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.     

Foregut Mesenchyme Contributes Cells to Islets during Pancreatic Development in a 3-Dimensional Avian Model

Current interest in the potential use of pancreatic stem-cells in the treatment of insulin dependent diabetes mellitus has led to increased research into normal pancreatic development. Pancreatic organogenesis involves branching morphogenesis of undifferentiated epithelium within surrounding mesenchyme. Current understanding is that the pancreatic islets develop exclusively from the epithelium of the embryonic buds. However, a cellular contribution to islets by mesenchyme has not been conclusively excluded. We present evidence that the mesenchyme of both the dorsal pancreatic bud and stomach rudiment make a substantial contribution of cells to islets during development in a three-dimensional avian model. These data suggest that mesenchyme can be a source not only of signals but also of cells for the definitive epithelia, making pancreatic organogenesis more akin to that of the kidney than to other endodermal organs. This raises the possibility for the use of mesenchymal cells as stem- or progenitor- cells for islet transplantation.     

Foregut Mesenchyme Contributes Cells to Islets during Pancreatic Development in a 3-Dimensional Avian Model

Current interest in the potential use of pancreatic stem-cells in the treatment of insulin dependent diabetes mellitus has led to increased research into normal pancreatic development. Pancreatic organogenesis involves branching morphogenesis of undifferentiated epithelium within surrounding mesenchyme. Current understanding is that the pancreatic islets develop exclusively from the epithelium of the embryonic buds. However, a cellular contribution to islets by mesenchyme has not been conclusively excluded. We present evidence that the mesenchyme of both the dorsal pancreatic bud and stomach rudiment make a substantial contribution of cells to islets during development in a three-dimensional avian model. These data suggest that mesenchyme can be a source not only of signals but also of cells for the definitive epithelia, making pancreatic organogenesis more akin to that of the kidney than to other endodermal organs. This raises the possibility for the use of mesenchymal cells as stem-or progenitor-cells for islet transplantation.Key Words: islets, stem-cells, development, epithelium, mesenchyme, pancreas, stomach, chick-quail, 3-dimensional, endocrine     

Patterning parameters associated with the branching of the ureteric bud regulated by epithelial-mesenchymal interactions

The mechanisms by which the branching of epithelial tissue occurs and is regulated to generate different organ structures are not well understood. In this work, image analyses of the organ rudiments demonstrate specific epithelial branching patterns for the early lung and kidney; the lung type typically generating several side branches, whereas kidney branching was mainly dichotomous. Parameters such as the number of epithelial tips, the angle of the first branch, the position index of the first branch (PIFB) in a module, and the percentage of epithelial module type (PMT) were analysed. The branching patterns in the cultured lung and kidney, and in homotypic tissue recombinants recapitulated their early in vivo branching patterns. The parameters were applied to heterotypic tissue recombinants between lung mesenchyme and ureteric bud, and tip number, PIFB and PMT values qualified the change in ureter morphogenesis and the reprogramming of the ureteric bud with lung mesenchyme. All the values for the heterotypic recombinant between ureteric bud and lung mesenchyme were significantly different from those for kidney samples but similar to those of the lung samples. Hence, lung mesenchyme can instruct the ureteric bud to undergo aspects of early lung-type epithelial morphogenesis. Different areas of the lung mesenchyme, except the tracheal region, were sufficient to promote ureteric bud growth and branching. In conclusion, our findings provide morphogenetic parameters for monitoring epithelial development in early embryonic lung and kidney and demonstrate the use of heterotypic tissue recombinants as a model for studying tissue-specific epithelial branching during organogenesis.     

Angioblast-mesenchyme induction of early kidney development is mediated by Wt1 and Vegfa

Most studies on kidney development have considered the interaction of the metanephric mesenchyme and the ureteric bud to be the major inductive event that maintains tubular differentiation and branching morphogenesis. The mesenchyme produces Gdnf, which stimulates branching, and the ureteric bud stimulates continued growth of the mesenchyme and differentiation of nephrons from the induced mesenchyme. Null mutation of the Wt1 gene eliminates outgrowth of the ureteric bud, but Gdnf has been identified as a target of Pax2, but not of Wt1. Using a novel system for microinjecting and electroporating plasmid expression constructs into murine organ cultures, it has been demonstrated that Vegfa expression in the mesenchyme is regulated by Wt1. Previous studies had identified a population of Flk1-expressing cells in the periphery of the induced mesenchyme, and adjacent to the stalk of the ureteric bud, and that Vegfa was able to stimulate growth of kidneys in organ culture. Here it is demonstrated that signaling through Flk1 is required to maintain expression of Pax2 in the mesenchyme of the early kidney, and for Pax2 to stimulate expression of Gdnf. However, once Gdnf stimulates branching of the ureteric bud, the Flk1-dependent angioblast signal is no longer required to maintain branching morphogenesis and induction of nephrons. Thus, this work demonstrates the presence of a second set of inductive events, involving the mesenchymal and angioblast populations, whereby Wt1-stimulated expression of Vegfa elicits an as-yet-unidentified signal from the angioblasts, which is required to stimulate the expression of Pax2 and Gdnf, which in turn elicits an inductive signal from the ureteric bud.     

