Signals Integrating Cellular Dynamics to Sculpt the Inner Ear (A1)

描述（由申请人提供）：在人类受试者和动物模型中，细胞间信号的错误调节会破坏内耳的形态发生，导致听力和平衡障碍。内耳膜迷路的正常形态形成需要区域和细胞命运规范与细胞行为变化的时间整合。过去的工作已经开始确定耳囊肿基因表达下游信号的变化，包括BMP， FGF和SHH，但对驱动形态发生的细胞行为以及这些行为如何与细胞命运的进行性限制协调以产生成熟的前庭和耳蜗室知之甚少。利用鸡和小鼠胚胎的时间和空间控制的功能丧失和获得，我们提出验证BMP/TGFss， FGF和HH信号整合调节耳囊肿区域和细胞命运规范和细胞行为，以启动发育中的膜迷路前庭和耳蜗室的正常形态发生的一般假设。我们的初步数据为我们的方法提供了原理证明。在对照鸡胚胎中，我们发现背外侧耳囊肿上皮发生柱状到鳞状细胞形状的变化，并伴随变薄和扩张形成原始管袋。时空控制的功能丧失和功能获得实验表明，BMP/SMAD信号对于这种细胞形状变化既是必要的，也是充分的。此外，类似的小鸡错误表达实验揭示了在早期耳囊肿背腹侧模式和形态发生过程中调节BMP/SHH信号的共同交叉点。在小鼠实验中，我们发现耳囊来源的FGF3和FGF10信号，除了它们在前庭形态发生中众所周知的作用外，也是启动耳蜗形态发生所必需的。然而，这些上皮信号并不需要早期耳囊肿区域模式，这在缺乏fgf的耳囊肿中是正常的。因此，我们提出验证以下假设：1)BMP/TGFss、FGF和HH信号的时间整合控制了耳囊肿形态发生的三个关键早期步骤：原始耳道生长、原基向垂直和外侧耳道囊的细分以及初始耳蜗生长；2)这些信号通过相关细胞行为的改变协调耳囊肿区域和/或细胞命运的规范。我们的建议利用了一组成熟的研究人员的独特专业知识，利用最先进的分子遗传学和胚胎学技术，在两种具有互补优势的动物模型中阐明了驱动生长因子信号下游关键形态发生步骤的机制的重要差异和相似性。这将通过提供信号通路如何整合以控制前庭和耳蜗形态发生的启动，以及这些信号如何协调细胞命运的规范和细胞行为的变化来启动和塑造正常功能的膜迷宫，从而推动该领域的发展。这些信息为理解和最终预防人类听力损失提供了重要的基础。

DESCRIPTION (provided by applicant): Misregulation of intercellular signaling disrupts inner ear morphogenesis in human subjects and animal models, leading to hearing and balance disorders. Normal morphogenesis of the inner ear's membranous labyrinth requires temporal integration of regional and cell fate specification with changes in cell behavior. Past work has begun to identify changes in otocyst gene expression downstream of signals, including BMP, FGF and SHH, but much less is known about the cell behaviors driving morphogenesis and how these behaviors are coordinated temporally with progressive restriction of cell fates to generate the mature vestibular and cochlear compartments. Using temporally and spatially controlled loss- and gain-of-function in chick and mouse embryos, we propose to test the general hypothesis that BMP/TGFss, FGF and HH signaling are integrated to regulate otocyst regional and cell fate specification and cell behavior to initiate normal morphogenesis of the vestibular and cochlear compartments of the developing membranous labyrinth. Our preliminary data provide proof-of-principle for our approach. In control chick embryos, we identified a columnar-to-squamous cell shape change in the dorsolateral otocyst epithelium that occurs concomitant with thinning and expansion to form the primordial canal pouch. Spatiotemporally controlled loss- and gain-of-function experiments showed that BMP/SMAD signaling is both necessary and sufficient for this cell shape change. In addition, similar chick misexpression experiments revealed a common intersection point regulating BMP/SHH signaling during early otocyst dorsoventral patterning and morphogenesis. In mouse, we found that otocyst-derived FGF3 and FGF10 signals, in addition to their well-known roles in vestibular morphogenesis, are required to initiate cochlear morphogenesis. However, these epithelial signals are not required for early otocyst regional patterning, which is normal in FGF-deficient otocysts. Therefore, we propose to test the specific hypotheses that 1) temporal integration of BMP/TGFss, FGF and HH signaling controls three key early steps of otocyst morphogenesis: primordial canal outgrowth, subdivision of the primordium into vertical and lateral canal pouches and initial cochlear outgrowth, and 2) that such signaling coordinates otocyst regional and/or cell fate specification with changes in relevant cell behaviors. Our proposal takes advantage of the unique expertise of a team of established investigators using state-of-the-art molecular genetic and embryologic techniques in two animal models with complementary strengths that will illuminate important differences and similarities in mechanisms driving key morphogenetic steps downstream of growth factor signaling. This will advance the field by providing a novel understanding of how signaling pathways are integrated to control initiation of vestibular and cochlear morphogenesis, and how these signals coordinate specification of cell fate and changes in cell behavior to initiate and sculpt a normally functioning membranous labyrinth. Such information provides an essential foundation for understanding and ultimately preventing human hearing loss.