Stabilization of {beta}-catenin impacts pancreas growth

doi:10.1242/dev.02366

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First published online 12 April 2006
doi: 10.1242/dev.02366

Development 133, 2023-2032 (2006)
Published by The Company of Biologists 2006

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Stabilization of ß-catenin impacts pancreas growth

Patrick W. Heiser¹, Janet Lau¹, Makoto M. Taketo², Pedro L. Herrera³ and Matthias Hebrok¹^,*

¹ Diabetes Center, Department of Medicine, University of California, San Francisco, CA 94143, USA.
² Department of Pharmacology, Graduate School of Medicine, Kyoto University, Kyoto, 606-8501, Japan.
³ Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva CH-1211, Switzerland.

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Fig. 1. Temporal and spatial differences in Cre recombinase activity in the PdxCre^early and PdxCre^late transgenic lines. Staining for lacZ expression marks cells that have undergone Cre-mediated recombination in PdxCre^late and PdxCre^early R26R mice. (A) Control E10.5 embryo. (B) No ß-galactosidase staining is detectable within the PdxCre^late R26R embryo at E10.5. (C) Strong ß-galacotosidase staining is present within the pancreas (arrow) of a PdxCre^early R26R E10.5 embryo. (D-F) Histological examination of pancreas sections immunostained for Pdx1 (brown) and enzymatically stained for ß-galactosidase activity (blue). (F) The majority of Pdx1⁺ cells in the PdxCre^early R26R animal, are also positive for lacZ at E10.5. (G-I) ß-Galactosidase (blue) and Pdx1 (brown) staining at E12.5. ß-Galactosidase staining is detectable within a subset of the Pdx1⁺ cells in the PdxCre^late R26R animals (H). (I) Equivalent pancreas sections of a PdxCre^early R26R animal show robust ß-galactosidase staining in the majority of Pdx1⁺ cells. (J-L) Adult animals stained with Eosin (red) and for ß-galactosidase (blue). Islets are outlined in black. (K) In the adult PdxCre^late R26R animal, only a subset of islet cells exhibits ß-galactosidase staining. Cre expression within the exocrine tissue is also mosaic in these animals. (L) A greater percentage of islet cells and exocrine cells exhibit strong ß-galactosidase staining in the adult PdxCre^early R26R pancreas. (M-O) Adult pancreatic ducts. (O)The majority of pancreatic ducts (stained with an antibody against mucin 1, red) are also ß-galactosidase ⁺ (green) in the PdxCre^early R26R pancreas (non-specific staining sometimes encountered within the center of ducts indicated with an asterisk). (N) ß-Galactosidase⁺ cells are seldom encountered within the PdxCre^late R26R pancreas. No ß-galactosidase staining is observed in control animals (A,D,G,J,M) at any of the time points characterized. Scale bars: 25 µm.

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Fig. 2. Stabilization of ß-catenin results in disruption of pancreas formation in PdxCre^early ß-cat^active mice. (A-C) Examination of pancreas gross morphology. The majority of the dorsal pancreas (dp) and ventral pancreas (vp) has been lost in PdxCre^early ß-cat^active mice (C) at E18.5. By contrast, PdxCre^late ß-cat^active pancreas morphology (B) appears equivalent to control (A) at E18.5. s, stomach; sp, spleen; d, duodenum. (D-F) Hematoxylin and Eosin staining was performed on paraffin sections from E18.5 tissue from either control (D), PdxCre^late ß-cat^active (E) or PdxCre^early ß-cat^active (F) animals. Whereas PdxCre^late ß-cat^active pancreas structure (E) is normal, the PdxCre^early ß-cat^active pancreatic remnant (F) contains very little exocrine tissue and exhibits multiple pronounced cyst structures (c). The majority of the remaining tissue appears mesenchymal in PdxCre^early ß-cat^active mutants. (G) Staining of pancreas sections for ß-catenin (green) and the nuclear co-stain DAPI (blue) reveals that ß-catenin is localized exclusively to the membrane in control animals. (H,I) Significant levels of nuclear ß-catenin can be detected in both the PdxCre^late ß-cat^active and PdxCre^early ß-cat^active mutants. Insets show increased magnification of equivalent fields in each image. Scale bars: 50 µm. (J-L) Staining for the epithelial marker E-cadherin (green) indicates the dramatic loss of epithelial tissue in the PdxCre^early ß-cat^active mutant (L) when compared with both the PdxCre^late ß-cat^active (K) and control (J). The PdxCre^early ß-cat^active cysts are lined by E-cadherin-positive cells. Insets show increased magnification of equivalent fields in each image to show detailed plasma membrane localization of E-cadherin in both PdxCre^late ß-cat^active and PdxCre^early ß-cat^active mutants. Scale bars: 100 µm. (M-O) Staining for glucagon (green), insulin (red) and the nuclear marker DAPI (blue), reveals islet structure in the PdxCre^late ß-cat^active mutant (N) that is equivalent to control (M). By contrast, insulin⁺ and glucagon ⁺ cells are scattered within the walls of the cysts and surrounding mesenchyme in the PdxCre^early ß-cat^active mutant tissue (O). Scale bars: 100 µm.

