ABSTRACT
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.
Subject(s)
Calcium-Binding Proteins/genetics , Intercellular Signaling Peptides and Proteins/genetics , Membrane Proteins/genetics , Pancreas/embryology , Receptors, Notch/physiology , Zebrafish/embryology , Animals , Base Sequence , Cell Differentiation/physiology , DNA Primers , Intracellular Signaling Peptides and Proteins , Jagged-1 Protein , Pancreas/cytology , Serrate-Jagged Proteins , Signal TransductionABSTRACT
Recent large-scale mutagenesis screens have made the zebrafish the first vertebrate organism to allow a forward genetic approach to the discovery of developmental control genes. Mutations can be cloned positionally, or placed on a simple sequence length polymorphism (SSLP) map to match them with mapped candidate genes and expressed sequence tags (ESTs). To facilitate the mapping of candidate genes and to increase the density of markers available for positional cloning, we have created a radiation hybrid (RH) map of the zebrafish genome. This technique is based on somatic cell hybrid lines produced by fusion of lethally irradiated cells of the species of interest with a rodent cell line. Random fragments of the donor chromosomes are integrated into recipient chromosomes or retained as separate minichromosomes. The radiation-induced breakpoints can be used for mapping in a manner analogous to genetic mapping, but at higher resolution and without a need for polymorphism. Genome-wide maps exist for the human, based on three RH panels of different resolutions, as well as for the dog, rat and mouse. For our map of the zebrafish genome, we used an existing RH panel and 1,451 sequence tagged site (STS) markers, including SSLPs, cloned candidate genes and ESTs. Of these, 1,275 (87.9%) have significant linkage to at least one other marker. The fraction of ESTs with significant linkage, which can be used as an estimate of map coverage, is 81.9%. We found the average marker retention frequency to be 18.4%. One cR3000 is equivalent to 61 kb, resulting in a potential resolution of approximately 350 kb.
