Neural Tube Defects in Disheveled Mutant Mice

描述（由申请人提供）：神经管缺陷（NTD）是人类常见的出生缺陷。虽然已知遗传易感性因素与人类NTD相关，但尚未确定。小鼠可作为研究神经管畸形相关遗传因素和神经管闭合机制的模型系统。在研究具有三种小鼠Disheveled（Dvl）基因中的每一种的失活的小鼠的过程中，我们发现Dvl 2-/-和Dvl 1-/-Dvl 2-/-显示NTD。Disheveled是进化上保守的Wnt/wingless信号转导途径和细胞平面极性（PCP）途径中的一个重要基因。所有真核生物Dvl蛋白都含有三个高度保守的结构域：DIX、PDZ和DEP。在果蝇和非洲爪蟾中的研究表明，虽然DIX和DEP结构域的N-末端部分是Wnt途径信号传导所必需的，但PDZ和C-末端DEP结构域是PCP/会聚延伸途径所必需的。Wnt或PCP途径的破坏可能导致NTD。我们建议确定Dvl蛋白在神经管关闭过程中的空间和时间要求，以及Dvl突变小鼠所显示的NTDs是否是由Writ信号传导缺陷和/或PCP通路缺陷引起的。我们将使用以下具体目标。1)为了进一步表征负责Dvl 1/2突变体中NTD的Dvl依赖性途径，我们将在野生型和Dvl 1/2双突变体中的神经管闭合期间在细胞中和体内检查Wnt和PCP/会聚延伸途径信号传导。2)我们将测试环尾/斜视（Stbm）和融合/轴蛋白与Dvl 1和Dvl 2在神经管闭合中的遗传相互作用，以分别在遗传上区分PCP和Wnt通路效应。3)为了确定神经管中需要Dvl 2功能用于正常闭合的位点，我们将产生具有Dvl 2的特异性功能丧失或Dvl 2以空间和时间限制模式特异性表达的小鼠。将评估这些突变对野生型和Dvl突变体中神经管闭合的影响。4)为了确定神经管闭合所需的Dvl 2蛋白的结构域，我们将使用BAC转基因策略在小鼠中产生Dvl 2突变体的等位基因系列。Dvl 2中的精确突变将使用细菌中BAC的同源重组来工程化，并且所得突变体Dvl 2BAC将用于产生转基因小鼠。将评估这些突变对Dvl -/-Dvl 2-/-突变体中神经管闭合的影响。

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.