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

LIN28B 和卵泡抑素在支持小鼠耳蜗细胞重编程和毛细胞再生中的功能

• 批准号: 10513325
• 负责人: ANGELIKA DOETZLHOFER
• 金额: $ 48.48万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-12-01 至 2026-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10513325
• 关键词: Ablation; Activins; Address; Adolescent; Adult; Alleles; Attenuated; Auditory; Biological Assay; CRISPR/Cas technology; Cell Proliferation; Cell Reprogramming; Cells; Cochlea; Cre lox recombination system; Cues; Data; Diphtheria Toxin; Disease; Doxycycline; Ear; Fluorescent in Situ Hybridization; Follistatin; Gene Expression Profile; Generations; Genes; Hair Cells; Hearing; Human; In Vitro; Knock-out; Labyrinth; LoxP-flanked allele; Mediating; Mitotic; Mus; Natural regeneration; Neonatal; Organoids; Persons; Publishing; RNA-Binding Proteins; Regenerative capacity; Regenerative response; Role; Signal Transduction; Supporting Cell; Testing; Therapeutic; Transforming Growth Factor Beta 2; Transgenes; Transgenic Mice; Transgenic Organisms; Trauma; Up-Regulation; antagonist; candidate selection; cell dedifferentiation; cell determination; cell injury; cochlear development; deafness; experimental study; gene function; gene network; hair cell regeneration; hearing impairment; hearing restoration; in vitro Model; in vivo; in vivo regeneration; inducible Cre; insight; mature animal; mouse genetics; mouse model; neonatal mice; notch protein; novel; overexpression; paralogous gene; pluripotency; progenitor; programs; regenerative; repair strategy; response; single molecule; single-cell RNA sequencing; sound; tool; transcriptome

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).

项目摘要 我们提出的研究旨在解决LIN 28 B和卵泡抑素在支持细胞重编程中的功能， 小鼠耳蜗毛细胞再生。由于疾病或创伤导致的听毛细胞（HC）损失是 永久性的，并且是人类听力损伤和耳聋的主要原因。听觉未成熟 支持细胞（SC）确实能对损伤做出反应，但它们迅速再生丢失的HC的能力 随着SC经历成熟而下降，并且在成年动物中几乎没有观察到HC再生。我们最近 揭示了RNA结合蛋白LIN 28 B和它的副产物LIN 28 A控制着细胞的再生能力。 新生儿耳蜗类器官和外植体中的耳蜗SC。LIN 28 A/B在耳蜗HC中是否具有类似的作用 体内再生还有待测试。此外，我们最近的体外研究表明，LIN 28 B促进了 HC再生通过将SC重编程为祖细胞样细胞，并且这种状态转变可以是 通过激活素拮抗剂卵泡抑素的共激活进一步增强。然而，SC是否真的激活了 在HC再生过程中过渡祖细胞样状态，如果是这样，LIN 28 B和FST如何影响这种状态， 状态转换仍未解决。在我们提出的研究中，我们将使用小鼠遗传工具来解决是否 LIN 28 A/B在体内调节未成熟耳蜗中的自发耳蜗HC再生（aim 1）。此外，委员会认为， 我们将使用单细胞RNA测序和单分子FISH来确定LIN 28 B是否重新编程， 在HC再生期间，耳蜗SC转化为祖细胞样细胞（aim 2）。此外，为了确定LIN 28 B和 FST增强SC重编程和随后的HC再生，我们将操纵潜在的功能 使用慢病毒过表达和CRISPR-Cas9介导的敲除策略的LIN 28 B和FST效应基因 耳蜗类器官（AIM 3）。最后，我们将确定LIN 28 B和FST与Atoh 1的共表达是否 使SC能够在体内成熟耳蜗中再生耳蜗HC（aim 4）。