RESUMO
AIM: To investigate the role of IKBKAP (inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase complex-associated protein) in the development of enteric nervous system (ENS) and Hirschsprung disease (HSCR). METHODS: In this study, we injected a morpholino that blocked the translation of ikbkap protein to 1-cell stage zebrafish embryos. The phenotype in the ENS was analysed by antibody staining of the pan-neuronal marker HuC/D followed by enteric neuron counting. The mean numbers of enteric neurons were compared between the morphant and the control. We also studied the expressions of ret and phox2bb, which are involved in ENS development, in the ikbkap morpholino injected embryos by quantitative reverse transcriptase polymerase chain reaction and compared them with the control. RESULTS: We observed aganglionosis (χ2, P<0.01) and a reduced number of enteric neurons (38.8±9.9 vs 50.2±17.3, P<0.05) in the zebrafish embryos injected with ikbkap translation-blocking morpholino (morphant) when compared with the control embryos. Specificity of the morpholino was confirmed by similar results obtained using a second non-overlapping morpholino that blocked the translation of ikbkap. We further studied the morphant by analysing the expression levels of genes involved in ENS development such as ret, phox2bb and sox10, and found that phox2bb, the ortholog of human PHOX2B, was significantly down-regulated (0.51±0.15 vs 1.00±0, P<0.05). Although we also observed a reduction in the expression of ret, the difference was not significant. CONCLUSION: Loss of IKBKAP contributed to HSCR as demonstrated by functional analysis in zebrafish embryos.
Assuntos
Proteínas de Transporte/genética , Sistema Nervoso Entérico/embriologia , Doença de Hirschsprung/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Modelos Animais de Doenças , Sistema Nervoso Entérico/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Predisposição Genética para Doença , Doença de Hirschsprung/embriologia , Doença de Hirschsprung/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Imuno-Histoquímica , Morfolinos/administração & dosagem , Fenótipo , Proteínas Proto-Oncogênicas c-ret/genética , Proteínas Proto-Oncogênicas c-ret/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/metabolismoRESUMO
Patterning of neural crest (NC) for the formation of specific structures along the anterio-posterior (A-P) body axis is governed by a combinatorial action of Hox genes, which are expressed in the neuroepithelium at the time of NC induction. Hoxb5 was expressed in NC at both induction and migratory stages, and our previous data suggested that Hoxb5 played a role in the NC development. However, the underlying mechanisms by which Hoxb5 regulates the early NC development are largely unknown. Current study showed that both the human and mouse Foxd3 promoters were bound and trans-activated by Hoxb5 in NC-derived neuroblastoma cells. The binding of Hoxb5 to Foxd3 promoter in vivo was further confirmed in the brain and neural tube of mouse embryos. Moreover, Wnt1-Cre mediated perturbation of Hoxb5 signaling at the dorsal neural tube in mouse embryos resulted in Foxd3 down-regulation. In ovo, Foxd3 alleviated the apoptosis of neural cells induced by perturbed Hoxb5 signaling, and Hoxb5 induced ectopic Foxd3 expression in the chick neural tube. This study demonstrated that Hoxb5 (an A-P patterning gene) regulated the NC development by directly inducing Foxd3 (a NC specifier and survival gene).