Studies On The Innate Immune Function Of Notch1a And Notch3 And Their Effects On The Development Of Swimbladder In Zebrafish

Notch signaling pathway is an important signaling pathway for cell-to-cell communication.Notch receptor was first reporter in 1919,and it was named after notch because the deletion of this gene caused a notch in the wings of Drosophila melanogaster.Notch receptors have highly conserved structural homology between different species and even different receptors of the same species.So far,four Notch receptors have been found in zebrafish,which are Notch1a,Notch1b,Notch2,and Notch3.Notch signaling pathways exchange information through the binding of receptors and ligands between adjacent cells,thereby participating in the regulation of individuals growth,development,disease and immunity.In our laboratory,the homozygous phenotypes of zebrafish notch1a^-/-and notch3^-/-mutants had been observed.It was found that the larvae of mutants died from 13 to 17days post polymerization?dpf?and have abnormal swimbladder development from 5 dpf,so we studied the mechanism of Notch1a and Notch3 participating in?innate?immune response and affecting the development of swimbladder.In order to study the effects that Notch1a and Notch3 have on the innate immune functions of zebrafish,the zebrafish NF-?B promoter dual luciferase reporter gene expression vector was firstly constructed and its transcriptional activity was tested.Secondly,the junction molecules myd88 and traf6eukaryotic expression vectors in the Toll-like receptor?TLR?signaling pathway were constructed.Finally,the transcriptional activity of NF-?B family gene promoters activated by N1a ICD and N3ICD via My D88 or TRAF6 pathway was detected in HEK293T cell line using the dual luciferase reporter gene system.The following are the main experimental results of this study:?1?We successfully constructed five gene promoter reporter vectors of NF-?B family in zebrafish.It was found by the dual-luciferase detection system that in the resting state of HEK293T cells,the activity of the p GL3-rela-Enhancer reporter plasmid was 50-60times higher than that of the p GL3-Enhancer reporter plasmid,and the activity of the p GL3-nf?b2-Enhancer reporter plasmid was 7-8 times higher than that of the p GL3-Enhancer control group.The activity of the p GL3-relb-Enhancer and p GL3-rel-Enhancer groups was 2 times higher than that of the control group,which indicated the activity was significantly enhanced.The activity of p GL3-nf?b1-Enhancer group was markedly 0.1times lower than that of the control group.After HEK293T cells were stimulated by LPS,it was found that the activity of the recombinant plasmids in each group was significantly increased by 2-3 times compared with that of no LPS.?2?We successfully cloned full-length fragments of CDS p CMV-myd88 and p CMV-traf6 gene in zebrafish,and constructed the eukaryotic expression vectors of them.After overexpression of myd88 gene in zebrafish,the transcriptional activity of rela,relb and nf?b1 were significantly increased,about 2.5 times that of the control group.After overexpression of traf6 gene in zebrafish,the transcriptional activity of rela reporter gene was about 2 times that of the control group,the transcriptional activity of relb and rel was about 1.5 times that of the control group,and the transcriptional activity of nf?b1 was significantly increased,which is about 8 times that of the control group.?3?After overexpression of zebrafia N1a ICD in HEK293T cells,the transcriptional activity of rela gene promoter in NF-?B family triggered by myd88 or traf6 was not significantly affected.After co-tranfection of zebrafish N1a ICD and myd88 or traf6,the transcriptional activity of the nf?b1 gene promoter was significantly activated.After overexpression of zebrafia N3ICD in HEK293T cells,there was no significant effect on the transcriptional activity of rela gene promoter in NF-?B family triggered by myd88,but the transcriptional activity of rela gene promoter triggered by traf6 was significantly activated,and it was increased about 2 times.At the same time,the transcriptional activity of nf?b1 gene promoter was increased by 4 times compared with the overexpression of myd88,and the transcriptional activity of nf?b1 gene promoter triggered by traf6 was also significantly increased by about 2 times.And Notch1a can significantly