Conditional ablation of Notch signaling in pancreatic development

The role of the Notch signaling members Notch1, Notch2 and Rbpj in exocrine pancreatic development is not well defined. We therefore analyzed conditional pancreas-specific Rbpj and combined Notch1/Notch2 knockout mice using Ptf1a(+/Cre(ex1)) mice crossed with floxed Rbpj or Notch1/Notch2 mice. Mice were analyzed at different embryonic stages for pancreatic exocrine and endocrine development. The absence of Rbpj in pancreatic progenitor cells impaired exocrine pancreas development up to embryonic day 18.5 and led to premature differentiation of pancreatic progenitors into endocrine cells. In Rbpj-deficient pancreata, amylase-expressing acini and islets formed during late embryonic and postnatal development, suggesting an essential role of Rbpj in early but not late development. Contrary to this severe phenotype, the concomitant inactivation of Notch1 and Notch2 only moderately disturbed the proliferation of pancreatic epithelial cells during early embryonic development, and did not inhibit pancreatic development. Our results show that, in contrast to Rbpj, Notch1 and Notch2 are not essential for pancreatogenesis. These data favor a Notch-independent role of Rbpj in the development of the exocrine pancreas. Furthermore, our findings suggest that in late stages of pancreatic development exocrine cell differentiation and maintenance are independent of Rbpj.     

Ductal cells of the pancreas

Ductal cells of the pancreas form the epithelial lining of the branched tubes that deliver enzymes produced by pancreatic acinar cells into the duodenum. In addition, these cells secrete bicarbonate that neutralizes stomach acidity. During development, epithelium of endodermal origin evaginates from the future duodenum area and invades the mesenchyme to form a complex branched network. All endocrine, acinar and ductal cells arise from common precursors in this epithelial structure. Adult ductal cells share some similarities with embryonic primitive ducts and may retain the ability to generate endocrine cells in the adult. Based on challenged pancreas regeneration experiments, the adult ductal cells have been proposed to be pancreatic stem cells but their role in normal endocrine cell turnover has recently been challenged. Manipulating their ability to give rise to endocrine cells may open new avenues in the treatment of diabetes and therefore they have recently been under scrutiny. In addition, in the main form of pancreatic cancer, pancreas adenocarcinoma, tumor cells share similarities with ductal cells. The secrets of an appropriate therapy for this deadly cancer may thus reside in the biology of ductal cells.     

Mesenchymal epimorphin is important for pancreatic duct morphogenesis

Epithelial-mesenchymal interactions are crucial for the proper development of many organs, including the pancreas. Within the pancreas, the ducts are thought to harbor stem/progenitor cells, and possibly to give rise to pancreatic ductal carcinoma. Little is known about the mechanism of formation of pancreatic ducts in the embryo. Pancreatic mesenchyme contains numerous soluble factors which help to sustain the growth and differentiation of exocrine and endocrine structures. Here, we report that one such morphoregulatory mesenchymal protein, epimorphin, plays an important role during pancreatic ductal proliferation and differentiation. We found that epimorphin is expressed in pancreatic mesenchyme during early stages of development, and at mesenchymal-epithelial interfaces surrounding the ducts at later stages. Strong upregulation of epimorphin expression was seen during in vitro pancreatic duct differentiation. Similarly, in vitro pancreatic duct formation was inhibited by a neutralizing antibody against epimorphin, whereas addition of recombinant epimorphin partially rescued duct formation. Together, our study demonstrates the role of epimorphin in pancreatic ductal morphogenesis.     

