Cell Type Specific Transcriptional Cascades in Inner Ear Development

残疾性年龄相关性听力损失（ARHL）困扰着超过50%的70岁以上的人口。 耳蜗的感觉毛细胞（HC），主要是外毛细胞（OHC）的进行性损失， 大多数形式的ARHL的病因或最终共同病理。然而，ARHL的分子基础是 未知，阻碍有针对性的干预措施的发展。此外，尝试重新编程祖细胞 细胞朝向耳蜗HC命运的分化主要导致不成熟的HC样细胞，类似于前庭HC。为这些 因此，研究OHC分化和存活的分子途径至关重要。 转录因子是基因表达的关键调节因子。当与疾病相关时， 确定TF的上游调节器和下游目标是确定TF的有效途径， 在同一进程中的其他关键角色。在这次充满竞争力的更新中，我们建立在以前的成功基础上， 确定RFX和IKZF 2 TF在OHC分化、功能和存活中的关键作用，并随访 有两个特定的目的，旨在阐明其在HC/OHC遗传程序中的分子机制： 第1组RFXTF（RFX 1/2/3）是纤毛发生的主要调节因子。然而，尽管缺乏Rx 1/3的OHC 在听力开始后12小时内死亡，他们有正常的神经束发育。RFX 2是瞬时的 在发育中的毛细胞中表达，可能防止了Rfx 1/3缺失的纤毛发生缺陷。因此我们 假设组1 RFXTF在HC发育中具有双重作用，在毛束中具有早期作用 形成，以及后来在终末分化和存活中的作用。具体目标1：通过以下方式确定机制： 所述RFXTF支持胚胎以及出生后早期耳蜗毛细胞分化。 IKZF 2是OHC基因表达的关键调节因子，精确调节仅在OHC起始时表达 从P4我们假设：（1）IKZF 2靶基因的缺失将揭示OHC功能所必需的基因 （2）IKZF 2是维持成熟OHC所必需的;（3）Ikzf 2的表达受一种免疫调节因子的调节。 OHC成熟的主要调节剂。具体目标2：确定IKZF 2导致OHC的机制 分化，以确定其在成熟OHC中的作用，并确定其上游调控网络。 为了实现这些目标，我们联合收割机将用于OHC特异性多组学分析的新方案与状态- 最先进的生物信息学，以及新型小鼠模型的表征，以验证候选基因的作用 在听觉上。成功完成这些目标将：（1）影响我们对OHC差异化的理解， 存活率;（2）功能性表征关键RFX和IKZF 2靶基因在听力中的作用;（3）导致 分化和成熟OHC的转录组学和表观遗传学图谱， 研究HC再生/鉴定ARHL的候选基因;和（4）产生许多小鼠模型 RFX和IKZF 2靶基因，这将很容易提供给科学界。此外，所有 作为该提案一部分而产生的数据将通过全球数据库（umgear.org）共享。

Disabling age-related hearing loss (ARHL) afflicts greater than 50% of the population over the age of 70. The progressive loss of sensory hair cells (HCs) of the cochlea, primarily the outer HCs (OHCs), is either the cause or the final common pathology of most forms of ARHL. However, the molecular basis of ARHL is unknown, impeding development of targeted interventions. Furthermore, attempts to reprogram progenitor cells towards cochlear HC fates result primarily in immature HC-like cells, similar to vestibular HCs. For these reasons, the study of the molecular pathways underlying OHC differentiation and survival is critical. Transcription factors (TFs) are key regulators of gene expression. When associated with disease, identifying the upstream regulators and downstream targets of a TF is an effective route to determine additional key players in the same process. In this competitive renewal we build upon our previous success, identifying critical roles for the RFX and IKZF2 TFs in OHC differentiation, function and survival, and follow up with two specific aims designed to elucidate their molecular mechanism within the HC/OHC genetic programs: Group 1 RFX TFs (RFX1/2/3), are master regulators of ciliogenesis. However, while OHCs lacking Rfx1/3 die within 12 hours from the onset of hearing, they have normal bundle development. RFX2 is transiently expressed in the developing HCs, possibly preventing a ciliogenesis defect with loss of Rfx1/3. We therefore hypothesize that group 1 RFX TFs have a dual role in HC development, with an early role in hair bundle formation, and later role in terminal differentiation and survival. Specific Aim 1: To identify the mechanisms by which RFX TFs support embryonic as well as early post-natal cochlear hair cell differentiation. IKZF2 is a key regulator of OHC gene expression, precisely regulated to express only in OHCs starting from P4. We hypothesize that: (1) deletion of IKZF2 target genes will reveal genes essential for OHC function and survival; (2) IKZF2 is required for maintenance of mature OHCs; and (3) Ikzf2 expression is regulated by a master-regulator of OHC maturation. Specific Aim 2: To identify the mechanism by which IKZF2 leads to OHC differentiation, to determine its role in mature OHCs, and to identify its upstream regulatory network. To accomplish these aims we combine novel protocols for OHC-specific multi-omic analyses with state- of-the-art bioinformatics, and characterization of novel mouse models to validate the roles of candidate genes in hearing. Successful completion of these aims will: (1) impact our understanding of OHC differentiation and survival; (2) functionally characterize the roles of key RFX and IKZF2 target genes in hearing; (3) result in transcriptomic and epigenetic maps of differentiating and mature OHCs which will be applied to inform studies for HC regeneration/identifying candidate genes for ARHL; and (4) generate numerous mouse models for RFX and IKZF2 target genes, which will be readily available to the scientific community. Additionally, all of the data generated as part of this proposal will be shared via the gEAR (umgear.org).