母源AXIN2基因变异对子代胚胎发育及WNT信号转导途径的影响

Maternal gene plays important role in the development of offspring. However, the mechanism of maternal gene remains unknown. Our prior study have: ①found that AXIN2 gene mutation is an important associated factor for female patients give birth to congenital defect offsprings. ②found that AXIN2 protein is down-regulated and the key protein β-catenin is up-regulated in the granulosa cells of those female patients and their congenital defected fetuses.③successfully constructed AXIN2 mutation C57BL/6 mouse model. ④ found that F1 generation AXIN2 mutation mice occurred the congenital phenotype of prehensile tail.And when F1 AXIN2 mutant female mice paired to wildtype male mice, their offspring suffered from open neural tube defects,abdominal wall defect,hydrocephalus,prehensile tail,intrauterine death and skeletal abnormality . The objective of this study is to investigate the variant effects and mechanism of maternal AXIN2 gene defect and key factors(WNTs、GSK3β、β-catenin,et al.) in WNT pathway through embryonic development via molecular,celluar and animal research level and research view. And to estimate the repair effect of wildtype AXIN2-mRNA in damaged embryo neural stem cell causing by maternal AXIN2 gene defect.This study has a de novo source of innovation and will provide important clues for understanding ,diagnosis and treatment for reproductive and congenital diseases.

母源基因在调控子代发育中发挥重要作用，但其作用机制尚不明。本项目前期研究：①在反复生育先天发育缺陷子代的女性中发现AXIN2基因突变；②证实该类女性卵泡颗粒细胞和其先天发育缺陷的子代胎儿病例中AXIN2表达均下调，WNT信号通路关键蛋白β-catenin均显著上调；③成功构建了AXIN2突变C57BL/6小鼠模型；④发现F1代杂合突变小鼠发生明显卷尾表型，且F1代杂合突变雌鼠与野生雄鼠配对生育的子代胚胎发生开放性神经管缺陷、腹壁缺损、脑积水、胎死宫内、卷尾及骨骼畸形等异常表型。本研究拟从细胞、分子、动物水平及正面和反面角度阐明母源AXIN2基因变异对子代发育障碍的影响；探讨母源AXIN2基因缺陷时WNT/β-catenin信号通路对胚胎发育的调节机制；并摸索应用活力型AXIN2-mRNA修复母源AXIN2基因缺陷引起的胚胎神经干细胞损伤，为阐明生育障碍和先天性疾病的发生机制及诊治提供线索。

母源基因在调控子代发育中发挥重要作用，但其作用机制尚不明。在本研究中，我们在反复生育先天发育缺陷子代的女性中发现AXIN2基因突变，通过细胞实验和动物模型探讨了AXIN2基因可能的致病机制。既往研究显示AXIN2作为β-catenin 降解复合体的一部分，可能通过限制Wnt/β-catenin信号通路的活性，在肿瘤发生和胚胎发育的过程中发挥重要作用。我们的研究结果表明：①AXIN2基因沉默通过调节线粒体相关的凋亡信号通路，在促进胚胎干细胞增殖和减少凋亡中发挥重要作用。AXIN2沉默的功能依赖于Wnt/β-catenin信号通路的调节，包括激活β-catenin/TCF4复合体和增强下游分子cyclin D1、c-myc和c-jun的表达。②证实斑马鱼的 axin2基因存在母源性表达，通过CRISPR-Cas9技术成功构建斑马鱼axin2杂合突变、纯合突变及基因敲除突变体模型。③对不同时期的斑马鱼axin2突变体胚胎进行显微形态学观察，发现斑马鱼axin2基因缺陷，表达产物缺失可导致子代胚胎发育异常，出现发育迟缓、心包水肿甚至胚胎死亡等异常表型。④对不同时期的斑马鱼突变体胚胎进行转录组测序，通过富集差异基因和通路后发现axin2的母源性缺失的转录组水平在胚层形成期呈现明显的异常，KEGG分析共发现14 条与人 AXIN2 基因相关的信号通路，其中Wnt signaling pathway受到直接影响，其余13条信号通路均是包含（或部分包含）了 Wnt signaling pathway。GO分析结果提示negative regulation of canonical Wnt signaling pathway、heart development、dorsal/ventral pattern formation 有差异基因富集，进一步筛选后考虑斑马鱼 apc2、bambia、ccnd1、dkk1b、fzd2、fzd8b、mych、notum1a、smad3a、sox17、tcf7l2、tp53、wnt11、wnt5b、frzb、rspo3、szl、tpbga这些基因在斑马鱼axin2母源性缺失的过程中发挥着重要作用。上述结果为了解AXIN2在人类生育障碍和先天性疾病的发生机制中的作用和诊治提供科学依据。