Genetic Analysis of Inner Ear Development in Xenopus tropicalis

描述(由申请人提供)：听力或平衡障碍是常见的，由发育和环境原因引起。为了了解正常的内耳发育，我们将使用遗传易驯化的热带爪蛙来研究耳朵发育的遗传网络。内耳的结构和发育在脊椎动物中表现出保守的特征，而四足动物的耳朵的发育与哺乳动物非常相似。在光学清晰的蝌蚪上可以观察到耳朵发育的相关阶段，这使得这种动物成为耳朵突变基因筛查的理想动物。此外，耳朵发育中的缺陷会导致异常的游泳行为和翻正反应的丧失，因此在解剖学上可能不明显的缺陷也可能被评分。作为对PA-06-365，“听力和平衡中的细胞谱系和发育研究”的回应，我们已经解决了“更全面地代表模型生物系统”来研究耳朵发育的声明需求。我们建议通过研究热带非洲爪哇的耳囊发育来推进我们对耳朵发育的理解，这是一种两栖动物，其内耳发育与哺乳动物一样保存得很好。在利用非洲爪哇进行的小规模正向遗传筛查中，我们恢复了扰乱耳朵形态、耳石形成和平衡/游泳行为的突变。我们已经在其中两个影响耳锥发育和耳囊大小的基因中分离出受影响的基因。因此，我们已经证明了筛选和恢复突变、分析表型以及定位和克隆受影响的基因是可能的。热带管圆线虫的基因组与哺乳动物基因组具有相当大的结构相似性和同步性，没有发现额外的全基因组重复的迹象，因此我们有信心在各种与哺乳动物具有保守功能的基因中发现隐性突变。随着基因组组装和注释的最新改进，以及外显子捕获和高通量序列分析的技术进步，我们相信可以快速定位其他突变，并分离受影响的基因。在下一个授权期，我们建议通过位置克隆来鉴定更多的突变等位基因，并将突变表型与潜在的分子和细胞缺陷联系起来。我们将分子方法与经典的胚胎移植相结合，以了解突变体的细胞自主性，以及相互作用以产生功能正常的耳朵的信号和反应组织。

DESCRIPTION (provided by applicant): Deficits in hearing or balance are common, and result from both developmental and environmental causes. In order to understand normal inner ear development, we will use the genetically tractable frog, Xenopus tropicalis, to investigate the genetic network underlying ear development. The structure and development of the inner ear shows conserved features among the vertebrates, and the development of the ear of the tetrapod is very similar to that of mammals. The relevant stages of ear development can be observed in the optically clear tadpole, making this animal ideal for genetic screens for ear mutants. Furthermore, defects in ear development lead to abnormal swimming behavior and loss of the righting response, so that defects that may not be anatomically obvious can also be scored. In response to PA-06-365, "Cell Lineage and Developmental Studies in Hearing and Balance" we have addressed the stated need for "more comprehensive representation of model organisms systems" to study ear development. We propose to advance our understanding of ear development by examining otic vesicle development in the tetrapod Xenopus tropicalis, an amphibian whose inner ear development is well-conserved with mammals. In a small-scale forward genetic screen using Xenopus tropicalis we have recovered mutants that disrupt ear morphology, otolith formation, and balancing/swimming behavior. We have isolated the affected gene in two of these that affect otoconial development and otocyst size. Thus we have shown that it is possible to screen for and recover mutants, analyze the phenotype, and map and clone the affected genes. The genome of X. tropicalis shows considerable structural similarity and synteny with mammalian genomes, with no sign of additional whole genome duplications, so we are confident that we can identify recessive mutants in a variety of genes which show conserved functions with the mammals. With recent improvements in the genome assembly and annotation, and technical advances in exon capture and high throughput sequence analysis, we are confident that other mutations can be rapidly mapped, and the affected genes isolated. In the next grant period, we propose to characterize additional mutant alleles by positional cloning, and link mutant phenotypes with the underlying molecular and cellular defects. We combine molecular approaches with classical embryological transplantations to understand the cel autonomy of mutants, and the signaling and responding tissues that interact to produce the functioning ear.