CONDITIONAL MUTAGENESIS DURING OTIC VESICLE ONTOGENY

我们建议使用cre/loxP重组系统在小鼠耳泡发育过程中诱导骨形态发生蛋白受体-1A(Bmpr)的组织特异性突变。前人的工作已经证明，Hmx3基因在耳泡发育的早期就有表达。我们建议利用Hmx3基因的转录调节区在内耳个体发育过程中在体内诱导Bmpr基因的条件突变。这项提案中开发的方法学将普遍适用于任何被假设为调节耳囊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来调节内耳的发育或功能。传统上对广泛表达的信号分子的基因敲除，如Bmpr，通常会导致早期胚胎死亡。在这项建议中开发的耳泡特异性Hmx3/cre“突变子”系谱将使在八囊泡中诱导任何感兴趣的基因的条件性突变成为可能。因此，整个研究内耳发育和功能的科学家都可以利用这个谱系来突变任何感兴趣的基因。这项建议的具体目的是：在Bmpr基因中诱导耳部小泡特异性突变，以检测其在感觉上皮形成中的作用。根据杂交组织化学分析，推测BMPs在内耳感觉上皮细胞的诱导中起关键作用。我们建议对BMPs最广泛表达的I型受体Bmpr介导的感官上皮形成诱导信号的假说进行遗传学检验。Bmpr等位基因的条件性突变将通过产生一个小鼠家系来完成，该家系将cre重组酶的表达引导到耳泡。这个家系将通过将cre重组酶基因导入内耳特定的同源框基因Hmx3的位置而产生。

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.