Hair Cell Regeneration: Molecular Regulation

听觉毛细胞是内耳中的机械感受器，将声波转化为神经信号。在人类中，毛细胞丢失是不可逆转的，并会导致严重的、永久性的听力障碍。相比之下，成熟的鸟在鸡的听觉上皮(基底乳头，或BP)中再生毛细胞。尽管有这种能力，但人们对鸟类毛细胞前体的身份、细胞谱系或分子调控知之甚少。为解决这些问题，提出了四个具体目标。在目标I中，使用体内和体外方法研究了祖细胞分裂的动力学。首先，使用两个核苷酸类似物，我将确定祖细胞是否在单个病变和相隔一个恢复期的两个病变后分裂不止一次。其次，我将在BP原代培养中进行细胞谱系分析，以评估毛细胞的数量 具有干细胞样行为(即形成含有毛细胞的集落)的祖细胞与那些在形成毛细胞和/或支持细胞之前只分裂一到两次的祖细胞相比。这些实验将有助于确定成熟禽类BP中存在的前体细胞的类型，如果证实存在干细胞，它们将为AIM II的实验提供基础。在AIM II中，提出了两种方法来产生毛细胞前体细胞的纯化培养：克隆扩增和病毒介导的具有有丝分裂潜力的细胞的选择。通过每种方法获得的祖细胞被检测：1)它们对支持细胞分裂的已知调节因子的反应；2)它们再生毛细胞和支持细胞表型的能力。 AIM III的实验将测试在完整的BP中静止和分裂的支持细胞之间是否存在系统性的形态变化。耳囊在卵子中感染编码GFP的逆转录病毒，雏鸡被允许成熟到孵化后。比较对照雏鸡和暴露于庆大霉素的雏鸡BP中GFP标记的支持细胞，以确定1)静止的支持细胞之间是否存在不同的形态亚群，以及2)分裂细胞是否表现出区别于生长停滞细胞的形态特征。在Aim IV中，我将测试一种潜在的祖细胞周期调节因子-同源盒样转录因子cProx1-在毛细胞祖细胞培养中的功能。培养的细胞感染编码全长或显性阴性Prox1的逆转录病毒，并评估cProx1错误表达或抑制对细胞分裂的影响。我假设cProx 1的活动是必要的 足以刺激细胞退出细胞周期。我希望这些实验中产生的信息将有助于促进哺乳动物物种的毛细胞再生。

Auditory hair cells are mechanoreceptors in the inner ear that transduce sound waves into neural signals. In humans, hair cell loss is irreversible and leads to profound, permanent hearing deficits. In contrast, mature birds regenerate hair cells in the chicken auditory epithelium (basilar papilla, or BP). Despite this ability, little is known about the identity, cell lineage, or molecular regulation of avian hair cell progenitors. Four Specific Aims are proposed to address these issues. In Aim I, the dynamics of progenitor cell division are examined using in vivo and in vitro approaches. First, using two nucleotide analogs, I will determine if progenitor cells divide more than once after a single lesion and after two lesions separated by a recovery period. Second, I will perform cell lineage analysis in primary BP cultures to assess the number of hair cell progenitors that have stem-cell like behavior (i.e., form colonies containing hair cells) versus those that divide only once or twice prior to forming hair cells and/or supporting cells. These experiments will help define the types of progenitor cells present in the mature avian BP, and if the presence of stem cells is confirmed, they will provide a foundation for experiments in Aim II. In Aim II, two methods are proposed to generate purified cultures of hair cell progenitors: clonal expansion and viral-mediated selection of cells with mitotic potential. Progenitor cells derived by each method are examined for 1) their response to known regulators of supporting cell division and 2) their ability to regenerate cells with hair-cell and supporting-cell phenotypes. Experiments in Aim III will test if systematic variations in morphology exist among quiescent and dividing supporting cells in the intact BP. Otocysts are infected with a retrovirus encoding GFP in ovo, and chicks are allowed to mature to post-hatch. GFP-labeled supporting cells in the BP of control chicks and of chicks exposed to gentamicin are compared to determine if 1) distinct morphological subsets exist among quiescent supporting cells and 2) if dividing cells exhibit morphological features that distinguish them from growth arrested cells. In Aim IV, I will test the function of a potential regulator of progenitor cell cycling - the homeobox-like transcription factor, cProxl - in cultures enriched for hair cell progenitors. Cultured cells are infected with a retrovirus encoding full-length or dominant-negative Proxl, and the effects of cProxl misexpression or inhibition on cell division are assessed. I hypothesize that cProx 1 activity is necessary and sufficient to stimulate cells to withdraw from the cell cycle. I hope the information generated in these experiments will be useful toward promoting hair cell regeneration in mammalian species.