Avian Hair Cell Regeneration: Molecular Regulation

描述(申请人提供)：鸟类是唯一已知的温血脊椎动物，在成熟时能再生内耳的感觉毛细胞(HC)。最近的研究表明，鸟类利用两种机制来产生新的HCs：祖细胞的重新分裂，非感觉支持细胞(SCs)的重新分裂，以及SCs直接转分化为HC，而不干预有丝分裂。禽类HC再生的研究为阐明SC对HC丢失的反应提供了重要的细胞和分子机制。这些研究是指导对哺乳动物HC上皮中SC行为的分析的关键前奏，以确定为什么哺乳动物的HC再生没有完成。我们将重点介绍两个分子Atoh1和Notch的作用，这两个分子对哺乳动物HC的发育至关重要。目标1将通过测试Notch激活拮抗转分化而Atoh1激活促进转分化的假设，阐述调控SC决定在HC损伤后直接转分化的信号。我们将检查受损的BP，以确定Notch途径中基因的时空表达模式是否支持Notch的角色。我们将激活或抑制Notch及其配体和效应器，并确定直接转分化是否会因此而改变。我们还将检验Atoh1功能对于直接转分化是必要和充分的假设。在目标2中，我们将通过检验Atoh1和Notch都抑制SC分裂的假设来解决调控SC决定在HC损伤后在禽类BP中分裂的信号。我们将确定Atoh1的过度表达是否会导致SC分裂的细胞自主抑制，以及Atoh1的抑制是否会促进分裂。我们将确定Notch途径中的哪些基因在SCs分裂及其周围细胞中发生了变化。我们将确定Notch功能的操作是否显著改变SC分裂的比率。在目标3中，我们将评估在HC损伤后成年小鼠椭圆形干细胞中Atoh1表达和直接转分化自发发生的程度。我们将研究在HC丢失后，成年小鼠卵圆囊中Notch途径中的基因表达是如何改变的。我们还将测试以下假设：抑制成年小鼠胞囊中的Notch信号可促进直接转分化和SC分裂。

DESCRIPTION (provided by applicant): Birds are the only warm-blooded vertebrates known to regenerate sensory hair cells (HCs) of the inner ear in maturity. Recent studies have demonstrated that birds utilize two mechanisms to generate new HCs: renewed cell division of progenitor cells, the non-sensory supporting cells (SCs), and direct transdifferentiation of SCs into HCs, without intervening mitosis. Studies of avian HC regeneration offer an important opportunity to elucidate cellular and molecular mechanisms governing these SC responses to HC loss. Such studies are critical preludes for directing analyses of SC behavior in mammalian HC-epithelia toward determining why HC regeneration is not completed in mammals. We will focus on the role of two molecules, Atoh1 and Notch, which are critical for mammalian HC development. Aim 1 will address signals regulating the SC decision to directly transdifferentiate after HC damage in mature chicken BPs by testing the hypotheses that Notch activation antagonizes transdifferentiation while Atoh1 activation promotes it. We will examine damaged BPs to determine if temporospatial expression patterns of genes in the Notch pathway support the proposed role for Notch. We will activate or inhibit Notch, its ligands, and its effectors and determine if direct transdifferentiation becomes altered in response. We will also test the hypothesis that Atoh1 function is necessary and sufficient for direct transdifferentiation. In Aim 2, we will address signals regulating the SC decision to divide after HC damage in avian BPs by testing the hypothesis that Atoh1 and Notch both inhibit SC division. We will determine if overexpression of Atoh1 leads to cell-autonomous inhibition of SC division and if inhibition of Atoh1 promotes division. We will determine which genes in the Notch pathway are altered in dividing SCs and in cells surrounding them. We will determine if manipulation of Notch function significantly alters rates of SC division. In Aim 3, we will assess the extent to which Atoh1 expression and direct transdifferentiation occur spontaneously in SCs of the adult mouse utricle after HC damage. We will examine how expression of genes in the Notch pathway is altered in adult mouse utricles after HC loss. We will also test the hypotheses that inhibition of Notch signaling in adult mouse utricles promotes direct transdifferentiation and SC division.