Genetic dissection of auditory circuit assembly

项目摘要 螺旋神经节神经元（SGN）编码动物听到的所有信息并将这些信息发送到大脑。在 为了实现快速和可靠的信号传输，SGN表现出许多专门的特性， 包括通过大的、富含AMPA受体的突触后密度对谷氨酸作出反应的能力。尽管所有 SGN是突触能的，它们的突触性质和对声音的反应的差异表明， 有三种不同的亚型。高自发放电率（SR）SGN具有低阈值，并且可能是 首先对声音做出反应。低SR SGN具有更高的阈值，并且被提议用于提高检测能力 噪声中的声音;中等SR SGN介于两者之间。这些生理差异伴随着平行 AMPA受体丰度和相对突触前带大小的变化。低SR SGN 突触更容易受到噪音的影响，这可能是为什么有些人有麻烦， 尽管他们的听觉阈值正常，但他们仍然能理解所听到的内容。该项目的长期目标是 了解SGN如何获得感知声音所需的属性。更直接的是，我们将 定义赋予SGN亚型独特性质和功能的内在转录网络。 我们假设SGN多样化取决于泛SGN Gata 3网络的综合活动， 由转录因子Runx 1驱动的亚型特异性程序。之前，我们证明了Gata 3 影响SGN分化的多种特征，部分通过转录因子Mafb起作用（Lu等， 2011; Appler等人，2013; Yu等人，2013年）。在Mafb突变小鼠中，SGN不发育正常的突触后神经元。 密度随后，我们发现I型SGN分为三种分子上不同的亚型（Ia，Ib和Ic）。 分别与高、中和低SRSGN的特征匹配（Shrestha等人，2018年）。新的初步 研究表明，I型SGN的多样化需要转录因子Runx 1， 仅限于Ib和Ic亚型的晚期胚胎发育，然后维持整个生命。此外，Ic SGN 从Runx 1条件性基因敲除（CKO）小鼠中似乎丢失，如基因表达的变化所示 和ABR反应的改变在这里，我们将定义Runx 1的角色及其与Gata 3的关系。我们将 对Runx 1CKO小鼠进行全面分析，检查SGN组成、突触异质性和 对听觉的影响。同时，我们将使用遗传和病毒过表达方法来了解Gata 3和 其Maf效应子影响体内亚型身份和突触异质性的出现。使用单 细胞和批量RNA测序，我们将确定开发Ib/c SGN的分子程序，并测试 这些程序是如何被Runx 1或Gata 3的丢失所改变的，以及Maf因子是如何起作用的。这些 研究将阐明驱动SGN多样化的分子程序，显示这种多样性如何影响 听力，并可能揭示一种方法，以取代失去的Ic SGN，从而恢复正常的听力后，声创伤。

Project Summary Spiral ganglion neurons (SGNs) encode everything an animal hears and send this information to the brain. In order to achieve rapid and reliable signal transmission, SGNs exhibit a number of specialized properties, including the ability to respond to glutamate via large, AMPA-receptor rich post-synaptic densities. Although all SGNs are glutamatergic, differences in the nature of their synapses and their responses to sound indicate that there are three distinct subtypes. High spontaneous firing rate (SR) SGNs have low thresholds and are likely the first to respond to sound. Low SR SGNs have higher thresholds and are proposed to improve the ability to detect sounds in noise; medium SR SGNs fall in between. These physiological differences are accompanied by parallel changes in the abundance of AMPA receptors and the size of the opposing pre-synaptic ribbon. Low SR SGN synapses are more vulnerable to the effects of noise exposure, which may be why some people have trouble understanding what they hear despite normal auditory thresholds. The long term goal of this project is to understand how SGNs acquire the properties needed for the perception of sound. More immediately, we will define the intrinsic transcriptional networks that endow SGN subtypes with their distinct properties and functions. We hypothesize that SGN diversification depends on the combined activities of a pan-SGN Gata3 network and a subtype-specific program driven by the transcription factor Runx1. Previously, we showed that Gata3 influences multiple features of SGN differentiation, acting in part through the transcription factor Mafb (Lu et al., 2011; Appler et al., 2013; Yu et al., 2013). In Mafb mutant mice, SGNs do not develop normal post-synaptic densities. Subsequently, we showed that Type I SGNs fall into three molecular distinct subtypes (Ia, Ib, and Ic) that match the features of high, medium, and low SR SGNs respectively (Shrestha et al., 2018). New preliminary studies suggest that diversification among Type I SGNs requires the transcription factor Runx1, which is restricted to Ib and Ic subtypes by late embryogenesis and then maintained throughout life. Further, Ic SGNs appear to be lost from Runx1 conditional knock-out (CKO) mice, as indicated by changes in gene expression and altered ABR responses. Here, we will define the role of Runx1 and its relationship with Gata3. We will perform a thorough analysis of Runx1CKO mice, examining SGN composition, synaptic heterogeneity, and the effects on hearing. In parallel, we will use genetic and viral overexpression approaches to learn how Gata3 and its Maf effectors influence the emergence of subtype identity and synaptic heterogeneity in vivo. Using single cell and bulk RNA-sequencing, we will define the molecular programs active in developing Ib/c SGNs and test how these programs are altered by loss of Runx1 or Gata3, as well as how the Maf factors contribute. These studies will elucidate the molecular programs driving SGN diversification, show how this diversity influences hearing, and may reveal a way to replace lost Ic SGNs and hence restore normal hearing after acoustic trauma.