RESUMO
Testis differentiation in zebrafish involves juvenile ovary to testis transformation initiated by an apoptotic wave. The molecular regulation of this transformation process is not fully understood. NF-κB is activated at an early stage of development and has been shown to interact with steroidogenic factor-1 in mammals, leading to the suppression of anti-Müllerian hormone (Amh) gene expression. Because steroidogenic factor-1 and Amh are important for proper testis development, NF-κB-mediated induction of anti-apoptotic genes could, therefore, also play a role in zebrafish gonad differentiation. The aim of this study was to examine the potential role of NF-κB in zebrafish gonad differentiation. Exposure of juvenile zebrafish to heat-killed Escherichia coli activated the NF-κB pathways and resulted in an increased ratio of females from 30 to 85%. Microarray and quantitative real-time-PCR analysis of gonads showed elevated expression of NF-κB-regulated genes. To confirm the involvement of NF-κB-induced anti-apoptotic effects, zebrafish were treated with sodium deoxycholate, a known inducer of NF-κB or NF-κB activation inhibitor (NAI). Sodium deoxycholate treatment mimicked the effect of heat-killed bacteria and resulted in an increased proportion of females from 25 to 45%, whereas the inhibition of NF-κB using NAI resulted in a decrease in females from 45 to 20%. This study provides proof for an essential role of NF-κB in gonadal differentiation of zebrafish and represents an important step toward the complete understanding of the complicated process of sex differentiation in this species and possibly other cyprinid teleosts as well.
Assuntos
NF-kappa B/metabolismo , Ovário/crescimento & desenvolvimento , Testículo/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Western Blotting , Linhagem Celular , Ácido Desoxicólico/farmacologia , Escherichia coli/imunologia , Feminino , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Temperatura Alta , Masculino , Modelos Genéticos , NF-kappa B/antagonistas & inibidores , NF-kappa B/genética , Análise de Sequência com Séries de Oligonucleotídeos , Ovário/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Diferenciação Sexual/efeitos dos fármacos , Diferenciação Sexual/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Testículo/metabolismo , Transcriptoma/genética , Transcriptoma/imunologia , Peixe-Zebra/genética , Peixe-Zebra/imunologia , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/genéticaRESUMO
The NR5A family of orphan nuclear receptors has been implicated in development of the vertebrate embryo, but their exact role remains largely unknown. To evaluate the regulation and developmental role for ff1a (NR5A2) in zebrafish (Danio rerio), we performed morpholino knockdown to block translation of the ff1a gene and the upstream located sox9a gene during embryogenesis. Using a newly developed antibody against Ff1a we could show that the ff1a morpholinos were functional and that a reduction in the expression of Ff1a correlated to altered phenotypes. The role of Sox9a in ff1a gene regulation and function was studied using sox9a morpholinos. Knock-down of sox9a resulted in abolished ff1a signals in the somites, mandibular arches and pharyngeal arches, while the pectoral fin signal remained. The reduction in Ff1a levels correlated to truncated tails and cranio-facial malformation. As Sox9a is involved in chondrocyte development we analysed for cartilage formation and found that blocking translation of either sox9a or ff1a also blocked cartilage formation. In light of the results, the present study suggests a novel function of ff1a in chondrocyte development.