Analyses of pancreas development by generation of gfp transgenic zebrafish using an exocrine pancreas-specific elastaseA gene promoter

In contrast to what we know on development of endocrine pancreas, the formation of exocrine pancreas remains poorly understood. To create an animal model that allows observation of exocrine cell differentiation, proliferation, and morphogenesis in living animals, we used the zebrafish elastaseA (elaA) regulatory sequence to develop transgenic zebrafish that display highly specific exocrine pancreas expression of GFP in both larvae and adult. By following GFP expression, we found that the pancreas in early development was a relatively compact organ and later extended posterior along the intestine. By transferring the elaA:gfp transgene into slow muscle omitted mutant that is deficient in receiving Hedgehog signals, we further showed that Hedgehog signaling is required for exocrine morphogenesis but not for cell differentiation. We also applied the morpholino knockdown and toxin-mediated cell ablation approaches to this transgenic line. We showed that the development of exocrine pancreas is Islet-1 dependent. Injection of the diphtheria toxin A (DTA) construct under the elastaseA promoter resulted in selective ablation of exocrine cells while the endocrine cells and other endodermal derivatives (liver and intestine) were not affected. Thus, our works demonstrated the new transgenic line provided a useful experimental tool in analyzing exocrine pancreas development.     

DTA-mediated targeted ablation revealed differential interdependence of endocrine cell lineages in early development of zebrafish pancreas

Cell lineage analysis is critical in understanding the relationship between progenitors and differentiated cells as well as the mechanism underlying the process of differentiation. In order to study the zebrafish endocrine pancreas cell lineage, transgenic expression of diphtheria toxin gene A chain (DTA) under two cell type-specific promoters derived from the insulin (ins) and somatostatin2 (sst2) genes was used to ablate the two types of endocrine cells: insulin-producing β-cells and somatostatin-producing δ-cells, respectively. We found that ablation of β-cells resulted in a reduction of not only β-cells but also glucagon-producing α-cells; in contrast, δ-cells were largely unaffected. Ablation of δ-cells led to reduction of all three types of endocrine cells: α-, β-, and δ. Interestingly, α-cells were more profoundly affected in both β- and δ-cell ablations and were frequently reduced together with β- and δ-cells. By taking advantage of Tg(ins:gfp) and Tg(sst2:gfp) lines, we also monitored the changes of different types of endocrine cells in vivo after ablation and found that both β- and δ-cell populations significantly recovered by 3 dpf after their ablation and it seemed that δ-cells had a better capability of recovery than β-cells. Thus, our current observations indicated differential interdependence of these three cell lineages. The development of zebrafish α-cells, but not δ-cells, is dependent on β-cells, while the development of both α- and β-cells is dependent on δ-cells. In contrast, the development of δ-cells was independent of β-cells.     

The endocrine pancreas of glucagon-and somatostatin-immunized rabbits

An active or passive immunization against hormones and the subsequent neutralization of hormones by circulating antibodies is a valuable tool for the identification of hormonal action. To recognize presumed local (autocrine, paracrine) effects exerted by pancreatic hormones, the endocrine pancreas of rabbits was investigated electron-microscopically after long-term immunization against glucagon or somatostatin. Glucagon immunization resulted in hyperplasia and hypertrophy of glucagon- (A-) cells and in their increased metabolic activities: They showed prominent nucleoli, increased amounts of endoplasmic reticulum, Golgi areas, and mitochondria. These changes were paralleled by alterations in secretion granules (increased size, decreased hormonal content), increased numbers of lysosomes (crinophagic bodies), and an increment of the filamentous system. Basically, these findings point to an autocrine regulation of A-cells. Following somatostatin immunization, somatostatin- (D-) cells were hyperplastic but unchanged in their metabolic state. Instead, insulin-(B-) cells and A-cells exhibited equivalents of increased cellular activities (parameters, see above). This stimulation most probably is caused by cancelled paracrine (inhibitory) effects of somatostatin. The changes observed after both immunizations were differently expressed in morphologically heterogeneous islet types (size, angioarchitecture, cellular composition, microtopology of the various cell types). It is concluded, therefore, that the regulation of islets is not uniform. Autocrine and paracrine effects exerted by islet hormones are of different significance in individual islets, or they interfere differently with other regulatory signals.     

