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：
10366493
负责人：
ANGELIKA DOETZLHOFER
金额：
$ 53.75万
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/10366493
关键词：
Activins Address Adolescent Adult Alleles Attenuated Auditory Biological Assay CRISPR/Cas technology Cell Proliferation 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 Transgenes Transgenic Mice Transgenic Organisms Trauma Up-Regulation antagonist base 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 insight mature animal mouse genetics mouse model neonatal mice notch protein novel overexpression paralogous gene pluripotency progenitor ranpirnase 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).

项目摘要我们提出的研究旨在解决Lin28b和Follistatin在支持细胞重编程和鼠耳蜗中的毛细胞再生。由于疾病或创伤引起的听觉毛细胞（HCS）的丧失是永久性，是人类听力障碍和耳聋的主要原因。未成熟的听觉支持细胞（SC）确实会重新生成HC，以响应损害，但它们迅速再生HC的能力随着SC经历成熟，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控制着再生能力新生儿人工耳蜗和外植体中的耳蜗SC。 Lin28a/b在人工耳蜗HC中是否具有相似的作用体内再生尚未进行测试。此外，我们最近的体外研究表明LIN28B促进 HC通过将SC重新编程成祖细胞样细胞而再生，并且这种状态过渡可以是通过激活素拮抗剂卵脂素的共激活进一步增强。但是，SC是否真的激活 HC再生期间类似过渡的祖细胞状状态，如果是的，LIN28B和FST如何影响此类国家过渡仍未解决。在我们提出的研究中，我们将使用鼠标遗传工具来解决是否是否 Lin28a/b调节体内未成熟耳蜗中的自发人耳蜗HC再生（AIM1）。此外，我们将使用单细胞RNA测序和单分子鱼来确定LIN28B是否重新编程在HC再生期间，人工耳蜗SC进入祖细胞样细胞（AIM2）。此外，确定Lin28b和 FST增强SC重新编程和随后的HC再生，我们将操纵电位功能 LIN28B和FST效应子基因使用慢病毒过表达和CRISPR-CAS9介导的敲除策略在人工耳蜗中（AIM3）。最后，我们将确定LIN28B和FST的共表达是否与ATOH1 使SC可以在体内成熟的耳蜗中再生耳蜗HC（AIM4）。