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

描述（由申请人提供）：细胞间信号传导的失调会扰乱人类受试者和动物模型的内耳形态发生，导致听力和平衡障碍。内耳膜迷路的正常形态发生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识别信号下游耳囊基因表达的变化，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逐步限制在时间上协调以产生成熟的前庭和耳蜗室的了解甚少。利用鸡和小鼠胚胎中时间和空间控制的功能丧失和获得，我们建议测试以下一般假设：BMP/TGFs、FGF和HH信号传导被整合以调节耳囊区域和细胞命运规范和细胞行为，以启动发育中的膜迷路的前庭和耳蜗室的正常形态发生。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上皮发生柱状细胞到鳞状细胞形状的变化，这种变化伴随着变薄和扩张而形成原始管袋。时空控制的功能丧失和功能获得实验表明，BMP/SMAD 信号传导对于这种细胞形状变化既是必要的也是充分的。此外，类似的雏鸡错误表达实验揭示了早期耳囊背腹模式和形态发生过程中调节 BMP/SHH 信号传导的共同交叉点。在小鼠中，我们发现耳囊衍生的 FGF3 和 FGF10 信号除了在前庭形态发生中发挥众所周知的作用外，也是启动耳蜗形态发生所必需的。然而，这些上皮信号并不是早期耳囊区域模式所必需的，这在 FGF 缺陷的耳囊中是正常的。因此，我们建议测试以下具体假设：1) BMP/TGFss、FGF 和 HH 信号传导的时间整合控制耳囊形态发生的三个关键早期步骤：原基管生长、原基细分为垂直和侧向管囊以及初始耳蜗生长，2) 这种信号传导协调耳囊区域和/或细胞命运规范与相关细胞行为的变化。我们的提案利用了一组成熟研究人员的独特专业知识，在两种具有互补优势的动物模型中使用最先进的分子遗传学和胚胎学技术，这将阐明驱动生长因子信号传导下游关键形态发生步骤的机制的重要差异和相似之处。这将通过提供对如何整合信号通路来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以启动和塑造正常功能的膜迷路的新理解来推进该领域的发展。此类信息为理解并最终预防人类听力损失提供了重要基础。 公共卫生相关性：听力损失是最常见的人类感觉缺陷，可能是由于发育过程中生长因子信号传导水平发生变化的突变引起的。我们使用小鼠和鸡胚胎作为实验模型来研究生长因子信号在内耳形成​​过程中的作用。我们的研究预计将贡献可能有助于开发预防、诊断和治疗听力损失的新方法的知识。

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. PUBLIC HEALTH RELEVANCE: Hearing loss is the most common human sensory deficit and can be caused by mutations that change the levels of growth factor signaling during development. We are using mice and chick embryos as experimental models to study the roles of growth factor signaling during inner ear formation. Our studies are expected to contribute knowledge that will likely be useful for developing new approaches for preventing, diagnosing, and treating hearing loss.