Development of endocrine pancreatic islets in embryos of the grass snake Natrix natrix (Lepidosauria,Serpentes)

Differentiation of the pancreatic islets in grass snake Natrix natrix embryos, was analyzed using light, transmission electron microscopy, and immuno-gold labeling. The study focuses on the origin of islets, mode of islet formation, and cell arrangement within islets. Two waves of pancreatic islet formation in grass snake embryos were described. The first wave begins just after egg laying when precursors of endocrine cells located within large cell agglomerates in the dorsal pancreatic bud differentiate. The large cell agglomerates were divided by mesenchymal cells thus forming the first islets. This mode of islet formation is described as fission. During the second wave of pancreatic islet formation which is related to the formation of the duct mantle, we observed four phases of islet formation: (a) differentiation of individual endocrine cells from the progenitor layer of duct walls (budding) and their incomplete delamination; (b) formation of two types of small groups of endocrine cells (A/D and B) in the wall of pancreatic ducts; (c) joining groups of cells emerging from neighboring ducts (fusion) and rearrangement of cells within islets; (d) differentiated pancreatic islets with characteristic arrangement of endocrine cells. Mature pancreatic islets of the grass snake contained mainly A endocrine cells. Single B and D or PP–cells were present at the periphery of the islets. This arrangement of endocrine cells within pancreatic islets of the grass snake differs from that reported from most others vertebrate species. Endocrine cells in the pancreas of grass snake embryos were also present in the walls of intralobular and intercalated ducts. At hatching, some endocrine cells were in contact with the lumen of the pancreatic ducts.     

Nestin is expressed in vascular endothelial cells in the adult human pancreas.

In this study we examined the expression of nestin in islets, the exocrine part, and the big ducts of the adult human pancreas by immunofluorescent double staining. Two different anti-nestin antisera in combination with various pancreatic and endothelial markers were employed. Nestin-immunoreactive cells were found in islets and in the exocrine portion. All nestin-positive cells co-expressed the vascular endothelial markers PECAM-1 (CD31), endoglin (CD105), and CD34 as well as vimentin. Endocrine, acinar, and duct cells did not stain for nestin. We also demonstrated that in the area of big pancreatic ducts, nestin-positive cells represent small capillaries scattered in the connective tissue surrounding the duct epithelium and do not reside between the duct cells. We detected nestin-expressing endothelial cells located adjacent to the duct epithelium where endocrine differentiation occurs. We have shown that nestin is expressed by vascular endothelial cells in human pancreas, and therefore it is unlikely that nestin specifically marks a subpopulation of cells representing endocrine progenitors in the adult pancreas.     

Effect of initial developmental stage on morphology of transplanted embryonic chick pancreas

Summary It has been reported that only certain types of pancreatic parenchymal cells survive transplantation. This study examines whether the extent of differentiation of the pancreas at the time of transplantation affects the resulting morphology or viability of its components. Segments of chick pancreas or its primordia from stages preceding formation of dorsal bud (60 h) through hatching (day 21) were implanted in the abdominal region of three-day chick embryos. After various periods of growth, grafts were examined by light- and electron microscopy. In all transplants, individual endocrine cells (A, B, D, PP) and islet structure were identical to those of normal embryos of comparable age. The exocrine portion also appeared normal in implants from embryos younger than seven days. In grafts from older donors, however, normal acini and ducts were replaced by dilated, irregularly shaped tubules from which new islets appeared to develop. These results suggest that transplantation causes structural modification of exocrine tissue, which may reflect its initial functional capabilities. These observations are compared with similar structural alterations that occur following experimental obstruction of ducts and in human pancreatic pathology.     

Postnatal development of the pancreas in the opossum. Light microscopy.

The pancreas of the newborn opossum consists of a central region of forming islets surrounded by primitive tubules that end in proacinar cells. Paratubular buds, which are outgrowths from the tubular epithelium, characterize the newborn pancreas and eventually give rise to both exocrine and endocrine units. 4 days after birth, definite intralobular ducts, acini and centroacinar cells are observed. In addition to the central expanding islets (primary islets), endocrine cells are observed singly or in small groups in the ductal epithelium. The endocrine cells are believed to originate from the terminal cells of the ductal epithelium and, throughout the entire postnatal period, retain a close association with the exocrine epithelium. With the simultaneous proliferation of both endocrine and exocrine components from the ductal system, the majority of the islets observed at 24 days (5.0 cm) appear to be surrounded by a single layer of acinar cells. As acini develop and the ducts expand toward the periphery, this layer of acinar cells separates from the developing islets, the majority of which have become localized within the centers of lobules to form the secondary islets by the 10.0-cm stage (59 days). A marked development of lobules is observed by the 13.0-cm stage and the majority of acinar cells now are filled with zymogen granules. Acinar cells continue to proliferate late into the postnatal period and the majority of acini exhibit a tubular form in the juvenile and adult opossum.     

