Neural Tube Defects in Disheveled Mutant Mice

描述（申请人提供）：神经管缺陷（NTDs）是人类常见的出生缺陷。虽然已知遗传易感性因素与人类被忽视热带病有关，但它们尚未被确定。该小鼠可作为研究ntd相关遗传因素和神经管闭合机制的模型系统。在研究三种小鼠Disheveled （Dvl）基因失活的小鼠过程中，我们发现Dvl2 -/-和Dvl1 -/- Dvl2 -/-显示NTDs。disheveld是进化保守的Wnt/无翼信号转导途径和平面细胞极性（PCP）途径中的一个重要基因。所有真核生物的Dvl蛋白都包含三个高度保守的结构域：DIX、PDZ和DEP。对果蝇和非洲爪蟾的研究表明，Wnt通路信号需要DIX和DEP结构域的n端部分，而PCP/收敛扩展通路则需要PDZ和c端DEP结构域。Wnt或PCP通路的破坏都可能导致NTDs。我们拟确定神经管闭合过程中对Dvl蛋白的时空需求，以及Dvl突变小鼠所显示的NTDs是否由Writ信号缺陷和/或PCP通路缺陷引起。我们将使用以下具体目标。1)为了进一步表征Dvl 1/2突变体中导致NTDs的Dvl依赖通路，我们将研究野生型和Dvl 1/2双突变体神经管闭合过程中细胞和体内的Wnt和PCP/趋同延伸通路信号。2)我们将检测环路尾/斜视（Stbm）和融合/轴蛋白与Dvl1和Dvl2在神经管闭合中的遗传相互作用，分别区分PCP和Wnt通路的作用。3)为了确定神经管中需要Dvl2功能才能正常闭合的部位，我们将产生Dvl2特异性功能丧失或在空间和时间限制模式下Dvl2特异性表达的小鼠。这些突变对野生型和Dvl突变体神经管闭合的影响将被评估。4)为了确定神经管闭合所需的Dvl2蛋白结构域，我们将使用BAC转基因策略在小鼠中产生一系列Dvl2等位基因突变体。将利用细菌中BACs的同源重组来设计Dvl2的精确突变，得到的突变Dvl2 BACs将用于生产转基因小鼠。这些突变对Dvl -/- dvl2 -/-突变的神经管闭合的影响将被评估。

DESCRIPTION (provided by applicant): Neural tube defects (NTDs) are common birth defects in humans. Although it is known that genetic susceptibility factors are associated with NTDs in humans, they have yet to be identified. The mouse can be used as a model system to study genetic factors associated with NTDs and the mechanism of neural tube closure. In the course of studying mice with inactivation of each of the three mouse Disheveled (Dvl) genes, we discovered that Dvl2 -/- and Dvl1 -/- Dvl2 -/- display NTDs. Disheveled is an important gene in the evolutionarily conserved Wnt/wingless signal transduction pathway and the planar cell polarity (PCP) pathways. All eukaryotic Dvl proteins contain three highly conserved domains: DIX, PDZ and DEP. Studies in Drosophila and Xenopus indicate that while the DIX and N-terminal portion of the DEP domain are required for Wnt pathway signaling, the PDZ and C-terminal DEP domain are essential for the PCP/convergent extension pathway. Disruption of either the Wnt or PCP pathways may result in NTDs. We propose to determine the spatial and temporal requirements for Dvl proteins during neural tube closure, and whether the NTDs displayed by Dvl mutant mice are caused by Writ signaling defects and/or PCP pathway defects. We will use the following specific aims. 1) To further characterize Dvl-dependent pathways responsible for the NTDs in Dvl 1/2 mutants, we will examine Wnt and PCP/convergent extension pathway signaling in cells and in vivo during neural tube closure in wild-type and Dvl 1/2 double mutants. 2) We will test for genetic interactions between Loop tail/strabismus (Stbm) and fused/axin with Dvl1 and Dvl2 in neural tube closure, to genetically distinguish PCP and Wnt pathway effects, respectively. 3) To determine the sites in neural tube that require Dvl2 function for normal closure, we will produce mice with specific loss-of-function of Dvl2 or specific expression of Dvl2 in spatially and temporally restricted patterns. The effect of these mutations on neural tube closure in wild type and Dvl mutants will be assessed. 4) To determine the domains of the Dvl2 protein that are required for neural tube closure, we will produce an allele series of Dvl2 mutants in mice using a BAC transgenic strategy. Precise mutations in Dvl2 will be engineered using homologous recombination of BACs in bacteria, and the resultant mutant Dvl2 BACs will be used to produce transgenic mice. The effect of these mutations on neural tube closure in Dvl -/-Dvl2 -/- mutants will be assessed.