The Function Research Of Dip2a In The Mouse Embryonic Stem Cell Neuronal Differentiation

DIP2A is a member of the Disco interacting protein homolog(DIP2)family,which are highly conserved in a variety of biological evolution,including DIP2 A,DIP2B and DIP2 C.Dip2a has been reported to be highly expressed in the nervous system of mouse embryos and adults,and its mutations and deletion are associated with a variety of neurological disorders,such as autism,Alzheimer’s disease,comorbidity anxiety,developmental dyslexia,schizophrenia,etc.In addition,studies have shown that Dip2 a knockout mice can reduce the postsynaptic density,resulting in mitochondrial metabolism disorders in the cerebral cortex and changes in the morphology of neurons in the hippocampus.However,the exact mechanisms of Dip2 a in neural differentiation remains unclear.Mouse embryonic stem cells are ideal models for studying gene function in vitro.Based on a homozygous Dip2 a gene knockout mouse embryonic stem cell line generated in our laboratory,we further investigated the effects of Dip2 a knockout on neural differentiation of mouse embryonic stem cells.The results showed that Dip2 a knockout after multiple passages had no effects on the maintenance of pluripotency of mouse embryonic stem cells,but it had a minor effects on the proliferation and cell cycle of embryonic stem cells,reducing cell proliferation and preventing cells from staying in G1 phase.Through N2B27 monolayer culture for neural differentiation,we found that Dip2 a knockout can affect the expression of nerve-related genes,resulting in significantly reduced expression of Sox1,Pax6,Nestin,Neurod1,β-Ⅲ Tubulin and Map2.Through in vitro KSR embryoid neural differentiation experiments,we further confirmed the observed the same phenotype during KSR differentiation,which was specifically manifested by reduced expression of nerve-related genes,weaker fluorescence intensity and smaller morphology of the formed embryoid Sox1-GFP.At the same time,we performed RNAseq on cells differentiated from wild type and Dip2 a knockout type.GO analysis showed that the terms associated with biological processes,such as nerve and synaptic development were highly enriched.KEGG pathway analysis showed the MYC targets v1 signaling pathway was mainly enriched.Heatmap showed the genes related to nerve differentiation and synaptic formation significantly down regulated.In this study,we used mouse embryonic stem cells as the model to investigate the function of Dip2 a knockout on the neuronal differentiation of mouse embryonic cells.Preliminary results demonstrated that Dip2 a knockout down regulated the expression of genes related to neural differentiation,such as synaptic formation,synaptic density and plasticity.Signaling pathways related to neural development,such as Wnt/β pathway was involved.This study lays a foundation for further research on the exact molecular mechanisms of Dip2 a in neural differentiation.