TGF-beta isoform signaling regulates secondary transition and mesenchymal-induced endocrine development in the embryonic mouse pancreas

Transforming growth factor-beta (TGF-beta) superfamily signaling has been implicated in many developmental processes, including pancreatic development. Previous studies are conflicting with regard to an exact role for TGF-beta signaling in various aspects of pancreatic organogenesis. Here we have investigated the role of TGF-beta isoform signaling in embryonic pancreas differentiation and lineage selection. The TGF-beta isoform receptors (RI, RII and ALK1) were localized mainly to both the pancreatic epithelium and mesenchyme at early stages of development, but then with increasing age localized to the pancreatic islets and ducts. To determine the specific role of TGF-beta isoforms, we functionally inactivated TGF-beta signaling at different points in the signaling cascade. Disruption of TGF-beta signaling at the receptor level using mice overexpressing the dominant-negative TGF-beta type II receptor showed an increase in endocrine precursors and proliferating endocrine cells, with an abnormal accumulation of endocrine cells around the developing ducts of mid-late stage embryonic pancreas. This pattern suggested that TGF-beta isoform signaling may suppress the origination of secondary transition endocrine cells from the ducts. Secondly, TGF-beta isoform ligand inhibition with neutralizing antibody in pancreatic organ culture also led to an increase in the number of endocrine-positive cells. Thirdly, hybrid mix-and-match in vitro recombinations of transgenic pancreatic mesenchyme and wild-type epithelium also led to increased endocrine cell differentiation, but with different patterns depending on the directionality of the epithelial-mesenchymal signaling. Together these results suggest that TGF-beta signaling is important for restraining the growth and differentiation of pancreatic epithelial cells, particularly away from the endocrine lineage. Inhibition of TGF-beta signaling in the embryonic period may thus allow pancreatic epithelial cells to progress towards the endocrine lineage unchecked, particularly as part of the secondary transition of pancreatic endocrine cell development. TGF-beta RII in the ducts and islets may normally serve to downregulate the production of beta cells from embryonic ducts.     

5-羟色胺在豚鼠胰腺分布的免疫组织化学研究

本研究用ABC免疫染色法,结合葡萄糖氧化酶-DAB-硫酸镍铵(Glucose oxidase-DAB-Nickle,GDN)显色技术,在Bouin液固定的常规石蜡切片上,研究了5-羟色胺(5-hydroxytryptamin,5-HT)在豚鼠胰腺内的定位和分布,并用相邻切片免疫双标记,观察了它与胰岛素的共存关系,结果发现,在豚鼠胰腺内,外分泌部均有5-HT免疫反应细胞分布。在胰腺内分泌部(胰岛)5-HT免疫反应细胞分布均匀,大部分胰岛细胞呈阳性5-HT样免疫反应,用相邻薄切片免疫双标记技术证明,胰岛内的5-HT免疫反应细胞主要是B细胞。在胰腺外分泌部,5-HT免疫反应细胞呈单个分散或聚集分布,主要位于腺泡和导管等处,偶见于结缔组织间隔中。本文对研究5-HT在胰腺的生理作用及其机制提供了形态学依据。     

Distribution of neuropeptide Y and vasoactive intestinal polypeptide immunoreactive nerves in normal and transplanted pancreatic tissue

Neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) immunoreactive nerves were demonstrated in 21-day-old embryonic pancreatic tissue fragments transplanted into the anterior eye chamber of rats for 22, 45 and 109 days and in 60-day-old normal adult pancreas using immunohistochemical technique. In normal adult tissue, NPY-positive neurons lie close to the basal and lateral walls of the acinar cells. NPY-containing nerve fiber plexuses were found around blood vessels. VIP-immunopositive nerves were also discernible in the outer parts of the islets of Langerhans and on pancreatic ducts. In the transplants, it is not only the neural elements that survived but also the pancreatic ducts and the endocrine cells. VIP- and NPY-positive neurons were found in the stroma of the surviving pancreatic tissue. The distribution of these neural elements is similar to that of normal tissue in the surviving pancreatic ducts but different with regards to the acinar tissue. This study confirms that intrinsic nerves can survive and synthesize polypeptides even after 109 days of transplantation into the anterior eye chamber.     

