初级性别决定因子。本项目首先结合定量PCR和荧光原位杂交方法获得了miR-2954在性分化前后早期鸡胚及性腺中雄性高表达，并表达于雌、雄性腺髓质部的特性；构建了慢病毒介导的miR-2954分子的 Crispr-cas9敲低重组质粒和超表达重组质粒，均能以质粒或慢病毒形式成功转染鸡胚原代性腺细胞；通过miR-2954敲低及芳香化酶抑制剂（AI）性反转鸡胚模型，探究了miR-2954主要通过影响雄性分化基因DMRT1及SOX9的表达，从而在雄性性腺的早期分化途径中起重要调控作用；结合miRNA pulldown和转录组测序在鸡胚性腺细胞及性腺组织中全面鉴定了miR-2954的靶基因，发现其在性腺中主要靶向非编码lncRNAs和cirRNAs起调控作用；进一步在性腺组织细胞和体细胞水平探究miR-2954介导的靶基因YY1的功能，免疫荧光结果表明YY1与miR-2954的表达定位相似，主要分布在不同阶段鸡胚睾丸髓质部的支持细胞、精原细胞和间质细胞中，原代睾丸支持细胞的超表达结果表明miR-2954可能通过调控YY1来影响支持细胞分泌相关的细胞因子和激素来影响雄性精原细胞生成及分化，从而调控性腺发育；在鸡胚成肌细胞中，miR-2954通过靶向YY1抑制成肌细胞的增殖并促进其分化，从而在不同性别鸡胚肌肉发育中起着重要调控作用。本项目研究结果首次阐明了miR-2954作为细胞自主性别决定因子在鸡早期雄性性腺分化与发育中的重要调控作用，并通过靶基因的鉴定和功能研究初步构建了miR-2954参与鸡胚早期性发育的调控机制，从非编码基因角度为完善鸡性别决定与性分化调控机制提供了支撑资料。