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细胞内因和外因共同控制的遗传程序精确地完成。我们以视网膜为模型系统，研究了控制中枢神经系统层压结构的分子机制。之前我们已经表明；1) Wnt2b是一种分泌信号分子，可以在旋转培养条件下诱导单解离的视网膜祖细胞形成正确的层流。2)在正常发育过程中，Wnt2b调控睫状体边缘区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周围视网膜干细胞维持的下游分子级联。首先，我们发现即使在Notch信号被阻断的情况下，Wnt2b也能维持视网膜祖细胞的未分化状态，这一直被认为在维持视网膜祖细胞的未分化状态中发挥着重要作用。Wnt2b可以不依赖Notch信号而下调多个前神经bHLH基因的表达，在外源启动子的控制下强制表达Cath5，从而抑制Wnt2b对神经元分化的负面影响。这些结果表明，Wnt2b通过降低前神经源性基因和神经源性基因的表达来维持边缘祖细胞的初始状态，从而阻止这些细胞进入由前神经源性基因和Notch调控的分化级联。为了进一步研究阻断原基因功能的精确分子级联，我们对来自wnt应答和非wnt应答细胞的文库进行了减法筛选。我们获得了几个响应Wnt信号激活而被激活的基因。然后，我们通过原位杂交检测了这些wnt应答基因在发育过程中的表达模式。值得注意的是，这些基因中的许多实际上在含有纤毛边缘区的干细胞中表达，这支持了我们的假设，即Wnt信号在这种特殊的细胞类型中起作用。我们还发现HES1作为Notch信号的下游效应因子，在视网膜干细胞中受到Wnt信号的调控，并且HES1的活性对于视网膜干细胞的维持是必要和充分的。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