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

¹ INSERM 457, Hospital Robert Debré, 75019 Paris, France
² Department of Genetic Biochemistry, Kyoto University, Kyoto, Japan
³ Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo, Japan
⁴ Institut Curie-UMR 144 CNRS, Paris, France

View larger version (65K): [in a new window]   Fig. 1. Fgf10 is expressed in the pancreatic mesenchyme during the early stages of pancreatic organogenesis. (A,B,E) Whole-mount in situ hybridisation on dissected gastrointestinal tracts (dorsal is to the right) showing (A) Fgf10 expression in the gut dissected from an E9.5 embryo. The first signs of Fgf10 expression in the posterior foregut were two distinct stripes where the dorsal and ventral pancreatic buds emerge. Expression of Fgf10 in the foregut anterior to the pancreas was also observed in the lung buds. (B) By E10.5, the dorsal epithelial bud was clearly visible and Fgf10 was expressed broadly in the surrounding mesenchyme. Expression of Fgf10 also extended into the posterior stomach mesenchyme. (C,D) Vibratome sections (20 µm) of the gut from the E10.5 embryo shown in B demonstrates that Fgf10 expression is confined to the mesenchyme adjacent to the dorsal (C) and ventral (D) bud. (E) Fgf10 continued to be expressed at E11.5 in a restricted area in the dorsal mesenchyme (indicated by an arrow). The uniform weak staining observed in the stomach epithelium was due to the trapping of the in situ probe in the lumen of the stomach. d, dorsal; v, ventral; db, dorsal bud; vb, ventral bud; lb, lung bud; st, stomach.

View larger version (88K): [in a new window]   Fig. 2. Pancreatic hypoplasia and absence of islet cells in Fgf10–/–embryos. (A) Gastrointestinal tract from an E17.5 wild-type embryo. (B) Gross appearance of pancreatic region dissected from an E17.5 wild-type embryo and (C) a schematic representation illustrating the pancreas. The pancreatic tissue at this stage is localised near the spleen, which derived from the dorsal bud (green), and along the duodenum, which derived from the ventral bud (blue). (D) Haematoxylin and Eosin staining of the pancreas tissue from an E17.5 wild-type embryo showing the presence of acini exocrine tissue and heavily nucleated clusters of islet cells. (E) The islet clusters express insulin (green) and glucagon (red). (F) Exocrine tissue expresses carboxypeptidaseA (green). (G) The gastrointestinal tract from E17.5 Fgf10–/– embryos were overtly similar to that of wild-type littermates except for a smaller stomach. (H) The pancreatic tissue in the mutant Fgf10 embryos was drastically reduced (I) although present in both the splenic (green) and duodenal (blue) locations. (J) Haematoxylin and Eosin staining shows the presence of acinar tissue but no islet clusters are evident. (K) Scattered insulin (green) and glucagon-expressing (red) cells are present. Co-expression of insulin and glucagon indicates that these endocrine cells are immature (arrow). (L) The acini from the mutant Fgf10 embryo stained for the exocrine marker, carboxypeptidaseA. CA, carboxypeptidaseA; ins, insulin; glu, glucagon; st, stomach; sp, spleen.

View larger version (63K): [in a new window]   Fig. 3. The size of the pancreatic epithelium in Fgf10–/– embryos is greatly reduced. (A) Hnf3ß whole-mount in situ hybridisation to the epithelium of the foregut of wild-type E11.5 embryos. (B) Hnf3ß expression in the pancreatic region and posterior stomach was greatly reduced in the foregut of Fgf10 mutant embryos. (C) ISL1 was expressed predominantly in the dorsal mesenchyme and a few differentiating endocrine cells in the pancreatic epithelium from E10.5 wild-type embryos. The dorsal bud is oriented to the top. (D) Strong expression of ISL1 was observed in the dorsal mesenchyme of Fgf10–/– embryos at E10.5, although very few scattered cells within the epithelium that expressed ISL1 were detected. (E) Sagittal sections of the dissected gut from wild-type embryo stained with carboxypeptidaseA (green) and glucagon (red). The branched morphology of both dorsal and ventral pancreatic buds is evident. (F) Sagittal section of dissected gut from an Fgf10–/– embryo shows that the formation of both dorsal and ventral buds occurred, however, no branching of the epithelium is apparent. (G) Transverse sections of the dorsal bud of E13.5 wild-type embryo stained for the pan-epithelial markers cytokeratin (red) and glucagon (green). The dorsal pancreatic bud of a wild-type embryo shows a characteristic highly branched epithelium. (H) The dorsal bud of the Fgf10–/– embryo has a small pancreatic epithelium and no branching of the epithelium is visible. Differentiation of early endocrine cells, as identified by glucagon staining, occurred in the mutant embryos and these cells are seen typically clustered together. db, dorsal pancreatic bud; vb, ventral pancreatic bud; ISL1, Islet1; CA, carboxypeptidaseA; du, duodenum.

