Posttranscriptional regulation of Dmrt1 during sexual development in Medaka

在后生动物中，雄性和雌性的发育和两性繁殖是最普遍的模式。尽管这一现象本身具有普遍性，但性别决定的机制在不同的群体中差异很大，并反复和独立地演变。潜在的分子途径可以在进化过程中迅速改变，即使在密切相关的有机体群体中也是如此。性别决定和性腺分化所涉及的因素也可能有本质上的不同。唯一的例外是转录因子DMRT1，它在所有后生动物中高度保守，已被证明参与了从苍蝇和蠕虫到人类的有性发育。在脊椎动物中，需要根据细胞类型特异性、发育时间和转录水平对基因表达进行精确调控，以确保性别决定中的适当功能。我们正在使用小型水族鱼模型Medaka(宽叶水稻)来研究DMRT1在脊椎动物性别发育中的作用，并在更广泛的水平上帮助理解性别决定机制的高度可塑性的进化。我们早些时候已经证明，在青竹中，dmrt1(Dmrt1bY)的复制副本是男性性别决定基因的主控。我们发现，dmrt1bY的表达调控最关键的发生在转录后水平上，通过细胞类型特异性和发育时间依赖的差异mRNA稳定性。我们在dmrt1bY的3‘UTR端发现了一个蛋白结合基序，负责这一调控。有趣的是，这个基序在无脊椎动物和脊椎动物(包括人类)的dmrt1同源物中高度保守。在这个项目中，我们想要识别与dmrt1 3‘UTR相互作用的蛋白质，并调节其表达，以及它们与其他生殖系和性腺决定基因的相互作用。一方面，我们建议研究两个候选蛋白质，这两个蛋白质已被报道与两个不同的序列结合，这两个序列都组成了dmrt1 3‘UTR共识结合基序。这将在这两种蛋白的重组表达和体外结合活性检测之后完成。同时，我们将采取公正的方法，通过亲和层析纯化筛选结合因子。在功能研究方面，我们将研究3‘UTR盒结合蛋白的表达模式，并进一步研究利用因子过表达、基因敲除和基因敲除的方法来验证它们在dmrt1转录后表达调控中的作用。最后，将在小鼠身上研究FISH模型中所描述的因素在性发育中的作用。这将有助于更好地理解高度保守的性别决定因子DMRT1如何在脊椎动物中发挥其功能，并增加关于导致脊椎动物性腺原基向睾丸或卵巢发育的复杂调控相互作用的知识。

The development of males and females and bisexual reproduction is the most prevalent mode in metazoans. Despite the generality of the phenomenon itself the mechanisms how sex is determined are very different among various groups and have evolved repeatedly and independently. The underlying molecular pathways can change quickly during evolution, even within closely related groups of organisms. The factors involved in sex determination and gonad differentiation can also be substantially different. A single exception is the transcription factor Dmrt1, which is highly conserved in all metazoans and has been shown to be involved in sexual development from flies and worms up to humans. In vertebrates a precise regulation of gene expression with respect to cell type specificity, time of development and transcript level is requested to ensure a proper function in sex determination. We are using the small aquarium fish model medaka (Oryzias latipes) to study the role of Dmrt1 in vertebrate sexual development and on a more general level to contribute to an understanding of the evolution of the high plasticity of sex determining mechanisms. We have shown earlier that in medaka a duplicated copy of dmrt1 (dmrt1bY) is the master male sex-determining gene. We found that regulation of dmrt1bY expression most critically occurs on the posttranscriptional level through a cell type specific and developmental time dependent differential mRNA stability. We identified a protein-binding motif in the 3' UTR of dmrt1bY to be responsible for this regulation. Interestingly, this motif is highly conserved in dmrt1 homologues of invertebrates and vertebrates, including human. In this project we want to identify the proteins which interact with the dmrt1 3' UTR and regulate its expression, as well as their interaction with other germline and gonad determining genes. On the one hand, we propose to study two candidate proteins that have been reported to bind to two different sequences, which both together make up the dmrt1 3' UTR consensus binding motif. This will be done after recombinant expression of both proteins and assaying their binding activities in vitro. In parallel, we will take an unbiased approach and screen for binding factors by affinity column purification. For functional characterization, we will study the expression pattern of the 3' UTR box binding proteins and further on use factor overexpression, gene knockdown and gene knockout approaches to validate their role in dmrt1 posttranscriptional expression regulation. Finally, the factors characterized in the fish model will be studied in mouse for their role in sexual development. This should contribute to a better understanding how the highly conserved sex determining factor Dmrt1 exerts its function in vertebrates and increase the knowledge about the complex regulatory interactions that lead to determination of the gonad anlage towards testis or ovary development in vertebrates.