The role of supporting cell de-differentiation in cochlear hair cell regeneration

项目摘要 耳蜗毛细胞（HC）的丧失是噪声性听力损失和年龄相关性听力损失的主要原因 国际吧在非哺乳动物物种中已经观察到HC对损伤的反应再生，例如 如鸟类和鱼类，但在成熟的哺乳动物耳蜗中没有观察到。虽然支持细胞（SC）， 为毛细胞提供代谢和结构支持的不同细胞群，显示出产生 在新生小鼠的毛细胞中，这种可塑性在出生后第5天丧失。我们实验室以前的工作已经确定了 作为HC再生的关键成分，SC去分化为更像祖细胞的状态， 在耳蜗发育过程中，毛细胞和支持细胞来自共同的祖细胞库。 支持细胞去分化，支持细胞特异性基因表达下调， 的表达上调允许祖细胞样细胞响应HC命运诱导线索。 初步研究已经鉴定了转录因子的NFI和ZBTB家族的成员， SC身份的潜在调节剂，维持SC处于终末分化状态并防止 内源性重编程NFI因子先前已经在视网膜病变的背景下进行了研究。 再生，其中它们已被证明可调节视网膜Müller神经胶质细胞分化。此外，本发明还 NFI因子功能的破坏已显示促进Müller神经胶质向视网膜神经元的转化。 Zbtb 20与星形细胞发生有关，并与报春花综合征有关， 这是一种已知会导致听力损失的发育障碍。 这项拨款提案的首要假设是，Nfia/B/c/x和Zbtb 20在以下方面至关重要： 维持SC身份，并且这些因素中的一个或多个的丧失将使SC重编程成为可能 和HC再生。将使用体外耳蜗测试这一假设。 来源于P2和P5期小鼠耳蜗感觉上皮细胞的类器官培养物。此外， 将在体内和HC损伤模型中研究Zbtb 20。我将使用RT-qPCR，荧光成像的HC 报告细胞系、免疫标记、EdU脉冲实验和目的1中的scRNA测序，以确定是否获得 NFIA/B/C/X或ZBTB 20的功能抑制耳蜗SC重编程和基于SC的HC形成 体外P2和目标2中的P2，以确定NFIA/B/C/X或ZBTB 20的功能丧失是否增强耳蜗SC 可塑性和基于SC的HC形成在体外P5阶段。在目标3中，我将使用免疫标记、RT-qPCR、EdU 脉冲实验和HC损伤模型，以确定Zbtb 20在维持终末SC中的作用。 差异化国家这些研究将扩大我们对SC身份维持和HC的认识 再生过程，并将有助于未来的转化研究使用再生疗法治疗听力 人类的损失。

PROJECT SUMMARY Cochlear Hair Cell (HC) loss is a leading cause of noise-induced and age-related hearing loss worldwide. Regeneration of HCs in response to damage has been observed in nonmammalian species, such as birds and fish, but is not observed in the mature mammalian cochlea. Although Supporting Cells (SCs), a diverse population of cells offering metabolic and structural support to hair cells, show the capacity to produce hair cells in neonatal mice, this plasticity is lost by postnatal day 5. Previous work from our lab has identified the de-differentiation of SCs into a more progenitor-like state as a key component of the HC regenerative process, as hair cells and supporting cells arise from a common progenitor pool during cochlear development. Supporting cell de-differentiation, the downregulation in expression of supporting cell-specific genes and upregulation in expression of, allows the progenitor-like cells to respond to HC fate-inducing cues. Preliminary studies have identified members of the NFI and ZBTB families of transcription factors as potential regulators of SC identity, maintaining SCs in a terminally differentiated state and preventing endogenous reprogramming. The NFI factors have previously been studies in the context of retinal regeneration, where they have been shown to regulate retinal Müller glial cell differentiation. Additionally, disruption of NFI factor function has been shown to promote conversion of Müller glia into retinal neurons. Zbtb20 has been implicated in astrocytogenesis, and has been linked to Primrose syndrome, a rare developmental disorder which is known to cause hearing loss. The overarching hypothesis of this grant proposal is that Nfia/b/c/x and Zbtb20 are crucial in maintaining SC identity, and that loss of one or more of these factors will enable SC reprogramming and HC regeneration in stage P5 mouse tissue. This hypothesis will be tested using in vitro cochlear organoid culture derived from stage P2 and P5 murine cochlear sensory epithelia. Additionally, the function of Zbtb20 will be studied in vivo and in a HC damage model. I will use RT-qPCR, fluorescence imaging of a HC reporter line, immunolabeling, EdU pulse experiments, and scRNA-sequencing in Aim 1 to determine if gain of function of NFIA/B/C/X or ZBTB20 inhibits cochlear SC reprogramming and SC-based HC formation at stage P2 in vitro and in Aim 2 to determine if loss of function of NFIA/B/C/X or ZBTB20 enhances cochlear SC plasticity and SC-based HC formation at stage P5 in vitro. In Aim 3, I will use immunolabeling, RT-qPCR, EdU pulse experiments, and a HC damage model to define the role of Zbtb20 in maintaining SCs in a terminally differentiated state. These studies will expand our knowledge of SC identity maintenance and the HC regenerative process and will aid future translational research using regenerative therapies to treat hearing loss in humans.