Formation of the digestive system in zebrafish. II. Pancreas morphogenesis

Recent studies have suggested that the zebrafish pancreas develops from a single pancreatic anlage, located on the dorsal aspect of the developing gut. However, using a transgenic zebrafish line that expresses GFP throughout the endoderm, we report that, in fact, two pancreatic anlagen join to form the pancreas. One anlage is located on the dorsal aspect of the developing gut and is present by 24 h postfertilization (hpf), the second anlage is located on the ventral aspect of the developing gut in a position anterior to the dorsal anlage and is present by 40 hpf. These two buds merge by 52 hpf to form the pancreas. Using heart and soul mutant embryos, in which the pancreatic anlagen most often do not fuse, we show that the posterior bud generates only endocrine tissue, while the anterior bud gives rise to the pancreatic duct and exocrine cells. Interestingly, at later stages, the anterior bud also gives rise to a small number of endocrine cells usually present near the pancreatic duct. Altogether, these studies show that in zebrafish, as in the other model systems analyzed to date, the pancreas arises from multiple buds. To analyze whether other features of pancreas development are conserved and investigate the influence of surrounding tissues on pancreas development, we examined the role of the vasculature in this process. Contrary to reports in other model systems, we find that, although vascular endothelium is in contact with the posterior bud throughout pancreas development, its absence in cloche mutant embryos does not appear to affect the early morphogenesis or differentiation of the pancreas.     

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.     

Bmp4 and Fgf10 play opposing roles during lung bud morphogenesis

Morphogenesis of the mouse lung involves reciprocal interactions between the epithelial endoderm and the surrounding mesenchyme, leading to an invariant early pattern of branching that forms the basis of the respiratory tree. There is evidence that Fibroblast growth factor 10 (Fgf10) and Bone Morphogenetic Protein 4 (Bmp4), expressed in the distal mesenchyme and endoderm, respectively, play important roles in branching morphogenesis. To examine these roles in more detail, we have exploited an in vitro culture system in which isolated endoderm is incubated in Matrigel(TM) substratum with Fgf-loaded beads. In addition, we have used a Bmp4(lacZ) line of mice in which lacZ faithfully reports Bmp4 expression. Analysis of lung endoderm in vivo shows a dynamic pattern of Bmp4(lacZ) expression during bud outgrowth, extension and branching. In vitro, Fgf10 induces both proliferation and chemotaxis of isolated endoderm, whether it is derived from the distal or proximal lung. Moreover, after 48 hours, Bmp4(lacZ) expression is upregulated in the endoderm closest to the bead. Addition of 30-50 ng/ml of exogenous purified Bmp4 to the culture medium inhibits Fgf-induced budding or chemotaxis, and inhibits overall proliferation. By contrast, the Bmp-binding protein Noggin enhances Fgf-induced morphogenesis. Based on these and other results, we propose a model for the combinatorial roles of Fgf10 and Bmp4 in branching morphogenesis of the lung.     

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.     

Cessation of renal morphogenesis in mice

The kidney develops by cycles of ureteric bud branching and nephron formation. The cycles begin and are sustained by reciprocal inductive interactions and feedback between ureteric bud tips and the surrounding mesenchyme. Understanding how the cycles end is important because it controls nephron number. During the period when nephrogenesis ends in mice, we examined the morphology, gene expression, and function of the domains that control branching and nephrogenesis. We found that the nephrogenic mesenchyme, which is required for continued branching, was gone by the third postnatal day. This was associated with an accelerated rate of new nephron formation in the absence of apoptosis. At the same time, the tips of the ureteric bud branches lost the typical appearance of an ampulla and lost Wnt11 expression, consistent with the absence of the capping mesenchyme. Surprisingly, expression of Wnt9b, a gene necessary for mesenchyme induction, continued. We then tested the postnatal day three bud branch tip and showed that it maintained its ability both to promote survival of metanephric mesenchyme and to induce nephrogenesis in culture. These results suggest that the sequence of events leading to disruption of the cycle of branching morphogenesis and nephrogenesis began with the loss of mesenchyme that resulted from its conversion into nephrons.     

