The fringe molecules induce endocrine differentiation in embryonic endoderm by activating cMyt1/cMyt3

Endocrine differentiation in the early embryonic pancreas is regulated by Notch signaling. Activated Notch signaling maintains pancreatic progenitor cells in an undifferentiated state, whereas suppression of Notch leads to endocrine cell differentiation. Yet it is not known what mechanism is employed to inactivate Notch in a correct number of precursor cells to balance progenitor proliferation and differentiation. We report that an established Notch modifier, Manic Fringe (Mfng), is expressed in the putative endocrine progenitors, but not in exocrine pancreatic tissues, during early islet differentiation. Using chicken embryonic endoderm as an assaying system, we found that ectopic Mfng expression is sufficient to induce endodermal cells to differentiate towards an endocrine fate. This endocrine-inducing activity depends on inactivation of Notch. Furthermore, ectopic Mfng expression induces the expression of basic helix-loop-helix gene, Ngn3, and two zinc finger genes, cMyt1 and cMyt3. These results suggest that Mfng-mediated repression of Notch signaling could serve as a trigger for endocrine islet differentiation.     

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.     

NRP1 and NRP2 cooperate to regulate gangliogenesis, axon guidance and target innervation in the sympathetic nervous system

During mouse pancreas development, the transient expression of Neurogenin3 (Neurog3) in uncommitted pancreas progenitors is required to determine endocrine destiny. However it has been reported that Neurog3-expressing cells can eventually adopt acinar or ductal fates and that Neurog3 levels were important to secure the islet destiny. It is not known whether the competence of Neurog3-induced cells to give rise to non-endocrine lineages is an intrinsic property of these progenitors or depends on pancreas developmental stage. Using temporal genetic labeling approaches we examined the dynamic of endocrine progenitor differentiation and explored the plasticity of Neurog3-induced cells throughout development. We found that Neurog3(+) progenitors develop into hormone-expressing cells in a fast process taking less then 10h. Furthermore, fate-mapping studies in heterozygote (Neurog3(CreERT/+)) and Neurog3-deficient (Neurog3(CreERT/CreERT)) embryos revealed that Neurog3-induced cells have different potential over time. At the early bud stage, failed endocrine progenitors can adopt acinar or ductal fate, whereas later in the branching pancreas they do not contribute to the acinar lineage but Neurog3-deficient cells eventually differentiate into duct cells. Thus these results provide evidence that the plasticity of Neurog3-induced cells becomes restricted during development. Furthermore these data suggest that during the secondary transition, endocrine progenitor cells arise from bipotent precursors already committed to the duct/endocrine lineages and not from domain of cells having distinct potentialities.     

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.     

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.     

Ngn3(+) endocrine progenitor cells control the fate and morphogenesis of pancreatic ductal epithelium

During pancreas development, endocrine and exocrine cells arise from a common multipotent progenitor pool. How these cell fate decisions are coordinated with tissue morphogenesis is poorly understood. Here we have examined ductal morphology, endocrine progenitor cell fate and Notch signaling in Ngn3^−/− mice, which do not produce islet cells. Ngn3 deficiency results in reduced branching and enlarged pancreatic duct-like structures, concomitant with Ngn3 promoter activation throughout the ductal epithelium and reduced Notch signaling. Conversely, forced generation of surplus endocrine progenitor cells causes reduced duct caliber and an excessive number of tip cells. Thus, endocrine progenitor cells normally provide a feedback signal to adjacent multipotent ductal progenitor cells that activates Notch signaling, inhibits further endocrine differentiation and promotes proper morphogenesis. These results uncover a novel layer of regulation coordinating pancreas morphogenesis and endocrine/exocrine differentiation, and suggest ways to enhance the yield of beta cells from stem cells.     

Combined activities of hedgehog signaling inhibitors regulate pancreas development

Hedgehog signaling is known to regulate tissue morphogenesis and cell differentiation in a dose-dependent manner. Loss of Indian hedgehog (Ihh) results in reduction in pancreas size, indicating a requirement for hedgehog signaling during pancreas development. By contrast, ectopic expression of sonic hedgehog (Shh) inhibits pancreatic marker expression and results in transformation of pancreatic mesenchyme into duodenal mesoderm. These observations suggest that hedgehog signaling activity has to be regulated tightly to ensure proper pancreas development. We have analyzed the function of two hedgehog inhibitors, Hhip and patched 1 (Ptch), during pancreas formation. Our results indicated that loss of Hhip results in increased hedgehog signaling within the pancreas anlage. Pancreas morphogenesis, islet formation and endocrine cell proliferation is impaired in Hhip mutant embryos. Additional loss of one Ptch allele in Hhip-/-Ptch+/- embryos further impairs pancreatic growth and endodermal cell differentiation. These results demonstrate combined requirements for Hhip and Ptch during pancreas development and point to a dose-dependent response to hedgehog signaling within pancreatic tissue. Reduction of Fgf10 expression in Hhip homozygous mutants suggests that at least some of the observed phenotypes result from hedgehog-mediated inhibition of Fgf signaling at early stages.     

