Neurogenesis in the Chick Embryo Spinal Cord

描述(由申请者提供)：激励这个特定项目的大问题是了解负责形成脊椎动物神经管背腹轴的发育过程是如何与调节增殖和神经发生的过程相交的。在脊髓，神经元的产生和分化遵循从腹侧到背侧的梯度，在背侧中间神经元和翼状前体细胞分化之前，来自基底板前体的运动神经元产生。初步结果表明，脊髓神经上皮细胞生成时间的差异与发育阶段和背腹轴上的位置有关。建议进行实验以进一步确定这些差异，并探索在胚胎学和分子水平上对这些事件的调节。申请人建议使用实验胚胎学方法测试来自旁轴中胚层和/或外胚层上皮的信号是否会对脊髓背侧和腹侧区域的细胞生成时间和神经发生产生不同的影响。累积BrdU标记和免疫细胞化学将被用来估计正常和手术操作胚胎中细胞周期参数和神经元分化的变化。手术操作对细胞死亡的可能影响将使用TUNEL分析来解决。我们将利用原位杂交技术评估四个ID家族成员的表达模式，以研究脊髓神经发生的分子调控。已知ID家族成员是正作用的碱性螺旋-环-螺旋转录因子的负调控因子，它们在其他系统中作为细胞分化的抑制因子发挥作用。ID蛋白也与细胞周期控制有关，它们是参与指定脊髓轴的分子的靶基因。申请人建议测试这样的假设，即这些基因中的一个或多个在增殖的脊髓神经上皮细胞中表达，当细胞退出细胞周期并开始分化时，表达下调。初步研究的结果将用于指导未来对神经发生及其停止的模式调控的研究，这些事件对正常的神经系统发育至关重要。神经发生缺陷可能是人类先天性脑异常和/或智力低下的潜在原因。

DESCRIPTION (provided by applicant): The large question motivating this specific project is to understand how developmental processes responsible for patterning the dorso-ventral axis of the vertebrate neural tube intersect with those regulating proliferation and neurogenesis. In the spinal cord neuron production and differentiation follows a ventral to dorsal gradient with motoneuron generation from basal plate precursors preceding differentiation of dorsal interneurons from alar precursor cells. Preliminary results suggest differences in cell generation time of the spinal cord neurepithelium that correlate with both developmental stage and position on the dorso-ventral axis. Experiments are proposed to further establish these differences and to explore regulation of these events at both embryological and molecular levels. The applicant proposes to test whether signals from the paraxial mesoderm and/or the ectodermal epithelium differentially influence cell generation time and neurogenesis in the dorsal and ventral regions of the spinal cord using experimental embryological methods. Cumulative BrdU labeling and immunocytochemistry will be used to estimate changes in cell cycle parameters and neuronal differentiation in normal and surgically manipulated embryos. Possible effects of surgical manipulations on cell death will be addressed using TUNEL assays. Molecular regulation of neurogenesis in the spinal cord will be studied by evaluating the expression patterns of four Id family members using in situ hybridization. Id family members are known to be negative regulators of the positive acting basic helix-loop-helix transcription factors, and they function in other systems as inhibitors of cell differentiation. Id proteins have also been implicated in cell cycle control and they are target genes of molecules involved in specifying the axes of the spinal cord. The applicant proposes to test the hypothesis that one or more of these genes is expressed in proliferating spinal cord neural epithelium, and that expression is down-regulated when cells withdraw from the cell cycle and begin to differentiate. The results of the initial studies will be used to guide future investigations on the patterned regulation of neurogenesis and its cessation, events that are critical for normal nervous system development. Deficiencies in neurogenesis may be underlying causes of human congenital brain abnormalities and/or mental retardation.