The development of zebrafish pancreas affected by deficiency of Hedgehog signaling

The pancreas development depends on complex regulation of several signaling pathways, including the Hedgehog (Hh) signaling via a receptor complex component, Smoothened, which deficiency blocks the Hh signaling. Such a defect in birds and mammals results in an annular pancreas. We showed that in developing zebrafish, the mutation of Smoothened or inhibition of Hh signaling by its antagonist cyclopamine caused developmental defects of internal organs, liver, pancreas, and gut. In particular, the pancreatic primordium was duplicated. The two exocrine pancreatic primordia surround the gut. This phenomenon correlates with a significant reduction of the gut's diameter, causing the annular pancreas phenotype.     

The endocrine pancreas of Alligator mississippiensis

Summary Immunocytochemical methods for light and electron microscopy were used to demonstrate the regulatory peptides present in the endocrine pancreas of thealligator, Alligator mississippiensis.The peptides studied included insulin, glucagon (pancreatic and enteric), somatostatin, pancreatic polypeptide (avian, bovine and human), vasoactive intestinal polypeptide, substance P, metenkephalin, -endorphin, C-terminal gastrin/CCK and gastric inhibitory polypeptide. Endocrine cells were detected using antisera to insulin, pancreatic glucagon, somatostatin and avian pancreatic polypeptide, whereas peptidergic nerves were stained with antisera to vasoactive intestinal polypeptide. All other antisera were unreactive in the alligator pancreas. The peptide-containing structures were identified ultrastructurally by both the semithin/thin and immuno-gold methods. The results showed that five of the regulatory peptides commonly detected in the mammalian pancreas were immunologically recognisable in the alligator. In addition, the ultrastructural appearance of the peptide-containing cells was clearly distinct from that reported in mammals.     

The endocrine pancreas of glucagon-and somatostatin-immunized rabbits

Summary Peptide antibodies raised in rabbits are widely used in biology and medicine. During immunization of the animals, the respective antibodies may affect the endocrine cells physiologically responsible for the synthesis of peptides used as antigens. Since corresponding morphological data are still sparse, the rabbit endocrine pancreas was systematically investigated by light microscopy and immunocytochemistry after long-term immunization against glucagon and somatostatin. Both immunizations led to an increase in the number of islets (nesidioblastosis), to the development of giant islets (macronesia), and to changes in the relative proportions of the major types of endocrine cells or their hormonal content. The latter changes differed after either immunization: glucagon immunization resulted in hypertrophy and hyperplasia of glucagon cells and a decrease in their hormonal content; somatostatin immunization led to an increased proportion of somatostatin cells and a lowered hormonal content of insulin cells. The various alterations were expressed differently according to islet type; islets of the rabbit pancreas differ in size or angioarchitecture, and in the proportion and distribution of endocrine cells. The present findings point to autocrine or paracrine effects of the respective peptides. These effects, however, are obviously of differing significance in morphologically heterogeneous islets.Dedicated to Professor Dr. Tsuneo Fujita, Niigata University, JapanPresented in part at the 30th Symposium of the Deutsche Gesellschaft für Endokrinologie (see Jörns et al. 1986)     

Notch-responsive cells initiate the secondary transition in larval zebrafish pancreas

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.     

Insulin-, glucagonand somatostatin-like immunoreactivity in the endocrine pancreas of the lungfish,Neoceratodus forsteri

Summary The endocrine pancreas of the Australian lungfish,Neoceratodus forsteri, was investigated immunocytochemically for the presence of polypeptide hormone-producing cells. Three cell types were identified, namely insulin-, glucagon-, and somatostatin-immunoreactive elements. The insulin cells are confined solely to the center of the islets. Glucagon and somatostatin cells are distributed peripherally around the central mass of the insulin cells. Isolated cells or clusters of glucagon and somatostatin cells are also dispersed within the exocrine parenchyma. The immunoreactive cell types are compared with those staining with standard histological procedures. The spatial relationships of the different cell populations are examined.     

