RESUMO
Haematopoietic stem and progenitor cells (HSPCs) have been the focus of developmental and regenerative studies, yet our understanding of the signalling events regulating their specification remains incomplete. We demonstrate that supt16h, a component of the Facilitates chromatin transcription (FACT) complex, is required for HSPC formation. Zebrafish supt16h mutants express reduced levels of Notch-signalling components, genes essential for HSPC development, due to abrogated transcription. Whereas global chromatin accessibility in supt16h mutants is not substantially altered, we observe a specific increase in p53 accessibility, causing an accumulation of p53. We further demonstrate that p53 influences expression of the Polycomb-group protein PHC1, which functions as a transcriptional repressor of Notch genes. Suppression of phc1 or its upstream regulator, p53, rescues the loss of both Notch and HSPC phenotypes in supt16h mutants. Our results highlight a relationship between supt16h, p53 and phc1 to specify HSPCs via modulation of Notch signalling.
Assuntos
Proteínas de Ciclo Celular/genética , Células-Tronco Hematopoéticas/metabolismo , Receptores Notch/genética , Fatores de Transcrição/genética , Proteína Supressora de Tumor p53/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Proteínas de Ciclo Celular/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento , Ontologia Genética , Células-Tronco Hematopoéticas/citologia , Mutação , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 1/metabolismo , Receptores Notch/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Proteína Supressora de Tumor p53/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/metabolismoRESUMO
Snail2 is a zinc-finger transcription factor best known to repress expression of genes encoding cell adherence proteins to facilitate induction of the epithelial-to-mesenchymal transition. While this role has been best documented in the developmental migration of the neural crest and mesoderm, here we expand on previously reported preliminary findings that morpholino knock-down of snai2 impairs the generation of hematopoietic stem cells (HSCs) during zebrafish development. We demonstrate that snai2 morphants fail to initiate HSC specification and show defects in the somitic niche of migrating HSC precursors. These defects include a reduction in sclerotome markers as well as in the Notch ligands dlc and dld, which are known to be essential components of HSC specification. Accordingly, enforced expression of the Notch1-intracellular domain was capable of rescuing HSC specification in snai2 morphants. To parallel our approach, we obtained two mutant alleles of snai2. In contrast to the morphants, homozygous mutant embryos displayed no defects in HSC specification or in sclerotome development, and mutant fish survive into adulthood. However, when these homozygous mutants were injected with snai2 morpholino, HSCs were improperly specified. In summary, our morpholino data support a role for Snai2 in HSC development, whereas our mutant data suggest that Snai2 is dispensable for this process. Together, these findings further support the need for careful consideration of both morpholino and mutant phenotypes in studies of gene function.