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.     

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.     

Activated Notch1 prevents differentiation of pancreatic acinar cells and attenuate endocrine development

Mice carrying loss-of-function mutations in certain Notch pathway genes display increased and accelerated pancreatic endocrine development, leading to depletion of precursor cells followed by pancreatic hypoplasia. Here, we have investigated the effect of expressing a constitutively active form of the Notch1 receptor (Notch1(ICD)) in the developing pancreas using the pdx1 promoter. At e10.5 to e12.5, we observe a disorganized pancreatic epithelium with reduced numbers of endocrine cells, confirming a repressive activity of Notch1 upon the early differentiation program. Subsequent branching morphogenesis is impaired and the pancreatic epithelium forms cyst-like structures with ductal phenotype containing a few endocrine cells but completely devoid of acinar cells. The endocrine cells that do form show abnormal expression of cell type-specific markers. Our observations show that sustained Notch1 signaling not only significantly represses endocrine development, but also fully prevents pancreatic exocrine development, suggesting that a possible role of Notch1 is to maintain the undifferentiated state of common pancreatic precursor cells.     

Interplay between FGF10 and Notch signalling is required for the self-renewal of pancreatic progenitors

Recent studies have shown that persistent expression of FGF10 in the developing pancreas of transgenic mice results in enhanced and prolonged proliferation of pancreatic progenitors, pancreatic hyperplasia and impaired pancreatic differentiation. These studies have also suggested that FGF10 prevents the differentiation of pancreatic progenitors by maintaining persistent Notch signalling. Here, we provide experimental evidence sustaining the capacity of FGF10 to induce the proliferation of pancreatic precursors, while preventing their differentiation. Using explant cultures of E10.5 isolated dorsal pancreatic epithelium, we found that FGF10 maintained Notch activation and induced the expansion of pancreatic precursors while blocking their differentiation. In addition, by using a gamma-secretase inhibitor, we were able to down-regulate the expression of Hes1, a target gene of the Notch pathway in explant cultures of pancreatic epithelium treated with FGF10. In such explants, the effect of FGF10 on the proliferation and maintenance of pancreatic progenitors was suppressed. These results demonstrate that activation of the Notch pathway is required as a downstream mediator of FGF10 signalling in pancreatic precursor cells.     

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.     

FGF10 signaling maintains the pancreatic progenitor cell state revealing a novel role of Notch in organ development

FGF10 plays an important role in the morphogenesis of several tissues by control of mesenchymal-to-epithelial signaling. In the pancreas, mesenchymal FGF10 is required to maintain the Pdx1-expressing epithelial progenitor cell population, and in the absence of FGF10 signaling, these cells fail to proliferate. Ectopic expression of FGF10 in the pancreatic epithelium caused increased proliferation of pancreatic progenitor cells and abrogation of pancreatic cell differentiation of all cell types. A hyperplastic pancreas consisting of undifferentiated cells expressing Pdx1, Nkx6.1, and cell adhesion markers normally characterizing early pancreatic progenitor cells resulted. Differentiation was attenuated even as proliferation of the pancreatic cells slowed during late gestation, suggesting that the trophic effect of FGF10 was independent of its effects upon cell differentiation. The FGF10-positive pancreatic cells expressed Notch1 and Notch2, the Notch-ligand genes Jagged1 and Jagged2, as well as the Notch target gene Hes1. This activation of Notch is distinct from the previously recognized mechanism of lateral inhibition. These data suggest that FGF10 signaling serves to integrate cell growth and terminal differentiation at the level of Notch activation, revealing a novel second role of this key signaling system during pancreatic development.     

Embryogenesis of the murine endocrine pancreas; early expression of pancreatic polypeptide gene.

By immunofluorescence on cytospin preparations and on semithin sections of mouse pancreatic buds, we have found glucagon and pancreatic polypeptide (PP)-containing cells at embryonal day 10.5 (E 10.5) in dorsal buds and at E 11.5 in ventral buds. Insulin-containing cells appear in dorsal buds at E 11.5, and one to two days later in ventral buds. Somatostatin-containing cells are detectable from E 13.5 in both dorsal and ventral buds. A quantitative analysis shows that up to E 15.5, PP-containing cells are relatively abundant in both buds. By PCR amplification of oligo(dT)-primed cDNAs prepared from total pancreatic RNA, we also detect PP mRNA from E 10.5 onwards, thus confirming the early expression of the PP gene in the developing mouse pancreas. Analysis of endocrine cells in situ suggests three major patterns of cell distribution in embryonic pancreas. First, individual hormone-containing cells are located within the epithelium of pancreatic ducts. In both dorsal and ventral buds, the majority of these endocrine cells contain PP, but many also contain glucagon, insulin or somatostatin. Secondly, clusters of endocrine cells are found in the pancreatic interstitium. Many of these cells contain both glucagon and PP which, by immunogold labelling of consecutive thin sections, can be shown to co-exist within individual secretory granules. Finally, starting on E 18.5, typical islets are formed with centrally located B cells and with the adult 'one cell-one hormone' phenotype. These results suggest an intriguing ontogenic relationship between A- and PP-cells, and also indicate that PP-containing cells may occupy a hitherto unexpected place in the lineage of endocrine islet cells.     

