MOLECULAR ANALYSES OF TEMPORAL BONE DEVELOPMENT

这项建议的主要目标是研究POU结构域基因-脑4(Brn4)在内耳发育过程中的功能。人类Brn4基因的同源突变(POU3F4)会导致“在砧骨手术中与外淋巴喷涌相关的混合性传导性和感觉神经性耳聋”(DFN3；McKusick目录号304400)。在这些患者中都会出现距骨和颞骨的畸形。最近，我们的实验室已经证明，在一个含有Brn4基因靶向“敲除”突变的小鼠家系的内耳中也出现了类似的畸形。对这种动物模型中发育异常的个体发生的分析将提供一种手段来表征导致突变表型的机制，以及洞察颞骨的正常发育。这项建议的具体目的是：1.通过Brn4/Pou3f4基因的靶向突变，全面描述小鼠发育异常的个体发育。2.研究Brn4基因敲除小鼠发生发育缺陷的细胞机制。我们建议测试两种可能的机制来解释突变体中观察到的颞骨质量下降：假设1)Brn4抑制耳囊形成过程中的细胞凋亡，或假设2)Brn4调节耳间充质细胞的增殖。3.为了充分描述Brn4突变等位基因的分子基础，性连锁坐立不安。4.鉴定Brn4基因中的生物特异性转录调控元件。5.对Brn4缺失家系的听觉功能进行功能分析。为了解决Brn4缺失突变导致听力损失的病理机制，我们建议更详细地评估Brn4缺失家系的听觉功能。

The broad goal of this proposal is to study the function of the POU-domain gene, Brain-4 (Brn4), during inner ear development. Mutations in the human ortholog of the Brn4 gene (POU3F4) produce "mixed conductive and sensorineural deafness associated with perilymphatic gusher during stapes surgery" (DFN3; McKusick catalog number 304400). Malformations of both the stapes and the temporal bone occur in these patients. Recently, our laboratory has demonstrated that similar malformations occur in the inner ear of a mouse pedigree containing a targeted "knockout" mutation in the Brn4 gene. Analysis of the ontogeny of developmental abnormalities in this animal model will provide a means to characterize the mechanisms that lead to the mutant phenotype, as well as, insight into the normal development of the temporal bone. The specific aims of this proposal are: 1. To fully characterize the ontogeny of developmental anomalies in mice with a targeted mutation in the Brn4/Pou3f4 gene. 2. To characterize the cellular mechanisms by which developmental defects occur in the Brn4 knockout mice. We propose to test two possible mechanisms that would explain the decreased temporal bone mass observed in the mutant: Hypothesis 1) Brn4 inhibits apoptosis during otic capsule formation, or Hypothesis 2) Brn4 regulates cell proliferation of otic mesenchyme cells. 3. To fully characterize the molecular basis of a Brn4 mutant allele, sex- linked fidget. 4. To characterize otic-specific transcriptional regulatory elements in the Brn4 gene. 5. To undertake functional analyses of auditory function of the Brn4 null pedigree. To address the pathological mechanisms leading to hearing loss of the Brn4 null mutants, we propose to assess auditory function of the Brn4 null pedigree in greater detail.