Analysis of mPygo2 mutant mice suggests a requirement for mesenchymal Wnt signaling in pancreatic growth and differentiation

Pygopus has recently been identified in Drosophila as an essential component of the nuclear complex required for canonical Wnt signaling. Here, we have investigated the role of the mammalian pygopus ortholog, mPygo2, in pancreas development. We show that a null mutation of mPygo2 in mice causes pancreas hypoplasia due to decreased progenitor cell proliferation after embryonic day (e) 12.5. During the same time window, mPygo2-deficient embryos begin to display a reduction in endocrine progenitors and consequently a decrease in islet endocrine cell mass. Consistent with its function after e12.5, late-developing endocrine cell types, such as beta, delta and PP cells, are specifically reduced, while the earlier-forming alpha cells develop normally. We find canonical Wnt signaling to be predominantly active in the mesenchyme at the time when mPygo2 is required and demonstrate the dependence of Wnt signal transduction on mPygo2. Furthermore, conditional deletion of mPygo2^flox allele in the pancreatic epithelium does not phenocopy the defects in mPygo2-null mutants. Since mPygo2 is expressed in the pancreatic mesenchyme and the role of the mesenchyme in epithelial progenitor cell expansion is well documented, our findings suggest an indirect role for mPygo2 in epithelial growth and differentiation through regulation of mesenchymal signals. Together, our data suggest a previously unappreciated role for mesenchymal Wnt signaling in regulating pancreatic organ growth and cell differentiation.     

Selective expansion of the beta-cell compartment in the pancreas of keratinocyte growth factor transgenic mice

Epithelial-mesenchymal interactions are essential for growth, differentiation, and regeneration of exocrine and endocrine cells in the pancreas. The keratinocyte growth factor (KGF) is derived from mesenchyme and has been shown to promote epithelial cell differentiation and proliferation in a paracrine fashion. Here, we have examined the effect of ectopic expression of KGF on pancreatic differentiation and proliferation in transgenic mice by using the proximal elastase promoter. KGF transgenic mice were generated following standard procedures and analyzed by histology, morphometry, immunohistochemistry, Western blot analysis, and glucose tolerance testing. In KGF transgenic mice, the number of islets, the average size of islets, and the relation of endocrine to exocrine tissue are increased compared with littermate controls. An expansion of the beta-cell population is responsible for the increase in the endocrine compartment. Ectopic expression of KGF results in proliferation of beta-cells and pancreatic duct cells most likely through activation of the protein kinase B (PKB)/Akt signaling pathway. Glucose tolerance and insulin secretion are impaired in transgenic animals. These results provide evidence that ectopic expression of KGF in acinar cells promotes the expansion of the beta-cell lineage in vivo through activation of the PKB/Akt pathway. Furthermore, the observed phenotype demonstrates that an increase in the beta-cell compartment does not necessarily result in an improved glucose tolerance in vivo.     

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.     

Pancreatic mesenchyme regulates epithelial organogenesis throughout development

The developing pancreatic epithelium gives rise to all endocrine and exocrine cells of the mature organ. During organogenesis, the epithelial cells receive essential signals from the overlying mesenchyme. Previous studies, focusing on ex vivo tissue explants or complete knockout mice, have identified an important role for the mesenchyme in regulating the expansion of progenitor cells in the early pancreas epithelium. However, due to the lack of genetic tools directing expression specifically to the mesenchyme, the potential roles of this supporting tissue in vivo, especially in guiding later stages of pancreas organogenesis, have not been elucidated. We employed transgenic tools and fetal surgical techniques to ablate mesenchyme via Cre-mediated mesenchymal expression of Diphtheria Toxin (DT) at the onset of pancreas formation, and at later developmental stages via in utero injection of DT into transgenic mice expressing the Diphtheria Toxin receptor (DTR) in this tissue. Our results demonstrate that mesenchymal cells regulate pancreatic growth and branching at both early and late developmental stages by supporting proliferation of precursors and differentiated cells, respectively. Interestingly, while cell differentiation was not affected, the expansion of both the endocrine and exocrine compartments was equally impaired. To further elucidate signals required for mesenchymal cell function, we eliminated β-catenin signaling and determined that it is a critical pathway in regulating mesenchyme survival and growth. Our study presents the first in vivo evidence that the embryonic mesenchyme provides critical signals to the epithelium throughout pancreas organogenesis. The findings are novel and relevant as they indicate a critical role for the mesenchyme during late expansion of endocrine and exocrine compartments. In addition, our results provide a molecular mechanism for mesenchymal expansion and survival by identifying β-catenin signaling as an essential mediator of this process. These results have implications for developing strategies to expand pancreas progenitors and β-cells for clinical transplantation.     

