Neural Stem Cells in the Otocyst

描述(申请人提供)：在发育过程中，细胞排列在 耳上皮头腹侧区域获得神经元的身份 前体从耳囊中迁移出来，形成第VIII(前庭耳蜗区) 神经节。目前对其相关的分子机制知之甚少。 III型神经节神经元的诱导。这项研究的主要目标是 确定对耳囊细胞有神经发生作用的分子因子 并验证这些因子在体内的生理功能。 体内神经诱导的过程。这项研究的长期目标是 描述耳囊中未分化细胞的分子机制 变得有能力致力于神经细胞，随后分化 转化为耳蜗神经节或前庭神经节神经元。以下两个具体目标 将致力于：(1)鉴定具有神经发生功能的可溶性蛋白质 对未分化的耳囊细胞的影响。(2)建立活体动物模型 操纵未分化的耳囊细胞基因表达的系统。为了达到目标 #1，将建立原代耳囊细胞培养，并进行神经发生 单独或联合应用FGF2、FGF8、FGF10、TGFb2、BMP4、Noggin或维甲酸的作用 联合后，将对分离的耳囊细胞进行体外实验。对于目标2， 将建立一种使用电穿孔的体内基因传递方法，并 在活体胚胎形成前，PHOX2B会在耳囊中过表达 第八神经节的成员。PHOX2B过表达对细胞增殖的影响 并将在体内测试耳囊细胞的基因表达。这些实验 代表了系统性尝试的第一步，即产生前庭和 耳囊干细胞来源的耳蜗神经节神经元替代受损或死亡 神经元。此外，对神经诱导和治疗中涉及的因素的知识 内耳的具体情况将促进我们对病因的理解 遗传性耳聋或平衡障碍。

DESCRIPTION (provided by applicant): During development, cells lining on the rostro-ventral region of the otic epithelium acquire their identity as neuron precursors and migrate out of the otocyst to form the VIII (vestibulocochlear) ganglion. Little is known about the molecular mechanisms involved in the induction of VIII ganglion neurons. The primary goals of this study are to identify molecular factors that have neurogenetic effects on otocyst cells in vitro, and also to verify the physiological function of these factors during the course of neural induction in vivo. The long-term goal of this study is to delineate molecular mechanisms by which undifferentiated cells in the otocyst become competent to commit to neuronal cells, and subsequently differentiate into cochlear or vestibular ganglion neurons. The following two specific aims will be addressed: (1) to identify soluble proteins that have neurogenetic effects on undifferentiated otocyst cells. (2) To establish an in vivo model system to manipulate gene expression in undifferentiated otocyst cells. For Aim #1, primary otocyst cell cultures will be established and the neurogenetic effects of FGF2, FGF8, FGF10, TGFb2, BMP4, Noggin or retinoic acid, alone or in combination, on dissociated otocyst cells will be tested in vitro. For Aim #2, an in vivo gene delivery method using electroporation will be established and Phox2b will be overexpressed in the otocyst of live embryos before the formation of the VIII ganglion. The effects of Phox2b overexpression on cell proliferation and gene expression in otocyst cells will be tested in vivo. These experiments represent the first step in a systematic attempt to generate vestibular and cochlear ganglion neurons from otocyst stem cells to replace damaged or dead neurons. Furthermore, knowledge of the factors involved in neural induction and specification in the inner ear would advance our understanding of the cause of genetically inherited deafness or balance disorders.