Isolation and characterization of a novel plasma membrane protein, osteoblast induction factor (obif), associated with osteoblast differentiation

Background

The exocrine pancreas is composed of a branched network of ducts connected to acini. They are lined by a monolayered epithelium that derives from the endoderm and is surrounded by mesoderm-derived mesenchyme. The morphogenic mechanisms by which the ductal network is established as well as the signaling pathways involved in this process are poorly understood.

Results

By morphological analyzis of wild-type and mutant mouse embryos and using cultured embryonic explants we investigated how epithelial morphogenesis takes place and is regulated by chemokine signaling. Pancreas ontogenesis displayed a sequence of two opposite epithelial transitions. During the first transition, the monolayered and polarized endodermal cells give rise to tissue buds composed of a mass of non polarized epithelial cells. During the second transition the buds reorganize into branched and polarized epithelial monolayers that further differentiate into tubulo-acinar glands. We found that the second epithelial transition is controlled by the chemokine Stromal cell-Derived Factor (SDF)-1. The latter is expressed by the mesenchyme, whereas its receptor CXCR4 is expressed by the epithelium. Reorganization of cultured pancreatic buds into monolayered epithelia was blocked in the presence of AMD3100, a SDF-1 antagonist. Analyzis of sdf1 and cxcr4 knockout embryos at the stage of the second epithelial transition revealed transient defective morphogenesis of the ventral and dorsal pancreas. Reorganization of a globular mass of epithelial cells in polarized monolayers is also observed during submandibular glands development. We found that SDF-1 and CXCR4 are expressed in this organ and that AMD3100 treatment of submandibular gland explants blocks its branching morphogenesis.

Conclusion

In conclusion, our data show that the primitive pancreatic ductal network, which is lined by a monolayered and polarized epithelium, forms by remodeling of a globular mass of non polarized epithelial cells. Our data also suggest that SDF-1 controls the branching morphogenesis of several exocrine tissues.     

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.     

Fgf10 is essential for maintaining the proliferative capacity of epithelial progenitor cells during early pancreatic organogenesis.

The importance of mesenchymal-epithelial interactions for the proper development of the pancreas has been acknowledged since the early 1960s, even though the molecule(s) mediating this process have remained unknown. We demonstrate here that Fgf10, a member of the fibroblast growth factor family (FGFs), plays an essential role in this process. We show that Fgf10 is expressed in the mesenchyme directly adjacent to the early dorsal and ventral pancreatic epithelial buds. In Fgf10(-/-) mouse embryos, the evagination of the epithelium and the initial formation of the dorsal and ventral buds appear normal. However, the subsequent growth, differentiation and branching morphogenesis of the pancreatic epithelium are arrested; this is primarily due to a dramatic reduction in the proliferation of the epithelial progenitor cells marked by the production of the homeobox protein PDX1. Furthermore, FGF10 restores the population of PDX1-positive cells in organ cultures derived from Fgf10(-/-) embryos. These results indicate that Fgf10 signalling is required for the normal development of the pancreas and should prove useful in devising methods to expand pancreatic progenitor cells.     

Identification of pleiotrophin as a mesenchymal factor involved in ureteric bud branching morphogenesis

Branching morphogenesis is central to epithelial organogenesis. In the developing kidney, the epithelial ureteric bud invades the metanephric mesenchyme, which directs the ureteric bud to undergo repeated branching. A soluble factor(s) in the conditioned medium of a metanephric mesenchyme cell line is essential for multiple branching morphogenesis of the isolated ureteric bud. The identity of this factor had proved elusive, but it appeared distinct from factors such as HGF and EGF receptor ligands that have been previously implicated in branching morphogenesis of mature epithelial cell lines. Using sequential column chromatography, we have now purified to apparent homogeneity an 18 kDa protein, pleiotrophin, from the conditioned medium of a metanephric mesenchyme cell line that induces isolated ureteric bud branching morphogenesis in the presence of glial cell-derived neurotrophic factor. Pleiotrophin alone was also found to induce the formation of branching tubules in an immortalized ureteric bud cell line cultured three-dimensionally in an extracellular matrix gel. Consistent with an important role in ureteric bud morphogenesis during kidney development, pleiotrophin was found to localize to the basement membrane of the developing ureteric bud in the embryonic kidney. We suggest that pleiotrophin could act as a key mesenchymally derived factor regulating branching morphogenesis of the ureteric bud and perhaps other embryonic epithelial structures.     

