RESUMO
From a differential display designed to isolate genes that are down-regulated upon differentiation of the central nervous system in Danio rerio embryos, we isolated d-asb11 (ankyrin repeat and suppressor of cytokine signaling box-containing protein 11). Knockdown of the d-Asb11 protein altered the expression of neural precursor genes sox2 and sox3 and resulted in an initial relative increase in proneural cell numbers. This was reflected by neurogenin1 expansion followed by premature neuronal differentiation, as demonstrated by HuC labeling and resulting in reduced size of the definitive neuronal compartment. Forced misexpression of d-asb11 was capable of ectopically inducing sox2 while it diminished or entirely abolished neurogenesis. Overexpression of d-Asb11 in both a pluripotent and a neural-committed progenitor cell line resulted in the stimulus-induced inhibition of terminal neuronal differentiation and enhanced proliferation. We conclude that d-Asb11 is a novel regulator of the neuronal progenitor compartment size by maintaining the neural precursors in the proliferating undifferentiated state possibly through the control of SoxB1 transcription factors.
Assuntos
Diferenciação Celular/fisiologia , Sistema Nervoso Central/embriologia , Sistema Nervoso Central/metabolismo , Neurônios/metabolismo , Células-Tronco/metabolismo , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem da Célula/fisiologia , Proliferação de Células , Sistema Nervoso Central/citologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas HMGB/metabolismo , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Grupo de Alta Mobilidade/metabolismo , Humanos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/metabolismo , Células PC12 , Ratos , Fatores de Transcrição SOXB1 , Proteínas Supressoras da Sinalização de Citocina/genética , Proteínas Supressoras da Sinalização de Citocina/isolamento & purificação , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/isolamento & purificaçãoRESUMO
The specific molecular determinants that govern progenitor expansion and final compartment size in the myogenic lineage, either during gestation or during regenerative myogenesis, remain largely obscure. Recently, we retrieved d-asb11 from a zebrafish screen designed to identify gene products that are downregulated during embryogenesis upon terminal differentiation and identified it as a potential regulator of compartment size in the ectodermal lineage. A role in mesodermal derivatives remained, however, unexplored. Here we report pan-vertebrate expression of Asb11 in muscle compartments, where it highly specifically localizes to the Pax7(+) muscle satellite cell compartment. Forced expression of d-asb11 impaired terminal differentiation and caused enhanced proliferation in the myogenic progenitor compartment both in in vivo and in vitro model systems. Conversely, introduction of a germline hypomorphic mutation in the zebrafish d-asb11 gene produced premature differentiation of the muscle progenitors and delayed regenerative responses in adult injured muscle. Thus, the expression of d-asb11 is necessary for muscle progenitor expansion, whereas its downregulation marks the onset of terminal differentiation. Hence, we provide evidence that d-asb11 is a principal regulator of embryonic as well as adult regenerative myogenesis.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Desenvolvimento Muscular , Regeneração , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Alelos , Animais , Blastômeros/citologia , Blastômeros/metabolismo , Contagem de Células , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Mutação em Linhagem Germinativa , Imuno-Histoquímica , Camundongos , Modelos Animais , Músculo Esquelético/citologia , Músculo Esquelético/lesões , Músculo Esquelético/metabolismo , Fator de Transcrição PAX7/genética , Fator de Transcrição PAX7/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células Satélites de Músculo Esquelético/citologia , Células Satélites de Músculo Esquelético/metabolismo , Proteínas Supressoras da Sinalização de Citocina/genética , Transfecção , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
In canonical Delta-Notch signalling, expression of Delta activates Notch in neighbouring cells, leading to downregulation of Delta in these cells. This process of lateral inhibition results in selection of either Delta-signalling cells or Notch-signalling cells. Here we show that d-Asb11 is an important mediator of this lateral inhibition. In zebrafish embryos, morpholino oligonucleotide (MO)-mediated knockdown of d-Asb11 caused repression of specific Delta-Notch elements and their transcriptional targets, whereas these were induced when d-Asb11 was misexpressed. d-Asb11 also activated legitimate Notch reporters cell-non-autonomously in vitro and in vivo when co-expressed with a Notch reporter. However, it repressed Notch reporters when expressed in Delta-expressing cells. Consistent with these results, d-Asb11 was able to specifically ubiquitylate and degrade DeltaA both in vitro and in vivo. We conclude that d-Asb11 is a component in the regulation of Delta-Notch signalling, important in fine-tuning the lateral inhibition gradients between DeltaA and Notch through a cell non-autonomous mechanism.
Assuntos
Proteínas de Membrana/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Embrião não Mamífero/metabolismo , Retroalimentação Fisiológica , Genes Reporter , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Ligação Proteica , Ativação Transcricional/genética , Peixe-Zebra/embriologiaRESUMO
Heparan sulfate proteoglycans function in development and disease. They consist of a core protein with attached heparan sulfate chains that are altered by a series of carbohydrate-modifying enzymes and sulfotransferases. Here, we report on the identification and characterization of a gene encoding zebrafish heparan sulfate 6-O-sulfotransferase (hs6st) that shows high homology to other heparan sulfate 6-O-sulfotransferases. When expressed as a fusion protein in cultured cells, the protein shows specific 6-O-sulfotransferase activity and preferentially acts on the iduronosyl N-sulfoglycosamine. In the developing embryo, hs6st is expressed in the brain, the somites, and the fins; the same structures that were affected upon morpholino-mediated functional knockdown. Morpholino injections significantly inhibited 6-O- but not 2-O-sulfation as assessed by HPLC. Morphants display disturbed somite specification independent of the somite oscillator mechanism and have impaired muscle differentiation. In conclusion, our results show that transfer of sulfate to specific positions on glycosaminoglycans is essential for muscle development.