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First published online May 23, 2008
doi: 10.1242/10.1242/dev.015859

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Signals from the neural crest regulate beta-cell mass in the pancreas

¹ Diabetes Center, Hormone Research Institute
² Department of Medicine, University of California at San Francisco, San Francisco, CA 94143, USA.

View larger version (17K): [in this window] [in a new window]   Fig. 1. The temporal pattern of Phox2b expression in the embryonic pancreas and stomach. Phox2b mRNA levels were determined by real-time RT-PCR (TaqMan) of RNA isolated from the pancreas (A) and stomach (B) of mouse embryos from E10.5 to E15.5. All values are expressed relative to the level in pancreatic RNA at E10.5 and represent the mean value of three independent experiments performed in triplicate ±s.e.m. In A, mRNA values that are below or at the limit of detection (E10.5, E14.5 and E15.5) are displayed at the value for the limit of detection.

View larger version (125K): [in this window] [in a new window]   Fig. 2. The pattern of Phox2b expression in embryonic pancreas and gut. (A-D) Mouse embryos harvested at E12.5 were stained by immunohistochemistry with peroxidase (A) for Phox2b (brown) or by immunofluorescence (B-D) for Phox2b (FITC, green in B; Cy3, red in C), Pdx1 (Cy3, red in B) and Isl1 (FITC, green in C,D). Dashed line outlines the pancreatic epithelium. Arrows indicate representative nuclei with Phox2b staining. Scale bars: 50 µm. p, pancreas; s, stomach; i, intestine.

View larger version (121K): [in this window] [in a new window]   Fig. 3. Lineage tracing of Phox2b-expressing cells. Lineage tracing was performed by crossing R26R mice with either Pdx1-cre (A,B) or Wnt1-cre (C-E) mouse lines. Mouse embryos harvested at E12.5 were stained for β-galactosidase activity with X-gal (blue), and stained for Pdx1 (A,C) or Phox2b (B,D,E) by immunohistochemistry with peroxidase labeling (brown). Scale bars: 50 µm.

View larger version (47K): [in this window] [in a new window]   Fig. 4. Sox10 expression in the pancreas. (A) Sox10 mRNA levels were determined by RT-PCR of RNA isolated from the pancreas of mouse embryos from E10.5 to E15.5 and compared with β-actin mRNA levels. Stomach (B-E) and pancreas (F-I) from mouse embryos harvested at E12.5 were stained by immunofluorescence for Sox10 (Cy3, red) and Phox2b (FITC, green), and co-stained with nuclear stain DAPI (blue). Scale bar: 25 µm.

View larger version (63K): [in this window] [in a new window]   Fig. 5. Nkx2.2 regulation of Phox2b. (A) Pancreas from a mouse embryo harvested at E12.5 stained by immunofluorescence for Nkx2.2 (Cy3, red) and Phox2b (FITC, green). (B) Lineage tracing was performed by crossing R26R mice with Wnt1-cre mice. Mouse embryos harvested at E12.5 were stained for β-galactosidase activity with X-gal (blue), and stained for Nkx2.2 by immunohistochemistry with peroxidase labeling (brown). (C) Phox2b mRNA levels were determined by real-time RT-PCR (TaqMan) with RNA isolated from pancreas (black bars) and stomach (white bars) from E13.5 embryos with the Nkx2.2 genotypes shown. Values for both pancreas and stomach are expressed separately relative to the levels of each in Nkx2.2+/+ embryos. The Phox2b mRNA level in the stomach was not tested for the Nkx2.2+/- embryos. Data represent the mean values of four independent experiments performed in triplicate ±s.e.m. (D-G) The pancreas (D,E) and stomach (F,G) of Nkx2.2+/+ (D,F) and Nkx2.2-/- (E,G) embryos at E13.5 were stained by immunofluorescence for Phox2b (Cy3, red) and Nkx2.2 (FITC, green). An increase in Phox2b staining was observed in the pancreas from Nkx2.2-/- embryos (compare D and E) but not in the stomach (compare F and G). *P<0.01; **P<0.001, compared with Nkx2.2+/+ embryos by Student's t-test. Scale bars: 50 µm.

View larger version (77K): [in this window] [in a new window]   Fig. 6. Turnover of Phox2b-expressing cells. Pancreas from Nkx2.2+/+ (A,B) and Nkx2.2-/- (C-F) mouse embryos at E12.5 (A), E13.5 (B,C), and E15.5 (D-F) were co-stained by immunofluorescence for Pdx1 (FITC, green) and the apoptosis marker cleaved caspase-3 (Cy3, red; A-C), or for Phox2b (Cy3, red; D,F) and the replication marker Ki67 (FITC, green; E,F). Scale bars: 50 µm.

View larger version (83K): [in this window] [in a new window]   Fig. 7. Differentiated neural crest cells in pancreas and stomach. Pancreas and stomach from Phox2b+/+ (A-C) and Phox2b-/- (E-G) mouse embryos at E13.5 (A,E) and E17.5 (B,C,F,G) were co-stained by immunofluorescence for Pdx1 (FITC, green) and the neural marker Pgp9.5 (Cy3, red). (D,H) Pancreas from Phox2b+/+ (D) and Phox2b-/- (H) embryos at E18.5 were co-stained by immunofluorescence for insulin (Cy3, red) and the glial marker Fabp7 (FITC, green). s, stomach; p, pancreas. Scale bars: 50 µm.

View larger version (40K): [in this window] [in a new window]   Fig. 8. Non-cell autonomous regulation of islet cells by Phox2b. Pancreas from Phox2b+/+ (A) and Phox2b-/- (B) mouse embryos at E17.5 co-stained by immunofluorescence for insulin (FITC, green) and glucagon (Cy3, red). (C) Insulin- and glucagon-positive cells from Phox2b+/+ (white bars) and phox2b-/- (black bars) embryos counted and expressed as the total number of cells per total pancreatic area in mm2. Each data point represents the mean of five embryos ±s.e.m. (D) Insulin and glucagon mRNA levels measured by real-time RT-PCR (TaqMan) from RNA from pancreas of Phox2b+/+ (white bars) and Phox2b-/- (black bars) embryos at E17.5. Each data point represents the mean of four independent litters ±s.e.m. (E) Pancreas from a Phox2b-/- mouse embryo at E17.5 co-stained by immunofluorescence for Ki67 (FITC, green) and insulin (Cy3, red). (F) The percentage of beta-cells replicating at E17.5 in embryos with the genotypes shown assessed by counting the number of cells co-staining for insulin and Ki67 and dividing by the total number of cells staining for insulin. Each data point represents the mean of five pancreas ±s.e.m. (G) Nkx2.2 (white bars) and neurogenin 3 (black bars) mRNA levels determined by real-time RT-PCR (TaqMan) with RNA isolated from pancreas from E15.5 embryos with the Phox2b genotypes shown. Data represent the mean values of three independent experiments performed in triplicate ±s.e.m. *P<0.01, **P<0.001, by Student's t-test. Scale bars: 50 µm.

View larger version (18K): [in this window] [in a new window]   Fig. 9. Proposed model for the non-cell-autonomous interactions between Phox2b and Nkx2.2 in the developing pancreas.

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