The Study Of The Effects Of Embigin Gene On The Migration Of Enteric Neural Crest-derived Cells

Part Ⅰ:Establishment of Emb knockout mice and its developmental disorders of enteric nervous system.Objective:Established a stable heritable mouse model with Emb gene knockout and detected the development of enteric nervous system.Methods:CRISPR-Cas9 genome editing tool was used to construct Emb knockout mice.Western blot and RT-qPCR experiments were used to detect the mRNA and protein expression of Emb gene in the gut of their offspring.The developmental disorders of enteric nervous system in knockout mice were studied from the aspects of morphology and immunofluorescence histochemistry.Results:we constructed an Emb knockout mouse model and verified the decrease of corresponding mRNA and protein expression in colon through a series of experiments.We found that the weight and appearance of mutant mice were no different from those of wild type mice,but Emb-/-die at or before birth.In addition,the Emb-/-mice displayed an intestinal blockage phenotype with an abnormal accumulation of material in the gut.Histological sections showed the defect of enteric neural crest-derived cells in the distal colon by immunofluorescence.Conclusion:The Emb knockout mouse model was successfully constructed,and it was found that Emb-/-mice had Hirschsprung’s disease phenotype and defect of enteric neural crest-derived cells in distal colon.Part Ⅱ:The Study of the disorders of enteric neural crest-derived cells migration in Emb knockout mice.Objective:Explore the migration ability of enteric neural crest-derived cells in Emb knockout mice in vitro.Methods:For cell migration assay,E11.5 wild type and M-deficient mouse guts(from stomach to ileocecal junction)were placed on a filter membrane.Explant was cultured for 48h and detected the migration of enteric neural crest-derived cells by immunofluorescence.Results:In vitro experiment,we found that the migration distance of enteric neural crest-derived cells in Emb-/-mice was significantly less than that in the normal control group,although the number of neural crest-derived cells within the range of 0～100μm from the explant edge was slightly more than that of the normal control group,the total number of cells was less.In contrast,there was no significant difference in total cell numbers and migratory distance between the Emb+/-mice and the normal control group.We also observed marked aggregation of neuronal fibers in wild type cells,while mutant enteric neural crest-derived cells had more random protrusions.Conclusion:Emb gene is necessary for the migration and axonal growth of enteric neural crest-derived cells.In Emb knockout mice,the migration ability of neural crest-derived cells was impaired,the number and distance of migration decreased,and axonal growth was limited.Part Ⅲ:Role of Emb in speed and directionality of enteric neural crest cellsObjective:Enteric neural crest cells were labeled with an fluorescein protein in Sox 10 transgenic mice.And recorded the rostral-to-caudal migration of enteric neural crest cells in Emb knockout mice using this model.Methods:The Cre-loxp system was used to image the enteric neural crest cells in Emb knockout mice.Then the migration of neural cells was imaging by confocal microscope.Results:Sox10-CreER mice were mated with Rosa-tdTomato mice.After injecting tamoxifen into pregnant mice,red fluorescence was observed in the gut of embryos.Detected enteric neural crest-derived cells migration along the gut by live imaging.Conclusion:Sox10 transgenic fluorescent mice were successfully constructed,and their intestinal neural crest cells specifically expressed red fluorescence.Lack of homozygosity mice,EMB knockout mice specifically expressing red fluorescent protein in enteric neural crest-derived cells have not been obtained,but the feasibility of time-lapse imaging has been verified...