Critical Roles for Fibroblast Growth Factor Receptors in Bladder Development

 描述(申请人提供)：发育性膀胱缺陷可在一生中导致许多健康问题，包括儿童肾功能衰竭、尿路感染、结石和尿潴留；然而，膀胱疾病的遗传决定因素在很大程度上是未知的。该应用程序的广泛长期目标是阐明膀胱发育的分子控制，以开发针对结构性膀胱疾病的有效疗法。目前，大部分对膀胱发育的分子调控尚不清楚。成纤维细胞生长因子受体2(FGFR2)在发育中的膀胱间充质(未来的肌肉和固有层)中表达；然而，在小鼠中，FGFR2的全局缺失导致在尿路发育开始之前的早期胚胎死亡，使得该受体在膀胱发育中的作用尚不清楚。为了避免全球基因敲除的早期致命性，使用Tbx18cre系有条件地删除了膀胱间充质中的FGFR2(Fgfr2BM-/-)。初步数据显示，虽然Fgfr2BM-/-胚胎膀胱的总体积没有变化，但与对照组相比，突变体的外浓缩间充质(未来的肌肉)的体积相对减少，肌肉标志物的表达减少。相反，Fgfr2BM-/-膀胱的内侧间充质(未来的固有层)的体积相对增加，早期胶原标志物的表达增加，并渗透到肌肉中。出生后早期突变小鼠的膀胱具有组织学异常和功能缺陷，包括收缩能力降低、顺应性差和高压；老年Fgfr2BM-/-小鼠出现严重的膀胱扩张并伴有纤维化和肌源性衰竭(类似无张力膀胱)，还出现肾脏损伤。从机制上讲，Fgfr2BM-/-胚胎膀胱似乎增加了声波刺猬(Shh)的活性(以及刺猬下游的其他途径，如Wnt和Bmp4)，这可能是对间充质的错配。FGFR2对刺猬的抑制似乎是通过调节HH共同受体Cdon和Boc来实现的。最后，初步的膀胱培养实验建议使用低剂量的刺猬抑制剂来修复突变的膀胱肌缺陷。最重要的假设是，膀胱间充质中FGFR信号的缺失会导致早期发育模式缺陷，这会对出生后产生重大影响。为了验证这一假说，提出了以下目标：目的1：描述Fgfr2BM-/-膀胱中进行性的组织学、功能和信号缺陷。结构和分子膀胱错误模式将在Fgfr2BM-/-胚胎和新生小鼠中被询问。发育性膀胱错构化的结构和功能后果将在出生后的小鼠中确定。FGFR2下游的信号缺陷也将在突变的膀胱中表现出来。目的2：确定FGFR突变体花纹缺陷的分子机制。在胚胎Fgfr2BM-/-膀胱中，刺猬信号的扰动(以及刺猬下游的其他潜在通路，如Bmp4和Wnt)将被询问。将使用化学和遗传方法在体外和体内尝试挽救Fgfr2BM-/-小鼠的膀胱缺陷。

 DESCRIPTION (provided by applicant): Developmental bladder defects can lead to numerous health problems throughout life including pediatric kidney failure, urinary tract infections, stone, and urinary retention; however, genetic determinants of bladder diseases are largely unknown. The application's broad long-term objectives are to elucidate the molecular control of bladder development to develop effective therapies for structural bladder disease. Presently, most of the molecular control of bladder development is unknown. Fibroblast growth factor receptor 2 (Fgfr2) is expressed in developing bladder mesenchyme (future muscle and lamina propria); however, global deletion of Fgfr2 in mice leads to early embryonic lethality prior to the onset of urinary tract development, making the roles of the receptor in bladder development unclear. To circumvent the early lethality of the global knockouts, a Tbx18cre line was used to conditionally delete Fgfr2 in the bladder mesenchyme (Fgfr2BM-/-). Preliminary data show that while Fgfr2BM-/- embryonic bladders total bladder volumes are unchanged, the mutants have a relative reduction in the volume of the outer condensing mesenchyme (future muscle) and a decrease in muscle marker expression vs. controls. Conversely, Fgfr2BM-/- bladders have a relative increase in volume of the inner mesenchyme (future lamina propria) and higher expression of early collagen markers with infiltration into the muscle. Early postnatal mutant bladders have histological abnormalities and functional defects including decreases in contractility, poor compliance, and high pressures; aged Fgfr2BM-/- mice develop severe bladder distention with fibrosis and myogenic failure (resembling atonic bladders) and also develop kidney injury. Mechanistically, Fgfr2BM-/- embryonic bladders appear to have increased sonic hedgehog (Shh) activity (and other pathways downstream of hedgehog such as Wnt and Bmp4), which likely mispatterns the mesenchyme. Fgfr2 suppression of hedgehog appears to by regulation of Hh co-receptors Cdon and Boc. Finally preliminary bladder culture experiments suggest a rescue of mutant bladder muscle defects with low doses of a hedgehog inhibitor. The overarching hypothesis is that loss of Fgfr signaling in bladder mesenchyme leads to early developmental patterning defects that have significant postnatal consequences. To test the hypothesis, the following aims are proposed: Aim1: Characterize the progressive histological, functional, and signaling defects in Fgfr2BM-/- bladders. Structural and molecular bladder mispatterning will be interrogated in Fgfr2BM-/- embryos and newborn mice. Structural and functional consequences of developmental bladder mispatterning will be determined in postnatal mice. Signaling defects downstream of Fgfr2 will also be characterized in mutant bladders. Aim 2: Determine the molecular mechanisms driving the patterning defects in the Fgfr mutants. Perturbations in hedgehog signaling (and other potential pathways downstream of hedgehog such as Bmp4 and Wnt) will be interrogated in embryonic Fgfr2BM-/- bladders. Chemical and genetic approaches will be used ex vivo and in vivo to attempt rescue of bladder defects in Fgfr2BM-/- mice.