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First published online 17 November 2004
doi: 10.1242/dev.01535

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GDF11 modulates NGN3⁺ islet progenitor cell number and promotes ß-cell differentiation in pancreas development

Erin B. Harmon^1,, Åsa A. Apelqvist^1,*,, Nora G. Smart¹, Xueying Gu¹, Douglas H. Osborne¹ and Seung K. Kim^1,2,

¹ Department of Developmental Biology, Stanford University, Stanford, CA 94305-5329, USA
² Department of Medicine (Oncology Division), Stanford University, Stanford, CA 94305-5329, USA

View larger version (47K): [in a new window]   Fig. 1. Pancreatic Gdf11 expression detected by in-situ hybridization. (A) Expression of Gdf11 (purple) in dorsal pancreas epithelium at E11.5. Little-to-no expression of Gdf11 mRNA is detected in pancreatic mesenchymal cells that surround the epithelium. (B) Expression of Gdf11 (purple) in dorsal pancreas, ventral pancreas and stomach epithelium, duodenal epithelium and kidney (inset) at E13.5. (C) Expression of Gdf11 (purple) in exocrine acinar cells and duodenal epithelium at E18.5. Little-to-no Gdf11 expression was detected in islets. Blood cells are stained black in this panel. (D) Sense control at E18.5. Labels are the same as in C. d, duodenal epithelium; dp, dorsal pancreas epithelium; e, exocrine acinar cells; i, islets; k, kidney; s, stomach epithelium; vp, ventral pancreas epithelium.

View larger version (71K): [in a new window]   Fig. 2. Defective stomach, spleen and pancreas development in embryos deficient for Gdf11. `Control' indicates that the observed phenotype is indistinguishable in Gdf11+/+ and Gdf11+/- embryos. (A,B) Sectioned stomachs stained with hematoxylin and eosin and whole mounted spleens (insets) from mice at P1. Note thinning of the stomach epithelial and mesenchymal layers and paucity of epithelial folds in the Gdf11-/- mouse. (A,B inset) Whole spleen preparations demonstrate spleen malformation in Gdf11-/- mice. (C,D) Whole mounted preparations of pancreas from Gdf11+/- and Gdf11-/- mice at P1. (E,F) Significantly reduced exocrine acinar cell development in Gdf11-deficient mice at E17.5. Acini are dark following immunoperoxidase staining with antiserum specific for carboxypeptidase A. (G) Quantification of morphometry showing significant reduction of exocrine acinar cell volume in Gdf11-/- mice relative to both wild-type and heterozygous Gdf11+/- mice. Data are shown as the average measurements from at least six mice of indicated genotypes±standard error of the mean. Asterisk indicates significance as P<0.01. (H,I) The endocrine cell compartment is significantly increased in Gdf11-/- mice relative to wild-type mice at E17, as assessed by immunofluorescent staining using the neuroendocrine marker, synaptophysin (red). (J) Quantification of synaptophysin staining in each of the indicated genotypes. The asterisk indicates significance as P<0.05 between wild-type and Gdf11-/- mice. There is no significant difference between wild-type and Gdf11+/- mice. (J) Duct cell number, as assessed by lectin DBA staining is normal in Gdf11-/- mice at E17 (green). (M) Quantification of DBA staining for all three genotypes. NS, not significant. (N,O) Merge of synaptophysin (red) and DBA (green) channels for wild-type and Gdf11-/- mice at E17.5, confirming that DBA+ cells are distinct from synaptophysin+ cells. (P,Q) Apoptotic pancreatic nuclei (green, arrows) labeled by TUNEL from E17.5 Gdf11+/+ (P) and Gdf11-/- (Q) mice. All nuclei are simultaneously co-stained by DAPI (blue).

View larger version (86K): [in a new window]   Fig. 3. Defective pancreatic islet ß-cell and -cell numbers in Gdf11-deficient mice. (A-C) Immunohistochemical detection of insulin (brown) at E17.5 in mice with the indicated genotypes. (D) Quantification of ß-cell mass by point-counting morphometry in Gdf11+/+, Gdf11+/- and Gdf11-/- pancreata at P1. Data are shown as the average measurements from at least four mice of indicated genotypes ± standard error of the mean. The asterisk indicates a significant difference between Gdf11-/- animals and Gdf11+ littermates at P<0.05. (E-G) Immunohistochemical detection of glucagon (brown) at E17.5 in mice with the indicated genotypes. (H) Quantification of -cell mass by point-counting morphometry in Gdf11+/+, Gdf11+/- and Gdf11-/- pancreata at P1. Data are shown as the average measurements from at least four mice of indicated genotypes ± standard error of the mean. Asterisks indicate significant differences between wild-type littermates and Gdf11 deficient animals at P<0.05. (I-K) Immunohistochemical detection of somatostatin (brown) at E17.5 in mice with the indicated genotypes. (L-N) Immunohistochemical detection of pancreatic polypeptide (brown) at E17.5 in mice with the indicated genotypes. (O-Q) Pancreatic islet cell expression of insulin (green) and glucagon (red) at P1. Representative samples are shown.

