Investigation of the mechanisms controlling formation of the laminated structures in the central nervous system

在中枢神经系统的组织发生过程中，大量的过程必须按照由细胞内在和外在因素控制的遗传程序精确地执行。我们以视网膜为模型系统研究了中枢神经系统中控制层状结构的分子机制。此前，我们已经展示了1）分泌的信号分子Wnt2b可以在旋转培养条件下诱导单个解离的视网膜祖细胞正确形成层状结构。2)在正常发育过程中，Wnt2b控制位于睫状体边缘区的视网膜干细胞的增殖和分化。在本研究项目中，我们进一步研究了导致视网膜干细胞在外周视网膜维持的下游分子级联。首先，我们发现Wnt2b即使在Notch信号被阻断的条件下也能维持未分化的祖细胞，这一直被认为是 关于我们 在维持视网膜祖细胞的未分化状态中起作用。Wnt2b下调多个前神经bHLH基因的表达，独立于Notch信号传导，并且在外源启动子的控制下强制表达Cath5抑制了Wnt2b对神经元分化的负面影响。这些结果表明，Wnt2b通过减弱前神经基因和神经源性基因的表达来维持边缘祖细胞的幼稚状态，从而阻止这些细胞进入由前神经基因和Notch调节的分化级联。我们对来源于Wnt应答和非Wnt应答细胞的文库进行了消减筛选。我们获得了响应于Wnt信号传导的激活而被激活的几个基因。然后，我们研究了这些Wnt反应基因的表达模式在发展过程中通过原位杂交。值得注意的是，这些基因中的许多实际上在含有干细胞的睫状边缘区中表达，这支持了我们的假设，即Wnt信号传导在这种专门的细胞类型中起作用。我们还发现，已被公认为Notch信号下游效应子的HES1在视网膜干细胞中受Wnt信号调节，并且HES1活性对于视网膜干细胞的维持是必要且足够的。少

During the histogenesis of the central nervous system, an enormous number of processes must be precisely executed according to the genetic program controlled by both cell-intrinsic and extrinsic factors. We have investigated the molecular mechanisms controlling laminated structures in the central nervous system using retina as a model system. Previously we have shown ; 1) Wnt2b, a secreted signaling molecule, can induce correct laminar formation from singly dissociated retinal progenitor cells in a rotation culture condition. 2) Wnt2b control proliferation and differentiation of the retina stem cells located in the ciliary marginal zones during normal development. In this research project, we further investigated downstream molecular cascade leading to the maintenance of the retinal stem cells at the peripheral retina.Firstly, we found that Wnt2b maintained the undifferentiated progenitor cells even under the conditions where Notch signaling was blocked, which has long been believed to … More play a role in maintaining undifferentiated state of retinal progenitor cells. Wnt2b downregulated the expression of multiple proneural bHLH genes independently of Notch signaling, and forced expression of Cath5 under the control of an exogenous promoter suppressed the negative effect of Wnt2b on neuronal differentiation. These result suggested that Wnt2b maintains the naive state of marginal progenitor cells by attenuating the expression of both proneural and neurogenic genes, thus preventing those cells from launching out into the differentiation cascade regulated by proneural genes and Notch.To further investigate the precise molecular cascade that blocks the function of proneural genes, we carried out subtractive screening of the library derived from Wnt-responding and non-Wnt-responding cells. We obtained several genes that are activated in response to the activation of the Wnt signaling. We then examined the expression pattern of these Wnt-responding genes during development by in situ hybridization. Notably, many of these genes were actually expressed in the stem cell-containing ciliary marginal zones, supporting our hypothesis that Wnt signaling is operating in this specialized cell type. We also found that HES1, which has been recognized as a downstream effector of Notch signaling, is regulated by Wnt signaling in the retinal stem cells, and that HES1 activity was necessary and sufficient for the maintenance of retinal stem cells. Less