Molecular basis of mammalian cochlear regeneration

摘要：感音神经性耳聋影响全球15亿人，其主要病因是 耳蜗毛细胞和支持细胞不可逆转的丧失。虽然助听器和人工耳蜗可以 改善听力，我们目前缺乏逆转听力损失的潜在病理-毛细胞和 支持细胞丢失。最近的研究发现，已定义的转录因子可以对内源性耳蜗重编程 支持细胞直接获得毛细胞命运，然而，形成的毛细胞在数量和数量上都是有限的 成熟度。此外，这种被称为直接转分化的非有丝分裂途径导致了 支持细胞的总体数量。因此，更好地了解1)支持细胞的有丝分裂再生 2)耳蜗中再生的毛细胞如何成熟是至关重要的。 在这项建议中，我们将测试转录因子的单一或组合应用是否可以 补充未成熟和成熟小鼠耳蜗中的毛细胞和支持细胞。在初步的和最近的 使用转基因小鼠模型发表的数据显示，1)大上皮脊(GER)细胞，而不是 作为发育过程中的一种暂时性结构，迁移到Corti器官中以再生丢失的支持细胞 并成熟成为支持细胞亚型的指骨内侧细胞，这对人的生存至关重要 内毛细胞，2)损伤诱导GER细胞强劲增殖并上调转录因子 3)Atoh1的过度表达强烈地诱导了GER中新的毛细胞的形成，这与增殖有关 成熟后成为内毛细胞和外毛细胞样细胞。 第一个目标是验证损伤反应转录因子促进有丝分裂的假设。 新生和受损的成熟耳蜗处的再生。在第二个目标中，我们将使用再生的毛细胞 在GER中作为一个模型系统来描述再生毛细胞成熟的时空特征 然后测试外毛细胞因子Ikzf2是否会增强外毛细胞的命运。此外，我们还将审查 毛细胞转录因子组合诱导毛细胞再生和成熟的能力 成熟的耳蜗骨受损。为了不偏不倚地了解异位支持细胞的遗传特征 毛细胞，第三个目标将探测GER来源的毛细胞和支持细胞的转录。我们会 使用生物信息学方法揭示他们的遗传图景，以定义标记祖先和 再生的支持细胞和毛细胞以及驱动再生的候选基因。 总之，我们将应用最先进的技术(活细胞成像、电生理学、SNRNA-SEQ、 内耳手术，病毒转导)，以研究支持细胞和毛细胞再生的机制。我们 已经组建了一个专家团队，他们共同努力收集有希望的初步数据。在最后 在这项为期5年的计划中，我们将确定1)转录因子是否可以增强耳蜗 再生2)决定哺乳动物支持细胞和毛细胞再生的机制。

Abstract: Sensorineural hearing loss affects 1.5 billion people worldwide, with the primary pathology being the irreversible loss of cochlear hair cells and supporting cells. Although hearing aids and cochlear implants can improve hearing, we currently lack the ability to reverse the underlying pathology of hearing loss-hair cell and supporting cell loss. Recent studies found that defined transcription factors can reprogram endogenous cochlear supporting cells to directly acquire a hair cell fate, however, the hair cells formed are limited both in number and degree of maturation. Moreover, this non-mitotic approach, coined direct transdifferentiation, leads to a loss of the overall supporting cell population. Thus, a better understanding of 1) mitotic regeneration of supporting cells and 2) how regenerated hair cells in the cochlea mature is critical. In this proposal, we will test whether singular or combinatorial application of transcription factors can replenish hair cells and supporting cells in the immature and mature mouse cochlea. In preliminary and recently published data using transgenic mouse models, we found that 1) greater epithelial ridge (GER) cells, instead of being a transient structure during development, migrate into the organ of Corti to regenerate lost supporting cells and mature to become the supporting cell subtype inner phalangeal cells, which are critical for the survival of inner hair cells, 2) damage induces GER cells to robustly proliferate and upregulate transcription factors associated with proliferation, 3) Atoh1 overexpression robustly induces new hair cell formation in the GER, which mature to become inner hair cell- and outer hair cell-like cells. The first aim will test the hypothesis that damage-responsive transcription factors promote mitotic regeneration in the neonatal and damaged mature cochlea. In the second aim, we will use regenerated hair cells in the GER as a model system to characterize the spatiotemporal features by which regenerated hair cells mature and then test whether the outer hair cell factor Ikzf2 enhances an outer hair cell fate. Moreover, we will examine the ability of combination of hair cell transcription factors to induce hair cell regeneration and maturation in the damaged mature cochlea. To gain an unbiased insight into the genetic signature of ectopic supporting cells and hair cells, the third aim will probe the transcriptomes of GER-derived hair cells and supporting cells. We will reveal their genetic landscape using bioinformatic approaches to define genes marking progenitors and regenerated supporting cells and hair cells and candidate genes driving regeneration. In summary, we will apply state-of-the-art technologies (live cell imaging, electrophysiology, snRNA-seq, inner ear surgery, viral transduction) to study the mechanisms of supporting cell and hair cell regeneration. We have assembled a team of experts who have worked together to collect promising preliminary data. At the end of this 5-year proposal, we will have determined 1) whether transcription factors can enhance cochlear regeneration 2) mechanisms dictating regeneration of supporting cells and hair cells in mammals.