The effect of pancreatic mesenchyme on the differentiation of endocrine cells from gastric endoderm

To determine whether mesenchyme plays a part in the differentiation of gut endocrine cells, proventricular endoderm from 4- to 5-day chick or quail embryos was associated with mesenchyme from the dorsal pancreatic bud of chick embryos of the same age. The combinations were grown on the chorioallantoic membranes of host chick embryos until they reached a total incubation age of 21 days. Proventricular or pancreatic endoderm of the appropriate age and species reassociated with its own mesenchyme provided the controls. Morphogenesis in the experimental grafts corresponded closely to that in proventricular controls, i.e. the pancreatic mesenchyme supported the development of proventricular glands from proventricular endoderm. Insulin, glucagon and somatostatin cells and cells with pancreatic polypeptide-like immunoreactivity differentiated in the pancreatic controls. The latter three endocrine cell types, together with neurotensin and bombesin/gastrin-releasing polypeptide (GRP) cells, developed in proventricular controls and experimental grafts. The proportions of the major types common to proventriculus and pancreas (somatostatin and glucagon cells) were in general similar when experimental grafts were compared with proventricular controls but different when experimental and pancreatic control grafts were compared. Hence pancreatic mesenchyme did not materially affect the proportions of these three cell types in experimental grafts, induced no specific pancreatic (insulin) cell type and allowed the differentiation of the characteristic proventricular endocrine cell types, neurotensin and bombesin/GRP cells. However, an important finding was a significant reduction in the proportion of bombesin/GRP cells, attributable in part to a decrease in their number and in part to an increase in the numbers of endocrine cells of the other types. This indicates that mesenchyme may well play a part in determining the regional specificity of populations of gut endocrine cells.     

Intestinal mesenchyme provokes differentiation of intestinal endocrine cells in gizzard endoderm

The gizzard (muscular stomach) of chicks is deficient in endocrine cells at hatching. It has previously been shown that proventricular types and proportions of endocrine cells can be induced in gizzard endoderm under the influence of proventricular (glandular stomach) mesenchyme. In order to test its capacity to form nongastric endocrine cell types, gizzard endoderm of 3.75- to 5-day chick embryos was combined with mesenchyme from the small intestine of 3.5- to 4-day quail embryos. The combinations were grown as chorio-allantoic grafts until they attained an incubation age comparable to that of hatching chicks. Controls comprised reassociated endoderm and mesenchyme of chick gizzard and of quail intestine. In the experimental grafts, morphogenesis was predominantly intestinal but some grafts showed gizzard-like features, particularly if the endoderm had been provided by older donors. All intestinal endocrine cell types, including those also found in the normal proventriculus (serotonin-, glucagon-, pancreatic polypeptide-, neurotensin- and somatostatin-immunoreactive cells) differentiated in experimental grafts, some even where morphogenesis was gizzard-like. Hence progenitors of not only gastric, but also intestinal, endocrine cells are indeed present in gizzard endoderm. The possibility that gizzard mesenchyme is inhibitory to endocrine cell differentiation is mooted. Motilin- and secretin-immunoreactive cells, which are characteristic of the intestine but not of the proventriculus of chicks at hatching, were respectively sparse or absent when the endoderm was derived from older donors. Thus the ability of gizzard endoderm to differentiate into nongastric endocrine cell types declines before its capacity to form gastric types. The unexpected appearance of gastrin-releasing peptide (GRP)-immunoreactive cells, a proventricular type not found in normal chick intestine, suggests that the intestinal mesenchyme, at least in this instance, was exercising a permissive role.     