Dorsal pancreas agenesis in N-cadherin- deficient mice.

Members of the cadherin family of cell adhesion molecules are thought to be crucial regulators of tissue patterning and organogenesis. During pancreatic ontogeny N-cadherin is initially expressed in the pancreatic mesenchyme and later in pancreatic endoderm. Analysis of N-cadherin-deficient mice revealed that these mice suffer from selective agenesis of the dorsal pancreas. Further analysis demonstrated that the mechanism for the lack of a dorsal pancreas involves an essential function of N-cadherin as a survival factor in the dorsal pancreatic mesenchyme.     

Epithelial-mesenchymal interactions regulate the stage-specific expression of a cell surface proteoglycan, syndecan, in the developing kidney

Morphogenesis of the kidney is regulated by reciprocal tissue interactions between the epithelial ureter bud and the metanephric mesenchyme. The differentiation of the kidney involves profound changes in the extracellular matrix, and therefore matrix receptors may have an important role in this process. We studied the expression of syndecan, a cell surface proteoglycan acting as a receptor for interstitial matrix materials, by using a monoclonal antibody against the core protein of the molecule. Syndecan was not detected in the uninduced metanephric mesenchyme. During the formation of the ureter bud from the Wolffian duct, syndecan appeared in the mesenchymal cells around the invaginating bud. Simultaneously with the first branching of the ureter bud, the whole nephric mesenchyme became syndecan positive, but a 3- to 10-cell-thick layer around the branching ureter bud, representing the presumptive tubular cells, was most intensely stained. During the assembly of the mesenchyme cells into pretubular aggregates, syndecan was detected in these aggregates and, to a lesser degree, in the morphologically undifferentiated mesenchyme. Thereafter syndecan was found only in the differentiating epithelium, from which it was gradually lost during maturation of the nephron. It was last detected in the periphery of the kidney, where tubulogenesis still continued. In transfilter cultures we showed that syndecan appeared in the nephric mesenchyme during the period when the mesenchyme becomes programmed to transform into epithelial structures. By using interspecies recombinations and a species-specific antibody we excluded the possibility that syndecan in the mesenchyme would originate from the inductor. We conclude that syndecan expression is regulated by epithelial-mesenchymal interactions. The findings that syndecan appeared as an early response to induction and that its distribution showed both spatial and temporal correlation with kidney morphogenesis suggest an important role for this molecule in development.     

Coordinated expression of Hoxb genes and signaling molecules during development of the chick respiratory tract

To elucidate the molecular mechanism for regulating the region-specific morphogenesis of the chicken respiratory tract, we analyzed the spatiotemporal expression patterns of the Hoxb genes, Bmp-2, Bmp-4, Wnt-5a, and Wnt-11 in the developing respiratory tract. We found region-specific expression of these genes in the mesenchymal layer of the respiratory tract. Before bronchial branching proceeds, Hoxb genes show nested expression patterns around the ventral-distal tip of the lung bud. As morphogenesis proceeds, these expression domains correspond to the morphological subdivisions of the chick respiratory tract. Hoxb-5 and Hoxb-6 expression domains demarcate the trachea, bronchial tree, and air sacs. Particularly the expression domains of Hoxb-6 to -9 correspond to the morphological subdivisions of the air sacs along the proximodistal axis. Bmp-4 and Bmp-2 are expressed throughout the entire pulmonary mesenchyme and its dorsal half, respectively. Wnt-5a and Wnt-11 are expressed in the tracheal mesenchyme. Interestingly, the expression domain of Bmp-2 is complementary to the Hoxb-6 domain. The respiratory mesenchyme influences the process of epithelial branching during morphogenesis. By tissue recombination experiments, we found that the dorsal and the ventral pulmonary mesenchyme, demarcated by Hoxb-6 expression, have different inductive capacities toward the tracheal epithelium. These observations suggest the possibility that Hoxb genes are involved in the system specifying regional differences in morphogenesis and cytodifferentiation of respiratory tract. In addition, it is possible that BMPs and WNTs mediate region-specific epithelial-mesenchymal interaction in this system.