| Objectives To clone zebrafish nrg1gene, and detect nrg1expresssion in adult zebrafishintestine and embryos.Methods Total RNA was extracted from adult zebrafish gut and embryos in differentperiods, primers were designed to amplify nrg1gene, then nrg1homology analysis wasperformed among different species. Nrg1was cloned into pGEM-T easy vector,digoxigenin-labeled RNA probes were synthesized in vitro, and in situ hybridization ofnrg1was carried out in adult zebrafish gut and embryos.Results Zebrafish nrg1gene was cloned correctly, zebrafish nrg1shared67%nucleotide identity with human nrg1. The phylogenetic tree analysis showed that thezebrafish nrg1evoluted faster than mouse, chicken, human and rat, zebrafish nrg1andmouse nrg1had closer relationship. Nrg1was expressed in adult zebrafish intestinalmucous layer, no expression was detected in muscularis and serosa. Nrg1was expressed inblastocyst stage, strong expression was detected in gastrulation stage and somite stage,mainly located in the animal pole. In pharyngula period, nrg1mRNA expression was weak,the nrg1expression level was increased from hatching period, and started to decrease from120hpf.Conclusion zebrafish nrg1shares high nucleotide identity with human, mouse and ratnrg1. Zebrafish nrg1is expressed in adult zebrafish gut and larvae gut, suggesting nrg1may be associated with the development of the gut in zebrafish. Objective To establish zebrafish nrg1knockdown and overexpression models, andanalysis embryonic phenotypes in nrg1knockdown and overexpression model.Methods Control/nrg1-morpholino and EGFP-nrg1lineared vector were co-injected intozebrafish embryos, the strength of green fluorescence was used to evaluate knockdownefficiency. Nrg1-MO and nrg1-RNA was injected into embryo to observe phenotypesresulted from nrg1expression changes.Results Strong fluorescence expression was detected in12hpf embryos injected bycontrol-MO and nrg1-EGFP linearized vector; embryos injected by nrg1-MO andnrg1-EGFP linearized vector manifested weakened green fluorescence. Compared withcontrol group, nrg1MO-injected embryos had delayed hatching, small head, pericardialoedema, shortened-twisted trunk and tail, swimming speed was lower than in controlmorpholino-injected embryos. The nrg1-MO phenotype could be partially rescued whenthe embryos were co-injected with nrg1mRNA. There was no significant phenotypicvariation in the nrg1mRNA group.Conclusion Nrg1protein can be knocked down by ATG-MO efficiently. Deformities,growth and developmental delay or even death can be observed by interfering nrg1expression of zebrafish embryos. Objective To determine whether nrg1is required for the of proliferation, differentiationand migration of enteric neurons.Methods Zebrafish embryos were divided into four groups: control group, nrg1knockdown group, nrg1overexpression group and nrg1rescue group, then the embryoswere collected at different developmental stages (66,72,88,96,120and144hpf). Embryoswere fixed in4%paraformaldehyde (containing1‰DEPC), then the embryos wasdigested and washed, anti-phosphohistone H3(enteric neurons proliferation) and anti-HuMAb16A11antibody (enteric neurons differentiation and migration) was addedrespectively, at last the embryos were incubated by fluorescent secondary antibody.Results The knockdown of nrg1caused a decrease in the number of ENS precursors. At66hpf, the differentiated enteric neurons were first observed with Hu C/D antibody at theanterior region in control and nrg1knockdown group. Hereafter, in control group, thefluorescence extended to the posterior region, and the number of the differentiated entericneurons increased. Knockdown of nrg1blocked the migration of enteric neurons, especiallyin the stage the precursors migrating along the intestine. Over-expression of nrg1,co-injected with nrg1-MO and nrg1-RNA resulted in normal ENS proliferation anddifferentiation.Conclusion Nrg1can affect the proliferation, differentiation process of enteric neurons.In nrg1knockdown embryos, the process of the enreic precursor cells migrating from vagalcrest to the anterior gut was not affected. Knockdown of nrg1blocked the migration of enteric neurons from the anterior to posterior region of intestine. Objective To analyzed the effect of nrg1on the expression profiles of the enteric nervousdevelopmental related gene during key stages of ENS development in zebrafsh.Methods Zebrafish embryos were divided into four groups: control group, nrg1knockdown group, nrg1overexpression group,nrg1rescue group, then the embryos werecollected at different developmental stages (36,48and60hpf). Embryos were fixed in4%paraformaldehyde (containing1‰DEPC), then the embryos was digested with proteinaseK, embryos were prehybridized and incubated in HYB+containing RNA probe, thenblocked with BSA and incubated with anti-DIG antibody and visualized with NBT/BCIP.The chromogenic embryos were preserved in PBST with30%glycerol.Results At48hpf, weaker mRNA levels of ret, gdnf, phox2b, sox10,shh were detected inknock-down model. Over expressed nrg1RNA did not increase the levels of the specificmarkers, but nrg1RNA rescued the nrg1-MO effect. These results indicated that nrg1wasrequired for the development of ENS in zebrafsh, but it was not indispensable when vagalneural crest cells migrated to the anterior gut. At36hpf, the expression pattern of crestinwas normal in nrg1morphants; however, the crestin expression was reduced when nrg1was knocked down at60hpf.Conclusion Abnormal nrg1expression resulted in decreased expression of ENS relatedgenes: ret, gdnf, phox2b, sox10, shh. Knockdown of nrg1zebrafish expressed normalcrestin when enteric precursors arrived in intestine, but crestin expression decreased asenteric precursors reached intestine, suggesting that nrg1may play a role mainly in the process ENS precursor cells migrating along intestine. Objective To study the role of nrg1in the development of intestinal vagal nerve fibers.Methods Zebrafish embryos were divided into two groups: control group and nrg1knockdown group, then the embryos were collected at96and7dpf. Embryos were fixed in4%paraformaldehyde (containing1‰DEPC), then the embryos was digested withproteinase K, after washed by PTD（PBS, triton X, DMSO）, acetylated α-tubulin antibodywas added,4℃overnight, at last the embryos were incubated by fluorescent secondaryantibody.Results At96hpf, intestinal vagal nerve bundles were thick, short and chaotic in nrg1knockdown zebrafish. At7dpf, vagal nerve bundles in control group zebrafish wasarranged reticularly and extended into intestinal tract; on the contrary, there was no nervebranch extended into intestinal tract in nrg1knockdown zebrafish.Conclusion Nrg1is not only involved in the development of enteric neurons, but alsoaffects intestinal vagal nerve bundle development. |
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