DEV02875 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Pancreas differentiation in Apc^P−/− mice is normal. (A) Immunofluorescence for amylase (two upper panels; note the nuclear staining for β-catenin in Apc^P−/−), and for PDX1, insulin, glucagon, somatostatin and pancreatic polypeptide on pancreatic islets of Langerhans. Anti-β-catenin and anti-amylase antibodies were used in combination (upper panels; dashed lines outline two acini). Scale bars are 20 μm. (B) Morphometrical analyses and immunoassays. The relative β-cell area and pancreatic insulin and glucagon concentrations in Apc^P−/− pancreas are significantly decreased. Because the total pancreatic mass is increased, the total β-cell mass is unchanged when compared with controls. Pancreatic insulin: control 44.9±8.29 μg, Apc^P−/− 34.2±7.74 μg; pancreatic glucagon: control 0.96±0.113 μg, Apc^P−/− 0.69±0.270 μg. n=4, P<0.05 (*) or P<0.01 (**).

• Supplemental Figure S2 -

  Fig. S2. Exocrine function in Apc^P−/− mice. Amylase activity was determined in pancreata (A,B) and plasma (C) of 2-month-old control and Apc^P−/− mice. Whereas the total pancreatic amylase content was unchanged in Apc^P−/− animals as compared to controls, their amylasemia was four- to five-times higher, corresponding to the difference in pancreatic size. n=4 mice per group.

• Supplemental Figure S3 -

  Fig. S3. Endocrine function in Apc^P−/− mice. (A,B) Basal (fasting) plasma insulin (A) and glucose (B) concentrations; n=4 mice per group. (C,D) Intraperitoneal glucose tolerance tests (GTT) in 2- and 12-month-old mice; young adults have higher peaks of glycemia after the glucose challenge, but recovery is optimal. n=7 mice per group. Dashed line, control; plain line, Apc^P−/−. (E) Plasma insulin (ELISA) after i.p. injection of glucose, reveals no change in insulin secretion between Apc^P−/− and control pancreata. n=4 mice per group.

• Supplemental Figure S4 -

  Fig. S4. Islet-specific inactivation of Apc. (A) H/E staining showing normal morphology of Apc^loxP/loxP;Pax6-Cre^+/− (Apc^i−/−) pancreata. β-catenin accumulates in the cytoplasm of Apc^i−/− islet cells. (B) Expression profile (real time PCR) of selected genes in Apc^i−/− isolated islets of 1-month-old animals. Expression levels are relative to transcripts in control islets, calculated using three different housekeeping genes (see Materials and methods), and normalized to 1 (100%). RNA was extracted from 130 islets per group, from at least two different individuals, and real-time RT-PCR reactions were performed in triplicate. Dashed lines outline islets. Confocal pictures are shown for anti-pan β-catenin immunofluorescence. i, islet. Scale bars are 20 μm

• Supplemental Figure S5 -

  Fig. S5. Expression of ICAT, a β-catenin inhibitor, is maintained in Apc^P−/− acinar cells. At 12 months of age, ICAT levels are strongly downregulated in acinar cells of control mice (left column), whereas high levels persist in acinar cells of Apc^P−/− pancreata (right column). ICAT expression remains high at all times in islet cells of both conditions. Note that some Apc^P−/− acinar cells are hypertrophic and have dysplastic nuclei at this stage. DAPI fields correspond to the respective upper panels, to show all nuclei. Dashed lines outline islets. i, islet. Scale bars: 20 μm (same magnification in all panels).

• Supplemental Figure S6 -

  Fig. S6. β-catenin signaling is refrained in pancreas and defines a window of competence. (A) The pancreas develops normally following Apc deletion. After birth and during 5-6 months, acinar cells proliferate more rapidly than in controls, so that the whole pancreas is oversized by 3 weeks of age. This enlargement remains stable throughout life, and no tumors develop. (B) Developing pancreatic primordia and islets lack the ability to accumulate unphosphorylated β-catenin in the nuclei of their cells. As a result, only the acinar pancreas is phenotypically affected by the mutation, after birth, whereas the endocrine pancreas remains normal. Sustained, for life, accumulation of nuclear β-catenin in acinar cells is not tumorigenic, probably because high levels of β-catenin target genes cannot be maintained after 5-6 months. This second brake thus defines the posterior limit of a β-catenin competence window, during which c-myc expression is upregulated.