Visualizing and elucidating the FGF-dependent mechanisms of urethral morphogenesis

项目摘要 尿道下裂是一种先天性缺陷，其中尿道口从阴茎远端移位 而是终止于阴茎的腹侧，或者在严重的情况下，终止于阴囊或会阴。 尿道下裂是最常见的出生缺陷之一，大约每125名活产男婴中就有1名患有尿道下裂。 手术是目前唯一的治疗方法，但有并发症和复发的风险。最近的工作 已经证明，遗传因素和环境因素，如胚胎暴露于 内分泌干扰物可增加尿道下裂的发病率。尽管有这些研究，我们仍然没有 很好地掌握生殖器形态发生的哪些方面被破坏并导致尿道下裂。 胚胎生殖结节（GT）产生阴茎或阴蒂，并含有来自 所有三个胚层。GT的内胚层成分主要产生尿道、中胚层、 细胞有助于外生殖器内的支持结构，外胚层产生 覆盖皮肤。在GT发育期间，内胚层和外胚层在内胚层/外胚层处连接， 在GT的腹侧上的连接（EEJ），形成身体中极少数这样的连接的部位之一， 发生。在阴茎发育和尿道内化的过程中，这个连接被重塑， 导致尿道口的远端定位;在阴蒂切除期间和之后，EEJ保持完整 尿道口在阴蒂和阴道之间的腹侧位置。这 导致假设EEJ重塑是必要的尿道内化在阴茎，和 对EEJ的破坏引起尿道下裂。为了验证这一假设，该提案使用了小鼠突变体， FGF信号通路作为测试EEJ破坏如何导致GT畸形的模型： Fgfr 2突变显示GT中的内胚层和外胚层缺陷，随后发展为严重的 尿道下裂本提案的目的1将测试Fgfr 2如何调节尿道内分泌过程中EEJ的细胞运动。 内化;该目的利用了一种新GT器官培养系统， 成像技术直接可视化小鼠EEJ的形态发生。目标2将审查 尿道内化的生物力学机制和测试细胞外基质粘附的作用 为EEJ的发展做出贡献。 这项提议的发现将产生一个细胞和组织运动的图谱， 在小鼠尿道形成期间，这是大多数哺乳动物器官系统无法获得的资源。 这里提出的研究不仅将照亮正常的形态发生机制， 外生殖器发育，但也将提供深入了解尿道下裂如何出现，并将有助于形状 潜在的预防措施，这种常见的先天性缺陷。

PROJECT SUMMARY Hypospadias is a birth defect wherein the urethral opening is displaced from the distal end of the penis and instead terminates on the ventral side of the penis, or in severe cases, on the scrotum or perineum. Hypospadias is one of the most common birth defects, affecting approximately 1 in 125 live male births. Surgery is currently the only treatment, which comes with a risk of complications and recurrence. Recent work has demonstrated that both genetic factors and environmental factors such as embryonic exposure to endocrine disrupting chemicals can increase the rate of hypospadias. Despite these studies, we still do not have a good grasp on what aspects of genitalia morphogenesis become disrupted and result in hypospadias. The embryonic genital tubercle (GT) gives rise to either the penis or clitoris, and contains tissues from all three germ layers. The endodermal component of the GT primarily gives rise to the urethra, mesodermal cells contribute to supportive structures within the external genitalia, and the ectoderm gives rise to the overlying skin. During GT development, the endoderm and ectoderm are connected at an endoderm/ectoderm junction (EEJ) on the ventral side of the GT, forming one of very few sites in the body where such a junction occurs. This junction is remodeled away over the course of penis development and urethral internalization, leading to distal positioning of the urethral meatus; the EEJ remains intact during and after clitoral development, resulting in the ventral position of the urethral opening between the clitoris and vagina. This leads to the hypothesis that EEJ remodeling is necessary for urethral internalization in the penis, and disruptions to the EEJ cause hypospadias. To test this hypothesis, this proposal uses mouse mutants in the Fgf signaling pathway as a model to test how disruptions of the EEJ result in GT malformations: mice with Fgfr2 mutations display endodermal and ectodermal defects in the GT and subsequently develop severe hypospadias. Aim 1 of this proposal will test how Fgfr2 regulates cell movements at the EEJ during urethral internalization; this aim makes use of a novel GT organ culture system coupled with four-dimensional live imaging techniques to directly visualize morphogenesis of the mouse EEJ. Aim 2 will examine the biomechanical mechanisms underlying urethral internalization and test the role of extracellular matrix adhesion in contributing to EEJ development. The findings from this proposal will generate an atlas of the cell and tissue movements which occur during urethral formation in the mouse, a resource which is not available for most mammalian organ systems. The research proposed here will not only shine a light onto the morphogenetic mechanisms underlying normal external genitalia development, but will also provide insight into how hypospadias arise and will help shape potential preventative measures for this common congenital defect.