Cell Type Specific Transcriptional Cascades in Inner Ear Development

年龄相关性失聪(ARHL)困扰着超过50%的70岁以上人口。 耳蜗感觉毛细胞(HCS)的进行性丧失，主要是外毛细胞(OHC)，是 大多数形式的急性淋巴细胞性白血病的病因或最终的共同病理。然而，ARHL的分子基础是 未知，阻碍了有针对性的干预措施的发展。此外，尝试对前体程序进行重新编程 走向耳蜗区HC命运的细胞主要导致未成熟的HC样细胞，类似于前庭HC。为了这些 因此，研究OHC分化和存活的分子途径是至关重要的。 转录因子是基因表达的关键调控因子。当与疾病联系在一起时， 确定信托基金的上游调节者和下游目标是确定 在同样的过程中，还有其他关键角色。在这场竞争性的更新中，我们建立在以往成功的基础上， 确定RFX和IKZF2转录因子在OHC分化、功能和生存中的关键作用，并进行随访 有两个特定的目的旨在阐明它们在HC/OHC遗传程序中的分子机制： 第1组RFX因子(RFX1/2/3)是纤毛发生的主要调节因子。然而，虽然OHC缺少Rfx1/3 在听力开始后12小时内死亡，他们有正常的束状发育。RFX2是暂时的 在发育中的HCS中表达，可能通过丢失Rfx1/3来防止纤毛发生缺陷。 假设第1组RFX转录因子在HC发育中具有双重作用，在毛束发育中具有早期作用 形成，以及后来在终末分化和生存中的作用。具体目标1：通过以下方式确定机制 这些RFX转录因子支持胚胎和出生后早期的耳蜗毛细胞分化。 IKZF2是OHC基因表达的关键调节者，被精确调控为仅在OHC启动时表达 从P4开始。我们假设：(1)IKZF2靶基因的缺失将揭示OHC功能所必需的基因 和存活；(2)IKZF2是维持成熟的OHC所必需的；以及(3)Ikzf2的表达受一种 OHC成熟的主调节因子。具体目标2：确定IKZF2导致OHC的机制 分化，以确定其在成熟的OHC中的作用，并确定其上游调控网络。 为了实现这些目标，我们将OHC特定多体分析的新协议与状态- 最先进的生物信息学，以及验证候选基因作用的新小鼠模型的特征 在听力方面。成功完成这些目标将：(1)影响我们对OHC区分和 存活；(2)关键的RFX和IKZF2靶基因在听力中的作用；(3)导致 分化和成熟的OHC的转录和表观遗传学图谱，将用于信息 HC再生/识别ARHL候选基因的研究；以及(4)建立大量的小鼠模型 对于RFX和IKZF2目标基因，科学界将很容易获得。此外，所有 作为这项提案的一部分产生的数据将通过装备(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).