Imaging pancreatic beta-cells in the intact pancreas

We have developed a method to visualize fluorescent protein-labeled beta-cells in the intact pancreas through combined reflection and confocal imaging. This method provides a 3-D view of the beta-cells in situ. Imaging of the pancreas from mouse insulin I promoter (MIP)-green (GFP) and red fluorescent protein (RFP) transgenic mice shows that islets, beta-cell clusters, and single beta-cells are not evenly distributed but are aligned along the large blood vessels. We also observe the solitary beta-cells in both fetal and adult mice and along the pancreatic and common bile ducts. We have imaged the developing endocrine cells in the embryos using neurogenin-3 (Ngn3)-GFP mice crossed with MIP-RFP mice. The dual-color-coded pancreas from embryos (E15.5) shows a large number of green Ngn3-expressing proendocrine cells with a smaller number of red beta-cells. The imaging technique that we have developed, coupled with the transgenic mice in which beta-cells and beta-cell progenitors are labeled with different fluorescent proteins, will be useful for studying pancreatic development and function in normal and disease states.     

Some characteristics of the histological structure of the fetal bovine pancreas

Some properties of histological structure of fetal bovine pancreas were demonstrated using light microscopic methods. The different forms of acino-insular complexes were described: 1) acino-insular complexes with single B-cells including epithelial layer of acini; 2) acino-insular complexes with segmental (sector) localization of insular cell groups; 3) acino-insular complexes with small and more large groups of endocrine cells timely contacted with acini; 4) acino-insular complexes at the stage of separation of endocrine cell groups (microislets) from acini. The consideration of acino-insular complexes in morphogenesis of bovine endocrine pancreas in discussed.     

Fetoacinar pancreatic protein in the developing human pancreas

The distribution of the 110-kilodalton fetoacinar pancreatic (FAP) protein was examined in 56 pancreases obtained from human embryos and fetuses (ranging 6 from weeks of gestation to full term) and 10 normal adult pancreases. This recently discovered protein is a concanavalin-A-binding glycoprotein that is specific for acinar cells of the pancreas. Using a murine monoclonal antibody for either immunoperoxidase or immunofluorescence procedures, FAP-protein expression was not found in embryos at less than 9 weeks of gestation. At 9-10 weeks, a clear staining was observed in the terminal portions of dilated buds in primitive pancreatic tubular structures (i.e., the site of the first development of the future acinus). At 11-12 weeks, acinar structuration began, and FAP-protein expression increased as shown by the higher number of stained acini and the greater staining intensity. Maximal expression occurred at 15-22 weeks and then gradually decreased; from 28 to 32 weeks until full term, the pancreas was almost negative for this protein. In the adult pancreases, the protein was either absent or only present in acinar cells surrounding the islets of Langerhans. The pancreatic ducts and endocrine cells remained negative throughout gestation and in adults. FAP-protein thus appears to be a marker of acinar-cell differentiation. Its function remains unknown at present. Its close association with the growth and development of the pancreas together with the fact that, in a previous study, it was found to be re-expressed in pancreatitis and in cancer, suggest that it may play a role in developmental regenerative and neoplastic processes in the pancreas.     

Expression and function of alpha(v)beta(3) and alpha(v)beta(5) integrins in the developing pancreas: roles in the adhesion and migration of putative endocrine progenitor cells

Cell-cell and cell-matrix interactions play a critical role in tissue morphogenesis and in homeostasis of adult tissues. The integrin family of adhesion receptors regulates cellular interactions with the extracellular matrix, which provides three-dimensional information for tissue organization. It is currently thought that pancreatic islet cells develop from undifferentiated progenitors residing within the ductal epithelium of the fetal pancreas. This process involves cell budding from the duct, migration into the surrounding mesenchyme, differentiation, and clustering into the highly organized islet of Langerhans. Here we report that alpha(v)beta(3) and alpha(v)beta(5), two integrins known to coordinate epithelial cell adhesion and movement, are expressed in pancreatic ductal cells and clusters of undifferentiated cells emerging from the ductal epithelium. We show that expression and function of alpha(v)beta(3) and alpha(v)beta(5) integrins are developmentally regulated during pancreatic islet ontogeny, and mediate adhesion and migration of putative endocrine progenitor cells both in vitro and in vivo in a model of pancreatic islet development. Moreover, we demonstrate the expression of fibronectin and collagen IV in the basal membrane of pancreatic ducts and of cell clusters budding from the ductal epithelium. Conversely, expression of vitronectin marks a population of epithelial cells adjacent to, or emerging from, pancreatic ducts. Thus, these data provide the first evidence for the contribution of integrins alpha(v)beta(3) and alpha(v)beta(5) and their ligands to morphogenetic events in the human endocrine pancreas.     