activate the transcriptional activity of nf?b1.Notch3 can significantly activate the transcriptional activity of rela and nf?b1.It was found that zebrafish N1a ICD and N3ICD can significantly activate the expression of p GL3-tp53.Our results show that the Notch1a and Notch3 of zebrafish can activate the NF-?B signaling pathway through the linker molecules myd88 and/or traf6,and then affect the expression of downstream genes such as cytokines to affect immune function.NF-?B transcription factor is a key regulator of inflammation and immune response,and it is also closely related to tumorigenesis.Studies have shown that the NF-?B signaling pathway may affect the mechanism of tumor cells by regulating the immune response.By observing the notch1a^-/-and notch3^-/-mutant zebrafish phenotypes,we found that the swimbladder of mutant zebrafish had defects in development,that is,they could not swell.So,as a homologous organ in mammalian lungs,is the developmental mechanism affected by cross-regulation between Notch and NF-?B signaling pathways?The previous reported gene results suggested that Notch1a and Notch3 can indeed significantly activate the transcriptional activity of rela or nf?b1.Therefore,we speculated that the NF-?B signaling pathway may affect the development of swimbladder.In order to study the effect of Notch on zebrafish swimbladder development,we first observed notch1a^-/-and notch3^-/-mutant zebrafish and found that the mutant fish had a defect in the tissue development of the zebrafish.The WT,notch1a^-/-and notch3^-/-mutant zebrafish were sliced for histopathological observation.The real-time quantitative PCR technology was used to detect the expression levels of swimbladder development-related genes in WT and mutant zebrafish on different days,and the differences in morphology,i.e.,length,width,and area,of WT and mutant zebrafish swimbladder were studied.With respect to the effects of Notch1a and Notch3 on the development of zebrafish swimbladders,we got the following results:?1?Histopathological sections and H-E staining were used to observe the development of the swimbladders of WT,notch1a^-/-and notch3^-/-mutants in zebrafish.According to the results of pathological dyeing WT zebrafish swimbladder developed normally,that is,in 4-5 days post fertilization?dpf?swimbladder formed inflated cavity filled with gas.And notch1a^-/-and notch3^-/-mutant zebrafish are dysplasia,namely in 4-5dpf swimbladder cannot be inflated,and they were still flat cystic structure as the same of the early stage of development,and between the gut and swim bladder,we found the pneumatic duct.The morphological data showed that the length of zebrafish swim bladders of WT,notch1a^-/-and notch3^-/-mutants were almost the same,while the width of zebrafish swim bladders of notch1a^-/-and notch3^-/-mutants were significantly shorter than that of WT,resulting in a smaller area of zebrafish swim bladders than that of WT.?2?The swimbladder development was mainly regulated by Wnt signaling pathway and the Hedgehog?Hh?signaling pathway.Through real-time fluorescent quantitative PCR,the expression levels of swimbladder development-related genes in the size of 6dpf WT,notch1a^-/-and notch3^-/-mutants was first tested.And we found that fz2,wnt2,dkk1,lef1,tcf3a,tcf3b and pbx1 genes in notch1a^-/-and notch3^-/-mutants were significantly down-regulated.The expression levels of fz2 and wnt2 in notch1a^-/-and notch3^-/-mutants were significantly down-regulated only at the size of 5dpf.To sum up,the immune function of zebrafish Notch1a and Notch3 molecules,which were confirmed by the transcriptional activity of NF-?B family genes mediated by TLR signaling pathway with the dual-luciferase detection system,will help us to clarify the role of Notch signaling pathway in TLR signaling pathway,and provide new ideas for further exploration of the immunological mechanism of tumor and other diseases and the discovery of potential new therapeutic approaches.Histopathological techniques were used to find out that the zebrafish swim bladders of notch1a^-/-and notch3^-/-mutants had bladder structures but could not inflate during the early development.Because the fish swimbladder tissue is the homologous organ of mammalian lungs,so the research of notch1a^-/-and notch3^-/-mutant swimbladder dysplasia can help us find solutions to certain human lung disease.