Genomic architecture of Shh dependent cochlear morphogenesis

描述（由申请人提供）：小鼠耳蜗源自耳泡的腹侧延伸。在胚胎发育的几天内，这种生长物经历了一系列复杂的形态发生变化，导致其延长、卷曲和分化为听觉所必需的感觉和非感觉细胞类型（格罗夫斯和Fekete，2012）。耳蜗的先天性畸形经常导致耳聋，强调了彻底了解其发育的重要性（Jackler等人，1987年）。我们先前描述了Sonic hedgehog（Shh）信号传导途径在促进耳泡内的腹侧身份（ventral identity）中的关键功能，这是耳蜗管随后生长所必需的（Riccomagno等人，2002; Bok等人，2007 b; Brown and Epstein，2011）。缺乏Shh或携带Smoothened（Smoecko）（一种重要的Shh信号转导成分）耳朵条件性敲除的小鼠胚胎表现出耳蜗发育不全。我们还将在耳蜗发育中具有关键作用的几种转录因子分类为腹侧耳上皮内Shh信号传导的转录靶（Pax 2、Otx 2、Gata 3）或效应子（Gli 2、Gli 3）。然而，尽管有这些进展，Shh依赖性转录因子促进耳蜗管生长的机制的详细了解仍然不清楚，主要是因为在这个转录级联下游作用的基因尚未确定。为了识别Shh信号的新靶点，我们比较了E11.5时对照组和Smoecko内耳的全基因组表达谱，此时耳蜗原基很明显，并发现了一组有趣的Shh响应基因，它们在耳蜗形态发生中具有已知和以前未表征的作用。有趣的是，这些基因中的几个在耳蜗导管的前感觉域内的发育后期保持其表达，这提高了Shh信号传导引发推定感觉上皮在其发育的后续步骤的可能性。我们建议根据以下实验计划来表征腹侧耳基因集：分类它们的时空表达模式和对Shh信号的依赖性（目的1）;解码它们的顺式调控结构（目的2）;并评估它们对耳蜗发育的功能贡献（目的3）。这些实验的结果应该推进我们的耳蜗形态发生和细胞命运规范内内耳的分子和细胞机制的基本理解。

DESCRIPTION (provided by applicant): The mouse cochlea derives from the ventral extension of the otic vesicle. Over the course of several days during embryonic development, this outgrowth undergoes a complex sequence of morphogenetic changes resulting in its lengthening, coiling and differential patterning into sensory and nonsensory cell types that are essential for hearing (Groves and Fekete, 2012). Congenital malformations of the cochlea often lead to deafness, emphasizing the importance of a thorough understanding of its development (Jackler et al., 1987). We previously described a critical function of the Sonic hedgehog (Shh) signaling pathway in promoting ventral identity within the otic vesicle that is necessary for the subsequent outgrowth of the cochlear duct (Riccomagno et al., 2002; Bok et al., 2007b; Brown and Epstein, 2011). Mouse embryos lacking Shh, or carrying an ear conditional knockout of Smoothened (Smoecko), an essential Shh signal transduction component, exhibit cochlear agenesis. We also classified several transcription factors with key roles in cochlear development as either transcriptional targets (Pax2, Otx2, Gata3) or effectors (Gli2, Gli3) of Shh signaling within the ventral otic epithelium. However, despite these advances, a detailed understanding of the mechanism by which Shh dependent transcription factors promote cochlear duct outgrowth remains unclear, primarily since the genes acting downstream in this transcriptional cascade have yet to be determined. To identify novel targets of Shh signaling we compared the genome-wide expression profiles of control and Smoecko inner ears at E11.5, when the cochlea anlage is evident, and uncovered an intriguing set of Shh responsive genes with a combination of known and previously uncharacterized roles in cochlear morphogenesis. Interestingly, several of these genes maintain their expression at later stages of development within the prosensory domain of the cochlear duct, raising the possibility that Shh signaling is priming the presumptive sensory epithelium for subsequent steps in its development. We propose to characterize the ventral otic gene set according to the following experimental plan: classify their spatiotemporal patterns of expression and dependency on Shh signaling (Aim 1); decode their cis-regulatory architecture (Aim 2); and assess their functional contribution to cochlear development (Aim 3). The results of these experiments should advance our fundamental understanding of the molecular and cellular mechanisms underlying cochlear morphogenesis and cell fate specification within the inner ear.