Wnt Signaling in Hair Cell Generation from Supporting Cells

 描述（由申请人提供）：我们已经表明，Notch信号传导的抑制导致毛细胞再生，并在毛细胞因噪声暴露而受损的成年耳中提供部分功能恢复。我们最近发现，Wnt信号提供了一个必要的刺激，推动胚胎祖细胞和成体干细胞的毛细胞的命运。Wnt信号传导对于暴露于Notch抑制的细胞中Atoh 1的诱导是必需的，如Wnt信号传导被阻断时Atoh 1上调的缺乏所证明的。这表明Wnt信号传导是耳蜗毛细胞分化所必需的， 切口抑制。与Notch抑制类似，由Wnt控制的基因表达刺激毛细胞分化，但它似乎也刺激导致支持细胞替换的基因。这些下游目标之一，Lgr 5，似乎标记耳蜗祖细胞能够出生后转分化成新的毛细胞。我们目前对信号通路的了解，以及我们对其操作所需模型的开发，允许进行一系列重要的实验来测试引起毛细胞再生和功能恢复所需的信号通路性质的假设。 虽然我们最近的工作提供了一个重要的证明原则的毛细胞替代成人，再生是有限的，转分化的支持细胞没有被取代。在这里，我们评估了毛细胞损伤后新生儿和成人耳蜗对Notch抑制的反应。我们假设毛细胞死亡后的Wnt信号导致新生儿耳蜗的部分再生反应，并且通过这些途径的信号没有达到启动成人耳蜗再生所需的水平。在目的1中，我们通过功能获得和丧失研究来测试我们关于新生儿耳蜗中Wnt信号传导的假设，以研究Wnt在响应Notch抑制的再生中的作用。在目标2中，我们测试了成年人受损耳蜗中Notch抑制或β-连环蛋白上调是否伴随着再生特征性的基因表达变化，在成年人受损耳蜗中，我们可以测量毛细胞再生。在目标3中，我们测试了我们的假设，即Wnt的强制激活与Notch抑制相结合将驱动支持细胞增殖和新毛细胞的转分化。我们进一步测试了这样的想法，即我们在损伤和γ-分泌酶抑制后看到的再生只有在Wnt信号传导活跃时才能发生。

 DESCRIPTION (provided by applicant): We have shown that inhibition of Notch signaling leads to regeneration of hair cells and provide partial functional recovery in the adult ear with hair cells damaged by noise-exposure. We have recently discovered that Wnt signaling provides a required stimulus for pushing both embryonic progenitor cells and adult stem cells to a hair cell fate. Wnt signaling is necessary for induction of Atoh1 in cells exposed to Notch inhibition, as demonstrated by an absence of Atoh1 upregulation when Wnt signaling is blocked. This suggests that Wnt signaling is required for hair cell differentiation in the cochlea in response to Notch inhibition. Similar to Notch inhibition, gene expression controlled by Wnt stimulates hair cell dfiferentiation, but it also appears to stimulate genes that lead to the replacement of supporting cells. One of these downstream targets, Lgr5, appears to mark cochlear progenitors capable of postnatal transdifferentiation into new hair cells. Our current knowledge of signaling pathways, and our development of the models needed for their manipulation allow a crucial series of experiments to test hypotheses on the nature of the signaling pathways required to elicit hair cell regeneration and recovery of function. Although our recent work has provided an important proof-of-principle for hair cell replacement in the adult, regeneration was limited and the transdifferentiated supporting cells were not replaced. Here, we assess the response of both the newborn and adult cochlea to inhibition of Notch after hair cell damage. We hypothesize that Wnt signaling after hair cell death leads to a partial regenerative response in the newborn cochlea and that signaling through these pathways does not reach levels necessary to initiate regeneration in the adult cochlea. In Aim 1, we test our hypothesis about Wnt signaling in the newborn cochlea by both gain and loss of function studies for a role of Wnt in regeneration in response to Notch inhibition. In Aim 2 we test whether Notch inhibition or β-catenin upregulation in the damaged cochlea in adults, where we can measure hair cell regeneration, is accompanied by gene expression changes characteristic of regeneration. In Aim 3, we test our hypothesis that forced activation of Wnt in combination with Notch inhibition will drive both supporting cell proliferation and transdifferentiation of new hair cells. We further test the idea that the regeneration we see after damage and inhibition of γ-secretase can only occur if Wnt signaling is active.