Nkx6.1 and nkx6.2 regulate α- and β-cell formation in zebrafish by acting on pancreatic endocrine progenitor cells

In mice, the Nkx6 genes are crucial to α- and β-cell differentiation, but the molecular mechanisms by which they regulate pancreatic subtype specification remain elusive. Here it is shown that in zebrafish, nkx6.1 and nkx6.2 are co-expressed at early stages in the first pancreatic endocrine progenitors, but that their expression domains gradually segregate into different layers, nkx6.1 being expressed ventrally with respect to the forming islet while nkx6.2 is expressed mainly in β-cells. Knockdown of nkx6.2 or nkx6.1 expression leads to nearly complete loss of α-cells but has no effect on β-, δ-, or ε-cells. In contrast, nkx6.1/nkx6.2 double knockdown leads additionally to a drastic reduction of β-cells. Synergy between the effects of nkx6.1 and nkx6.2 knockdown on both β- and α-cell differentiation suggests that nkx6.1 and nkx6.2 have the same biological activity, the required total nkx6 threshold being higher for α-cell than for β-cell differentiation. Finally, we demonstrate that the nkx6 act on the establishment of the pancreatic endocrine progenitor pool whose size is correlated with the total nkx6 expression level. On the basis of our data, we propose a model in which nkx6.1 and nkx6.2, by allowing the establishment of the endocrine progenitor pool, control α- and β-cell differentiation.     

An immunocytochemical study of endocrine pancreas of snakes

Summary The pancreas from eleven species of snakes representing both advanced and primitive families has been investigated for the presence of eleven regulatory peptides reported to occur in the mammalian endocrine pancreas. Of the eleven peptides studied, insulin, pancreatic glucagon and somatostatin were present in endocrine cells within the islets of all the species investigated. The neuropeptide, vasoactive intestinal polypeptide, was located within nerve terminals innervating the islets in the Boidinae, Colubrinae, Elaphidae and Crotalidae but absent from the Natricinae investigated.No immunoreactivity was demonstrable with the antisera to substance P, met-enkephalin, C-terminal gastrin, bombesin, glicentin and gastric inhibitory polypeptide. Pancreatic polypeptide-like immunoreactivity was demonstrable only in the boid snakes and exclusively stained by a C-terminal specific antiserum.     

On argyrophil reactions of endocrine cells in the human fetal pancreas

Summary The endocrine cells in the pancreas of five human fetuses with gestational ages of 18–20 weeks were examined by light and electron microscopy with special regard to argyrophil reactions. B-cells and typical A and D-cells were easily identified electron microscopically on the basis of their typical secretory granules. In the Grimelius argyrophil silver stain, a concentration of silver grains over the less electron dense peripheral mantle of the A-cell secretory granules was observed by electron microscopy. In the Hellerström and Hellman modification of the argyrophil Davenport alcoholic silver stain, silver grains were concentrated over the internal structures of the D-cell secretory granules. With this stain an accumulation of silver grains was also seen at the surface of the A-cell secretory granules. The argyrophil reaction of the A-granules was less pronounced than in the D-cells. In addition to B-cells and A- and D-cells, two other types of endocrine cell were observed by electron microscopy. These cells were argyrophil with the silver impregnation method of Grimelius. The electron microscopic findings at least partly explain the frequent overlapping between the two staining methods observed at the light microscope level.This study was supported by the Swedish Medical Research Council (Project No. 102)     

Immunohistochemical localization of insulin-like growth factor 1 and 2 in the endocrine pancreas of rat,dog, and man,and their coexistence with classical islet hormones

