Research On Molecular Mechanisms Of The SHROOM3 Gene Mutations Leading To The Neural Tube Defects In Macaca

The neural tube（neural tube）is a part of the nervous system that originates from the neuroectoderm of the embryonic body.During embryonic development,the notochord induces the neuroectoderm on the dorsal midline to form a neural plate and develop into a neural tube.Neural tube defects are also called neural tube defects（NTDs）.During early embryonic development,the neural tube cannot be closed or closed completely.It is a common congenital malformation second only to congenital heart disease in incidence.The cause of the disease is very complex,including environmental and genetic factors and their interactions.At present,the incidence of NTDs can be effectively reduced by 70%by supplementing proper amount of folic acid before pregnancy and early pregnancy in clinical practice.In order to find a more effective treatment method,a large number of studies have focused on the genotype study of patients with NTDs and found that the SHROOM3 gene is involved in the process of neural tube closure.The animal models of NTDs established by mice,zebrafish and Xenopus also further confirmed that Shroom3 gene affects neural tube development.However,existing studies have found that neural tube defects caused by SHROOM3 gene deletion cannot be prevented and treated by folic acid compensation.It has even been found that too much folic acid compensation can lead to increased lethality of embryos with SHROOM3 gene loss.Due to the lack of suitable animal models,the role and mechanism of this gene in the development of NTDs patients are still unclear.Non-human primates and human brains are highly similar in physiological structure,developmental space and timing.Therefore,the establishment of artificially induced primate NTDs models will be used to study the pathogenesis of human NTDs,drugs and stem cell transplantation for preclinical treatment The effectiveness and safety research provides an important research platform.In order to explore how the SHROOM3 gene mutation affects neural tube defects in primates,we carried out related experiments.The main research contents and results are as follows:1.Use CRISPR/Cas9 gene editing technology to construct SHROOM3 gene knockout cynomolgus monkey embryos and obtain SHROOM3 gene mutation cynomolgus monkey embryonic stem cells（cmESC）.The sgRNA was designed for exon 5 of the cynomolgus SHROOM3 gene,and the targeting efficiency was verified at the embryo level.The two most efficient sgRNA and sp Cas9 mRNA were selected and injected into the cynomolgus monkey 1-cell stage embryos to obtain SHROOM3 gene knockout Embryo.After confirming that embryos with SHROOM3 gene deletion can be obtained,the embryos that have developed into blastocysts are used to establish ES cell lines to obtain cm ES with SHROOM3 gene mutation.2.The expression time window of SHROOM3 gene in the process of neural differentiation in vitroAfter establishing an in vitro neuroepithelial stem differentiation system,five time points were set in the process to explore the time window and expression level of SHROOM3 gene expression during the entire development process.The experimental results show that the SHROOM3 gene has been expressed in embryonic stem cells and continued to be expressed during the process of neural differentiation in vitro,and the SHROOM3 gene expression level reached its peak during the formation of Rosettes structure.These data indicate that the SHROOM3 gene plays a very important role in the development of the nervous system,and it is speculated that the closure of the neural tube has an important relationship with the SHROOM3 gene.3.To study the in vitro neural differentiation potential of normal and SHROOM3gene knockout cmESC.Based on the establishment of neuroepithelial stem cell differentiation system in vitro,ES was induced into neuroepithelial stem cells（NESC）.Through immunofluorescence staining,RT-PCR,Western blot and q PCR techniques,ES,Neural Rosettes（NRs）were obtained from the in vitro differentiation of embryonic stem cells in wild type（WT）and SHROOM3^mut-ESC,Neural tube-like structures（NTs）and cells differentiated by NESC in the later stage were tested at these time points to analyze the differentiation ability of normal and mutant cells in vitro.It was found that SHROOM3 promotes the neural-directed differentiation of ES cells;the loss of SHROOM3 gene affects the ability of embryonic stem cells to differentiate into the ectoderm;the loss of SHROOM3 gene enhances the ability of neuroepithelial stem cells to differentiate into neurons and glial cells,but reduces expression of OTX2which effect the plasticity of the brain.Without SHROOM3,Wnt signaling has not been significantly disrupted;SHROOM3 gene deletion will affect cell connections,cytoskeleton and cell movement.Conclusion:The SHROOM3 gene mutation of cmesc can affect the cell growth rate,cell morphology and the ability of ectodermal differentiation of cmesc in vitro.SHROOM3 gene mutation may affect the potential of neural differentiation,but it does not directly block Wnt signaling pathway to affect neural tube closure.The specific molecular mechanism of neural tube defects caused by SHROOM3 gene mutation remains to be further studied.