Project III - Modeling meningomyelocele alleles and response to folic acid diet in mouse

项目III模拟小鼠脑膜脊髓膨出的等位基因和对叶酸饮食的反应 计划项目拨款的总体假设是从头基因突变与脑膜脊髓膨出有关 (Mm)风险以及叶酸(FA)通过染色质可及性和基因调控影响基因组 表情，来改变这种风险。小鼠被认为是人类神经管缺陷的金标准模型 (NTDS)，因为哺乳动物的发育高度保守，导致类似类型的头骨和尾骨 NTDS，利用相似的基因，并对相似的环境因素做出反应。鼠标一直是无价之宝 在定义神经管关闭所需的基因方面，目前已有250多个基因，并强调了 精确的细胞增殖、肌动蛋白调控、平面细胞极性/WNT信号、纤毛/刺猬 信号转导和FA代谢。许多小鼠NTD模型显示出等位基因特异性效应，例如， 纯合子缺失小鼠是胚胎致死的，杂合子缺失小鼠是健康的，但特定的点突变， 有些杂合子，有些纯合子，表现为NTD。在许多这样的小鼠模型中，NTD通常发生在较少的 超过100%的后代，即使在完全纯的遗传背景下，也反映了背景无关的部分 洞察力。和人类一样，NTD表型的外显率和表现力也会受到FA的影响 到目前为止，大约一半的测试基因显示出FA的反应性。FA已在 自1998年以来美国饮食中的谷物，调节核苷酸的生物合成和S-腺苷蛋氨酸的普遍存在 DNA甲基化的甲基供体。为了解释这些观察，我们假设NTD风险是 由一组核心基因的突变建立，风险由FA依赖的 表观基因组。项目III已经收集了以下初步数据：1)执行隐性ENU- 突变筛选神经管闭合所必需的基因。2)组装了越来越多的基因集合 以及来自30多个NTD突变体的等位基因。3]显示了潜在的初级纤毛基因突变 几个小鼠NTD突变体。4]证实了FA对小鼠NTDS的外显率和表达能力的影响。 5)开发了实时观察哺乳动物神经管闭合的活细胞成像平台。6]建模 人MM基因WLS在小鼠脑脊膜膨出中的表达。这项建议的重点是评估基因和 项目I和项目II中出现的等位基因是MM的危险因素，以及 甲基组和转录组可以影响这种风险。总而言之，项目III的目标是定义 哺乳动物MM基因突变与FA对外显性和外显性的影响 表现力。 目的1.建立人类脑膜脊髓膨出(MM)变异和遗传交互作用的模型，以评估其表达能力。 目的2.在逐个基因的基础上评估叶酸对NTD表达的影响。 目的3.在单细胞转录组、甲基组水平上评估叶酸对神经管的影响 和染色质无障碍景观。

Abstract – Project III Modeling meningomyelocele alleles and response to folic acid diet in mouse The overall hypothesis of the Program Project grant is that de novo mutations contribute to meningomyelocele (MM) risk and that folic acid (FA) influences the genome through chromatin accessibility and modulation of gene expression, to alter this risk. The mouse is considered the gold standard model for human neural tube defects (NTDs), because development is highly conserved in mammals, leading to similar types of cranial and caudal NTDs, utilizing similar genes, and responding to similar environmental factors. The mouse has been invaluable in defining genes required for neural tube closure, now numbering over 250 genes, and highlighting the need for exquisite precision of cell proliferation, actin regulation, planar cell polarity/WNT signaling, cilia/Hedgehog signaling, and FA metabolism. Many mouse NTD models show allele-specific effects, where for example, homozygous null mice are embryonic lethal, heterozygous null mice are healthy, but specific point mutations, some heterozygous, some homozygous, show NTD. In many of these mouse models, NTDs often occur in less than 100% of offspring, even in an entirely pure genetic background, reflecting background-independent partial penetrance. Like in human, the penetrance and expressivity of NTD phenotypes can be influenced by FA exposure, with about half of genes tested to date showing FA responsiveness. FA has been supplemented in grains in the US diet since 1998, regulating biosynthesis of nucleotides and S-adenosylmethionine, the universal methyl donor for DNA methylation. To account for these observations, we hypothesize that NTD risk is established by mutations in a core set of genes, with risk modified by FA-dependent changes in the epigenome. Project III has already assembled the following preliminary data: 1] Performed recessive ENU- mutagenesis screens for genes essential for neural tube closure. 2] Assembled a growing collection of genes and alleles from more than 30 NTD mutants. 3] Demonstrated mutations in primary ciliary genes underlying several mouse NTD mutants. 4] Demonstrated an impact of FA on penetrance and expressivity of murine NTDs. 5] Developed live-cell imaging platform to observe mammalian neural tube closure in real-time. 6] Modeled meningomyelocele in mouse for the human MM gene WLS. This proposal focuses on evaluating genes and alleles emerging from Project I and II as risk factors for MM, as well as the FA-dependent changes in the methylome and transcriptome that can influence this risk. Altogether, the goal of Project III is to define the relationships and interplay between mammalian MM mutations and FA influences on penetrance and expressivity. Aim 1. Model human meningomyelocele (MM) variants and genetic interactions to assess expressivity. Aim 2. Assess impact of folic acid on NTD expressivity on a gene-by-gene basis. Aim 3. Assess impact of folic acid on the neural tube at the level of single cell transcriptome, methylome and chromatin accessibility landscape.