MicroRNA miR-7 is preferentially expressed in endocrine cells of the developing and adult human pancreas

MicroRNAs (miRNA) are small non-coding RNAs that inhibit gene expression through binding to complementary messenger RNA sequences. miRNAs have been predicted to target genes important for pancreas development, proper endocrine cell function and metabolism. We previously described that miRNA-7 (miR-7) was the most abundant and differentially expressed islet miRNA, with 200-fold higher expression in mature human islets than in acinar tissue. Here we have analyzed the temporal and spatial expression of miR-7 in human fetal pancreas from 8 to 22 weeks of gestational age (wga). Human fetal (8–22 wga) and adult pancreases were processed for immunohistochemistry, in situ hybridization, and quantitative RT-PCR of miRNA and mRNA. miR-7 was expressed in the human developing pancreas from around 9 wga and reached its maximum expression levels between 14 and 18 wga, coinciding with the exponential increase of the pancreatic endocrine hormones. Throughout development miR-7 expression was preferentially localized to endocrine cells and its expression persisted in the adult pancreas. The present study provides a detailed analysis of the spatiotemporal expression of miR-7 in developing human pancreas. The specific localization of miR-7 expression to fetal and adult endocrine cells indicates a potential role for miR-7 in endocrine cell differentiation and/or function. Future functional studies of a potential role for miR-7 function in islet cell differentiation and physiology are likely to identify novel targets for the treatment of diabetes and will lead to the development of improved protocols for generating insulin-producing cells for cell replacement therapy.     

Recapitulation of embryonic neuroendocrine differentiation in adult human pancreatic duct cells expressing neurogenin 3

Regulatory proteins have been identified in embryonic development of the endocrine pancreas. It is unknown whether these factors can also play a role in the formation of pancreatic endocrine cells from postnatal nonendocrine cells. The present study demonstrates that adult human pancreatic duct cells can be converted into insulin-expressing cells after ectopic, adenovirus-mediated expression of the class B basic helix-loop-helix factor neurogenin 3 (ngn3), which is a critical factor in embryogenesis of the mouse endocrine pancreas. Infection with adenovirus ngn3 (Adngn3) induced gene and/or protein expression of NeuroD/beta2, Pax4, Nkx2.2, Pax6, and Nkx6.1, all known to be essential for beta-cell differentiation in mouse embryos. Expression of ngn3 in adult human duct cells induced Notch ligands Dll1 and Dll4 and neuroendocrine- and beta-cell-specific markers: it increased the percentage of synaptophysin- and insulin-positive cells 15-fold in ngn3-infected versus control cells. Infection with NeuroD/beta2 (a downstream target of ngn3) induced similar effects. These data indicate that the Delta-Notch pathway, which controls embryonic development of the mouse endocrine pancreas, can also operate in adult human duct cells driving them to a neuroendocrine phenotype with the formation of insulin-expressing cells.     

Pathology of the endocrine pancreas.

An immunocytochemical analysis of 94 pancreatic endocrine tumors revealed that 73 tumors were multicellular. Significant amounts of somatostatin and human pancreatic polypeptide were found by radioimmunoassay in extracts of 19 and 17 tumors resp., in addition to the hormone causing the clinical syndrome. Numerous tumors contained ductular structures. In the surrounding pancreatic parenchyma a proliferation of small ducts and budding-off from the ductular epithelium of endocrine cells was often observed. These features are hallmarks of nesidioblastosis of the endocrine pancreas which is a hyperplasia. In multiple endocrine neoplasia I hyperplasia of the endocrine pancreas is combined with larger nodules, currently labeled tumors. On the basis of these findings it is conceivable that pancreatic endocrine tumors are not primarily neoplastic and autonomous but that they are rather of hyperplastic origin.     

Differentiation of prospective mouse pancreatic islet cells during development in vitro and during regeneration

The pancreatic islets of mouse embryos are comprised of four different endocrine cell types and of cells containing a hormone (i.e., glucagon) and a catecholamine enzyme (tyrosine hydroxylase, TH) which appear sequentially during development in vivo. The presence of TH in glucagon cells, however, is transient, since adult pancreatic A cells do not express the enzyme. In this study we sought to determine whether the endocrine precursor cells are primed to differentiate and express catecholamine enzymes during their maturation following a predetermined sequence or whether these processes are regulated by environmental cues. To answer this question, we used immunocytochemical procedures to examine the differentiation of pancreatic rudiments removed from E11 mouse embryos and maintained in culture and of pancreases that regenerated in vitro from E11 pancreatic ducts. We found that although all the endocrine cell types differentiate in the gland in culture, the sequence of their appearance is different from that in vivo, suggesting that the timing of differentiation may be regulated by environmental factors. We also found that, in vitro, the pancreas contains TH-glucagon cells, indicating that the expression of the enzyme by pancreatic A cells is independent of factors present in vivo. Moreover, the fact that the TH-glucagon cells also differentiate during pancreatic regeneration suggests that the expression of the enzyme may be a characteristic stage of endocrine cell precursors during maturation.     

