RESUMO
ARP/ASCL transcription factors are key determinants of cell fate specification in a wide variety of tissues, coordinating the acquisition of generic cell fates and of specific subtype identities. How these factors, recognizing highly similar DNA motifs, display specific activities, is not yet fully understood. To address this issue, we overexpressed different ARP/ASCL factors in zebrafish ascl1a-/- mutant embryos to determine which ones are able to rescue the intestinal secretory lineage. We found that Ascl1a/b, Atoh1a/b and Neurod1 factors are all able to trigger the first step of the secretory regulatory cascade but distinct secretory cells are induced by these factors. Indeed, Neurod1 rescues the enteroendocrine lineage while Ascl1a/b and Atoh1a/b rescue the goblet cells. Gain-of-function experiments with Ascl1a/Neurod1 chimeric proteins revealed that the functional divergence is encoded by a 19-aa ultra-conserved element (UCE), present in all Neurod members but absent in the other ARP/ASCL proteins. Importantly, inserting the UCE into the Ascl1a protein reverses the rescuing capacity of this Ascl1a chimeric protein that cannot rescue the goblet cells anymore but can efficiently rescue the enteroendocrine cells. This novel domain acts indeed as a goblet cell fate repressor that inhibits gfi1aa expression, known to be important for goblet cell differentiation. Deleting the UCE domain of the endogenous Neurod1 protein leads to an increase in the number of goblet cells concomitant with a reduction of the enteroendocrine cells, phenotype also observed in the neurod1 null mutant. This highlights the crucial function of the UCE domain for NeuroD1 activity in the intestine. As Gfi1 acts as a binary cell fate switch in several tissues where Neurod1 is also expressed, we can envision a similar role of the UCE in other tissues, allowing Neurod1 to repress Gfi1 to influence the balance between cell fates.
Assuntos
Células Caliciformes , Peixe-Zebra , Animais , Diferenciação Celular/genética , Células Caliciformes/metabolismo , Fatores de Transcrição/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
BACKGROUND: In contrast to mammals, the zebrafish has the remarkable capacity to regenerate its pancreatic beta cells very efficiently. Understanding the mechanisms of regeneration in the zebrafish and the differences with mammals will be fundamental to discovering molecules able to stimulate the regeneration process in mammals. To identify the pancreatic cells able to give rise to new beta cells in the zebrafish, we generated new transgenic lines allowing the tracing of multipotent pancreatic progenitors and endocrine precursors. RESULTS: Using novel bacterial artificial chromosome transgenic nkx6.1 and ascl1b reporter lines, we established that nkx6.1-positive cells give rise to all the pancreatic cell types and ascl1b-positive cells give rise to all the endocrine cell types in the zebrafish embryo. These two genes are initially co-expressed in the pancreatic primordium and their domains segregate, not as a result of mutual repression, but through the opposite effects of Notch signaling, maintaining nkx6.1 expression while repressing ascl1b in progenitors. In the adult zebrafish, nkx6.1 expression persists exclusively in the ductal tree at the tip of which its expression coincides with Notch active signaling in centroacinar/terminal end duct cells. Tracing these cells reveals that they are able to differentiate into other ductal cells and into insulin-expressing cells in normal (non-diabetic) animals. This capacity of ductal cells to generate endocrine cells is supported by the detection of ascl1b in the nkx6.1:GFP ductal cell transcriptome. This transcriptome also reveals, besides actors of the Notch and Wnt pathways, several novel markers such as id2a. Finally, we show that beta cell ablation in the adult zebrafish triggers proliferation of ductal cells and their differentiation into insulin-expressing cells. CONCLUSIONS: We have shown that, in the zebrafish embryo, nkx6.1+ cells are bona fide multipotent pancreatic progenitors, while ascl1b+ cells represent committed endocrine precursors. In contrast to the mouse, pancreatic progenitor markers nkx6.1 and pdx1 continue to be expressed in adult ductal cells, a subset of which we show are still able to proliferate and undergo ductal and endocrine differentiation, providing robust evidence of the existence of pancreatic progenitor/stem cells in the adult zebrafish. Our findings support the hypothesis that nkx6.1+ pancreatic progenitors contribute to beta cell regeneration. Further characterization of these cells will open up new perspectives for anti-diabetic therapies.
