Growth-limiting role of endothelial cells in endoderm development

Endoderm development is dependent on inductive signals from different structures in close vicinity, including the notochord, lateral plate mesoderm and endothelial cells. Recently, we demonstrated that a functional vascular system is necessary for proper pancreas development, and that sphingosine-1-phosphate (S1P) exhibits the traits of a blood vessel-derived molecule involved in early pancreas morphogenesis. To examine whether S1P₁-signaling plays a more general role in endoderm development, S1P₁-deficient mice were analyzed. S1P₁ ablation results in compromised growth of several foregut-derived organs, including the stomach, dorsal and ventral pancreas and liver. Within the developing pancreas the reduction in organ size was due to deficient proliferation of Pdx1⁺ pancreatic progenitors, whereas endocrine cell differentiation was unaffected. Ablation of endothelial cells in vitro did not mimic the S1P₁ phenotype, instead, increased organ size and hyperbranching were observed. Consistent with a negative role for endothelial cells in endoderm organ expansion, excessive vasculature was discovered in S1P₁-deficient embryos. Altogether, our results show that endothelial cell hyperplasia negatively influences organ development in several foregut-derived organs.     

Hedgehog signaling regulates expansion of pancreatic epithelial cells

Embryonic Hedgehog signaling is essential for proper tissue morphogenesis and organ formation along the developing gastrointestinal tract. Hedgehog ligands are expressed throughout the endodermal epithelium at early embryonic stages but excluded from the region that will form the pancreas. Ectopic activation of Hedgehog signaling at the onset of pancreas development has been shown to inhibit organ morphogenesis. In contrast, Hedgehog signaling components are found within pancreatic tissue during subsequent stages of development as well as in the mature organ, indicating that a certain level of pathway activation is required for normal organ development and function. Here, we ectopically activate the Hedgehog pathway midway through pancreas development via expression of either Sonic (Shh) or Indian Hedgehog (Ihh) under control of the human Pax4-promoter. Similar pancreatic defects are observed in both Pax4-Shh and Pax4-Ihh transgenic lines, suggesting that regulation of the overall level of Hedgehog activity is critical for proper pancreas development. We also show that Hedgehog signaling controls mesenchymal vs. epithelial tissue differentiation and that pathway activation impairs formation of epithelial progenitors. Thus, tight control of Hedgehog pathway activity throughout embryonic development ensures proper pancreas organogenesis.     

Dynamics of embryonic pancreas development using real-time imaging

Current knowledge about developmental processes in complex organisms has relied almost exclusively on analyses of fixed specimens. However, organ growth is highly dynamic, and visualization of such dynamic processes, e.g., real-time tracking of cell movement and tissue morphogenesis, is becoming increasingly important. Here, we use live imaging to investigate expansion of the embryonic pancreatic epithelium in mouse. Using time-lapse imaging of tissue explants in culture, fluorescently labeled pancreatic epithelium was found to undergo significant expansion accompanied by branching. Quantification of the real-time imaging data revealed lateral branching as the predominant mode of morphogenesis during epithelial expansion. Live imaging also allowed documentation of dynamic beta-cell formation and migration. During in vitro growth, appearance of newly formed beta-cells was visualized using pancreatic explants from MIP-GFP transgenic animals. Migration and clustering of beta-cells were recorded for the first time using live imaging. Total beta-cell mass and concordant aggregation increased during the time of imaging, demonstrating that cells were clustering to form "pre-islets". Finally, inhibition of Hedgehog signaling in explant cultures led to a dramatic increase in total beta-cell mass, demonstrating application of the system in investigating roles of critical embryonic signaling pathways in pancreas development including beta-cell expansion. Thus, pancreas growth in vitro can be documented by live imaging, allowing visualization of the developing pancreas in real-time.     

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels¹. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation². A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo^3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ^2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing β-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium^7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.     

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.     

Embryonic mouse blood flow and oxygen correlate with early pancreatic differentiation

The mammalian embryo represents a fundamental paradox in biology. Its location within the uterus, especially early during development when embryonic cardiovascular development and placental blood flow are not well-established, leads to an obligate hypoxic environment. Despite this hypoxia, the embryonic cells are able to undergo remarkable growth, morphogenesis, and differentiation. Recent evidence suggests that embryonic organ differentiation, including pancreatic β-cells, is tightly regulated by oxygen levels. Since a major determinant of oxygen tension in mammalian embryos after implantation is embryonic blood flow, here we used a novel survivable in utero intracardiac injection technique to deliver a vascular tracer to living mouse embryos. Once injected, the embryonic heart could be visualized to continue contracting normally, thereby distributing the tracer specifically only to those regions where embryonic blood was flowing. We found that the embryonic pancreas early in development shows a remarkable paucity of blood flow and that the presence of blood flow correlates with the differentiation state of the developing pancreatic epithelial cells in the region of the blood flow.     