Immunohistochemical techniques were used to study the occurrence and distribution of insulin-like growth factor 1 (IGF-1) and IGF-2 in the pancreas of man, dog, and rat and their possible coexistence with insulin (INS), glucagon (GLUC), somatostatin (SOM) and pancreatic polypeptide (PP). All control experiments, including pre-absorption of the antisera with synthetic peptide hormones, indicated the specificity of the immunoreactions obtained. In all species investigated, IGF-2-immunoreactivity occurred exclusively in INS-immunoreactive cells as was found by the use of consecutive sections and double immunofluorescence on identical sections. In contrast, IGF-1-immunoreactivity co-existed with GLUC-immunoreactivity. In man, singular SOM-immunoreactive cells also contained IGF-1-immunoreactivity. Thus, IGF-1 and IGF-2 can be localized by means of immunohistochemistry in the mammalian pancreas, and can be shown to occur in different islet cell populations. It is presumed that IGF-1 derived from A-cells and/or D-cells acts on the B-cells in a paracrine manner. The co-existence of IGF-2-immunoreactivity and INS-immunoreactivity in the human, rat, and dog endocrine pancreas indicates that mammalian IGF-2 and INS genes are regulated simultaneously.     

Visualization of monoaminergic neurons and neurotoxicity of MPTP in live transgenic zebrafish

We describe an enhancer trap transgenic zebrafish line, ETvmat2:GFP, in which most monoaminergic neurons are labeled by green fluorescent protein (GFP) during embryonic development. The reporter gene of ETvmat2:GFP was inserted into the second intron of vesicular monoamine transporter 2 (vmat2) gene, and the GFP expression pattern recapitulates that of the vmat2 gene. The GFP positive neurons include the large and pear-shaped tyrosine hydroxylase positive neurons (TH populations 2 and 4) in the posterior tuberculum of ventral diencephalon (PT neurons), which are thought to be equivalent to the midbrain dopamine neurons in mammals. We found that these PT neurons and two other GFP labeled non-TH type neuronal groups, one in the paraventricular organ of the posterior tuberculum and the other in the hypothalamus, were significantly reduced after exposure to MPTP, while the rest of GFP-positive neuronal clusters, including those in telencephalon, pretectum, raphe nuclei and locus coeruleus, remain largely unchanged. Furthermore, we showed that the effects of hedgehog signaling pathway inhibition on the development of monoaminergic neurons can be easily visualized in individual living ETvmat2:GFP embryos. This enhancer trap line should be useful for genetic and pharmacological analyses of monoaminergic neuron development and processes underlying Parkinson's disease.     

Development of a rod photoreceptor mosaic revealed in transgenic zebrafish

The number and distribution of neurons within the vertebrate retina are tightly regulated. This is particularly apparent in the highly ordered, crystalline-like arrangement of the cone photoreceptors in the teleost. In this report, using a transgenic line of zebrafish, a novel and developmentally regulated mosaic pattern of the rod photoreceptors is described. The spatial and temporal expression of EGFP, under the control of the Xenopus rhodopsin gene promoter, was nearly identical to the endogenous rhodopsin. EGFP was first detected in the ventral nasal retinal in an area of precocious neurogenesis referred to as the "ventral patch". Subsequent expression of EGFP was observed in isolated cells sporadically distributed across the dorsal and central retina. However, confocal microscopy and spatial analysis of larval eyes or retinal explants from adults revealed a precise arrangement to the rod photoreceptors. The rod terminals were arranged in regularly spaced rows with clearly identifiable telodendria linking neighboring cells. The rod inner segments projected through the cone mosaic in a predictable pattern. In the adult, the rod mosaic originated near the retinal margin where clusters of rods differentiated around the immature short single cone. In the embryo, the sporadic differentiation of the rods led to the gradual formation of the mosaic pattern. With the growing interest in neuronal stem cells, revisiting this model of neurogenesis provides an avenue to uncover mechanisms underlying the precise integration of new neuronal elements into a preexisting neural network.     

