Insm1 in Development of Spiral and Vestibular Ganglia

描述（由申请人提供）：目前，在美国，有42,600名成人和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。然而，在没有毛细胞刺激的情况下，sgn可能会受损或退化，这使得人工耳蜗的效果降低。了解sgn是如何产生和发展的，可以为如何维持听神经以获得最佳的人工耳蜗功能提供方法。Insm1是一种锌指转录因子，在整个发育中的神经系统中表达于神经元祖细胞和新生神经元，但不表达于成熟神经元。它在神经发生过程中表达，促进未固定的顶端分裂祖细胞向基部迁移，并在发育中的皮层和嗅觉上皮中决定神经元细胞的命运。Insm1也在新生神经元中表达，是某些神经元类型分化所必需的。在发育中的耳囊肿中，一些顶端分裂的祖细胞向基底移动，分层并成为静听神经节（SAG）的神经元承诺祖细胞。我们假设(1)耳囊肿中的Insm1促进了从顶端未固定的祖细胞向分层神经元祖细胞的转变，(2)新生SAG神经元中Insm1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：确定Insm1基因在发育中的小鼠耳中的时空表达模式。我们将使用原位杂交技术（ISH）检测胚胎发育过程中不同时间点发育中的耳中Insm1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是通过顶端分裂祖细胞、分层祖细胞还是两者表达的。目的2：确定Insm1是否促进顶端分裂祖细胞向脱层神经元祖细胞的转变。我们将确定Insm1缺失对耳囊肿增殖和凋亡的影响。我们将使用IHC比较Insm1敲除（KO）和野生型（WT）的窝胚耳朵中增殖和凋亡（ACC3阳性）细胞的数量。我们还将通过在子宫内电穿孔驱动过表达来确定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是否为SAG神经元分化所必需。在Insm1 KO中，我们将寻找少数已知促进SAG神经元分化或通过IHC和ISH由新生SAG神经元表达的因子的表达变化。我们还将研究来自SAG的神经突生长作为分化的指标，使用IHC检测Tuj或神经丝，这清楚地标记了SAG的过程。我们将进行RNA测序来比较KO和WT增生后SAG的表达模式。这将为Insm1调控的基因提供彻底和公正的搜索。

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.