Genetic analysis of early endocrine pancreas formation in zebrafish

Endocrine pancreas of zebrafish consist of at least four different cell types that function similarly to mammalian pancreatic islet. No mutants specifically affecting formation of the endocrine pancreas have been identified during the previous large-scale mutagenesis screens in zebrafish due to invisibility of a pancreatic islet. We combined in situ hybridization method to visualize pancreatic islet with an ethyl-nitroso-urea mutagenesis screen to identify novel genes involved in pancreatic islet formation in zebrafish. We screened 900 genomes and identified 11 mutations belonging to nine different complementation groups. These mutants fall into three major phenotypic classes displaying severely reduced insulin expression, reduced insulin expression with abnormal islet morphology, or abnormal islet morphology with relatively normal number of insulin expressing cells. Seven of these mutants do not have any other visible phenotypes associated. These mutations affect different processes in pancreatic islet development. Additional analysis on glucagon and somatostatin cell specification revealed that somatostatin cells are specified at a separate domain from insulin cells whereas glucagon cells are specified adjacent to insulin cells. Furthermore, glucagon cells and somatostatin cells are always associated with insulin cells in mutants that have scattered insulin expression. These data indicate that there are separate mechanisms regulating endocrine cell migration, proliferation, and differentiation. Further study on these mutants will reveal important information on novel genes involved in pancreatic islet cell specification and morphogenesis.     

Genetic inducible fate mapping in larval zebrafish reveals origins of adult insulin-producing β-cells

The Notch-signaling pathway is known to be fundamental in controlling pancreas differentiation. We now report on using Cre-based 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 β-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.     

Zebrafish sox9b is crucial for hepatopancreatic duct development and pancreatic endocrine cell regeneration

Recent zebrafish studies have shown that the late appearing pancreatic endocrine cells are derived from pancreatic ducts but the regulatory factors involved are still largely unknown. Here, we show that the zebrafish sox9b gene is expressed in pancreatic ducts where it labels the pancreatic Notch-responsive cells previously shown to be progenitors. Inactivation of sox9b disturbs duct formation and impairs regeneration of beta cells from these ducts in larvae. sox9b expression in the midtrunk endoderm appears at the junction of the hepatic and ventral pancreatic buds and, by the end of embryogenesis, labels the hepatopancreatic ductal system as well as the intrapancreatic and intrahepatic ducts. Ductal morphogenesis and differentiation are specifically disrupted in sox9b mutants, with the dysmorphic hepatopancreatic ducts containing misdifferentiated hepatocyte-like and pancreatic-like cells. We also show that maintenance of sox9b expression in the extrapancreatic and intrapancreatic ducts requires FGF and Notch activity, respectively, both pathways known to prevent excessive endocrine differentiation in these ducts. Furthermore, beta cell recovery after specific ablation is severely compromised in sox9b mutant larvae. Our data position sox9b as a key player in the generation of secondary endocrine cells deriving from pancreatic ducts in zebrafish.     

Generation of living color transgenic zebrafish to trace somatostatin-expressing cells and endocrine pancreas organization

In the present study, both gfp and rfp transgenic zebrafish lines using a 2.5-kb zebrafish somatostain2 (sst2) promoter were generated. During embryonic development, expression of GFP/RFP in the endocrine pancreas of transgenic embryos was initiated at ∼20 hpf and the number of GFP/RFP positive cells in the pancreas increased in subsequent stages; thus, our newly generated Tg(sst2:gfp) and Tg(sst2:rfp) lines faithfully recapitulated sst2 expression in endocrine pancreatic cells and provided a useful tool in analyzing the development of Sst2-producing δ-cells in the pancreas. By crossing these new transgenic lines with previously available transgenic lines targeted in insulin (Ins)-producing β-cells, Tg(ins:gfp) and Tg(ins:rfp), in combination with immunodetection of glucagon (Gcg)-producing α-cells and pancreatic polypeptide (PP)-producing PP-cells, the organization and composition of endocrine islets were investigated in both embryonic and adult pancreas. We found that there was always a big cluster of endocrine cells (principal islet) in the anterior-dorsal pancreas, followed by numerous smaller clusters (variable in size) of endocrine cells (secondary islets) along the anterior–posterior axis of the pancreas. All four types of endocrine cells were found in the principal islet, but secondary islets may or may not contain PP-cells. In addition, there were also discrete endocrine cells throughout the pancreas. In all co-localization experiments, we did not find any endocrine cells positive for more than one hormone markers, suggesting that these endocrine cells produce only a single hormone. In both principal and secondary islets, we found that β-cells were generally located in the center and non-β cells in the periphery; reminiscent of the “mantel–core” organization of islets of Langerhans in mammals where β-cells form the core and non-β-cells the mantel. In zebrafish primary islet, β-cells constitute most of the mass (∼50%), followed by δ-cells and α-cells (20–25% each), and PP-cells (1–2%); this is also similar to the composition of mammalian islets.     

