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。其他人之前的研究表明，Hmx3基因在耳小泡发育的早期表达。我们建议利用Hmx3基因的转录调控区域在内耳个体发育过程中诱导Bmpr基因的条件突变。本提案中开发的方法将普遍适用于任何假设调节耳囊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生中发挥重要作用的基因将被用来调节内耳的发育或功能。传统的敲除广泛表达的信号分子基因，如Bmpr，通常会导致早期胚胎死亡。本研究中开发的耳膜囊泡特异性Hmx3/cre“突变”谱系将使在任何相关基因中诱导条件突变成为可能，特别是在耳膜囊泡中。因此，这个谱系可以被整个研究内耳发育和功能的科学家社区用来诱变任何感兴趣的基因。该建议的具体目的是：诱导Bmpr基因的耳泡特异性突变，以研究其在感觉上皮形成过程中的作用。基于杂交组织化学分析，假设bmp在内耳感觉上皮的诱导中起关键作用。我们建议对感觉上皮形成的诱导信号是由bmp最广泛表达的I型受体Bmpr介导的这一假设进行基因测试。有条件的Bmpr等位基因突变将通过产生直接表达cre重组酶到耳泡的小鼠系谱来完成。通过将cre重组酶基因引入内耳特异同源盒基因Hmx3的位点，将产生该家系。

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.