Molecular regulators of innervation and patterning across the developing cochlea

发育中耳蜗神经支配和模式的分子调节因子

• 批准号: 9258032
• 负责人: Mary K Scott
• 金额: $ 4.36万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-12 至 2018-08-11
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9258032
• 关键词: Afferent Neurons; Auditory; Basilar Papilla; Brain; Cell Differentiation process; Cells; Cellular Morphology; Chickens; Cochlea; Cochlear duct; Code; Communication; Culture Techniques; Data; Data Analyses; Development; Ectopic Expression; Efferent Neurons; Embryonic Development; Future; Ganglia; Genes; Genetic Transcription; Hair Cells; Hearing; Housing; Human; Immunohistochemistry; In Situ Hybridization; In Vitro; Knowledge; Label; Laboratories; Labyrinth; Mediating; Methods; Molecular; Morphology; Mus; Natural regeneration; Neurites; Neurons; Organ; Pattern; Proteins; RNA; Radial; Reporter; Reporting; Role; Semaphorins; Sensory; Side; Signal Transduction; Signaling Molecule; Surveys; TCF Transcription Factor; Techniques; Testing; Transcript; Transmission Electron Microscopy; Width; Work; axon guidance; beta catenin; contactin; deep sequencing; design; differential expression; experience; hearing impairment; in vivo; innovation; insight; nerve supply; overexpression; relating to nervous system; research study; response; sound; training opportunity; transcriptome

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.

基底乳头 (BP) 是鸡的听觉器官，里面有感觉细胞（称为毛细胞），这些细胞受传入和传出神经元的支配，沿着 BP 的径向轴，毛细胞的组织及其神经模式。神经支配使得 BP 毛细胞的一侧呈现高形态（高毛细胞）并且主要受传入神经支配。虽然许多研究已经研究了轴突引导因子对 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定的神经支配模式，先前的工作表明 Wnt9a 在发育中的 BP 结果中过度表达。毛细胞的增加呈现出高毛细胞形态，并对这些毛细胞的传入神经支配进行了控制和 Wnt9a 过表达 BP 的 RNA 深度测序，发现了轴突引导基因以及其他基因，这些基因位于毛细胞的下游。 Wnt9a 可能介导观察到的变化。本研究旨在检查其中三个基因对神经神经支配的影响，第一个和第二个目标将分别检查 Semaphorin-3F (Sema3F) 和 Contactin-6 (Cntn6) 的作用。 ，关于神经支配的径向模式，第三个目标将检查 Slit2 对 Roundabout (Robo) 介导的激活 β-catenin (pY489-β-catenin) 的影响。在这些目标中，原位杂交将用于确定这些转录物在耳蜗管中的内源性空间和时间表达模式，将用于检查这些转录物的效果。对于体外实验，SAG 将在纯化的轴突引导蛋白存在下培养。对于体内实验，轴突引导基因将被过度表达。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 转录因子报告基因将被电穿孔到 BP 中，以确定 pY489-β-连环蛋白是否具有激活转录的功能。这些实验将提供对 BP 神经支配的深入了解，这些基因能够影响神经支配的径向模式，可以进一步研究它们在已受损或破坏的毛细胞中再生神经支配的能力。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。 。