Determination of Hair Cell Fate from Postnatal Cochlear Supporting Cells

总结 在成年哺乳动物中，毛细胞的再生受到一种未知机制的阻碍， 最近的研究表明耳蜗毛细胞在出生后的最初几年内具有自发再生的能力， 天我们最近发现Wnt信号刺激毛细胞从祖细胞生成 在新生儿耳蜗中，我们假设Wnt通路的下游靶点变得更少， 随着耳蜗年龄的增长，我们的初步数据显示，组蛋白去乙酰化酶抑制剂， 保护组蛋白上的乙酰基，增加新生耳蜗支持细胞的增殖， 促进新生儿毛细胞分化，并在较小程度上促进成人内耳毛细胞分化。我们寻求学习两者 染色质变化的性质以及用表观遗传药物和CRISPR dCas 9逆转它们的潜力 介导的表观遗传修饰。 虽然我们最近的工作为成人毛细胞替代提供了重要的原理证明， 再生是有限的。在这里，我们评估了对两种表观遗传修饰剂抑制的反应， 新生儿和成人耳蜗，重点是转录因子Atoh 1的表达控制及其 下游目标我们评估了3种表观遗传修饰剂的作用，我们认为它们是增强子的关键- 基于HC分化所需基因的激活。在目标1中，我们评估了这些修饰符的效果， Tcf 4，Setd 7和Lsd 1，关于耳蜗类器官的表观遗传标记和染色质可及性，我们问 操纵它们的表达水平是否可以逆转染色质不可及性， 毛细胞的分化。在目标2中，我们评估了Lsd 1和HDAC抑制介导的染色质修饰 对Atoh 1激活和毛细胞分化的影响。在目标3中，我们检验了我们的假设， 通过这些染色质修饰剂操纵表观遗传变化将增加毛细胞分化， 受损的耳蜗我们测试了表观遗传修饰剂和抑制剂对毛细胞再生的影响， 噪声损伤小鼠耳聋模型。通过这些实验，我们提出了一系列至关重要的问题， 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.