Distinct delta and jagged genes control sequential segregation of pancreatic cell types from precursor pools in zebrafish.

The different cell types of the vertebrate pancreas arise asynchronously during organogenesis. Beta-cells producing insulin, alpha-cells producing glucagon, and exocrine cells secreting digestive enzymes differentiate sequentially from a common primordium. Notch signaling has been shown to be a major mechanism controlling these cell-fate choices. So far, the pleiotropy of Delta and Jagged/Serrate genes has hindered the evaluation of the roles of specific Notch ligands, as the phenotypes of knock-out mice are lethal before complete pancreas differentiation. Analyses of gene expression and experimental manipulations of zebrafish embryos allowed us to determine individual contributions of Notch ligands to pancreas development. We have found that temporally distinct phases of both endocrine and exocrine cell type specification are controlled by different delta and jagged genes. Specifically, deltaA knock-down embryos lack alpha cells, similarly to mib (Delta ubiquitin ligase) mutants and embryos treated with DAPT, a gamma secretase inhibitor able to block Notch signaling. Conversely, jagged1b morphants develop an excess of alpha-cells. Moreover, the pancreas of jagged2 knock-down embryos has a decreased ratio of exocrine-to-endocrine compartments. Finally, overexpression of Notch1a-intracellular-domain in the whole pancreas primordium or specifically in beta-cells helped us to refine a model of pancreas differentiation in which cells exit the precursor state at defined stages to form the pancreatic cell lineages, and, by a feedback mediated by different Notch ligands, limit the number of other cells that can leave the precursor state.

Early appearance of pancreatic hormone-expressing cells in the zebrafish embryo.

Adult pancreatic islets comprise four cell types, alpha, beta, delta and PP, expressing glucagon, insulin, somatostatin and pancreatic-polypeptide, respectively, arising from cell lineages whose relationships during endocrine pancreas differentiation are still uncertain [Edlund, 1998. Diabetes 47, 1817-1823]. As zebrafish (Danio rerio) represents an attractive vertebrate model to study mutants affecting pancreatic organogenesis [Pack et al., 1996. Development 123, 321-328], we have investigated the expression patterns of islet hormones in zebrafish embryos, from the 16-somite (17 h) to 48-h stages, by whole-mount in situ hybridization and immunofluorescence. Results showed that in the zebrafish pancreatic primordium (a) insulin is the first hormone gene to be expressed, and (b) somatostatin colocalizes with insulin while glucagon-expressing cells, since their appearance, are distinct from insulin- or insulin/somatostatin-expressing cells. Notably, both somatostatin and glucagon, but not insulin, are first expressed in extrapancreatic regions.