CONDITIONAL MUTAGENESIS DURING OTIC VESICLE ONTOGENY

我们建议采用cre/loxP重组系统诱导骨形态发生蛋白受体-1A（Bmpr）在小鼠耳泡发育过程中的组织特异性突变。其他人先前的工作已经证明Hmx 3基因在耳囊发育的早期表达。 我们建议利用Hmx 3基因的转录调控区在内耳个体发育过程中体内诱导Bmpr基因的条件突变。 在这个建议中开发的方法将普遍适用于任何假设调节耳泡发育或功能的基因的分析。 毫无疑问，许多在其他器官系统和早期胚胎发育中发挥重要作用的基因将被用于调节内耳发育或功能。 传统的基因敲除广泛表达的信号分子，如Bmpr，往往导致早期胚胎死亡。 在该建议中开发的耳囊特异性Hmx 3/cre“增变因子”谱系将使得在任何感兴趣的基因中诱导条件突变成为可能，特别是在耳囊中。因此，这个谱系可以被整个研究内耳发育和功能的科学家群体用来诱变任何感兴趣的基因。 该建议的具体目的是：诱导Bmpr基因的耳囊特异性突变，以检查其在感觉上皮形成过程中的作用。 基于杂交组织化学分析，已经假设BMPs在内耳感觉上皮的诱导中起关键作用。 我们建议从遗传学上检验这一假设，即感觉上皮形成的诱导信号是由Bmpr介导的，Bmpr是BMPs最广泛表达的I型受体。 floxed Bmpr等位基因的条件突变将通过产生将cre重组酶表达引导至耳囊的小鼠谱系来完成。 该谱系将通过将cre重组酶基因引入内耳特异性同源框基因Hmx 3的基因座中来产生。

We propose to employ the cre/loxP recombination system to induce a tissue-specific mutation in the Bone Morphogenetic Protein Receptor-1A (Bmpr) during mouse otic vesicle development. Previous work by others has demonstrated that the Hmx3 gene is expressed early in the development of the otic vesicle. We propose to harness the transcriptional regulatory regions of the Hmx3 gene to induce conditional mutations in the Bmpr gene in vivo during inner ear ontogeny. The methodology developed in this proposal will be generally applicable to the analysis of any gene hypothesized to regulate otic vesicle development or function. Undoubtedly, many genes that play important roles in other organ systems and in early embryogenesis will be utilized to regulate inner ear development or function. The traditional knockout of genes for widely-expressed signaling molecules, such as the Bmpr, often leads to early embryonic death. The otic vesicle-specific Hmx3/cre "mutator" pedigree developed in this proposal will make it feasible to induce conditional mutations in any gene of interest specifically in the octic vesicle. Therefore, this pedigree can be utilized by the whole community of scientists studying inner ear development and function to mutagenize in any gene of interest. The specific aim of this proposal is: To induce an otic vesicle-specific mutation in the Bmpr gene to examine its role during sensory epithelial formation. Based on hybridization histochemical analyses, it has been hyposthesized that BMPs play a critical role in the induction of sensory epithelial of the inner ear. We propose to genetically test the hypothesis that the inductive signal for sensory epithelial formation is mediated by Bmpr, the most widely expressed type I receptor for BMPs. Conditional mutation of the floxed Bmpr allele will be accomplished by generating a pedigree of mice that direct expression of cre recombinase to the otic vesicle. This pedigree will be generated by introducing the cre recombinase gene into the locus of an inner-ear specific homeobox gene, Hmx3.