Sox9在罗非鱼性腺分化发育中的功能及作用机制

Sox9 gene undergoes duplication in teleost fish and forms two copies of Sox9a and Sox9b. Previous report showed that Medaka Sox9b mutants had a reduction in the germ cell number and heterozygous females XX mutants that lack Sox9b function exhibited sex-reversed to male. Our primary investigation indicated Sox9a can repress the expression of Cyp19a1a and was also down-regulated in the testis of Dmrt1 knock-out of Nile tilapia (Oreochromis niloticus). These results suggested that Sox9a and Sox9b may play different roles in the regulations on the differentiation and development of gonad in teleost fish. In this study, based on the TALEN knock-out technology and sex-reverse model of Nile tilapia, and using the methods including in Situ Hybridization, immunohistochemical staining and luciferase assays, we are planning to survey the functions of Sox9a and Sox9b in the gonadal differentiation and development in teleost fish, and to decipher the molecular relationship between Sox9a or Sox9b and sex-differentiation related genes (such as Dmrt1 and Foxl2) or sex-hormone synthesis-related genes(Cyp19a1a and 11β-HSD). We hope our results from this study will elucidate the functions and signaling pathway of sox9 gene in the gonadal differentiation and development in teleost fish.

Sox9在鱼类中有Sox9a和Sox9b两个拷贝。已有研究报道证实青鳉Sox9b突变会导致生殖细胞数目减少并引起雌向雄性逆转；我们的前期研究显示，尼罗罗非鱼Sox9a既可抑制Cyp19a1a的表达，又可在Dmrt1敲除后下调表达。这暗示Sox9a和Sox9b在鱼性腺分化发育中发挥了不同的功能。本研究拟以尼罗罗非鱼为研究对象，以TALEN基因敲除技术和尼罗罗非鱼性逆转模型为基础，综合应用原位杂交、免疫组化及启动子荧光素酶报告实验等方法，系统分析Sox9a和Sox9b在尼罗罗非鱼性腺分化与发育中的功能，重点探究Sox9a和Sox9b与性别分化相关基因（Dmrt1和Foxl2）及性激素合成关键酶基因（Cyp19a1a和11β-HSD）等分子间的相互作用关系。相关研究结果有助于揭示Sox9在鱼性腺分化与发育调控中的功能和信号传导机制。

Sox基因家族是动物特有的一类包含高迁移率族盒（HMG-box）结构域的转录因子，目前关于罗非鱼Sox基因的研究报道非常少。我们首先利用Sox蛋白较保守的HMG-box结构域对罗非鱼基因组序列及相关的预测基因集进行BLAST同源性检索分析，共鉴定获得27个Sox基因。比较基因组学分析发现，罗非鱼等硬骨鱼的部分Sox基因伴随全基因组复制而发生复制扩张，其中哺乳动物雄性性别决定与分化相关Sox9基因的同源基因在罗非鱼中有Sox9a和Sox9b两个拷贝。表达谱分析发现，Sox9a基因在罗非鱼发育早期（孵化后5天和30天）的精巢和卵巢中都高表达，Sox9b在罗非鱼发育早后期（孵化后30天至180天）的精巢中高表达。基于TALEN基因敲除的功能分析发现，Sox9a的敲除突变不仅可导致罗非鱼精巢中的精原细胞减少和体细胞增生等，还可影响卵巢生殖细胞的存活，使卵巢Cyp19a1a和Cyp11b2的表达水平下降；Sox9b的敲除突变可能导致罗非鱼胚胎期致死。鉴于罗非鱼Sox9b主要在精巢发育的后期高表达，我们进一步克隆了Sox9b的启动子，并以此为基础，综合应用荧光素酶报告基因实验、凝胶阻滞实验（EMSA）和免疫共沉淀实验（ChIP）分析了Sox9b基因转录的调控机制。结果表明，罗非鱼精巢特异表达的Dmrt1对Sox9b的转录具有正调控作用，而且这种调控作用是通过Dmrt1直接识别结合Sox9b启动子的两个顺式作用元件（CRE）来实现的。这些创新性发现为解析Sox9在罗非鱼性腺分化发育中的作用奠定了关键基础，也为阐释罗非鱼生殖发育与性别分化分子调控机制提供了新的理论支撑。