Endoderm- and mesenchyme-dependent commitment of the differentiated epithelial cell types in the developing intestine of rat

During organogenesis, the intestinal tract progressively acquires a functional regionalization along the antero-posterior axis. Positional information needed for enterocytes has been studied, but the mechanisms that control Paneth and endocrine cell differentiation are poorly understood. We have used a model of endoderm/mesenchyme cross-associations to evaluate the respective roles of endoderm and mesenchyme in the cytodifferentiation of these epithelial cells. Heterotopic cross-associations comprising endoderm and mesenchyme from the presumptive proximal jejunum and colon were developed as xenografts in nude mice. Our results show that endoderm from the presumptive proximal jejunum when associated with colonic mesenchyme generate small intestinal enterocytes. Interestingly, no lysozyme-producing cells were generated. On the other hand, associations comprising colon endoderm and jejunal mesenchyme showed heterodifferentiation with typical small intestinal morphology with sucrase-isomaltase expression and Paneth cell differentiation. Heterotopic associations developed enteroendocrine cell patterns according to the normal fate of the endodermal moiety. As enteroendocrine cell commitment seems to occur before the other intestinal cell types, we cannot exclude a role of instructive signals from the mesenchyme on endocrine cell differentiation earlier in the development. These results identified a complex pattern of cell commitment, dependent of the differentiation type of the epithelial cell, on the regional origin of the endoderm and the associated mesenchyme.     

Dorsal pancreas agenesis in retinoic acid-deficient Raldh2 mutant mice

During embryogenesis, the pancreas arises from dorsal and ventral pancreatic protrusions from the primitive gut endoderm upon induction by different stimuli from neighboring mesodermal tissues. Recent studies have shown that Retinoic Acid (RA) signaling is essential for the development of the pancreas in non-mammalian vertebrates. To investigate whether RA regulates mouse pancreas development, we have studied the phenotype of mice with a targeted deletion in the retinaldehyde dehydrogenase 2 (Raldh2) gene, encoding the enzyme required to synthesize RA in the embryo. We show that Raldh2 is expressed in the dorsal pancreatic mesenchyme at the early stage of pancreas specification. RA-responding cells have been detected in pancreatic endodermal and mesenchymal cells. Raldh2-deficient mice do not develop a dorsal pancreatic bud. Mutant embryos lack Pdx 1 expression, an essential regulator of early pancreas development, in the dorsal but not the ventral endoderm. In contrast to Pdx 1-deficient mice, the early glucagon-expressing cells do not develop in Raldh2 knockout embryos. Shh expression is, as in the wild-type embryo, excluded from the dorsal endodermal region at the site where the dorsal bud is expected to form, indicating that the dorsal bud defect is not related to a mis-expression of Shh. Mesenchymal expression of the LIM homeodomain protein Isl 1, required for the formation of the dorsal mesenchyme, is altered in Raldh2--/-- embryos. The homeobox gene Hlxb9, which is essential for the initiation of the pancreatic program in the dorsal foregut endoderm, is still expressed in Raldh2--/-- dorsal epithelium but the number of HB9-expressing cells is severely reduced. Maternal supplementation of RA rescues early dorsal pancreas development and restores endodermal Pdx 1 and mesenchymal Isl 1 expression as well as endocrine cell differentiation. These findings suggest that RA signaling is important for the proper differentiation of the dorsal mesenchyme and development of the dorsal endoderm. We conclude that RA synthesized in the mesenchyme is specifically required for the normal development of the dorsal pancreatic endoderm at a stage preceding Pdx 1 function.     

Fetal gut mesenchyme induces differentiation of cultured intestinal endodermal and crypt cells

An experimental model was designed to analyze the effect of fetal gut mesenchyme on the cytodifferentiation of crypt cells and of embryonic progenitor cells. The cells used were the rat intestinal crypt cell line, IEC-17, and primary cell cultures prepared form isolated 14-day-old fetal intestinal endoderm (EC). Both cultures prepared from isolated 14-day-old fetal rat intestinal endoderm (EC). Both types of cells were associated with 14-day-old fetal rat gut mesenchyme (Rm) and grafted under the kidney capsule of adult rats. Seventy percent of the Rm/EC and ten percent of the Rm/IEC recombinants, recovered after 9 days, exhibited well-vascularized structures in which the mesenchyme had induced morphogenesis of the cells into a villus epithelium. The four main intestinal epithelial cell types, absorptive, goblet, endocrine, and Paneth cells, were identified using electron microscopy. Biochemical determinations of enzyme activities associated with brush border membranes revealed that alkaline phosphatase, lactase, sucrase, and maltase were expressed in both types of associations. These results were confirmed by immunofluorescence staining using monoclonal antibodies to brush border enzymes. Both enzyme assays and immunocytochemistry showed that the amount of enzymes present in the brush border membrane of Rm/IEC grafts was in general lower than that of the Rm/EC recombinants. The results indicate that fetal rat gut mesenchyme enables morphogenesis and cytodifferentiation of both crypt and embryonic progenitor cells.     

Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells

Of paramount importance for the development of cell therapies to treat diabetes is the production of sufficient numbers of pancreatic endocrine cells that function similarly to primary islets. We have developed a differentiation process that converts human embryonic stem (hES) cells to endocrine cells capable of synthesizing the pancreatic hormones insulin, glucagon, somatostatin, pancreatic polypeptide and ghrelin. This process mimics in vivo pancreatic organogenesis by directing cells through stages resembling definitive endoderm, gut-tube endoderm, pancreatic endoderm and endocrine precursor--en route to cells that express endocrine hormones. The hES cell-derived insulin-expressing cells have an insulin content approaching that of adult islets. Similar to fetal beta-cells, they release C-peptide in response to multiple secretory stimuli, but only minimally to glucose. Production of these hES cell-derived endocrine cells may represent a critical step in the development of a renewable source of cells for diabetes cell therapy.     

Fetal endoderm primarily holds the temporal and positional information required for mammalian intestinal development

In rodents, the intestinal tract progressively acquires a functional regionalization during postnatal development. Using lactase-phlorizin hydrolase as a marker, we have analyzed in a xenograft model the ontogenic potencies of fetal rat intestinal segments taken prior to endoderm cytodifferentiation. Segments from the presumptive proximal jejunum and distal ileum grafted in nude mice developed correct spatial and temporal patterns of lactase protein and mRNA expression, which reproduced the normal pre- and post-weaning conditions. Segments from the fetal colon showed a faint lactase immunostaining 8-10 d after transplantation in chick embryos but not in mice; it is consistent with the transient expression of this enzyme in the colon of rat neonates. Heterotopic cross-associations comprising endoderm and mesenchyme from the presumptive proximal jejunum and distal ileum developed as xenografts in nude mice, and they exhibited lactase mRNA and protein expression patterns that were typical of the origin of the endodermal moiety. Endoderm from the distal ileum also expressed a normal lactase pattern when it was associated to fetal skin fibroblasts, while the fibroblasts differentiated into muscle layers containing alpha-smooth- muscle actin. Noteworthy, associations comprising colon endoderm and small intestinal mesenchyme showed a typical small intestinal morphology and expressed the digestive enzyme sucrase-isomaltase normally absent in the colon. However, in heterologous associations comprising lung or stomach endoderm and small intestinal mesenchyme, the epithelial compartment expressed markers in accordance to their tissue of origin but neither intestinal lactase nor sucrase-isomaltase. A thick intestinal muscle coat in which cells expressed alpha-smooth- muscle actin surrounded the grafts. The results demonstrate that: (a) the temporal and positional information needed for intestinal ontogeny up to the post-weaning stage results from an intrinsic program that is fixed in mammalian fetuses prior to endoderm cytodifferentiation; (b) this temporal and positional information is primarily carried by the endodermal moiety which is also able to change the fate of heterologous mesodermal cells to form intestinal mesenchyme; and (c) the small intestinal mesenchyme in turn may deliver instructive information as shown in association with colonic endoderm; yet this effect is not obvious with nonintestinal endoderms.     