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Fig. 3. Stabilization of ß-catenin at E11.5, but not E13.5, results in pancreas hypoplasia. All images are from E18.5 embryos. (A-D) Whole-mount views of control (A) and PdxCre^ER ß-cat^active mutant (B-D) tamoxifen-injected embryos. (B) After injection at E11.5, mutants display a dramatic loss of pancreas mass. (C) When injected at E12.5, the dorsal and ventral pancreas appear moderately reduced in size in the majority of cases, but after E13.5 injection (D), gross pancreatic morphology appears normal. (E-H) Hematoxylin and Eosin-stained control (E) and mutant (F-H) pancreata after tamoxifen treatment. Mutants display the formation of large cysts (F, cysts indicated with black arrows) after injection at E11.5, but appear normal when injected at E12.5 (G) and E13.5 (H). (I-L) Pancreatic epithelia in control (I) and PdxCre^ER ß-cat^active mutant (J-L) tamoxifen-injected embryos. (J) Tamoxifen injection at E11.5 effectively targets a large number of cells within the PdxCre^ER ß-cat^active pancreatic epithelium, indicated by the accumulation of nuclear ß-catenin (compare with I). (K) After injection at E12.5, ß-catenin can be detected within the nuclei of a significant number of cells within the mutant pancreatic epithelium. (L) A large number of cells are present that exhibit nuclear ß-catenin within the pancreatic epithelium after injection at E13.5 (compare with morphology and histology in D and H). (M) The number of mutants seen at each tamoxifen injection time point, and the severity of the pancreas phenotype observed. Scale bars: 50 µm. s, stomach; sp, spleen; d, duodenum; dp, dorsal pancreas; vp, ventral pancreas.

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Fig. 4. Pancreatic defects in PdxCre^early ß-cat^active correlate with changes in FGF and hedgehog signaling, and loss of Pdx1⁺ progenitor cells. (A-C) Whole-mount in situ hybridization. Fgf10 expression is downregulated in the dorsal pancreatic (dp) and ventral pancreatic (vp) mesenchyme of the PdxCre^early ß-cat^active mutants (C) at E10.5. Fgf10 expression within the pancreatic mesenchyme of PdxCre^late ß-cat^active mutants (B) is equivalent to control (A). s, stomach. (D-F) Immunohistochemical staining. Pancreatic sections at E12.5 stained for the hedgehog (Hh) receptor Ptch1 reveal increased levels of Ptch1 within the pancreatic epithelium (circled in blue) of the PdxCre^early ß-cat^active mutants (F) when compared with control pancreas (D). Ptch1 staining in PdxCre^late ß-cat^active (E) pancreatic sections is equivalent to control (D). (G-I) Pancreatic sections at E12.5 stained for Hh. The PdxCre^early ß-cat^active pancreatic epithelium (circled in blue, I) also exhibits increased levels of Hh when compared with control (G) and PdxCre^late ß-cat^active pancreas tissue (H). (J-L) Staining of E12.5 pancreata with E-cadherin (green) to mark the pancreatic epithelium and Pdx1 (red) reveals a dramatic loss of Pdx1+ progenitor cells in the PdxCre^early ß-cat^active mutants (L). By contrast, the PdxCre^late ß-cat^active (K) display Pdx1⁺ cell numbers that are equivalent to control (J). Scale bars: 50 µm in J-L.

