EXTERNAL GENITALIA DEVELOPMENT AND HYPOSPADIAS

描述（由申请人提供）：生殖器畸形包括尿道下裂是仅次于心脏缺陷的第二常见男性出生缺陷。在过去的40年里，尿道下裂的发病率和其他男性生殖问题一起翻了一番。人们怀疑胎儿接触内分泌干扰物可能导致了这种增加。然而，我们对尿道下裂或生殖器发育的病因学的了解仍然非常有限。我们的初步研究令人信服地证明了Shh和Wnt两种信号通路在外生殖器发育的早期雄激素非依赖性和晚期两性二态期的重要性。当典型的Wnt通路在早期生殖器结节（GT）发育期间在尿道上皮中受到干扰时，严重的生殖器畸形就会发生。在生殖器雄性化过程中，在2-catenin基因敲除的外胚层和缺乏2-catenin的小鼠中观察到尿道下裂样表型。更重要的是，我们的初步数据揭示了Wnt拮抗剂的两性二态表达以及Shh和Wnt通路在男性外生殖器男性化过程中的功能作用。这些令人兴奋的结果解决了一个高度未被研究的领域，涉及非性腺和局部产生的男性因素，如生长因子信号级联介导两性二形外生殖器发育。基于这些观察结果，本研究将继续揭示调节生殖器发育和男性化的信号级联，重点关注Shh和Wnt通路。在目的1中，我们将通过尝试用Fgf8挽救Wnt突变体中的GT缺陷来验证Fgf8是GT发育过程中Wnt信号传导的直接靶标的假设。外胚层2-连环蛋白在GT形态发生中的作用也将被研究。在aim II中，我们将通过详细分析Shh和Smoothened条件敲除GT表型来验证在GT模式形成过程中多个步骤都需要Shh的假设。我们将描绘出GT中Shh应答组织，并研究Shh与Wnt通路之间的相互作用。最后，在aim III中，我们将通过分析几种条件敲除突变体的GT表型来验证Wnt/Shh信号参与外生殖器两性二态发育调节的假设。我们的长期目标是利用小鼠分子遗传学来了解生殖器发育和男性化的过程，以及尿道下裂等生殖器畸形的病因。公共卫生相关性：本项目拟研究Shh和Wnt两种信号通路在外生殖器发育的早期雄激素不依赖期和晚期两性二态期的功能。包括尿道下裂在内的生殖器畸形发生率很高，但其病因尚不清楚。本项目将利用几只条件敲除小鼠，揭示这两条重要通路的下游信号事件，以及它们如何相互作用，以及雄激素信号通路如何调节生殖器形态发生和雄性化。我们的研究将有助于了解外生殖器的发育和尿道下裂的病因。

DESCRIPTION (provided by applicant): Genital malformation including hypospadias represents the second most common male birth defect after cardiac defect. In the past 40 years, hypospadias incidence has doubled along with other male reproductive problems. It is suspected that fetal exposure to endocrine disruptors may have contributed to this increase. However, our understandings of the etiology of hypospadias or genital development in general are still very limited. Our preliminary studies convincingly demonstrated that the importance of two signaling pathways, Shh and Wnt in both early androgen-independent and late sexually dimorphic phases of external genitalia development. When the canonical Wnt pathway is perturbed in the urethral epithelium during early genital tubercle (GT) development, severe genital malformations developed. Hypospadias-like phenotype is observed in ectodermal 2-catenin knockouts and in mice lacking 2-catenin during genital masculinization. More importantly, our preliminary data revealed sexually dimorphic expression of Wnt-antagonists and the functional roles of Shh and Wnt pathways during the masculinization of male external genitalia. These exciting results address a highly understudied area involving non-gonadal and locally produced masculine factors, such as growth factor signaling cascade in mediating sexually dimorphic external genital development. Based on these observations, this proposal will continue to uncover the signaling cascade regulating genital development and masculinization, focusing on Shh and Wnt pathways. In aim I, we will test the hypothesis that Fgf8 is a direct target of Wnt signaling during GT development by attempting to rescue GT defects in Wnt mutants with FGF8. The role of ectodermal 2-catenin in GT morphogenesis will also be studied. In aim II, we will test the hypothesis that Shh is required at multiple steps during GT patterning by analyzing in detail Shh and Smoothened conditional knockout GT phenotype. Shh-responding tissues in the GT will be delineated and the interaction between Shh and Wnt pathway will be studied. Finally in aim III, we will test the hypothesis that Wnt/Shh signaling is involved in the regulation of sexually dimorphic development of external genitalia by analyzing GT phenotype of several conditional knockout mutants. Our long term goal is to use mouse molecular genetics to understand the process of genital development and masculinization and the etiology of genital malformations, such as hypospadias. PUBLIC HEALTH RELEVAVANCE: This project proposes to study the function of two signaling pathways, Shh and Wnt, in both early androgen-independent and late sexually dimorphic phases of external genitalia development. Genital malformations including hypospadias occur at a very high rate but their etiology remains largely unknown. Using several conditional knockout mice, this project will reveal the downstream signaling events of these two important pathways and how they interact with each other and the androgen signaling pathway to regulate genital morphogenesis and masculinization. Our studies will contribute greatly to the understanding of external genitalia development and the etiology of hypospadias.