Determination of Hair Cell Fate from Postnatal Cochlear Supporting Cells

摘要 成年哺乳动物中存在一种未知机制阻止毛细胞再生的现象，但我们发现 最近的研究表明，耳蜗毛细胞在出生后的最初几个阶段具有自发再生的能力 几天。我们最近发现Wnt信号可以刺激祖细胞产生毛细胞 在新生的耳蜗里，我们假设Wnt途径的下游靶子变得更少 随着耳蜗龄的增长而可接近。我们的初步数据显示，组蛋白脱乙酰酶抑制剂 保护组蛋白上的乙酰基，促进新生耳蜗支持细胞的增殖和 促进新生儿的毛细胞分化，对成人内耳也有促进作用。我们试图同时学习这两种知识 染色质改变的本质以及表观遗传药物和CRISPR dCas9逆转它们的可能性 介导性表观遗传修饰。 尽管我们最近的工作为成人毛细胞置换提供了重要的原理证明， 再生是有限的。在这里，我们评估了两种表观遗传修饰物对抑制的反应 转录因子Atoh1及其受体Atoh1和Atoh1的表达调控 下游目标。我们评估了3种表观遗传修饰物的效果，我们认为这3种修饰物是增强- 基于对HC分化所需基因的激活。在目标1中，我们评估了这些修饰物的效果， TCF4，Setd7和LSD1，关于耳蜗器中的表观遗传标记和染色质可及性，我们要求 控制它们的表达水平是否能逆转染色质不可及和增加 毛细胞的分化。在目标2中，我们评估了LSD1和HDAC抑制介导的染色质修饰 对毛细胞Atoh1活化和分化的影响。在目标3中，我们测试了我们的假设 通过这些染色质修饰物操纵表观遗传变化将促进毛细胞在 受损的耳蜗骨。我们测试了表观遗传修饰物和抑制物对毛细胞再生的影响。 一种小鼠耳聋的噪声损伤模型。通过这些实验，我们提出了一系列至关重要的问题 论毛细胞再生和功能恢复的表观遗传机制。

Summary Blocks to regeneration of hair cells by an unknown mechanism exist in the adult mammal, but we have recently shown that cochlear hair cells have a capacity for spontaneous regeneration in the first few postnatal days. We have recently discovered that Wnt signaling stimulates generation of hair cells from progenitor cells in the newborn cochlea, and we hypothesize that downstream targets of the Wnt pathway become less accessible with age of the cochlea. Our preliminary data show that histone deacetylase inhibitors, which preserve acetyl groups on histones, increase the proliferation of newborn cochlear supporting cells and promote hair cell differentiation in the newborn and to a lesser extent the adult inner ear. We seek to learn both the nature of chromatin changes and the potential to reverse them with epigenetic drugs and CRISPR dCas9 mediated epigenetic modifications. Although our recent work has provided an important proof-of-principle for hair cell replacement in the adult, regeneration was limited. Here, we assess the response to inhibition of epigenetic modifiers of both the newborn and adult cochlea with a focus on the control of expression of transcription factor Atoh1 and its downstream targets. We assess the effects of 3 epigenetic modifiers that we propose to be key to enhancer- based activation of genes required for HC differentiation. In Aim 1, we assess the effects of these modifiers, Tcf4, Setd7 and Lsd1, on epigenetic marks and chromatin accessibility in cochlear organoids and we ask whether manipulation of their level of expression can reverse chromatin inaccessibility and increase differentiation of hair cells. In Aim 2 we assess chromatin modification mediated by Lsd1 and HDAC inhibition for effects on Atoh1 activation and differentiation of hair cells. In Aim 3, we test our hypothesis that manipulation of epigenetic changes through these modifiers of chromatin will increase hair cell differentiation in the damaged cochlea. We test the epigenetic modifiers and inhibitors for their effect on hair cell regeneration in a noise damage model of mouse deafness. Through these experiments, we ask a crucial series of questions on epigenetic mechanisms in hair cell regeneration and recovery of function.