Function and regulation of zebrafish nkx2.2a during development of pancreatic islet and ducts

In the mouse Nkx2.2 is expressed in the entire pancreatic anlage. Nevertheless, absence of Nkx2.2 only perturbs the development of endocrine cell types, notably beta-cells which are completely absent. In order to test the possibility that Nkx2.2 might fulfil additional functions during pancreas development we analysed its zebrafish homologue nkx2.2a using gene targeting and GFP-transgenic fish lines. Our results suggest similar roles for nkx2.2a and Nkx2.2 during the development of the endocrine pancreas. Morpholino-based knock-down of nkx2.2a leads to a reduction of alpha- and beta-cell number and an increase of ghrelin-producing cells but, as in mice, does not affect delta-cells. Moreover, like in the mouse, two spatially distinct promoters regulate expression of nkx2.2a in precursors and differentiated islet cells. In addition we found that in zebrafish nkx2.2a is also expressed in the anterior pancreatic bud and, later, in the differentiated pancreatic ducts. A nkx2.2a-transgenic line in which pancreatic GFP expression is restricted to the pancreatic ducts revealed that single GFP-positive cells leave the anterior pancreatic bud and move towards the islet where they form intercellular connections between each other. Subsequently, these cells generate the branched network of the larval pancreatic ducts. Morpholinos that block nkx2.2a function also lead to the absence of the pancreatic ducts. We observed the same phenotype in ptf1a-morphants that are additionally characterized by a reduced number of nkx2.2a-positive duct precursors. Whereas important details of the molecular program leading to the differentiation of endocrine cell types are conserved between mammals and zebrafish, our results reveal a new function for nkx2.2a in the development of the pancreatic ducts.     

Pancreas development in zebrafish: early dispersed appearance of endocrine hormone expressing cells and their convergence to form the definitive islet

To begin to understand pancreas development and the control of endocrine lineage formation in zebrafish, we have examined the expression pattern of several genes shown to act in vertebrate pancreatic development: pdx-1, insulin (W. M. Milewski et al., 1998, Endocrinology 139, 1440-1449), glucagon, somatostatin (F. Argenton et al., 1999, Mech. Dev. 87, 217-221), islet-1 (Korzh et al., 1993, Development 118, 417-425), nkx2.2 (Barth and Wilson, 1995, Development 121, 1755-1768), and pax6.2 (Nornes et al., 1998, Mech. Dev. 77, 185-196). To determine the spatial relationship between the exocrine and the endocrine compartments, we have cloned the zebrafish trypsin gene, a digestive enzyme expressed in differentiated pancreatic exocrine cells. We found expression of all these genes in the developing pancreas throughout organogenesis. Endocrine cells first appear in a scattered fashion in two bilateral rows close to the midline during mid-somitogenesis and converge during late-somitogenesis to form a single islet dorsal to the nascent duodenum. We have examined development of the endocrine lineage in a number of previously described zebrafish mutations. Deletion of chordamesoderm in floating head (Xnot homolog) mutants reduces islet formation to small remnants, but does not delete the pancreas, indicating that notochord is involved in proper pancreas development, but not required for differentiation of pancreatic cell fates. In the absence of knypek gene function, which is involved in convergence movements, the bilateral endocrine primordia do not merge. Presence of trunk paraxial mesoderm also appears to be instrumental for convergence since the bilateral endocrine primordia do not merge in spadetail mutants. We discuss our findings on zebrafish pancreatogenesis in the light of evolution of the pancreas in chordates.     

