SHROOM3基因缺失导致神经管畸形的分子机制研究

Neural tube defects (NTDs), seriously affect the health of the population, are severe congenital deformity due to failure of neural tube closure during early nervous system development. The cause of disease includes environment, nutritious, genetics and interaction among them. SHROOM3 gene, as a genetic factor, regulates neural tube development and loss of SHROOM3 causes NTDs. Due to lack of ideal animal model, how this gene controls brain development in primates, including in human, is still unknown. .Previously, we used clustered regularly interspaced short palindromic repeat sequences (CRISPR)-Cas9 gene editing system and constructed single guide RNA of SHROOM3. And gene targeting efficiency was analyzed on cellular and embryonic level. Importantly, a SHROOM3 knockout embryonic stem (ES) cell line was generated. In the present project, SHROOM3 knockout cynomolgus monkey embryos will be constructed. Pregnant monkeys will also be obtained through embryo transfer. Neural differentiation potential from ES cells will also be assessed in vitro. Neural tube closure and genes expression, and signal pathways such as PCP pathway will be analyzed in vitro. These results will be helpful to explain the pathological mechanism of NTDs caused by SHROOM3 gene knockout, and will supply important scientific clues to prevention and treatment of NTDs in human.

神经管畸形（Neural tube defects，NTDs）是神经管闭合不全造成的先天缺陷，严重影响人口健康。除环境因素外，遗传缺陷如SHROOM3基因的缺失也会导致NTDs的发生。由于缺乏合适的动物模型，人类遗传性NTDs发生的机制尚不清楚。前期我们已经利用CRISPR/Cas9基因修饰体系，构建了SHROOM3基因的sgRNA，在细胞及胚胎水平测试了打靶效率，并已建立了1株SHROOM3敲除的胚胎干细胞系。本项目拟在前期工作基础上：1，构建SHROOM3基因敲除的食蟹猴胚胎，通过胚胎移植得到怀孕猴；2，体外研究ES神经分化潜能，在体外探讨SHROOM3缺失导致神经管闭合不全、进而造成神经管缺陷的分子机制。研究结果将对人类NTDs的预防和治疗提供重要参考依据。

神经管缺陷（neural tube defects, NTDs）是由于胚胎发育过程中早期神经管闭合不全或者完全不闭合导致的。世界范围内发病率为千分之一，在我国发病率甚至高达千分之五左右，严重影响优生优育及人口健康。该病的发病机制十分复杂，包括环境、营养和遗传等因素及这些因素相互作用导致的。现已发现，补充叶酸可以有效预防70%左右的NTDs的发生，说明环境及营养因素是导致NTDs的重要因素。然而，叶酸补充不能防止全部NTDs的发生，提示遗传缺陷是另一个导致NTDs发生的重要因素。科学家们通过对小鼠和斑马鱼等动物模型的研究发现， SHROOM3、VANGL2和WNT5A这三个基因在平面细胞极性（planar cell polarity, PCP）通路中， 对神经褶向中间靠拢形成管状结构也就是神经管过程中起着很重要的作用。但是这些动物模型都不能很好的反映人类的NTDs，因此灵长类动物模型就显得至关重要。灵长类胚胎干细胞（ESCs）向神经分化的过程与体内胚胎发育具有极其类似的时间模式，因此，利用灵长类ESCs 在体外模拟情况下研究体内胚胎发育过程意义重大。.项目实施期间共发表SCI论文3篇，培养博硕士研究生及专业技术人员10余人。项目执行期间积极开展学术交流，共参加国内外学术会议10次。

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