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Fig. 5. Stabilization of ß-catenin causes increased pancreas organ size in PdxCre^late ß-cat^active mice. (A) Gross morphology of pancreata from a control (left) and PdxCre^late ß-cat^active mutant (right). (B) Quantitative measurements revealed a 4.6-fold increase in pancreas mass in PdxCre^late ß-cat^active mutants between birth and 1 year (n $>=$ 7 for each time point analyzed; control, blue; PdxCre^late ß-cat^active, orange). (C,D) High levels of ß-catenin (green) can be detected in the DAPI stained (blue) nuclei of adult PdxCre^late ß-cat^active exocrine cells (D), identified by amylase staining (red). By contrast, ß-catenin is localized exclusively to the plasma membrane of control cells (C). Scale bars: 10 µm. (E,F) Staining of pancreatic sections for the proliferation marker, phospho-histone H3 (PH3, red) and co-stained for DAPI (blue) reveals an increase in the number of proliferating cells in the PdxCre^late ß-cat^active mutant (F) when compared with control (E). Scale bars: 100 µm. (G,H) Hematoxylin and Eosin staining of adult pancreas sections from PdxCre^late ß-cat^active mice (H) reveals an increase in cell density within the exocrine pancreas, but not the endocrine islet (at center) when compared with control (G). (I) Quantification of PH3 positive cells (n=4 per genotype analyzed) indicates that the relative number of proliferating cells in the PdxCre^late ß-cat^active mutant (orange) is increased 2.5-fold over control (blue) 21 days postnatally. (J) Quantification of the relative number of nuclei per field confirms that the density of cells within the exocrine pancreas of PdxCre^late ß-cat^active mice (orange) is increased 1.8-fold over control (blue), while the endocrine cell density is normal (n=4). Confidence intervals calculated using Student's t-test. #, not significant; ^*, P<0.05; ^**, P<0.01. Error bars represent s.e.m.

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Fig. 6. Pancreas insulin content and islet function appear normal in the PdxCre^late ß-cat^active mouse. (A,B) The islet architecture of adult PdxCre^late ß-cat^active mutant mice (B) is comparable with control (A) as revealed by glucagon (green) and insulin (red) staining of pancreas sections. DAPI stained nuclei are indicated in blue. Scale bars: 50 µm. (C-H) The majority of ß-cells, identified by insulin staining (red), in the adult PdxCre^late ß-cat^active mutant (D; at higher magnification in G) display localization of ß-catenin (green) to the plasma membrane that is equivalent to control (C; at higher magnification in F). However, a subset of ß-cells in the PdxCre^late ß-cat^active mutants (E; at higher magnification in H) exhibit high levels of cytoplasmic ß-catenin staining. Scale bars: 100 µm in C-E; 15 µm in F-H. (I,J) The ß-cells that exhibit strong cytoplasmic ß-catenin localization (green) in the PdxCre^late ß-cat^active mutant (J) still stain positive for the adult ß-cell transcription factor Pdx1 (red), a characteristic of properly differentiated ß-cells. ß-Cells present in equivalent adult pancreas sections from control animals are shown stained for Pdx1 (I). (K) Resolution of a fasting glucose challenge is equivalent in control (blue) and PdxCre^late ß-cat^active (orange) animals (n=6 for each genotype, error bars represent s.e.m.).

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Fig. 7. Abnormal cells with nuclear localization of ß-catenin are present in a subset of islets in 1-year-old PdxCre^late ß-cat^active mice. (A-H) Nuclear localization of ß-catenin in control (A,C,E,G) and mutant (B,D,F,H). Cells within a small subset of islets in 1-year-old PdxCre^late ß-cat^active mutant pancreata (B,D,F,H) exhibit nuclear localization of ß-catenin. These cells do not express insulin or Pdx1 (B,F; at higher magnification in D,H) seen in control islets (A,E; at higher magnification C,G). Scale bars: 50 µm in A,B,E,F; 25 µm in C,D,G,H.

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