WNT3 AND AXIS FORMATION AND CNS DEVELOPMENT

在过去的十年里，Wnt基因家族的成员被证明在脊椎动物胚胎中具有轴诱导活性。不幸的是，没有任何表达或遗传数据表明这些基因在正常发育中扮演着这种角色。然而，最近发现Wnt基因Wnt-3在原肠形成之前就表达了，并且该基因突变纯合子的小鼠缺乏体轴。尽管有这些期待已久的结果，但仍有许多问题没有得到回答。为此，建议进行以下实验：特异性靶点1.确定Wnt-3突变小鼠胚胎体轴缺陷的基础。1.1将在组织学水平上检测Wnt-3在e5.5-e7.5胚胎中的表达。1.2将产生野生型和Wnt-3突变细胞的嵌合胚胎，以确定早期胚胎中哪些Wnt-3表达区域是体轴形成所必需的。1.3将WNT-3mRNA注入非洲爪哇胚胎，检测WNT-3的轴向诱导活性。确定Wnt-3和Axin(抑制物或轴的形成和Wnt信号通路的关系)在轴形成过程中的关系。2.1将产生Wnt-3/Axin双突变小鼠，以确定Axin-/-小鼠中存在的多个轴是否依赖于Wnt-3信号。具体目标3.确定Wnt-3在胚胎发育过程中的后期功能。3.1使用Wnt-3(Wnt-3c)的条件等位基因，基因产物将在原始条纹中被消除，以确定是否需要Wnt-3来维持原始条纹的形成。3.2利用Wnt-3c等位基因，Wnt-3将从神经管中被特异性地消除，以确定其在中枢神经系统模式中的作用。

Within the last decade, members of the Wnt gene family have been shown to possess axis-inducing activity in vertebrate embryos. Unfortunately, there is no expression or genetic data that would suggest that these genes play this role in normal development. Very recently, however, it was found that a Wnt gene, Wnt-3, is expressed prior to gastrulation and that mice homozygous for a mutation at this locus lack a body axis. Notwithstanding these long awaited results, there are many questions that remain unanswered. Therefore the following experiments are proposed: Specific aim number 1. Determine the basis for the body axis defects in Wnt-3 mutant mouse embryos. 1.1 Wnt-3 expression will be examined at the histological level in E5.5-E7.5 embryos. 1.2 Chimeric embryos derived from wildtype and Wnt-3 mutant cells will be generated to determine which Wnt-3 expressing regions from early embryos are essential for body axis formation. 1.3 The axis inducing activity of Wnt-3 will be tested by injected Wnt-3 mRNA into Xenopus embryos. Specific aim number 2. Determine the relationship between Wnt-3 and Axin ( a repressor or axis formation and of the Wnt signaling pathway) in the process of axis formation. 2.1 Wnt-3/Axin double mutant mice will be generated to determine whether the supernumerary axes present in Axin-/- mice is dependent of Wnt-3 signaling. Specific aim number 3. Determine later functions of Wnt-3 during embryogenesis. 3.1 Using a conditional allele of Wnt-3 (Wnt-3c) the gene product will be eliminated in the primitive streak to determine whether Wnt-3 is required for the maintenance of primitive streak formation. 3.2 Using the Wnt-3c allele, Wnt-3 will be eliminated specifically from the neural tube to determine its role in patterning the central nervous system.