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

中枢神经系统层状结构形成控制机制的研究

基本信息

批准号：
16570184
负责人：
NAKAGAWA Shinichi
金额：
$ 2.3万
依托单位：
The Institute of Physical and Chemical Research
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-16570184/
关键词：
Retina Wnt2b Notch HES Stem cells Laminated structure Differentiation Proneural gene Wnt グリア神経細胞神経前駆細胞プロニューラル遺伝子 Delta 細胞周期層形成

项目摘要

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

在中枢神经系统的组织发生过程中，必须根据细胞内在和外在因素控制的遗传程序精确地执行大量的过程。我们以视网膜为模型系统，研究了控制中枢神经系统层状结构的分子机制。我们先前已经证明：1)Wnt2b是一种分泌型信号分子，在旋转培养条件下可以诱导单个分离的视网膜前体细胞正确地形成板层。2)Wnt2b在正常发育过程中对位于睫状缘的视网膜干细胞的增殖和分化具有调控作用。在本研究项目中，我们进一步研究了导致周边视网膜干细胞维持的下游分子级联。首先，我们发现Wnt2b即使在Notch信号被阻断的情况下也能维持未分化的前体细胞，这一直被认为是…More在维持视网膜祖细胞未分化状态中发挥作用。Wnt2b不依赖于Notch信号而下调多个原神经bHLH基因的表达，并且在外源启动子的控制下强制表达CAS5抑制了Wnt2b对神经元分化的负面影响。这些结果表明，Wnt2b通过减弱原神经和神经源性基因的表达来维持边缘前体细胞的幼稚状态，从而阻止这些细胞进入原神经基因和Notch调控的分化级联。为了进一步研究阻断原神经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消减筛选。我们获得了几个响应Wnt信号激活而激活的基因。然后，我们通过原位杂交检测了这些Wnt反应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是在含有干细胞的纤毛边缘区域表达的，这支持了我们的假设，即Wnt信号在这种特殊类型的细胞中起作用。我们还发现，HES1被认为是Notch信号的下游效应者，在视网膜干细胞中受Wnt信号的调节，HES1的活性是维持视网膜干细胞所必需的和充分的。较少