Genomic architecture of Shh dependent cochlear morphogenesis

描述（申请人提供）：小鼠耳蜗来源于耳囊的腹侧延伸。在胚胎发育的几天过程中，这种生长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（Groves和Fekete， 2012）。先天性耳蜗畸形常常导致耳聋，这就强调了彻底了解耳蜗发育的重要性（Jackler et al., 1987）。我们之前描述了Sonic hedgehog （Shh）信号通路在耳囊内促进腹侧身份的关键功能，这对于耳蜗管的后续生长是必要的（Riccomagno et al., 2002; Bok et al., 2007b; Brown and Epstein, 2011）。小鼠胚胎缺乏Shh，或携带一个重要的Shh信号转导成分Smoothened （Smoecko）的耳条件敲除，表现出耳蜗发育。我们还分类了几种在耳蜗发育中起关键作用的转录因子，作为耳腹上皮内Shh信号传导的转录靶点（Pax2, Otx2, Gata3）或效应器（Gli2, Gli3）。然而，尽管取得了这些进展，对Shh依赖性转录因子促进耳蜗管生长的机制的详细了解仍不清楚，主要是因为在这一转录级联反应的下游作用的基因尚未确定。为了确定Shh信号的新靶点，我们比较了对照组和Smoecko内耳在E11.5时的全基因组表达谱，当耳蜗样本明显时，发现了一组有趣的Shh应答基因，这些基因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，这些基因中的一些在耳蜗管前感觉区域的后期发育阶段保持其表达，这提高了Shh信号在其发育的后续步骤中启动假定的感觉上皮的可能性。我们建议根据以下实验计划来描述腹侧耳基因集：分类它们的时空表达模式和对Shh信号的依赖性（Aim 1）；解码它们的顺式监管架构（目标2）；并评估它们对耳蜗发育的功能贡献（目标3）。这些实验的结果将促进我们对内耳内耳蜗形态发生和细胞命运规范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.