Assuntos
Células Secretoras de Insulina/fisiologia , Células-Tronco Multipotentes/fisiologia , Pâncreas/fisiologia , Regeneração/fisiologia , Fatores de Transcrição/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Diferenciação Celular , Regulação da Expressão Gênica no Desenvolvimento , Células Secretoras de Insulina/citologia , Células-Tronco Multipotentes/citologia , Pâncreas/citologia , Receptores Notch/genética , Receptores Notch/metabolismo , Fatores de Transcrição/genética , Via de Sinalização Wnt/genética , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
BACKGROUND: NEUROG3 is a key regulator of pancreatic endocrine cell differentiation in mouse, essential for the generation of all mature hormone producing cells. It is repressed by Notch signaling that prevents pancreatic cell differentiation by maintaining precursors in an undifferentiated state. RESULTS: We show that, in zebrafish, neurog3 is not expressed in the pancreas and null neurog3 mutant embryos do not display any apparent endocrine defects. The control of endocrine cell fate is instead fulfilled by two basic helix-loop-helix factors, Ascl1b and Neurod1, that are both repressed by Notch signaling. ascl1b is transiently expressed in the mid-trunk endoderm just after gastrulation and is required for the generation of the first pancreatic endocrine precursor cells. Neurod1 is expressed afterwards in the pancreatic anlagen and pursues the endocrine cell differentiation program initiated by Ascl1b. Their complementary role in endocrine differentiation of the dorsal bud is demonstrated by the loss of all hormone-secreting cells following their simultaneous inactivation. This defect is due to a blockage of the initiation of endocrine cell differentiation. CONCLUSIONS: This study demonstrates that NEUROG3 is not the unique pancreatic endocrine cell fate determinant in vertebrates. A general survey of endocrine cell fate determinants in the whole digestive system among vertebrates indicates that they all belong to the ARP/ASCL family but not necessarily to the Neurog3 subfamily. The identity of the ARP/ASCL factor involved depends not only on the organ but also on the species. One could, therefore, consider differentiating stem cells into insulin-producing cells without the involvement of NEUROG3 but via another ARP/ASCL factor.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem da Célula , Células Endócrinas/citologia , Proteínas do Tecido Nervoso/metabolismo , Pâncreas/citologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem da Célula/efeitos dos fármacos , Linhagem da Célula/genética , Embrião não Mamífero/citologia , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Células Endócrinas/efeitos dos fármacos , Células Endócrinas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Proteínas HMGB/metabolismo , Camundongos , Modelos Biológicos , Morfolinos/farmacologia , Mutação/genética , Proteínas do Tecido Nervoso/genética , Pâncreas/efeitos dos fármacos , Pâncreas/embriologia , Pâncreas/metabolismo , Filogenia , Receptores Notch/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fatores de Transcrição , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
Forward genetics using zebrafish is a powerful tool for studying vertebrate development through large-scale mutagenesis. Nonetheless, the identification of the molecular lesion is still laborious and involves time-consuming genetic mapping. Here, we show that high-throughput sequencing of the whole zebrafish genome can directly locate the interval carrying the causative mutation and at the same time pinpoint the molecular lesion. The feasibility of this approach was validated by sequencing the m1045 mutant line that displays a severe hypoplasia of the exocrine pancreas. We generated 13 Gb of sequence, equivalent to an eightfold genomic coverage, from a pool of 50 mutant embryos obtained from a map-cross between the AB mutant carrier and the WIK polymorphic strain. The chromosomal region carrying the causal mutation was localized based on its unique property to display high levels of homozygosity among sequence reads as it derives exclusively from the initial AB mutated allele. We developed an algorithm identifying such a region by calculating a homozygosity score along all chromosomes. This highlighted an 8-Mb window on chromosome 5 with a score close to 1 in the m1045 mutants. The sequence analysis of all genes within this interval revealed a nonsense mutation in the snapc4 gene. Knockdown experiments confirmed the assertion that snapc4 is the gene whose mutation leads to exocrine pancreas hypoplasia. In conclusion, this study constitutes a proof-of-concept that whole-genome sequencing is a fast and effective alternative to the classical positional cloning strategies in zebrafish.
Assuntos
Alquilantes/toxicidade , Mapeamento Cromossômico/métodos , Análise Mutacional de DNA/métodos , Etilnitrosoureia/toxicidade , Homozigoto , Peixe-Zebra/genética , Algoritmos , Animais , Sequência de Bases , Códon sem Sentido , Feminino , Masculino , Dados de Sequência Molecular , Pâncreas Exócrino , Polimorfismo de Nucleotídeo Único/efeitos dos fármacos , Fatores de Transcrição/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
Pax6 is a well conserved transcription factor that contains two DNA-binding domains, a paired domain and a homeodomain, and plays a key role in the development of eye, brain, and pancreas in vertebrates. The recent identification of the zebrafish sunrise mutant, harboring a mutation in the pax6b homeobox and presenting eye abnormalities but no obvious pancreatic defects, raised a question about the role of pax6b in zebrafish pancreas. We show here that pax6b does play an essential role in pancreatic endocrine cell differentiation, as revealed by the phenotype of a novel zebrafish pax6b null mutant and of embryos injected with pax6b morpholinos. Pax6b-depleted embryos have almost no beta cells, a strongly reduced number of delta cells, and a significant increase of epsilon cells. Through the use of various morpholinos targeting intron-exon junctions, pax6b RNA splicing was perturbed at several sites, leading either to retention of intronic sequences or to deletion of exonic sequences in the pax6b transcript. By this strategy, we show that deletion of the Pax6b homeodomain in zebrafish embryos does not disturb pancreas development, whereas lens formation is strongly affected. These data thus provide the explanation for the lack of pancreatic defects in the sunrise pax6b mutants. In addition, partial reduction of Pax6b function in zebrafish embryos performed by injection of small amounts of pax6b morpholinos caused a clear rise in alpha cell number and in glucagon expression, emphasizing the importance of the fine tuning of the Pax6b level to its biological activity.