Ghrelin Expression in the Mouse Pancreas Defines a Unique Multipotent Progenitor Population

Pancreatic islet cells provide the major source of counteractive endocrine hormones required for maintaining glucose homeostasis; severe health problems result when these cell types are insufficiently active or reduced in number. Therefore, the process of islet endocrine cell lineage allocation is critical to ensure there is a correct balance of islet cell types. There are four endocrine cell types within the adult islet, including the glucagon-producing alpha cells, insulin-producing beta cells, somatostatin-producing delta cells and pancreatic polypeptide-producing PP cells. A fifth islet cell type, the ghrelin-producing epsilon cells, is primarily found during gestational development. Although hormone expression is generally assumed to mark the final entry to a determined cell state, we demonstrate in this study that ghrelin-expressing epsilon cells within the mouse pancreas do not represent a terminally differentiated endocrine population. Ghrelin cells give rise to significant numbers of alpha and PP cells and rare beta cells in the adult islet. Furthermore, pancreatic ghrelin-producing cells are maintained in pancreata lacking the essential endocrine lineage regulator Neurogenin3, and retain the ability to contribute to cells within the pancreatic ductal and exocrine lineages. These results demonstrate that the islet ghrelin-expressing epsilon cells represent a multi-potent progenitor cell population that delineates a major subgrouping of the islet endocrine cell populations. These studies also provide evidence that many of hormone-producing cells within the adult islet represent heterogeneous populations based on their ontogeny, which could have broader implications on the regulation of islet cell ratios and their ability to effectively respond to fluctuations in the metabolic environment during development.     

Development of hormonal peptides and processing enzymes in the embryonic avian pancreas with special reference to co-localisation

Studies on the developing mammalian pancreas have suggested that insulin and glucagon co-exist in a transient cell population and that peptide YY (PYY) marks the earliest developing endocrine cells. We have investigated this in the embryonic avian pancreas, which is characterised by anatomical separation of insulin and glucagon islets. Moreover, we have compared the development of the endocrine cells to that of processing enzymes involved in pancreatic hormone biosynthesis. PYY-like immunoreactivity occurred in islet cells from the youngest stages examined: it increased in amount from approximately 5 days of incubation and was co-localised with glucagon and to a lesser extent with insulin. Insulin and glucagon cells were numerous: co-existence of the two peptides in the same cells was but rarely observed. From the youngest stages examined, prohormone convertase (PC) 1/3-like immunoreactivity was detected in insulin cells and PC2-, 7B2- and carboxypeptidase E-like immunoreactivity in both glucagon and insulin cells. We conclude that: (1) PYY-like immunoreactivity occurs in avian islet cells but generally in lesser amounts than in mammals at the earlier stages, (2) the paucity of cells co-expressing insulin and glucagon indicate that all avian insulin cells do not pass through a stage where they co-express glucagon and (3) the early expression of the enzymes responsible for the processing of prohormones suggests that this process is initiated soon after islet cells first differentiate.     

Polypeptide YY- and neuropeptide Y-immunoreactive cells and nerves in the endocrine and exocrine pancreas of some vertebrates: An onto- and phylogenetic study

Summary The occurrence of polypeptide YY- and neuropeptide Y-immunoreactive cells and nerves in the pancreas of some species from all the eight main vertebrate groups (cyclostomes, cartilaginous fish, bony fish, amphibia, reptiles, birds, and mammals) was investigated. In addition, an ontogenetic study of these neurohormonal peptides was performed, using the rat pancreas. The distribution of these two peptides was compared with that of the structurally closely related pancreatic polypeptide.Polypeptide YY-immunoreactive cells were found to occur in the endocrine pancreas and neuropeptide Y-immunoreactivity was observed both in neurons and nerve fibres. The polypeptide YY-immunoreactive cells were limited to mammals and reptiles only. Neuropeptide Y-immunoreactive neurons and nerves were observed in reptiles, birds, and mammals only. One reptilian species (out of three) and one mammalian (out of six) failed to show any kind of immunoreactivity for the polypeptide or neuropeptide. Pancreatic polypeptide-immunoreactive cells were found in all the species examined except in the hagfish islet.In rat foetuses, polypeptide YY-immunoreactive cells and neuropeptide Y-immunoreactive nerve elements were first demonstrated at the seventeenth day of gestation, whereas pancreactic peptide-immunoreactive cells did not appear until postnatally, namely in two day-old rats. The polypeptide-containing cells, a new cell type in the endocrine pancreas, are rare. In contrast to the pancreatic peptide cells, they do not seem to have any kind of regional distribution.     