Origin of exocrine pancreatic cells from nestin-positive precursors in developing mouse pancreas

During pancreatic development, endocrine and exocrine cell types arise from common precursors in foregut endoderm. However, little information is available regarding regulation of pancreatic epithelial differentiation in specific precursor populations. We show that undifferentiated epithelial precursors in E10.5 mouse pancreas express nestin, an intermediate filament also expressed in neural stem cells. Within developing pancreatic epithelium, nestin is co-expressed with pdx1 and p48, but not ngn3. Epithelial nestin expression is extinguished upon differentiation of endocrine and exocrine cell types, and no nestin-positive epithelial cells are observed by E15.5. In E10.5 dorsal bud explants, activation of EGF signaling results in maintenance of undifferentiated nestin-positive precursors at the expense of differentiated acinar cells, suggesting a precursor/progeny relationship between these cell types. This relationship was confirmed by rigorous lineage tracing studies using nestin regulatory elements to drive Cre-mediated labeling of nestin-positive precursor cells and their progeny. These experiments demonstrate that a nestin promoter/enhancer element containing the second intron of the mouse nestin locus is active in undifferentiated E10.5 pancreatic epithelial cells, and that these nestin-positive precursors contribute to the generation of differentiated acinar cells. As in neural tissue, nestin-positive cells act as epithelial progenitors during pancreatic development, and may be regulated by EGF receptor activity.     

Fate maps of ventral and dorsal pancreatic progenitor cells in early somite stage mouse embryos

The origins of liver progenitor cells have been extensively studied, but evidence on the origin of pancreatic precursor cells is currently limited. Pancreatic and duodenal homeobox gene 1 (Pdx1) is one of the earliest known markers for the pancreas. A transgenic mouse line expressing green fluorescent protein (GFP) under the control of the Pdx1 promoter showed that Pdx1/GFP expression was first observed in the mid-region of the anterior intestinal portal (AIP) lip at embryonic day (E) 8.5 at the 5-6 somite stage (ss). The liver progenitors were confirmed to originate from separate domains at the lateral endoderm and the inner part of the medial AIP as previously reported (Tremblay and Zaret, 2005), which turned out to lie caudally to the Pdx1/GFP-expressing domain. To confirm if the early Pdx1/GFP-positive cells give rise to the pancreatic bud, we labeled the cells on the lip of the AIP using the carbocyanine dye CM-DiI and traced their fates in 1-4 ss, 5-6 ss and 7-9 ss E8.5 embryos using an ex utero whole embryo culture method. At 1 ss, the ventral pancreas progenitors were observed in the lateral endoderm, not yet being segregated from the liver or gut progenitors. Cells that contributed solely to the ventral pancreas first appeared at the AIP lip from 5 ss. At 5-6 ss, cells from the medial of the AIP lip contributed to the ventral pancreas. The pancreas fate region become narrower as development progresses. At 7-9 ss, the cells contributing to the ventral pancreas resided in a narrow region of the AIP lip. From 5 ss, the right flanking region contributes to the posterior gut, and the left flanking region contributes to the anterior gut. Dorsal pancreatic progenitors originate from the dorsal endoderm at the 3-6 somite level at 7-9 ss, though they have not yet diverged from the dorsal gut progenitors at this stage.     

Extraocular dorsal signal affects the developmental fate of the optic vesicle and patterns the optic neuroepithelium

Dorsal-ventral (DV) specification in the early optic vesicle plays a crucial role in the proper development of the eye. To address the questions of how DV specification is determined and how it affects fate determination of the optic vesicle, isolated optic vesicles were cultured either in vitro or in ovo. The dorsal and ventral halves of the optic vesicle were fated to develop into retinal pigment epithelium (RPE) and neural retina, respectively, when they were separated from each other and cultured. In optic vesicles treated with collagenase to remove the surrounding tissues, the neuroepithelium gave rise to cRax expression but not Mitf, suggesting that surrounding tissues are necessary for RPE specification. This was also confirmed in in ovo explant cultures. Combination cultures of collagenase-treated optic vesicles with either the dorsal or ventral part of the head indicated that head-derived factors have an important role in the fate determination of the optic vesicle: in the optic vesicles co-cultured with the dorsal part of the head Mitf expression was induced in the neuroepithelium, while the ventral head portion did not have this effect. The dorsal head also suppressed Pax2 expression in the optic vesicle. These observations indicate that factors from the dorsal head portion have important roles in the establishment of DV polarity within the optic vesicle, which in turn induces the patterning and differentiation of the neural retina and pigment epithelium.     

Development of the pancreas in medaka

In the present work, pancreatic organogenesis has been studied in the medaka (Oryzias latipes), a teleost fish with several advantages as an experimental system in developmental biology. We demonstrated that the pancreas develops from three primordia budding from the dorsal and ventral faces of the gut epithelium. Such buds then fuse to form a single endocrine islet surrounded by exocrine tissue. Interestingly, the endocrine tissue forms only from the dorsal bud. We next analyzed a collection of medakas that had been hybridized with cDNAs derived from an anterior brain library. We found new clones expressed in the pancreatic region demonstrating that the medaka can be used to define new genes expressed in the pancreatic region that follow a specific spatial and temporal pattern of expression.