SEL1L deficiency impairs growth and differentiation of pancreatic epithelial cells

Background  

The vertebrate pancreas contains islet, acinar and ductal cells. These cells derive from a transient pool of multipotent pancreatic progenitors during embryonic development. Insight into the genetic determinants regulating pancreatic organogenesis will help the development of cell-based therapies for the treatment of diabetes mellitus. Suppressor enhancer lin12/Notch 1 like (Sel1l) encodes a cytoplasmic protein that is highly expressed in the developing mouse pancreas. However, the morphological and molecular events regulated by Sel1l remain elusive.     

Blood vessels restrain pancreas branching, differentiation and growth

How organ size and form are controlled during development is a major question in biology. Blood vessels have been shown to be essential for early development of the liver and pancreas, and are fundamental to normal and pathological tissue growth. Here, we report that, surprisingly, non-nutritional signals from blood vessels act to restrain pancreas growth. Elimination of endothelial cells increases the size of embryonic pancreatic buds. Conversely, VEGF-induced hypervascularization decreases pancreas size. The growth phenotype results from vascular restriction of pancreatic tip cell formation, lateral branching and differentiation of the pancreatic epithelium into endocrine and acinar cells. The effects are seen both in vivo and ex vivo, indicating a perfusion-independent mechanism. Thus, the vasculature controls pancreas morphogenesis and growth by reducing branching and differentiation of primitive epithelial cells.     

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest^1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal''s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.     

GDF11 modulates NGN3+ islet progenitor cell number and promotes beta-cell differentiation in pancreas development

Identification of endogenous signals that regulate expansion and maturation of organ-specific progenitor cells is a major goal in studies of organ development. Here we provide evidence that growth differentiation factor 11 (GDF11), a member of the TGF-beta ligand family, governs the number and maturation of islet progenitor cells in mouse pancreas development. Gdf11 is expressed in embryonic pancreatic epithelium during formation of islet progenitor cells that express neurogenin 3. Mice deficient for Gdf11 harbor increased numbers of NGN3+ cells, revealing that GDF11 negatively regulates production of islet progenitor cells. Despite a marked expansion of these NGN3+ islet progenitors, mice lacking Gdf11 have reduced beta-cell numbers and evidence of arrested beta-cell development, indicating that GDF11 is also required for beta-cell maturation. Similar precursor and islet cell phenotypes are observed in mice deficient for SMAD2, an intracellular signaling factor activated by TGF-beta signals. Our data suggest that Gdf11 and Smad2 regulate islet cell differentiation in parallel to the Notch pathway, which previously has been shown to control development of NGN3+ cells. Thus, our studies reveal mechanisms by which GDF11 regulates the production and maturation of islet progenitor cells in pancreas development.     

The molecular basis and prospects in pancreatic development

Studies on the signaling mechanism that control the specification of endoderm-derived organs have only recently begun. While many studies revealed genes involved in the differentiation, growth and morphogenesis of the pancreas through studies of mutant mice, still little is known about how endoderm give rise to specific domains. Although many genes are known to have a role in pancreatic differentiation, growth and morphogenesis, few genes are known to take part in the specification of the pancreas so far. Hallmarks as well as gene markers for early development of the pancreas, which are however still very limited, will be useful for dissecting early events in pancreatic specification. Here, I give a summary on the origin of the dorsal and ventral pancreatic progenitors, signals for inductions, and genes so far known to function in pancreatic differentiation. I also give a future prospect in the use of ES cells and other experimental models, towards a comprehensive understanding of gene networks in the progenitor cells or intermediate cell types which arise during various stages of differentiation.     

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.     

Expression pattern for adrenomedullin during pancreatic development in the rat reveals a common precursor with other endocrine cell types

Adrenomedullin is an α-amidated 52-amino acid peptide involved in many physiological actions, among others the regulation of insulin secretion. Using immunohistochemical methods, we found that adrenomedullin immunoreactivity first appears at day 11.5 of embryonic development in the rat, coinciding with the appearance of pancreatic glucagon. The early appearance of adrenomedullin in the developing pancreas may indicate an active involvement in either the morphogenesis of the organ or its endocrine/paracrine/autocrine hormone regulation during intrauterine life. We also investigated the pattern of colocalizations of adrenomedullin with the other pancreatic hormones. At some point during development all the cell types express adrenomedullin, progressively evolving towards the adult pattern where only the pancreatic polypeptide cells contain a strong immunoreactivity for adrenomedullin. At this point the remaining cells of the islet are, in general, weakly stained. This sequential and time-dependent expression of adrenomedullin suggests a tight regulation similar to that observed for other modulatory substances responsible for embryonic morphogenesis.