MOLECULAR ANALYSES OF TEMPORAL BONE DEVELOPMENT

描述（由申请人提供）：该提案的主要目标是了解骨形态发生蛋白（BMP）信号传导在内耳发育过程中的作用。这一信号通路对骨性和膜性脊椎动物的个体发育至关重要。然而，BMP信号传导在颞骨软骨形成、半规管和前庭感觉上皮形成中的具体作用知之甚少。因此，为了更好地表征这一途径在内耳个体发育过程中的作用，我们开发了几种分子遗传学工具，用于分析内耳发育过程中BMP信号传导。我们将使用Cre介导的I型BMP受体基因失活来研究内耳胚胎发生过程中的BMP信号传导;采用Brn 4基因的转录调控元件来指导转基因小鼠耳间充质的基因表达;并利用介导Cre诱导的耳间充质基因重排的小鼠谱系来解决以下特定目标： 目标1。为了表征内耳发育过程中磷酸化SMAD的表达，我们建议进行磷酸化SMAD定位的泛免疫组化分析，以确定在内耳发育过程中活性BMP信号传导的区域。 目标2。为了从遗传学上检测BMP信号在颞骨形成中的作用，我们将检测BMP I型受体基因Bmprla和Bmprlb在耳间质中的功能。这些突变体的功能分析将通过记录听觉脑反应，耳蜗内电位和中耳传递函数与激光干涉仪进行评估。 目标3。为了从遗传学上测试BMP I型受体基因在感觉性和非感觉性上皮异常个体发育过程中的作用，我们将敲除耳泡中的这些受体。分析突变表型阐明内耳发育的基本分子机制。

DESCRIPTION (provided by applicant): The broad goal of this proposal is to understand the roles of Bone Morphogenetic Protein (BMP) signaling during inner ear development. This signaling pathway is crucial to the ontogeny of the bony and membranous labyrinths. However, the specific roles that BMP signaling plays in the chondrogenesis of the temporal bone, and in the formation of the semicircular canals and vestibular sensory epithelia are poorly understood. Thus, to better characterize the role of this pathway during inner ear ontogeny, we have developed several molecular genetic tools for analysis of BMP signaling during inner ear development. We will use Cre-mediated inactivation of type I BMP receptor genes to study BMP signaling during inner ear embryo genesis; employ transcriptional regulatory elements of the Brn4 gene to direct the expression of genes to the otic mesenchyme of transgenic mice; and exploit a pedigree of mice that mediates Cre-induced gene rearrangements in the otic mesenchyme to address the following specific aims: Aim #1. To characterize phospho-SMAD expression during inner ear development, we propose to undertake pan immunohistochemical analysis of phospho-SMAD localization to identify regions of active BMP signaling during inner ear development. Aim #2. To genetically test the role of BMP signaling during temporal bone formation, we will inactivate thepfunction of both BMP type I receptor genes, Bmprla and Bmprlb, in the otic mesenchyme. Functional analyses of these mutants will be assessed by recording auditory brain responses, endocochlear potential and middle ear transfer function with laser interferometer. Aim #3. To genetically test the role of BMP type I receptor genes during the ontogeny of sensory and nonsensorypepithelia, we will knockout these receptors in the otic vesicle. Analysis of mutant phenotypesporomises to elucidate fundamental molecular mechanisms governing inner ear development.