foxl2a 和 foxl2b 基因在斑马鱼卵巢发育和维持中的调控机制研究

Sex has always been one of the hot topics in life science research, but for the complexity of sex determination system in fish and the poor sex-controlling technology in aquatic animals, the mechanism of sexual development for fish are largely unknown. Foxl2 is the earliest known marker of ovarian differentiation in mammals. However, its effects on gonadal development in fish still remain poorly defined. In the past few years, we have established foxl2a-deficient (foxl2a-/-), foxl2b-deficient (foxl2b-/-) and foxl2a/foxl2b-deficient (foxl2a-/-/foxl2b-/-) lines with TALEN approach in zebrafish. Premature ovarian failure (POF), partial sex reversal and complete sex reversal were observed in the female gonads of these three mutant lines respectively. However, the specific molecular regulation mechanisms underlying this are not known yet. In this project, the cell events, gene regulatory networks, content of sex hormones and rescue effects will be compared among the gonad tissues of foxl2a-/- mutants, foxl2b-/- mutants, foxl2a-/-/foxl2b-/- mutants and control fishes at different developmental stages, with the aim to find out the mechanisms of foxl2a and foxl2b in regulating ovarian development at cellular level, molecular level and hormone level. The results will provide guide lines for the construction of gene networks on sex regulation in zebrafish, which may be potentially applied in other teleosts, and advance the research of genetic basis on sexual dimorphism in fish.

性别是生命科学研究的热门话题之一，但由于鱼类性别决定系统的复杂性和水产性别控制技术的滞后性，目前人们对鱼类性别调控机制的研究依然十分薄弱。Foxl2是哺乳动物卵巢分化中最早的标记基因，但它对鱼类性腺发育的影响鲜有研究。近年来，我们利用TALEN技术分别构建了敲除foxl2a和foxl2b的斑马鱼单突变系和双突变系，组织学分析显示这三种突变体的雌性性腺分别出现了卵巢早衰、不完全性逆转和完全性逆转的现象，但具体的分子调控机制仍不得而知。本项目拟通过对foxl2a-/-、foxl2b-/-、foxl2a-/-/foxl2b-/-三种突变体和WT不同发育时期的性腺进行细胞事件分析、基因调控网络分析、相关激素含量及回救效应分析，从细胞水平、分子水平和激素水平系统解析foxl2a和foxl2b在卵巢发育中的功能机制。本项目将为斑马鱼性控基因层级网络的构建和鱼类性别异形遗传基础的研究提供丰富的参考资料。

Foxl2是哺乳动物卵巢分化中最早的标记基因，敲除foxl2s的雌性斑马鱼会出现卵巢早衰（foxl2a-/-）、不完全性逆转（foxl2b-/-）和完全性逆转（foxl2a-/-/foxl2b-/-）的现象，但具体的分子调控机制仍不清楚。本项目通过对foxl2s突变体和WT的性腺进行细胞类型分析发现，在foxl2a-/-和foxl2b-/-突变卵巢形态发生改变过程中MO、VO和PVO最先退化凋亡，DO呈现出先升后降的趋势，在270dpf时，PO完全消失， foxl2b-/ -Ⅳ型卵巢中出现大量的SCs，占整个组织面积的13.15%。对突变体卵巢进行性控相关基因的动态变化模式分析发现，在foxl2a-/-卵巢发生形态变化过程中凋亡类marker急剧上调和雌激素合成的关键基因cyp19a1a下调导致卵母细胞大量退化，精巢发育的上游关键基因sox9a和dmrt1在150dpf时显著上调，但由于合成雄激素的关键基因cyp17a2剧烈下调致使精巢分化的相关信号通路未被启动；在foxl2b-/-卵巢发生形态变化过程中，雄激素合成的关键基因cyp17a1和cyp17a2均未受到抑制，促进精巢分化的上游基因dmrt1成功的启动了精巢发育的信号通路，致使foxl2b-/-卵巢向精巢方向转化，形成了精小囊结构。对类固醇合成的关键基因CYP进行系统分析发现，cyp27b1和cyp24a1促进了foxl2a-/- 和 foxl2b-/- 卵巢中Vitamin D3的分解，cyp11a2和cyp17a1促进了foxl2b-/- 卵巢中雄激素的合成。此外，我们还发现foxl2a和foxl2b协同调控斑马鱼MHB和第四脑室的发育。该研究为进一步解析鱼类性别调控网络奠定了基础。