Islet hypertrophy following pancreatic disruption of Smad4 signaling

To investigate the role of transforming growth factor (TGF)-beta family signaling in the adult pancreas, a transgenic mouse (E-dnSmad4) was created that expresses a dominant-negative Smad4 protein driven by a fragment of the elastase promoter. Although E-dnSmad4 mice have normal growth, pancreas weight, and pancreatic exocrine and ductal histology, beginning at 4-6 wk of age, E-dnSmad4 mice show an age-dependent increase in the size of islets. In parallel, an expanded population of replicating cells expressing the E-dnSmad4 transgene is found in the stroma between the enlarged islets and pancreatic ducts. Despite the marked enlargement, E-dnSmad4 islets contain normal ratios and spatial organization of endocrine cell subtypes and have normal glucose homeostasis. Replication of cells derived from primary duct cultures of wild-type mice, but not E-dnSmad4 mice, was inhibited by the addition of TGF-beta family proteins, demonstrating a cell-autonomous effect of the transgene. These data show that, in the adult pancreas, TGF-beta family signaling plays a role in islet size by regulating the growth of a pluripotent progenitor cell residing in the periductal stroma of the pancreas.     

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.     

Impaired differentiation of endocrine and exocrine cells of the pancreas in transgenic mouse expressing the truncated type II activin receptor

Activin A is expressed in endocrine precursor cells of the fetal pancreatic anlage. To determine the physiological significance of activins in the pancreas, a transgenic mouse line expressing the truncated type II activin receptor under the control of beta-actin promoter was developed. Histological analyses of the pancreas revealed that the pancreatic islets of the transgenic mouse were small in size and were located mainly along the pancreatic ducts. Immunoreactive insulin was detected in islets, some acinar cells, and in some epithelial cells in the duct. In addition, there were abnormal endocrine cells outside the islets. The shape and the size of the endocrine cells varied and some of them were larger than islets. These cells expressed immunoreactive insulin and glucagon. In the exocrine portion, there were morphologically abnormal exocrine cells, which did not form a typical acinar structure. The cells lacked spatial polarity characteristics of acinar cells but expressed immunoreactive amylase, which was distributed diffusely in the cytoplasm. Plasma glucose concentration was normal in the transgenic mouse before and after the administration of glucose. The insulin content of the pancreas in transgenic and normal mice was nearly identical. These results suggest that activins or related ligands regulate the differentiation of the pancreatic endocrine and exocrine cells.     

Persistent expression of HNF6 in islet endocrine cells causes disrupted islet architecture and loss of beta cell function

We used transgenesis to explore the requirement for downregulation of hepatocyte nuclear factor 6 (HNF6) expression in the assembly, differentiation, and function of pancreatic islets. In vivo, HNF6 expression becomes downregulated in pancreatic endocrine cells at 18. 5 days post coitum (d.p.c.), when definitive islets first begin to organize. We used an islet-specific regulatory element (pdx1(PB)) from pancreatic/duodenal homeobox (pdx1) gene to maintain HNF6 expression in endocrine cells beyond 18.5 d.p.c. Transgenic animals were diabetic. HNF6-overexpressing islets were hyperplastic and remained very close to the pancreatic ducts. Strikingly, alpha, delta, and PP cells were increased in number and abnormally intermingled with islet beta cells. Although several mature beta cell markers were expressed in beta cells of transgenic islets, the glucose transporter GLUT2 was absent or severely reduced. As glucose uptake/metabolism is essential for insulin secretion, decreased GLUT2 may contribute to the etiology of diabetes in pdx1(PB)-HNF6 transgenics. Concordantly, blood insulin was not raised by glucose challenge, suggesting profound beta cell dysfunction. Thus, we have shown that HNF6 downregulation during islet ontogeny is critical to normal pancreas formation and function: continued expression impairs the clustering of endocrine cells and their separation from the ductal epithelium, disrupts the spatial organization of endocrine cell types within the islet, and severely compromises beta cell physiology, leading to overt diabetes.