NEURAL FATE DETERMINATION IN THE MOUSE DORSAL ROOT GANGLION

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 对神经系统的感觉输入由位于脊髓侧面的背根神经节（DRG）中的特化神经元介导。在胚胎发育期间，这些神经元由迁移的神经嵴细胞产生，最终产生DRG中的所有神经元和神经胶质细胞。当神经嵴细胞到达DRG并分裂时，它们产生祖细胞，祖细胞必须首先决定是成为神经元还是神经胶质（神经命运决定）。决定神经命运的祖细胞必须决定分化成几种神经元亚型中的一种，这些神经元可以感知触觉、温度、疼痛、肢体运动和肢体位置（亚型规范）。虽然对基因敲除动物的分析表明，DRG中不同亚型神经元的正常发育需要几个基因，但尚不清楚它们是否参与神经细胞命运决定或神经亚型特化的过程。为了了解更多关于DRG神经命运决定和亚型规范的分子调控，我们建议研究基本螺旋-环-螺旋转录因子neurogenin 1（ngn 1）的作用，这是DRG神经元的一个亚型的发展所需的;疼痛感觉伤害性神经元。ngn 1基因敲除动物中伤害感受神经元的缺乏表明该基因是这些细胞发育所必需的，但不能告诉我们ngn 1是否参与神经命运决定或亚型特化。基于实验室先前的工作，证明相关的碱性螺旋-环-螺旋转录因子（mash 1）参与嗅觉上皮的神经命运决定，我们假设ngn 1参与DRG祖细胞的神经命运决定。为了检验这一假设，将追求以下具体目标：1）表达ngn 1的细胞的发育命运将使用条件谱系追踪方法来确定，以确定除神经元以外的细胞是否源自这些祖细胞。如果ngn 1参与神经命运决定，则可以在产生其他细胞类型（神经胶质）的细胞中发现低水平的ngn 1表达，而如果ngn 1参与神经亚型特化，则应该仅产生神经元; 2）将使用具有细胞类型特异性标志物的发育时间过程来确定野生型和ngn 1敲除胚胎的DRG中不同细胞类型的比例。如果ngn 1参与神经命运的决定，那么在敲除中ngn 1功能的丧失应该导致神经元比例的减少和其他（神经胶质）细胞数量的增加。另一方面，如果ngn 1参与神经亚型的特化，那么在敲除中ngn 1功能的丧失应该导致一种神经元亚型的减少和其他亚型的增加。3)第三个具体的目标将被追求，以确定在转基因小鼠系中表达髓鞘蛋白脂质蛋白-lacZ（PLP-LacZ）融合基因产物的DRG细胞类型，以确定该系是否可用于跟踪DRG中神经胶质细胞的发育。总之，这些特定的目标将有助于我们理解哺乳动物神经系统中的细胞命运决定，这是在发育胚胎中产生神经元和神经胶质正确平衡的关键步骤。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Sensory input to the nervous system is mediated by specialized neurons that reside in the dorsal root ganglia (DRG) that flank the spinal cord. During embryonic development, these neurons are generated from migrating neural crest cells that ultimately give rise to all of the neurons and glial cells in the DRG. As the neural crest cells reach the DRG and divide, they give rise to progenitor cells that must first decide whether to become neurons or glia (neural fate determination). The progenitors that become committed to a neural fate then must decide to differentiate into one of several subtypes of neurons that sense touch, temperature, pain, limb movements, and limb position (subtype specification). While the analysis of knockout animals has shown that several genes are required for the normal development of the different subtypes of neurons in the DRG, it is not clear if they are involved in the process of neural cell fate determination or neural subtype specification. To learn more about the molecular regulation of neural fate determination and subtype specification in the DRG, we propose to investigate the role of the basic helix-loop-helix transcription factor neurogenin1 (ngn1) which is required for the development of one subtype of DRG neuron; the pain-sensing nociceptive neurons. The lack of nociceptive neurons in ngn1 knockout animals shows that this gene is required for the development of these cells but does not tell us whether ngn1 is involved in neural fate determination or subtype specification. Based on previous work from the lab demonstrating that a related basic helix-loop-helix transcription factor (mash1) is involved in neural fate determination in the olfactory epithelium, we hypothesize that ngn1 is involved in neural fate determination in DRG progenitor cells. To test this hypothesis, the following specific aims will be pursued: 1) the developmental fate of cells that express ngn1 will be determined using a conditional lineage tracing approach to determine if cells other than neurons are derived from these progenitors. If ngn1 is involved in neural fate determination, a low level of ngn1 expression could be found in cells that give rise to other cell types (glia), whereas if ngn1 is involved in neural subtype specification, only neurons should be generated; 2) the proportion of different cell types in the DRG of wild-type and ngn1 knockout embryos will be determined using a developmental time course with cell-type specific markers. If ngn1 is involved in neural fate determination then the loss of ngn1 function in the knockout should result in a decrease in the proportion of neurons and an increase in the number of other (glial) cells. On the other hand, if ngn1 is involved in neural subtype specification, the loss of ngn1 function in the knockout should result in a decrease of one subtype of neuron and an increase in other subtypes. 3) A third specific aim will be pursued to identify the DRG cell types that express a myelin proteolipid protein-lacZ (PLP-LacZ) fusion gene product in a transgenic mouse line to determine if this line could be used to follow the development of glial cells in the DRG. Together, these specific aims will contribute to our understanding of cell fate determination in the mammalian nervous system which is a crucial step in generating the correct balance of neurons and glia in the developing embryo.