Study Of The Effect Of JAG1 On Enteric Nervous System Devlopment And Underlying Mechanisms

Aims This study aimed to explore the effect and potential underlying mechanisms of JAG1 on the development of enteric nervous system in a JAG1 knocking down zebrafish model and cytological experiments.The effect of JAG1 on the proliferation and migration of intestinal neural progenitor cells and its internal molecular mechanism were also studied.We tried to illustrate whether JAG1 can affect the proliferation and migration of intestinal neural progenitor cells through Notch pathway,and then affect the normal formation of ENS,resulting in the occurrence of Hirschsprung's disease.It might provide a new direction for further understanding the pathogenesis of Hirschsprung's disease.Methods Firstly,the expression of JAG1 protein in normal colon tissue from non-Hirschsprung's disease patients and embryonal mouse gut was verified.The expression level of Jag1 and Notch pathway signaling molecules during development was detected by q RT-PCR.Then,by constructing zebrafish JAG1-SplicingMorpholino probe to knock down the expression of jag1 m RNA in zebrafish,the mature neurons expression protein Hu,was labeled by immunofluorescence staining to observe the enteric nervous system development.Finally,the intestinal neural progenitor cells were obtained from the intestine of embryonic rats(E16.5 days)and cultured,and then interfered with recombinant proteins of JAG1,JAG1 Si RNA and DAPT(Notch pathway inhibitor).The migration ability of enteric neural progenitor cells was evaluated by Transwell test,the level of cell proliferation and differentiation was measured by EDU staining,and the expression level of Notch signaling pathway molecules were compared among different treatment groups.Results 1.There was no expression of JAG1 protein in myenteric plexus or submucosal plexus in normal colon tissue or adult rat colon tissue,while a small amount of JAG1 protein was expressed in stroma.The immunohistochemical results of embryonic rat intestine on E15 showed that the staining range of JAG1 was diffuse and the staining was light,but part of the range overlaps with PGP9.5.2.The Notch signaling molecules dynamically expressed during the development of enteric nervous system: the expression of Jag1,Notch1,Sox 10 at E11.5 days was significantly lower than that at E13.5 days and E15.5 days,while the expression levels of Dll1,Hes1 and Mash1 increased gradually along the development.The expression levels of Hey1 and GDNF reached the peak at E13.5 days 3.After using JAG1 Splicing Morpholino to knock down the expression level of jag1 m RNA in zebrafish,the Hu protein staining was not found in the whole intestine of some zebrafish,suggesting that no mature ganglion cells were found in the whole intestine.In some zebrafish,the Hu protein staining was absent only in the distal end of the intestine and indicates that the distal intestinal nerve development is abnormal 4.Cytological experiments confirmed that JAG1 can inhibit the expression of Hey-1 and Mash-1 and promote the proliferation and migration of intestinal neural progenitor cells through Notch signaling pathway.This process could be blocked by Notch pathway inhibitor DAPT.Conclusions 1.The expression of JAG1 protein and Notch pathway signaling molecules changed dynamically during embryonic intestinal development.2.The injection of JAG1-Splicing morpholino could cause Hirshchprung disaesa phenotye in zebrafish,suggesting that JAG1 was involved in the regulation of intestinal nerve system development.3.JAG1 could regulate the expression of Hey-1 and Mash-1 and promote the proliferation and migration of intestinal neural progenitor cells,suggesting that JAG1 may be involved in the regulation of intestinal nerve development by negatively regulating the expression of Hey-1 and Mash-1.