Cell-surface markers for the isolation of pancreatic cell types derived from human embryonic stem cells

Using a flow cytometry-based screen of commercial antibodies, we have identified cell-surface markers for the separation of pancreatic cell types derived from human embryonic stem (hES) cells. We show enrichment of pancreatic endoderm cells using CD142 and of endocrine cells using CD200 and CD318. After transplantation into mice, enriched pancreatic endoderm cells give rise to all the pancreatic lineages, including functional insulin-producing cells, demonstrating that they are pancreatic progenitors. In contrast, implanted, enriched polyhormonal endocrine cells principally give rise to glucagon cells. These antibodies will aid investigations that use pancreatic cells generated from pluripotent stem cells to study diabetes and pancreas biology.     

A possible role for the canonical Wnt pathway in endocrine cell development in chicks

Wnt signalling is involved in many developmental processes such as proliferation, differentiation, cell fate decisions, and morphogenesis. However, little is known about Wnt signalling during pancreas development. Multiple Wnt ligands and Frizzled receptors are expressed in the embryonic mouse pancreas, the surrounding mesenchyme, and have also been detected in the chicken endoderm during development. The aim of this study was to investigate the role of canonical Wnt signalling on endocrine cell development by use of the in ovo electroporation of the chicken endoderm. Overexpression with a constitutive active form of beta-catenin in combination with Ngn3 resulted in reduced numbers of glucagon cells. dnLEF-1 or naked-1 did not alter endocrine cell differentiation when co-expressed with Ngn3, but dnLEF-1 appeared to have some potential for inhibiting delamination of Ngn3 cells. In addition, neuronal beta-III-tubulin, which had previously been considered a specific marker for neuronal cells, was observed in the pancreas and was upregulated in the electroporated Ngn3 cells and thus may be a new endocrine marker in the chicken.     

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.     

Beta-cell differentiation from nonendocrine epithelial cells of the adult human pancreas

The nature and even existence of adult pancreatic endocrine stem or progenitor cells is a subject of controversy in the field of beta-cell replacement for diabetes. One place to search for such cells is in the nonendocrine fraction of cells that remain after islet isolation, which consist of a mixture of epithelia and mesenchyme. Culture in G418 resulted in elimination of the mesenchymal cells, leaving a highly purified population of nonendocrine pancreatic epithelial cells (NEPECs). To evaluate their differentiation potential, NEPECs were heritably marked and transplanted under the kidney capsule of immunodeficient mice. When cotransplanted with fetal pancreatic cells, NEPECs were capable of endocrine differentiation. We found no evidence of beta-cell replication or cell fusion that could have explained the appearance of insulin positive cells from a source other than NEPECs. Nonendocrine-to-endocrine differentiation of NEPECs supports the existence of endocrine stem or progenitor cells within the epithelial compartment of the adult human pancreas.     

Analysis of pancreatic development using a cell lineage label

We have devised a new culture system for in vitro culture of pancreatic buds from mouse embryos which enables the organ to grow as a flat branched structure suitable for wholemount immunostaining. This system has been used to analyze pancreatic development. We have also used the ROSA-26 gene trap mouse strain as a source of tissue which expresses lacZ in a stable manner, in all cell types, during in vitro culture. Combinations of lacZ epithelium and unlabeled mesenchyme show that both exocrine and endocrine cells arise from the epithelium, and smooth muscle cells from the mesenchyme. Although previously suspected, this is the first formal proof that both exocrine and endocrine cells are of endodermal origin. Combinations of lacZ epithelium with unlabeled stomach mesenchyme give similar results and show that stomach mesenchyme has the same trophic effect as pancreatic mesenchyme. When a lacZ and an unlabeled epithelium are combined with an unlabeled mesenchyme, both acini and islets in the resulting culture can be of mixed cell composition. This shows that neither of the chief structural units of the pancreas is formed by clonal growth from a single cell.     

