Zebrafish provide a highly versatile model in which to study vertebrate development. Many recent studies have elucidated early events in the organogenesis of the zebrafish pancreas; however, several aspects of early endocrine pancreas formation in the zebrafish are not homologous to the mammalian system. To better identify mechanisms of islet formation in the zebrafish, with true homology to those observed in mammals, we have temporally and spatially characterized zebrafish secondary islet formation. As is the case in the mouse, we show that Notch inhibition leads to precocious differentiation of endocrine tissues. Furthermore, we have used transgenic fish expressing fluorescent markers under the control of a Notch-responsive element to observe the precursors of these induced endocrine cells. These pancreatic Notch-responsive cells represent a novel population of putative progenitors that are associated with larval pancreatic ductal epithelium, suggesting functional homology between secondary islet formation in zebrafish and the secondary transition in mammals. We also show that Notch-responsive cells persist in the adult pancreas and possess the classical characteristics of centroacinar cells, a cell type believed to be a multipotent progenitor cell in adult mammalian pancreas.

Study of the cyclooxygenases (COXs) has been limited by the role of COX-2 in murine reproduction and renal organogenesis. We sought to characterize COX expression and function in zebrafish (z). Full-length cDNAs of zCOX-1 and zCOX-2 were cloned and assigned to conserved regions of chromosomes 5 and 2, respectively. The deduced proteins are 67% homologous with their human orthologs. Prostaglandin (PG) E 2 is the predominant zCOX product detected by mass spectrometry. Pharmacological inhibitors demonstrate selectivity when directed against heterologously expressed zCOX isoforms. Zebrafish thrombocyte aggregation ex vivo and hemostasis in vivo are sensitive to inhibition of zCOX-1, but not zCOX-2. Both zCOXs were widely expressed during development, and knockdown of zCOX-1 causes growth arrest during early embryogenesis. zCOX-1 is widely evident in the embryonic vasculature, whereas zCOX-2 exhibits a more restricted pattern of expression. Both zCOX isoforms are genetically and functionally homologous to their mammalian orthologs. The zebrafish affords a tractable model system for the study of COX biology and development.C yclooxygenases (COXs), also known as prostaglandin (PG) endoperoxide G͞H synthases (EC 1.14.99.1), catalyze the conversion of arachidonate to PGs. Two different COX isozymes, COX-1 and COX-2, encoded by separate genes, have been identified (1, 2). COX-1 is expressed constitutively and is primarily responsible for PGs that maintain homeostatic function. Conversely, COX-2 is highly regulated by growth factors, tumor promoters, and cytokines. The recognition of such segregated actions of the COX isozymes has rationalized the development of selective COX-2 inhibitors.Detailed elucidation of the role of COX isozymes in rodent model systems has been constrained by the importance of COX-2 during development (3). The zebrafish (Danio rerio) has emerged as an informative model organism for studies of vertebrate biology and genetics. Multiple phenotypes have been identified in chemical mutagenesis screens (4), of which some are strikingly reminiscent of human disease. For example, mutation of the novel basic helix-loop-helix transcription factor gridlock results in a stenosis of the aorta, which can be visualized by confocal microangiography and may be analogous to coarctation of the aorta in humans (5). The recent development of morpholino-based gene targeting technology allows for specific gene inactivation in zebrafish. Given the existence of phospholipase A 2 , an enzyme that liberates arachidonic acid for subsequent COX metabolism in zebrafish (6), we hypothesized that the prostanoid biosynthetic pathway might be expressed and functional in this model system. Materials and MethodsIsolation of zCOX-1 and zCOX-2. Zebrafish embryos were obtained from wild-type AB strain fish and raised at 28.5°C as described (7). Zebrafish expressed sequence tag (EST) clones with high homology to human COX-1 (clone fc62e06.x1) and human COX-2 (clone fa92e05.y1) were identified in the Washington University (St. Louis) G...

SUMMARYThe Notch-signaling pathway is known to be fundamental in controlling pancreas differentiation. We now report on using Crebased fate mapping to indelibly label pancreatic Notch-responsive cells (PNCs) at larval stages and follow their fate in the adult pancreas. We show that the PNCs represent a population of progenitors that can differentiate to multiple lineages, including adult ductal cells, centroacinar cells (CACs) and endocrine cells. These endocrine cells include the insulin-producing -cells. CACs are a functional component of the exocrine pancreas; however, our fate-mapping results indicate that CACs are more closely related to endocrine cells by lineage as they share a common progenitor. The majority of the exocrine pancreas consists of the secretory acinar cells; however, we only detect a very limited contribution of PNCs to acinar cells. To explain this observation we re-examined early events in pancreas formation. The pancreatic anlage that gives rise to the exocrine pancreas is located in the ventral gut endoderm (called the ventral bud). Ptf1a is a gene required for exocrine pancreas development and is first expressed as the ventral bud forms. We used transgenic marker lines to observe both the domain of cells expressing ptf1a and cells responding to Notch signaling. We do not detect any overlap in expression and demonstrate that the ventral bud consists of two cell populations: a ptf1-expressing domain and a Notch-responsive progenitor core. As pancreas organogenesis continues, the ventral bud derived PNCs align along the duct, remain multipotent and later in development differentiate to form secondary islets, ducts and CACs.