Hair Cell Regeneration: Molecular Regulation

听觉毛细胞是内耳中的机械感受器，可将声波转化为神经信号。在人类中，毛细胞的损失是不可逆转的，并导致严重的、永久性的听力缺陷。相比之下，成熟的鸟类再生毛细胞在鸡的听觉上皮（基底乳头，或BP）。尽管有这种能力，但人们对禽类毛细胞祖细胞的身份、细胞谱系或分子调控知之甚少。提出了四个具体目标来解决这些问题。在Aim I中，使用体内和体外方法检查祖细胞分裂的动力学。首先，使用两个核苷酸类似物，我将确定祖细胞在一次损伤后是否分裂不止一次，在两次损伤后是否分裂不止一次。其次，我将在原代BP培养中进行细胞谱系分析，以评估毛细胞的数量

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.