Insm1 in Development of Spiral and Vestibular Ganglia

Insm1 在螺旋神经节和前庭神经节发育中的作用

• 批准号: 8317151
• 负责人: Sarah M Lorenzen
• 金额: $ 3.46万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-02-08 至 2015-02-07
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8317151
• 关键词: Acoustic Nerve; Adrenal Glands; Adult; Apical; Apoptosis; Apoptotic; Attention; Auditory; Automobile Driving; Bromodeoxyuridine; Cell Death; Cells; Child; Cleaved cell; Cochlear Implants; Commit; Development; Ear; Ectopic Expression; Electroporation; Embryo; Embryonic Development; Ganglia; Genes; Hair Cells; Hearing; Human; Immunohistochemistry; In Situ Hybridization; Knock-out; Knockout Mice; Label; Medicine; Messenger RNA; Methods; Mus; Nervous system structure; Neurites; Neurons; Olfactory Epithelium; Patients; Pattern; Phenotype; Play; Process; Production; Proliferating; Proliferation Marker; RNA; Role; Speech; Staging; Staining method; Stains; Stem cells; Techniques; Testing; Time; United States; Vestibular ganglion; Zinc Fingers; base; cell injury; improved; in utero; nerve stem cell; neurofilament; neurogenesis; neuron development; novel; otoconia; postnatal; progenitor; spatiotemporal; spiral ganglion; tool; transcription factor

DESCRIPTION (provided by applicant): Currently, within the United States, 42,600 adults and 28,400 children have cochlear implants, which depend on the spiral ganglion neurons (SGNs) for conducting auditory information including human speech. However, the SGNs can be damaged or degenerate in the absence of stimulation from the hair cells, rendering cochlear implants less effective. Understanding how SGNs are generated and develop can inform methods for how to maintain the auditory nerve for optimal cochlear implant function. Insm1 is a zinc finger transcription factor expressed throughout the developing nervous system in neuronal progenitors and nascent, but not mature neurons. It is expressed during neurogenesis and promotes uncommitted, apically-dividing progenitors to migrate basally and commit to neuronal cell fate in both the developing cortex and olfactory epithelium. Insm1 is also expressed in nascent neurons and necessary for differentiation of certain neuron types. In the developing otocyst, some apically-dividing progenitors move basally, delaminate and become neuronally-committed progenitors of the statoacoustic ganglion (SAG). We hypothesize that (1) Insm1 in the otocyst promotes the transition from apically uncommitted progenitors to delaminated neuronal progenitors, and (2) that continued expression of Insm1 in nascent SAG neurons promotes their differentiation. We will test this hypothesis by using three specific aims. Aim 1: To determine the spatiotemporal expression pattern of Insm1 in the developing mouse ear. We will use in situ hybridization (ISH) to detect the expression of Insm1 in the developing ear at different time points during embryonic development. We will also use ISH followed by immunohistochemistry (IHC) for proliferation markers (Ph3, Ki67, and BrdU) to determine if Insm1 is expressed by apically dividing progenitors, delaminated progenitors, or both. Aim 2: To determine if Insm1 promotes a transition of apically-dividing progenitors to delaminated neuronal progenitors. We will determine the effects of Insm1 deletion on proliferation and apoptosis in the otocyst. We will compare the number of proliferating and apoptotic (ACC3 positive) cells in the ears of Insm1 knockout (KO) and wildtype (WT) littermate embryos using IHC. We will also determine the effects of Insm1 ectopic expression on delamination, proliferation, and neuron production by driving over expression using in utero electroporation. Aim 3: To establish if Insm1 is necessary for differentiation of SAG neurons. In the Insm1 KO we will look for changes in expression of the few factors that are either known to promote differentiation of SAG neurons or be expressed by nascent SAG neurons by IHC and ISH. We will also look at neurite outgrowth from the SAG as an indicator of differentiation, using IHC for Tuj or neurofilaments, which clearly label SAG processes. We will perform RNA Seq to compare expression patterns of the KO and WT post-proliferative SAG. This will afford a thorough and unbiased search for genes regulated by Insm1. PUBLIC HEALTH RELEVANCE: Currently, within the United States, 42,600 adults and 28,400 children have cochlear implants, which depend on the spiral ganglion neurons (SGNs) for conducting auditory information including human speech. Understanding how SGNs are generated and develop can inform methods for how to maintain the auditory nerve for optimal cochlear implant function. We believe that the transcription factor Insm1 promotes the production and differentiation of SGNs.

描述(申请人提供)：目前，在美国，42,600名成年人和28,400名儿童接受了人工耳蜗术，依靠螺旋神经节神经元(SGN)传递包括人类语言在内的听觉信息。然而，在没有毛细胞刺激的情况下，SGN可能会受损或退化，从而降低人工耳蜗术的效果。了解SGN是如何产生和发展的，可以为如何维持听神经以实现最佳的人工耳蜗术功能提供信息。Insm1是一种锌指转录因子，在发育中的神经系统中广泛表达，在神经前体和新生神经元中表达，但在成熟神经元中不表达。它在神经发生过程中表达，并促进未定向的、顶端分裂的祖细胞在发育中的皮质和嗅觉上皮细胞的基础上迁移并致力于神经细胞的命运。Insm1在新生神经元中也有表达，是某些神经元分化所必需的。在发育中的耳囊中，一些顶端分裂的前体细胞基底部移动、分层，成为状态听神经节(SAG)的神经元承诺的前体细胞。我们假设(1)Insm1在耳囊中促进从顶端未固定的前体细胞向分层的神经前体细胞的转变，以及(2)Insm1在新生SAG神经元中的持续表达促进其分化。我们将通过三个具体目标来检验这一假设。目的1：研究Insm1基因在小鼠耳发育过程中的时空表达模式。我们将使用原位杂交技术(ISH)检测Insm1在胚胎发育不同时间点在发育中的耳朵中的表达。我们还将使用ISH和免疫组织化学(IHC)检测增殖标记物(Ph3、Ki67和BrdU)，以确定Insm1是通过顶端分裂的祖细胞表达，还是通过分离的祖细胞表达，还是两者都表达。目的2：确定Insm1是否促进顶端分裂的祖细胞向分层的神经前体细胞转变。我们将确定Insm1缺失对耳囊细胞增殖和凋亡的影响。我们将使用IHC比较Insm1基因敲除(KO)和野生型(WT)产仔胚胎耳朵中增殖和凋亡(ACC3阳性)细胞的数量。我们还将通过使用宫内电穿孔驱动过度表达来确定Insm1异位表达对分层、增殖和神经元产生的影响。目的3：确定Insm1是否对SAG神经元的分化是必需的。在Insm1KO中，我们将寻找少数已知促进SAG神经元分化或由新生SAG神经元通过IHC和ISH表达的因子的表达变化。我们还将观察SAG突起的突起生长作为分化的指标，对TuJ或神经丝使用IHC，它们清楚地标记SAG突起。我们将进行RNA序列分析，以比较KO和WT在增殖后SAG的表达模式。这将使人们能够彻底和公正地寻找受Insm1调控的基因。 与公共健康相关：目前，在美国，有42,600名成年人和28,400名儿童植入了人工耳蜗，这依赖于螺旋神经节神经元(SGN)来传递包括人类语言在内的听觉信息。了解SGN是如何产生和发展的，可以为如何维持听神经以实现最佳的人工耳蜗术功能提供信息。我们认为转录因子Insm1促进SGN的产生和分化。