Molecular regulators of innervation and patterning across the developing cochlea

基底乳头（BP）是鸡的听觉器官，并且容纳被称为毛细胞的感觉细胞，其由传入和传出神经元支配。沿着BP的径向轴，毛细胞的组织和它们的神经支配模式是这样的，即在BP的一侧毛细胞呈现高的形态（高毛细胞）并且主要由传入神经支配。在BP的另一侧，毛细胞呈现短的形态（短毛细胞），并且主要由传出神经支配。虽然许多研究已经检查了轴突导向因子对BP神经支配的影响，但目前尚不清楚是什么因素驱动这种特定的神经支配模式。先前的研究表明，在发育中的BP中Wnt 9a过表达导致毛细胞的增加，这些毛细胞具有高毛细胞形态和这些毛细胞的传入神经支配。对照和Wnt 9a过表达BP的RNA深度测序已经鉴定了轴突引导基因，以及其他基因，这些基因位于Wnt 9a的下游，并且可能介导观察到的变化。本研究旨在研究这些基因中的三个对神经支配的影响。第一个和第二个目标将分别检查Semaphorin-3F（Sema 3F）和Contactin-6（Cntn 6）对神经支配的放射状图案的作用。第三个目的将检查Slit 2对BP和平衡听神经节（SAG）中的Roundabout（Robo）介导的活化β-连环蛋白（pY 489-β-连环蛋白）的作用。在这些目标中，原位杂交将用于确定这些转录本在耳蜗导管中的内源性空间和时间表达模式。在体外和体内的方法将被用来检查这些基因对神经支配的影响。对于体外实验，将在纯化的轴突引导蛋白存在下培养SAG。对于体内实验，轴突引导基因将过表达。免疫组织化学将被用来标记神经突，毛细胞，和下游因素，以检测这些操作的反应。在Slit 2实验中，将TCF转录因子报告基因电穿孔到BP中以确定pY 489-β-连环蛋白是否起激活转录的作用。这些实验的结果将提供对BP神经支配的深入了解。基因能够影响神经支配的辐射模式，可能会进一步研究它们在受损或破坏的毛细胞中再生神经支配的能力。这些知识将成为开发治疗听力损失的分子疗法的基础。该提案还将为申请人提供透射电子显微镜和体外培养技术等技术的培训机会，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.