Molecular regulators of innervation and patterning across the developing cochlea

基底乳头（BP）是鸡的听觉器官，里面有感觉细胞，被称为毛细胞，由传入和传出神经元支配。沿BP的径向轴，毛细胞的组织及其神经支配的模式是这样的，在BP的一侧毛细胞呈高形态（高毛细胞），主要受传入神经支配。另一侧的BP毛细胞呈短形态（短毛细胞），主要受传出神经支配。虽然许多研究已经检查了轴突引导因子对BP神经支配的影响，但目前尚不清楚是什么因素驱动了这种特殊的神经支配模式。先前的研究表明，Wnt9a在发育中的BP中过表达导致毛细胞的增加，这些毛细胞呈现高毛细胞形态和这些毛细胞的传入神经支配。对照和过表达Wnt9a的bp的RNA深度测序鉴定出轴突引导基因，以及其他基因，这些基因位于Wnt9a的下游，可能介导观察到的变化。本研究旨在研究其中三种基因对神经支配的影响。第一个和第二个目标将分别检查信号蛋白3f （Sema3F）和接触蛋白6 （Cntn6）在神经支配的径向模式中的作用。第三个目的是研究Slit2对BP和静声神经节（SAG）中Roundabout （Robo）介导的活化β-catenin （pY489-β-catenin）的影响。在这些目的中，原位杂交将用于确定这些转录本在耳蜗管内的内源性空间和时间表达模式。体外和体内方法将用于检查这些基因对神经支配的影响。在体外实验中，SAGs将在纯化的轴突引导蛋白存在下培养。在体内实验中，轴突引导基因会过表达。免疫组织化学将用于标记神经突、毛细胞和下游因子，以检测对这些操作的反应。在Slit2实验中，TCF转录因子报告因子将被电穿孔到bp中，以确定pY489-β-catenin是否具有激活转录的功能。这些实验的结果将为BP的神经支配提供见解。基因能够影响神经支配的径向模式，其在受损或破坏的毛细胞中再生神经支配的能力有待进一步研究。这些知识将成为发展分子疗法来治疗听力损失的基础。本项目将为申请人提供传输电子显微镜、体外培养技术等方面的培训机会，以及数据量化、数据分析和科学交流方面的经验。

The basilar papilla (BP) is the auditory organ of the chicken and houses sensory cells, known as hair cells, that are innervated by afferent and efferent neurons. Along the radial axis of the BP, the organization of hair cells and the pattern their neural innervation is such that on one side of the BP hair cells take on a tall morphology (tall hair cells) and are primarily innervated by afferents. On the other side of the BP hair cells take on a short morphology (short hair cells) and are primarily innervated by efferents. While many studies have examined the effects of axon guidance factors on innervation of the BP, it is currently unknown what factors drive this particular innervation pattern. Previous work has shown that Wnt9a overexpression in the developing BP results in an increase in hair cells that take on tall hair cell morphology and afferent innervation of those hair cells. RNA deep sequencing of control and Wnt9a overexpressing BPs has identified axon guidance genes, among other genes, that are downstream of Wnt9a and may be mediating the observed changes. This study proposes to examine the impact of three of these genes on neural innervation. The first and second aim will examine the role of Semaphorin-3F (Sema3F) and Contactin-6 (Cntn6), respectively, on the radial pattern of innervation. The third aim will examine the effects of Slit2 on Roundabout (Robo)-mediated activated β-catenin (pY489-β-catenin) in the BP and statoacoustic ganglion (SAG). In these aims, in-situ hybridization will be used to determine the endogenous spatial and temporal expression pattern of these transcripts in cochlear ducts. In vitro and in vivo methods will be used to examine the effect of these genes on innervation. For in vitro experiments, SAGs will be cultured in the presence of purified axon guidance protein. For in vivo experiments, axon guidance genes will be overexpressed. Immunohistochemistry will be used to label neurites, hair cells, and downstream factors to detect responses to these manipulations. In Slit2 experiments, a TCF transcription factor reporter will be electroporated into the BPs to determine if pY489-β-catenin functions to activate transcription. The results from these experiments will provide insights into innervation of the BP. Genes are able to influence the radial pattern of innervation may be further studied for their ability to regenerate innervation in hair cells that have been damaged or destroyed. This knowledge will be foundational for the development of molecular therapies to treat hearing loss. This proposal will additionally provide training opportunities for the applicant in techniques such as transmission electron microscopy and in vitro culturing techniques, as well as experience in data quantification, data analysis, and scientific communication.