The IIIb isoform of fibroblast growth factor receptor 2 is required for proper growth and branching of pancreatic ductal epithelium but not for differentiation of exocrine or endocrine cells

Fibroblast growth factors (Fgfs) and their receptors have been implicated in embryonic pancreas development. Recently it was shown that Fgf10, a major ligand for the IIIb isoform of fibroblast growth factor receptor 2 (Fgfr2b), has an important regulatory role in early pancreas development. The aim of our study was to define the role of Fgfr2b in pancreas development by analyzing the phenotype of Fgfr2b (-/-) mice. Pancreases of Fgfr2b (-/-) embryos were noticeably smaller than the wild type littermates during embryogenesis, and pancreatic ductal branching as well as duct cell proliferation was significantly reduced. However, both exocrine and endocrine pancreatic differentiation occurred relatively normally. Exogenous addition of Fgfr2b ligands (Fgf7 and Fgf10) stimulated duct cell proliferation and inhibited endocrine cell differentiation in the ex vivo embryonic organ cultures of wild type pancreas. Our results thus suggest that Fgfr2b-mediated signaling plays a major role in pancreatic ductal proliferation and branching morphogenesis, but has little effect on endocrine and exocrine differentiation.     

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.     

Direct lineage tracing reveals the ontogeny of pancreatic cell fates during mouse embryogenesis

Lineage tracing follows the progeny of labeled cells through development. This technique identifies precursors of mature cell types in vivo and describes the cell fate restriction steps they undergo in temporal order. In the mouse pancreas, direct cell lineage tracing reveals that Pdx1- expressing progenitors in the early embryo give rise to all pancreatic cells. The progenitors for the mature pancreatic ducts separate from the endocrine/exocrine tissues before E12.5. Expression of Ngn3 and pancreatic polypeptide marks endocrine cell lineages during early embryogenesis, and these cells behave as transient progenitors rather than stem cells. In adults, Ngn3 is expressed within the endocrine islets, and the NGN3+ cells seem to contribute to pancreatic islet renewal. These results indicate the stage at which each progenitor population is restricted to a particular fate and provide markers for isolating progenitors to study their growth, differentiation, and the genes necessary for their development.     

Cre-loxp fate-mapping of Pax6 enhancer active retinal and pancreatic progenitors

Pax6 plays important roles in the control of ocular and pancreatic development. We identified a 450 bp Pax6 enhancer that contains two interacting sequences: a 274 bp fragment sufficient for expression in retinal progenitors and an adjacent 156 bp fragment required for expression in pancreatic progenitors. Since this enhancer is only transiently expressed during embryogenesis, a Cre-loxP fate-mapping strategy was used to investigate the developmental potential of these progenitors. Surprisingly, the labeled retinal precursors predominantly gave rise to horizontal cells, indicating a cell lineage role in horizontal cell differentiation. In the pancreas, all enhancer-specific cells were restricted to endocrine and ductal cell lineages. This result lends support to a model whereby Pax6-expressing progenitors contribute to the adult pancreatic islets and ducts. The progenitor cell-specificity of this enhancer will be useful in studies that require either cell-specific expression or conditional gene inactivation in these cell populations.     

Lectin as a marker for staining and purification of embryonic pancreatic epithelium

The embryonic pancreatic epithelium, and later the ductal epithelium, is known to give rise to the endocrine and exocrine cells of the developing pancreas, but no specific surface marker for these cells has been identified. Here, we utilized Dolichos Biflorus Agglutinin (DBA) as a specific marker of these epithelial cells in developing mouse pancreas. From the results of an immunofluorescence study using fluorescein-DBA and pancreatic specific cell markers, we found that DBA detects specifically epithelial, but neither differentiating endocrine cells nor acinar cells. We further applied this marker in an immunomagnetic separation system (Dynabead system) to purify these putative multi-potential cells from a mixed developing pancreatic cell population. This procedure could be applied to study differentiation and cell lineage selections in the developing pancreas, and also may be applicable to selecting pancreatic precursor cells for potential cellular engineering.