Visualizing and elucidating the FGF-dependent mechanisms of urethral morphogenesis

项目总结 尿道下裂是一种出生缺陷，其特征是尿道口从阴茎远端移位。 取而代之的是终止于阴茎的腹侧，或在严重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名男性活产儿中就有一名受到影响。 手术是目前唯一的治疗方法，但存在并发症和复发的风险。近期工作 已经证明了遗传因素和环境因素，如胚胎暴露于 内分泌干扰物会增加尿道下裂的发生率。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受到干扰，从而导致尿道下裂。 胚胎生殖器结节(GT)形成阴茎或阴蒂，包含来自 这三层细菌都是。GT的内胚层成分主要形成尿路、中胚层。 细胞对外生殖器内的支持结构起作用，外胚层产生 覆盖的皮肤。在GT发育过程中，内胚层和外胚层在一个内胚层/外胚层相连。 位于GT腹侧的连接(EEJ)，是人体内极少数连接的部位之一 发生。在阴茎发育和尿路内化的过程中，这个连接处被重新塑造， 导致尿道口的远端定位；在阴蒂期间和之后，EEJ保持完好 发育，导致阴蒂和阴道之间的尿道口的腹侧位置。这 导致假设EEJ重塑对于阴茎的尿路内化是必要的，并且 EEJ受阻会导致尿道下裂。为了验证这一假设，这一提议使用了小鼠突变体 以成纤维细胞生长因子信号通路为模型测试EEJ的破坏如何导致GT畸形：小鼠 FGFR2突变在GT中表现为内胚层和外胚层缺陷，随后发展为严重的 尿道下裂。这项提案的目标1将测试FGFR2如何在尿道期调节EEJ的细胞运动 内化；这一目标利用了一种新的GT器官培养系统，与四维LIVE相结合 直接可视化小鼠EEJ形态发生的成像技术。Aim 2将研究 尿路内化的生物力学机制及细胞外基质粘连的作用 为EEJ的发展作出贡献。 这项提案的发现将生成细胞和组织发生运动的图谱 在小鼠的尿路形成过程中，一种大多数哺乳动物器官系统所不具备的资源。 这里提出的研究不仅将揭示正常的形态发生机制 外生殖器发育，但也将提供洞察如何发生和将有助于形成的尿道下裂 对这种常见的先天缺陷可能采取的预防措施。

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.