The adult mouse and human pancreas contain rare multipotent stem cells that express insulin

The search for putative precursor cells within the pancreas has been the focus of extensive research. Previously, we identified rare pancreas-derived multipotent precursor (PMP) cells in the mouse with the intriguing capacity to generate progeny in the pancreatic and neural lineages. Here, we establish the embryonic pancreas as the developmental source of PMPs through lineage-labeling experiments. We also show that PMPs express insulin and can contribute to multiple pancreatic and neural cell types in vivo. In addition, we have isolated PMPs from adult human islet tissue that are also capable of extensive proliferation, self-renewal, and generation of multiple differentiated pancreatic and neural cell types. Finally, both mouse and human PMP-derived cells ameliorated diabetes in transplanted mice. These findings demonstrate that the adult mammalian pancreas contains a population of insulin(+) multipotent stem cells and suggest that these cells may provide a promising line of investigation toward potential therapeutic benefit.     

The ghrelin cell: a novel developmentally regulated islet cell in the human pancreas

OBJECTIVES: Ghrelin, an endogenous ligand of the growth hormone secretagogue receptor (GHS-R), was recently identified in the stomach. Ghrelin is produced in a population of endocrine cells in the gastric mucosa, but expression in intestine, hypothalamus and testis has also been reported. Recent data indicate that ghrelin affects insulin secretion and plays a direct role in metabolic regulation and energy balance. On the basis of these findings, we decided to examine whether ghrelin is expressed in human pancreas. Specimens from fetal to adult human pancreas and stomach were studied by immunocytochemistry, for ghrelin and islet hormones, and in situ hybridisation, for ghrelin mRNA. RESULTS: We identified ghrelin expression in a separate population of islet cells in human fetal, neonatal, and adult pancreas. Pancreatic ghrelin cells were numerous from midgestation to early postnatally (10% of all endocrine cells). The cells were few, but regularly seen in adults as single cells at the islet periphery, in exocrine tissue, in ducts, and in pancreatic ganglia. Ghrelin cells did not express any of the known islet hormones. In fetuses, at midgestation, ghrelin cells in the pancreas clearly outnumbered those in the stomach. CONCLUSIONS: Ghrelin is expressed in a quite prominent endocrine cell population in human fetal pancreas, and ghrelin expression in the pancreas precedes by far that in the stomach. Pancreatic ghrelin cells remain in adult islets at lower numbers. Ghrelin is not co-expressed with any known islet hormone, and the ghrelin cells may therefore constitute a new islet cell type.     

Notch inhibits Ptf1 function and acinar cell differentiation in developing mouse and zebrafish pancreas

Notch signaling regulates cell fate decisions in a variety of adult and embryonic tissues, and represents a characteristic feature of exocrine pancreatic cancer. In developing mouse pancreas, targeted inactivation of Notch pathway components has defined a role for Notch in regulating early endocrine differentiation, but has been less informative with respect to a possible role for Notch in regulating subsequent exocrine differentiation events. Here, we show that activated Notch and Notch target genes actively repress completion of an acinar cell differentiation program in developing mouse and zebrafish pancreas. In developing mouse pancreas, the Notch target gene Hes1 is co-expressed with Ptf1-P48 in exocrine precursor cells, but not in differentiated amylase-positive acinar cells. Using lentiviral delivery systems to induce ectopic Notch pathway activation in explant cultures of E10.5 mouse dorsal pancreatic buds, we found that both Hes1 and Notch1-IC repress acinar cell differentiation, but not Ptf1-P48 expression, in a cell-autonomous manner. Ectopic Notch activation also delays acinar cell differentiation in developing zebrafish pancreas. Further evidence of a role for endogenous Notch in regulating exocrine pancreatic differentiation was provided by examination of zebrafish embryos with homozygous mindbomb mutations, in which Notch signaling is disrupted. mindbomb-deficient embryos display accelerated differentiation of exocrine pancreas relative to wild-type clutchmate controls. A similar phenotype was induced by expression of a dominant-negative Suppressor of Hairless [Su(H)] construct, confirming that Notch actively represses acinar cell differentiation during zebrafish pancreatic development. Using transient transfection assays involving a Ptf1-responsive reporter gene, we further demonstrate that Notch and Notch/Su(H) target genes directly inhibit Ptf1 activity, independent of changes in expression of Ptf1 component proteins. These results define a normal inhibitory role for Notch in the regulation of exocrine pancreatic differentiation.