The Function Research Of CHD8 In The Embryonic Stem Cell And Neuroectoderm Differentiation

CHD8 protein,a member of the ATP-dependent chromatin remodeling factor CHD family,is widely expressed in the brain.And the CHD8 homozygous knockout in mouse resulted in embryo death,indicating the important role of CHD8 in early embryo development.With the development of research and technology,a large number of whole exome sequencing have been explored in autism patients which identified CHD8 was one of the high-risk mutation genes in autism spectrum disorders.The autistic patients with CHD8 mutations always displayed deletion or insertion of the CHD8 allele leads to CHD8 haploinsufficiency.Although the basic function and mechanisms of CHD8 are poorly understood.Human embryonic stem cells,derived from the inner cell mass of blastocysts,possess indefinite proliferative capacity and can differentiate into all three germ layers cell type which can serve as a good development model in vitro to research the key events in early embryonic development and to better understand the signaling pathways,epigenetic and cell state change in embryonic development process.What’s more,embryonic stem cells can also be used as a good tool for disease model to mutant disease related-genes by gene editing techniques.In vitro differentiation to simulate the process of disease occurrence helps people to study the process and mechanism of disease occurrence,and provides ideas or therapeutic targets for the treatment of disease.Some studies based on CHD8 heterozygote and knockdown models have been reported to show that down-regulation of CHD8 expression can be associated with abnormal expression of autism-related genes as well as genes involved in neurodevelopmental processes and the heterozygous mice were also found to exhibit autism-related traits and the ability to promote the proliferation of neural progenitor cells.However,the function of CHD8 in embryonic development and the mechanisms of CHD8 in autism occurrence remain unclear.Especially CHD8 complete knockout embryonic stem cells have not been obtained,the remaining CHD8 in these models can still perform some functions.Thus,the CHD8 complete knockout embryonic stem cell model is emergingly required.Here,we generate the CHD8 knockout human embryonic stem cells by CRISPR/Cas9 technology and characterize the effect of complete loss-of-function of CHD8 on pluripotency maintenance and lineage determination by utilizing efficient directed differentiation protocols.The results showed that the deletion of CHD8 gene had no effect on the self-renewal and maintenance of human embryonic stem cells,but slightly increased the level of apoptosis and had a slight effect on the biological processes of embryonic stem cells such as proliferation and cell cycle,with promoting cell proliferation and changing G1 and G2/M phases of the cell cycle.Interestingly,in vitro spontaneous differentiation in embryoid body formation experiments showed that the deletion of CHD8 specifically affected the expression of neuroectoderm gene PAX6 but did not affect the expression of endoderm and mesoderm genes.By the direct differentiation assay towards definitive endoderm and neuroectoderm,it was further confirmed that the loss of CHD8 specifically led to the deficiency of neuroectoderm differentiation,resulting in the failure of neuroectoderm differentiation and a large number of cell deaths during the differentiation process.RNA-seq analysis based on embryonic stem cells and differentiation neural progenitor cells also indicated CHD8 did not alter the expression of pluripotent genes in stem cell stage,but in neural progenitor cells depletion of CHD8 induced the abnormal expression of the apoptosis genes and suppressed neuroectoderm-related genes.These results provide the evidence that CHD8 plays an essential role in the pluripotency exit and early neuroectoderm differentiation as well as the regulation of apoptosis during neurogenesis,and thus offer new insights into the pathological role of role of CHD8.