Genetic dissection of auditory circuit assembly

项目摘要 螺旋神经节神经元(SGN)编码动物听到的一切，并将这些信息发送到大脑。在……里面 为了实现快速和可靠的信号传输，SGN表现出许多专门的属性， 包括通过大量富含AMPA受体的突触后密度对谷氨酸做出反应的能力。尽管所有人 SGN是谷氨酸能的，它们突触的性质和对声音的反应的不同表明 有三种不同的亚型。高自发放电率(SR)SGN的阈值较低，很可能是 最先对声音做出反应。低SR SGN具有更高的阈值，建议用于提高检测能力 噪音中的声音；中型SR SGN介于两者之间。这些生理上的差异伴随着类似的 AMPA受体的丰度和相对突触前带的大小的变化。低SR SGN 突触更容易受到噪音的影响，这可能是一些人有问题的原因 理解他们听到的东西，而不是正常的听觉阈值。这个项目的长期目标是 了解SGN如何获得感知声音所需的特性。更紧迫的是，我们将 定义赋予SGN亚型不同属性和功能的内在转录网络。 我们假设，SGN多样化取决于泛SGN GATA3网络和 转录因子RUNX1驱动的亚型特异性程序。在此之前，我们展示了GATA3 影响SGN分化的多种特征，部分通过转录因子Mafb发挥作用(Lu等人， 2011年；Appler等人，2013年；Yu等人，2013年)。在Mafb突变小鼠中，SGN不发育正常的突触后 密度。随后，我们发现I型SGN分为三种分子亚型(Ia、Ib和Ic)。 分别与高、中和低SR SGN的特征匹配(Shrestha等人，2018年)。新的初稿 研究表明，I型SGN的多样化需要转录因子RUNX1，即 胚胎发育后期仅限于Ib和Ic亚型，并在一生中保持不变。此外，IC SGN 从基因表达的变化来看，RUNX1条件基因敲除(CKO)小鼠似乎丢失了 并改变了ABR反应。在这里，我们将定义RUNX1的作用及其与GATA3的关系。我们会 对Runx1CKO小鼠进行彻底分析，检查SGN组成、突触异质性和 对听力的影响。同时，我们将使用基因和病毒过度表达的方法来了解GATA3和 它的MAF效应器影响体内亚型同一性和突触异质性的出现。使用Single 细胞和批量RNA测序，我们将定义在开发Ib/c SGN和测试中活跃的分子程序 这些程序是如何因RUNX1或GATA3的缺失而改变的，以及MAF因子是如何发挥作用的。这些 研究将阐明推动SGN多样化的分子程序，展示这种多样性如何影响 并可能揭示一种替代丢失的IC SGN的方法，从而在听觉创伤后恢复正常的听力。

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.