View larger version (68K): [in a new window]   Fig. 4. Fgf10–/– embryos have a small pancreatic primordium because the Pdx1-expressing epithelial progenitor cell population is not maintained. Dorsal is to the right. (A) Pdx1 is expressed uniformly in undifferentiated cells throughout the developing pancreatic buds in wild-type embryos at E10.5 (B) Immunofluorescence analysis of PDX1 expression in transverse section of an E10.5 wild-type embryo shows the buds emerging from the foregut. (C) By 12.5, Pdx1 expression is no longer uniform (due to differentiation of precursor cells) and the branching of the epithelium is evident in the dorsal bud. (D) By E13.5, Pdx1 expression is increasingly restricted within the epithelium and accentuates the lobulated structure of both the pancreatic buds. As compared to the wild-type littermates the Pdx1 expression in the Fgf10–/– embryos at E10.5 (E) identifies the formation of two small but distinct pancreatic buds. (F) Immunofluorescence analysis confirmed the reduced expression of PDX1 in these two pancreatic buds in mutant embryos. Pdx1 expression is no longer observed later in development at E12.5 (G) and E13.5 (H). Arrows indicate the area within the gut where the pancreatic buds normally form. Occasionally, some weak expression of Pdx1 was observed in the ventral bud of mutant embryos (H). v, ventral; d, dorsal.

View larger version (82K): [in a new window]   Fig. 5. The smaller pancreatic epithelium in the Fgf10 mutants was primarily due to the decreased proliferation of progenitor cells that are marked by expression of PDX1. (A,B) Immunofluorescence analysis for the expression of PDX1 (green) and glucagon (red) in the pancreatic epithelium of E11.5; (A) wild-type littermate and (B) Fgf10–/– embryo. The pancreatic epithelium in the Fgf10 mutant embryo is reduced in size, although no concomitant increase in glucagon expression is evident. In addition, no glucagon-positive cells are evident in the ventral bud of the Fgf10–/– embryo. (C-H) Analysis of proliferating precursor epithelial cells in the dorsal bud of Fgf10–/– (D,F,H) and wild-type (C,E,G) littermate embryos at E10.5. PDX1 labelling identifies the dorsal pancreatic epithelium, which is smaller in Fgf10–/– (D) compared to wild-type littermates (C). (F) BrdU labelling shows very few proliferating cells within the dorsal pancreatic epithelium of Fgf10–/– embryos. (E) In wild type a large fraction of dorsal pancreatic epithelial cells have gone through S-phase and stained BrdU positive. The outline designates the boundary of the dorsal epithelial bud. (G,H) Merged images show double labelling for PDX1 and BrdU and confirm that the proliferating epithelial cells also express PDX1 (magnified 2x). To quantify the difference in proliferation of precursor epithelial cells between Fgf10–/– embryos and their wild-type littermates, four consecutive sections from each of four wild-type and four Fgf10 mutants were used to calculate the proliferative index (BrdU+/PDX1+)x100. Using such analysis, wild-type embryos displayed an average proliferative index of 50.4±3.8 (n=4) as compared with 15.1±2.9 (n=4) for Fgf10–/– embryos.

View larger version (77K): [in a new window]   Fig. 6. Exogenously added FGF10 can rescue the PDX1-expressing pool of epithelial cells in cultured pancreas derived from Fgf10 mutants. Consecutive sections of explanted cultured tissue were stained for PDX1 (green) and glucagon (red). (A) Cultured explanted tissue derived from an Fgf10 mutant stained for PDX1. Very few stained cells were detected. (B) Section adjacent to A showing that glucagon-positive cells were present. (C) Explanted tissue derived from Fgf10 mutants, cultured with 50 ng/ml FGF10 and stained for PDX1. The PDX1-positive cells are present in the bud outgrowth from the foregut epithelium. (D) Glucagon-expressing cells in an adjacent section to C. (E) An additional population of PDX1-positive cells detected in the explanted tissue from Fgf10 mutants that was cultured with FGF10. (F) No glucagon-positive cells were detected in the section adjacent to that in E. (G) Cultured explanted tissue derived from a wild-type littermate and stained for PDX1. The arrow indicates an additional population of PDX1-stained cells. (H) Glucagon-positive cells in the section adjacent to that in G. The white dashed lines in D, F and H indicate the position of the PDX1-positive cells in C, E and G, respectively.