Neural Tube Defects in Disheveled Mutant Mice

描述(申请人提供)：神经管缺陷(NTD)是人类常见的出生缺陷。虽然已知遗传易感因素与人类NTDS有关，但它们尚未被识别。该小鼠可作为研究NTDS相关遗传因素和神经管闭合机制的模型系统。在研究三种小鼠杂乱(DVL)基因失活的小鼠过程中，我们发现Dvl2-/-和Dvl1-/-Dvl2-/-显示NTD。Disheveled是进化上保守的Wnt/Wingless信号转导通路和平面细胞极性(PCP)通路中的一个重要基因。所有真核DVL蛋白都含有三个高度保守的结构域：DIX、PDZ和DEP。对果蝇和非洲爪哇的研究表明，虽然DEP结构域的DIX和N-末端部分是Wnt途径信号所必需的，但PDZ和C-末端DEP结构域对于PCP/收敛延伸途径是必不可少的。Wnt或PCP通路的中断可能导致NTDS。我们建议确定DVL蛋白在神经管关闭过程中的空间和时间需求，以及DVL突变小鼠显示的NTDS是否由WRIT信号缺陷和/或PCP途径缺陷引起。我们将使用以下具体目标。1)为了进一步确定DVL 1/2突变体中导致NTDS的依赖于DVL的通路，我们将检测野生型和DVL 1/2双突变体神经管关闭过程中细胞和体内的Wnt和PCP/收敛延伸通路信号。2)我们将在神经管闭合过程中检测环尾/斜视(Stbm)和FUSED/Axin与Dvl1和Dvl2之间的遗传交互作用，以分别从基因上区分PCP和Wnt通路的影响。3)为了确定神经管中需要Dvl2功能才能正常闭合的部位，我们将建立Dvl2功能特定缺失或Dvl2在空间和时间受限模式下表达的小鼠。这些突变对野生型和DVL突变的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蛋白的结构域，我们将使用BAC转基因策略在小鼠身上产生一系列Dvl2突变的等位基因。Dvl2的精确突变将利用细菌中BAC的同源重组进行工程设计，由此产生的突变Dvl2 BAC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.