Reciprocal endoderm-mesoderm interactions mediated by fgf24 and fgf10 govern pancreas development

In amniotes, the pancreatic mesenchyme plays a crucial role in pancreatic epithelium growth, notably through the secretion of fibroblast growth factors. However, the factors involved in the formation of the pancreatic mesenchyme are still largely unknown. In this study, we characterize, in zebrafish embryos, the pancreatic lateral plate mesoderm, which is located adjacent to the ventral pancreatic bud and is essential for its specification and growth. We firstly show that the endoderm, by expressing the fgf24 gene at early stages, triggers the patterning of the pancreatic lateral plate mesoderm. Based on the expression of isl1, fgf10 and meis genes, this tissue is analogous to the murine pancreatic mesenchyme. Secondly, Fgf10 acts redundantly with Fgf24 in the pancreatic lateral plate mesoderm and they are both required to specify the ventral pancreas. Our results unveil sequential signaling between the endoderm and mesoderm that is critical for the specification and growth of the ventral pancreas, and explain why the zebrafish ventral pancreatic bud generates the whole exocrine tissue.     

Vascular function and sphingosine-1-phosphate regulate development of the dorsal pancreatic mesenchyme

Early growth and differentiation of the pancreatic endoderm is regulated by soluble factors from the pancreatic mesenchyme. Previously, we demonstrated that N-cadherin-deficient mice lack a dorsal pancreas, due to a critical role of N-cadherin in dorsal pancreatic mesenchymal cell survival. Here, we show that restoring cardiac and circulatory function in N-cadherin null mice by cardiac-specific expression of N-cadherin, rescues formation of the dorsal pancreas, indicating that the phenotype is secondary to defects related to cardiac/vascular function. Based on this observation, we demonstrate that soluble factors present in plasma, such as sphingosine-1-phosphate, rescue formation of the dorsal pancreas in N-cadherin-deficient mice. We also show that sphingosine-1-phosphate indirectly promotes budding of the pancreatic endoderm by stimulating pancreatic mesenchymal cell proliferation. Finally, we identify sphingosine-1-phosphate receptors within the mesenchyme and show that pertussis toxin blocks the sphingosine-1-phosphate-induced actions, suggesting the involvement of G-protein-coupled sphingosine-1-phosphate receptors. Thus, we propose a new model where blood vessel-derived sphingosine-1-phosphate stimulates growth and budding of the dorsal pancreatic endoderm by induction of mesenchymal cell proliferation.     

Pancreatic beta-cells: Role of glycerol and aquaglyceroporin 7

Pancreatic β-cells originate from gut endoderm during development. Pancreatic endocrine cells represent about 10% of the mature pancreatic cells, and β-cells represent the majority of endocrine cells. β-cells secrete insulin in response to elevation of nutrient concentrations. Insulin maintains glucose homeostasis by stimulating glucose uptake into muscle and adipose tissue. Aquaglyceroporin 7, permeable to water, glycerol and urea, is expressed in pancreatic β-cells and was recently described as being involved in the control of insulin secretion.     

Effect of subcutaneous pancreatic tissue transplants on streptozotocin-induced diabetes in rats. I. Morphological studies on normal, diabetic and transplanted pancreatic tissues.

The present study examines the morphological changes occurring in subcutaneous pancreatic tissue grafts (SPTG) and its effect on the host pancreatic islet cells in streptozotocin (STZ)-induced diabetic rats using morphological techniques. SPTG survived after 15 weeks of transplantation. Its acinar cells degenerated but the ducts and endocrine cells survived. The surviving and newly formed pancreatic tubules and endocrine cells filled the spaces left by degenerated acinar cells. Compartmentalization of the surviving parenchymatic tissues was observed, with the pancreatic tubules lying in the periphery of the graft and the endocrine tissue in the inner portion of the graft. Lymphocytes invaded the inner portion of the graft, conglomerating around endocrine cells. It was interesting, however, that, lymphocytes where not observed in the periphery of the grafts where most of the surviving pancreatic tubules lie. In addition to this, necrotic tissues were observed in the inner part of the graft. Fifteen weeks after transplantation into the subcutaneous region, insulin, glucagon, somatostatin and pancreatic polypeptide-immunoreactive cells were observed in many parts of the graft. In the peripheral parts of the grafts, large numbers of pancreatic tubules differentiated into endocrine cells. In conclusion, the ductal and endocrine cells of pancreatic tissue fragments survived in the subcutaneous region of rat with normal pattern of distribution.