The distribution of endocrine cells along the mouse small intestine

The topographical distribution and incidence of endocrine cells in the crypt and villus epithelium and along the length of the mouse intestine was studied. Cells containing somatostatin and bombesin like reactivity were stained by immunocytochemical techniques using polyclonal antiserum. Most of the somatostatin cells were found in the duodenum, jejunum and ileum, and these cells were generally more frequent on the villus compared to the crypts. This may indicate that the somatostatin cells develop late in the endocrine cell lineage. Bombesin like cells were rare in occurence, and were only present in measureable numbers in the ileum, where they were observed in the crypt and villi. The application of ELISA assays to determine the specificity of the antisera for these peptides is also discussed.     

Characterization of somatostatin receptors and associated signaling pathways in pancreas of R6/2 transgenic mice

The present study describes the status of somatostatin receptors (SSTRs) and their colocalization with insulin (β), glucagon (α) and somatostatin (δ) producing cells in the pancreatic islets of 11 weeks old R6/2 Huntington's Disease transgenic (HD tg) and age-matched wild type (wt) mice. We also determined expression of tyrosine hydroxylase (TH), glutamic acid decarboxylase (GAD) and presynaptic marker synaptophysin (SYP) in addition to signal transduction pathways associated with diabetes. In R6/2 mice, islets are relatively smaller in size, exhibit enhanced expression and nuclear inclusion of mHtt along with the loss of insulin, glucagon and somatostatin expression. In comparison to wt, R6/2 mice display enhanced mRNA for all SSTRs except SSTR2. In the pancreatic lysate, SSTR1, 4 and 5 immunoreactivity decreases whereas SSTR3 immunoreactivity increases with no discernible changes in SSTR2 immunoreactivity. Furthermore, at the cellular level, R6/2 mice exhibit a receptor specific distributional pattern of SSTRs like immunoreactivity and colocalization with β, α and δ cells. While GAD expression is increased, TH and SYP immunoreactivity was decreased in R6/2 mice, anticipating a cross-talk between the CNS and pancreas in diabetes pathophysiology. We also dissected out the changes in signaling pathway and found decreased activation and expression of PKA, AKT, ERK1/2 and STAT3 in R6/2 mice pancreas. These findings suggest that the impaired organization of SSTRs within islets may lead to perturbed hormonal regulation and signaling. These interconnected complex events might shed new light on the pathogenesis of diabetes in neurodegenerative diseases and the role of SSTRs in potential therapeutic intervention.     

Generation and characterization of transgenic zebrafish lines using different ubiquitous promoters

Two commonly used promoters to ubiquitously express transgenes in zebrafish are the Xenopus laevis elongation factor 1 α promoter (XlEef1a1) and the zebrafish histone variant H2A.F/Z (h2afv) promoter. Recently, transgenes utilizing these promoters were shown to be silenced in certain adult tissues, particularly the central nervous system. To overcome this limitation, we cloned the promoters of four zebrafish genes that likely are transcribed ubiquitously throughout development and into the adult. These four genes are the TATA box binding protein gene, the taube nuss-like gene, the eukaryotic elongation factor 1-gamma gene, and the beta-actin-1 gene. We PCR amplified approximately 2.5 kb upstream of the putative translational start site of each gene and cloned each into a Tol2 expression vector that contains the EGFP reporter transgene. We used these four Tol2 vectors to independently generate stable transgenic fish lines for analysis of transgene expression during development and in the adult. We demonstrated that all four promoters drive a very broad pattern of EGFP expression throughout development and the adult. Using the retina as a well-characterized component of the CNS, all four promoters appeared to drive EGFP expression in all neuronal and non-neuronal cells of the adult retina. In contrast, the h2afv promoter failed to express EGFP in the adult retina. When we examined EGFP expression in the various cells of the blood cell lineage, we observed that all four promoters exhibited a more heterogenous expression pattern than either the XlEef1a1 or h2afv promoters. While these four ubiquitous promoters did not express EGFP in all the adult blood cells, they did express EGFP throughout the CNS and in broader expression patterns in the adult than either the XlEef1a1 or h2afv promoters. For these reasons, these four promoters will be valuable tools for expressing transgenes in adult zebrafish.