View larger version (100K): [in a new window]   Fig. 4. Premature expansion and increased numbers of Ngn3+ pancreatic cells in Gdf11-deficient mice. Pancreatic Ngn3 expression (blue) detected by antisense RNA in-situ hybridization in Gdf11+/+ (A,D,G), Gdf11+/- (B,E,H) and Gdf11-/- (C,F,I) embryos at the indicated embryonic stages. (G-I) Ngn3-expressing cells at E17.5 are marked by black arrowheads for clarity. (J,K) Pancreatic expression of insulin and glucagon (green) and Ngn3 (red) at E17.5 in mice with indicated genotypes. White arrowheads (K) mark three of the numerous Ngn3+ nuclei observed in Gdf11-/- mice. (L) Quantification of Ngn3+ pancreatic nuclei per mm2 tissue at E15.5 The increase of Ngn3+ pancreatic cells in Gdf1+/– and Gdf11-/- embryos compared with wild-type littermates is significant (asterisks) at P<0.05. Data from three to four embryos per genotype are presented as the average ± standard error of the mean. (M-O) Expression of HNF6 (black nuclei) in Gdf11+/+, Gdf11+/- and Gdf11-/- mice at E17.5. Black arrowheads in N and O mark a subset of ductal cells surrounding lumen that are heavily stained by anti-HES1 antisera. (P) Quantification of HNF6+ nuclei per mm2 tissue at E17.5. Data from three to four embryos per genotype are presented as the average ± standard error of the mean. Asterisks indicate that the difference in numbers of HNF6+ nuclei in Gdf11+/- and Gdf11-/- mice compared with Gdf11+/+ mice is significant at P<0.05.

View larger version (84K): [in a new window]   Fig. 5. Defects of ß-cell maturation in Gdf11-/- mice at E17.5. (A-B) Accumulation of cells expressing NKX6.1 (brown nuclei) in Gdf11-/- mice. (C) Quantification of NKX6.1+ nuclei per mm2 tissue. Data here and in panels F and K are shown as the average measurements ± standard error of the mean (n=3 mice per genotype). Asterisk indicates that the measured difference in NKX6.1+ nuclei in Gdf11-/- compared with Gdf11+/+ mice is significant at P<0.05. (D,E) Accumulation of cells expressing NKX2.2 (brown nuclei) in Gdf11-/- mice. (F) Quantification of NKX2.2+ nuclei per mm2 tissue. The asterisk indicates that the measured difference in NKX2.2+ nuclei in Gdf11-/- compared with Gdf11+/+ mice is significant, at P<0.05. (G,H) NKX6.1 positive cells retain their neuroendocrine identity in Gdf11-/- mice at E17.5. NKX6.1 (red nuclear staining) is co-expressed with synaptophysin (green cytoplasmic staining) in both wild-type and null animals. (I,J) Absence of insulin expression (green) in a subset of NKX6.1+ cells (red nuclear staining) in Gdf11-/- mice at E17.5. (K) Quantification of the percentage of NKX6.1+ cells lacking immunostainable insulin in wild-type and Gdf11-/- mice. The asterisk indicates that the difference in values is significant, at P<0.05. (L,M) Absence of glucagon expression (green) in NKX6.1+ cells (red nuclear staining) in wild-type and Gdf11-/- mice at E17.5. (N,O) Normal numbers of ghrelin+ cells in wild-type and Gdf11-/- mice at E17.5 (red cytoplasmic staining). Panel N and O insets demonstrate cytoplasmic localization of ghrelin.

View larger version (59K): [in a new window]   Fig. 6. Pancreatic defects in E17.5 Smad2 mutant embryos. (A,E) Ngn3 expression (blue) detected by antisense RNA in-situ hybridization. (B,F) NKX2.2 expression (brown nuclei) adjacent to ductal epithelium. (C,G) NKX6.1 expression (brown nuclei marked by arrows). (D,H) Insulin expression (green) detected by IHC and confocal microscopy. Inset, H: NGN3+ nuclei (red) adjacent to insulin+ cells (green). Images are representative of four or more animals per genotype.

View larger version (72K): [in a new window]   Fig. 7. Pancreatic expression of HES1 in Gdf11-deficient mice. (A-C) Expression of HES1 (black nuclei) in Gdf11+/+, Gdf11+/- and Gdf11-/- mice at E17.5. (D) Quantification of HES1+ nuclei per mm2 tissue. The difference in numbers of HES1+ nuclei in Gdf11+/+, Gdf11+/- and Gdf11-/- mice is not significant (NS). (E,F) Gdf11 expression (rust brown) detected by in-situ hybridization in Hes1+/+ and Hes1–/– pancreata at E15.5.