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

基本信息

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
项目类别：
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

项目摘要

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).

项目概要我们提出的研究旨在解决 LIN28B 和卵泡抑素在支持细胞重编程和小鼠耳蜗中的毛细胞再生。由于疾病或创伤导致听毛细胞 (HC) 损失永久性的，是人类听力障碍和耳聋的主要原因。听觉不成熟支持细胞 (SC) 确实会再生 HC 以应对损伤，但它们能够快速再生丢失的 HC 随着 SC 的成熟而下降，并且在成年动物中几乎没有观察到 HC 再生。我们最近发现 RNA 结合蛋白 LIN28B 及其旁系同源物 LIN28A 控制细胞的再生能力新生儿耳蜗类器官和外植体中的耳蜗 SC。 LIN28A/B在人工耳蜗HC中是否具有相似的作用体内再生尚待测试。此外，我们最近的体外研究表明 LIN28B 促进通过将 SC 重新编程为类祖细胞来实现 HC 再生，并且这种状态转变可以激活素拮抗剂卵泡抑素的共同激活进一步增强。然而，SC 是否真正激活了 HC 再生过程中的过渡祖细胞样状态，如果是这样，LIN28B 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 LIN28A/B 调节体内未成熟耳蜗的自发耳蜗 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是否重编程在 HC 再生过程中将耳蜗 SC 转化为类祖细胞 (aim2)。此外，要建立 LIN28B 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 LIN28B 和 FST 效应基因在耳蜗类器官中（目标3）。最后，我们将确定 LIN28B 和 FST 是否与 Atoh1 共表达使 SC 能够在体内成熟的耳蜗中再生耳蜗 HC（目标4）。