Notch/Rbp-j signaling prevents premature endocrine and ductal cell differentiation in the pancreas

To investigate the precise role of Notch/Rbp-j signaling in the pancreas, we inactivated Rbp-j by crossing Rbp-j floxed mice with Pdx.cre or Rip.cre transgenic mice. The loss of Rbp-j at the initial stage of pancreatic development induced accelerated alpha and PP cell differentiation and a concomitant decrease in the number of Neurogenin3 (Ngn3)-positive cells at E11.5. Then at E15, elongated tubular structures expressing ductal cell markers were evident; however, differentiation of acinar and all types of endocrine cells were reduced. During later embryonic stages, compensatory acinar cell differentiation was observed. The resultant mice exhibited insulin-deficient diabetes with both endocrine and exocrine pancreatic hypoplasia. In contrast, the loss of Rbp-j specifically in beta cells did not affect beta cell number and function. Thus, our analyses indicate that Notch/Rbp-j signaling prevents premature differentiation of pancreatic progenitor cells into endocrine and ductal cells during early development of the pancreas.     

Exocrine pancreas development in zebrafish

Although many of the genes that regulate development of the endocrine pancreas have been identified, comparatively little is known about how the exocrine pancreas forms. Previous studies have shown that exocrine pancreas development may be modeled in zebrafish. However, the timing and mechanism of acinar and ductal differentiation and morphogenesis have not been described. Here, we characterize zebrafish exocrine pancreas development in wild type and mutant larvae using histological, immunohistochemical and ultrastructural analyses. These data allow us to identify two stages of zebrafish exocrine development. During the first stage, the exocrine anlage forms from rostral endodermal cells. During the second stage, proto-differentiated progenitor cells undergo terminal differentiation followed by acinar gland and duct morphogenesis. Immunohistochemical analyses support a model in which the intrapancreatic ductal system develops from progenitors that join to form a contiguous network rather than by branching morphogenesis of the pancreatic epithelium, as described for mammals. Contemporaneous appearance of acinar glands and ducts in developing larvae and their disruption in pancreatic mutants suggest that common molecular pathways may regulate gland and duct morphogenesis and differentiation of their constituent cells. By contrast, analyses of mind bomb mutants and jagged morpholino-injected larvae suggest that Notch signaling principally regulates ductal differentiation of bipotential exocrine progenitors.     

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.     

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.     

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.     

Evidence for Epithelial�CMesenchymal Transition in Adult Human Pancreatic Exocrine Cells

It has been shown that adult pancreatic ductal cells can dedifferentiate and act as pancreatic progenitors. Dedifferentiation of epithelial cells is often associated with the epithelial–mesenchymal transition (EMT). In this study, we investigated the occurrence of EMT in adult human exocrine pancreatic cells both in vitro and in vivo. Cells of exocrine fraction isolated from the pancreas of brain-dead donors were first cultured in suspension for eight days. This led to the formation of spheroids, composed of a principal population of cells with duct-like phenotype. When cultivated in tissue culture-treated flasks, spheroid cells exhibited a proliferative capacity and coexpressed epithelial (cytokeratin7 and cytokeratin19) and mesenchymal (vimentin and α-smooth muscle actin) markers as well as marker of progenitor pancreatic cells (pancreatic duodenal homeobox factor-1) and surface markers of mesenchymal stem cells. The switch from E-cadherin to N-cadherin associated with Snail1 expression suggested that these cells underwent EMT. In addition, we showed coexpression of epithelial and mesenchymal markers in ductal cells of one normal adult pancreas and three type 2 diabetic pancreases. Some of the vimentin-positive cells were found to coexpress glucagon or amylase. These results point to the occurrence of EMT, which may take place on dedifferentiation of ductal cells during the regeneration or renewal of human pancreatic tissues. (J Histochem Cytochem 58:807–823, 2010)     

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.