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: 8607175
• 负责人: Sarah M Lorenzen
• 金额: $ 3.5万
• 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/8607175
• 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; 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; transcriptome sequencing

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.

描述（由申请人提供）：目前，在美国，42，600名成人和28，400名儿童植入了人工耳蜗，人工耳蜗依赖于螺旋神经节神经元（SGN）来传导包括人类语音在内的听觉信息。然而，在没有毛细胞刺激的情况下，SGN可能会受损或退化，从而使耳蜗植入物效果不佳。了解SGN是如何产生和发展的，可以为如何维持听觉神经以实现最佳人工耳蜗功能提供信息。Insm 1是一种锌指转录因子，在神经元祖细胞和新生但不成熟的神经元中表达。它在神经发生过程中表达，并促进未定型的、顶端分裂的祖细胞向基底迁移，并在发育中的皮质和嗅上皮中定型为神经元细胞。Insm 1也在新生神经元中表达，并且对于某些神经元类型的分化是必需的。在发育中的耳囊中，一些顶端分裂的祖细胞向基部移动，分层并成为平衡声神经节（SAG）的神经元定向祖细胞。我们假设：（1）耳囊中的Insm 1促进了从顶端未定型祖细胞向分层神经元祖细胞的转变;（2）新生SAG神经元中Insm 1的持续表达促进了它们的分化。我们将通过三个具体目标来检验这一假设。目的1：研究Insm 1在小鼠耳发育过程中的时空表达模式。我们将利用原位杂交技术检测Insm 1在胚胎发育过程中不同时间点在发育耳中的表达。我们还将使用ISH，然后免疫组织化学（IHC）增殖标志物（Ph 3，Ki 67和BrdU），以确定是否Insm 1是由顶部分裂的祖细胞，分层的祖细胞，或两者表达。目的2：确定Insm 1是否促进顶端分裂的祖细胞向分层的神经元祖细胞的转变。我们将确定Insm 1缺失对耳囊肿增殖和凋亡的影响。我们将使用IHC比较Insm 1敲除（KO）和野生型（WT）同窝胚胎耳中增殖和凋亡（ACC 3阳性）细胞的数量。我们还将确定Insm 1异位表达的分层，增殖和神经元生产的影响，通过驾驶过表达使用子宫内电穿孔。目的3：研究Insm 1在SAG神经元分化中的作用。在Insm 1 KO中，我们将通过IHC和ISH寻找已知促进SAG神经元分化或由新生SAG神经元表达的少数因子表达的变化。我们还将研究SAG的神经突生长作为分化的指标，使用Tuj或神经丝的IHC，其清楚地标记SAG过程。我们将进行RNA Seq以比较KO和WT增殖后SAG的表达模式。这将为Insm 1调控的基因提供一个彻底和公正的搜索。