The function of LIN28B and follistatin in supporting cell reprogramming and hair cell regeneration in the murine cochlea

项目总结 我们建议的研究旨在探讨Lin28b和Folistatin在支持细胞重新编程和 小鼠耳蜗毛细胞再生。由于疾病或创伤而导致的听觉毛细胞(Hcs)丧失 永久性的，是人类听力障碍和耳聋的主要原因。不成熟的听觉 支持细胞(SCs)确实会在损伤后再生HC，但其再生HC的能力会迅速丧失 随着干细胞的成熟而减少，在成年动物中很少或根本没有观察到HC再生。我们最近 发现RNA结合蛋白Lin28b及其类似的Lin28a控制着细胞的再生能力 新生儿耳蜗器和外植体中的耳蜗干细胞。LIN28A/B在耳蜗性HC中是否具有类似的作用 体内的再生还有待测试。此外，我们最近的体外研究表明，Lin28b促进 通过将干细胞重新编程为类祖细胞来再生HC，并且这种状态转换可以 激活素拮抗剂Folistatin的共同激活进一步增强了这一作用。然而，SC是否真的激活了 在HC再生过程中的过渡类祖细胞状态，如果是这样的话，LIN28b和FST如何影响这种状态 状态转换仍未解决。在我们提议的研究中，我们将使用小鼠遗传工具来解决 Lin28A/B调节体内未成熟耳蜗内自发的HC再生(Aim1)。此外， 我们将使用单细胞RNA测序和单分子FISH来确定Lin28b是否会重新编程 在HC再生过程中，耳蜗干细胞分化为类祖细胞(AIM2)。此外，为了确定LIN28B和LIN28B FST增强SC重新编程和随后的HC再生，我们将操纵电势的函数 慢病毒过表达LIN28b和FST效应基因及CRISPR-Cas9介导的基因敲除策略 在耳蜗器中(Aim3)。最后，我们将确定Lin28b和FST是否与Atoh1共表达 使干细胞能够在体内成熟的耳蜗内再生耳蜗生毛细胞(Aim4)。

Project summary Our proposed study aims to address the function of LIN28B and follistatin in supporting cell reprogramming and hair cell regeneration in the murine cochlea. Loss of auditory hair cells (HCs) due to disease or trauma is permanent and is a leading cause for hearing impairments and deafness in humans. Immature auditory supporting cells (SCs) do regenerate HCs in response to damage but their ability to regenerate lost HCs rapidly declines as SCs undergo maturation and little to no HC regeneration is observed in adult animals. We recently uncovered that the RNA binding protein LIN28B and its paralog LIN28A control the regenerative capacity of cochlear SCs in neonatal cochlear organoids and explants. Whether LIN28A/B has a similar role in cochlear HC regeneration in vivo has yet to be tested. Furthermore, our recent in vitro studies suggest that LIN28B promotes HC regeneration through reprogramming SCs into progenitor-like cells and that such state transition can be further enhanced by the co-activation of the Activin antagonist follistatin. However, whether SCs truly activate a transitional progenitor-like state during HC regeneration and if so, how LIN28B and FST may influence such state transition are still unresolved. In our proposed study we will use mouse genetic tools to address whether LIN28A/B regulates spontaneous cochlear HC regeneration in the immature cochlea in vivo (aim1). Furthermore, we will use single cell RNA sequencing and single molecule FISH to determine whether LIN28B reprograms cochlear SCs into progenitor-like cells during HC regeneration (aim2). Moreover, to establish how LIN28B and FST enhance SC reprogramming and subsequent HC regeneration we will manipulate the function of potential LIN28B and FST effector genes using lentiviral overexpression and CRISPR-Cas9-mediated knockout strategies in cochlear organoids (aim3). Finally, we will determine whether co-expression of LIN28B and FST with Atoh1 enables SCs to regenerate cochlear HCs in the mature cochlea in vivo (aim4).