Article Figures & Data
Figures
- Figure S1. Fzd9 deficiency does not produce phenotypic abnormalities in pancreatic islets of adult mice.
Pancreatic islets from 12-wk-old Fzd9WT/WT and Fzd9KO/KO stained using H&E (4×, scale bar: 200 μm).
- Figure 1. The absence of Fzd9 impairs the development of Myc-driven pancreatic insulinomas.
(A, B) H&E staining (4×) of pancreas sections from Fzd9-proficient and (B) Fzd9-deficient mice treated with tamoxifen for 3 wk. (C) Quantification of the islet size from Fzd9KO/KO;BclXL, Fzd9WT/WT;MycER;BclXL and Fzd9KO/KO;MycER;BclXL mice. (D) Presence of MycER detected by immunofluorescence against ER (red) in these 3-wk treated islets. Yellow dotted lines define the periphery of the pancreatic islets. Representative images for each of the groups are shown (10×). Data information: in (C), data are represented as mean ± SD. * and ** indicate P-values below 0.05 and 0.01, respectively (Kruskal–Wallis followed by Dunn’s test). (A, D) Scale bars: 200 μm in (A), 100 μm in (D).
- Figure 2. Sustained proliferation of pancreatic islets upon MycER activation requires Fzd9.
(A, B) Incorporated BrdU (green) detected by immunofluorescence (10×) after 3 d of tamoxifen treatment and (B) its quantification. Percentages of BrdU-positive cells per islet from at least three mice per group are represented. (C) Representative H&E staining of pancreas sections from mice treated with tamoxifen for 3 d. Yellow dotted lines define the periphery of the pancreatic islets (4×). (D, E) Incorporated BrdU detected by immunofluorescence (10×) after 3 wk of tamoxifen treatment and (E) its quantification. Percentages of BrdU-positive cells per islet from at least three mice per group are represented. (F) Quantification of the proliferation marker Ki67 in islets from mice treated for 1 and 3 wk. Percentages of Ki67-positive cells per islet from three mice per group are represented. Data information: in (B, E, F), data are represented as mean ± SD. (B, E, F) Statistical significance of differences was examined using one-way, in (B, E), or two-way (in F) ANOVA followed by Tukey’s test. ** and *** indicate P-values below 0.01 and 0.001, respectively. (A, C, D) Scale bars: 100 μm in (A, D), 200 μm in (C).
- Figure S2. Fzd9 deficiency does not impair either the recruitment of mast cells or the induction of angiogenesis.
(A) Toluidine blue staining of metachromatic mast cells (purple) in the mesenchyme surrounding the pancreas of mice injected with tamoxifen for 3 d (20×, scale bar: 50 μm). (B) Double immunofluorescence of endothelial cells (stained with anti-Meca32; green) and proliferating cells (stained with anti-Ki67; red) (20×, scale bar: 50 μm). Yellow boxes (i–iv) show amplified regions.
- Figure 3. Fzd9 mediates Myc-induced dedifferentiation of insulin-expressing β-cells.
(A, B, C) Insulin (green) and glucagon (red) detected by immunofluorescence (10×) in pancreatic tissue sections of Fzd9KO/KO;BclXL, (B) Fzd9WT/WT;MycER;BclXL, and (C) Fzd9KO/KO;MycER;BclXL treated with tamoxifen for 3 wk. (A, B, C, D) Quantification of total insulin intensity in the islets from (A, B, C). (E) Quantification of glucagon positive cells in the periphery of the islets. Percentages of glucagon positive cells are shown. Data information: in (D, E), data are represented as mean ± SD. (D, E) Statistical significance of differences was examined using Kruskal–Wallis followed by Dunn’s test (D) and one-way ANOVA followed by Tukey’s test (E). * and ** indicate P-values below 0.05 and 0.01, respectively. Scale bar: 100 μm.
- Figure S3. Fzd9 absence produces pockets of differentiated β-cells in pancreatic islets upon MycER activation.
H&E staining of pancreas sections from mice treated with tamoxifen for 2 wk. Yellow dotted lines define the periphery of the pancreatic islets. Arrows indicate pockets of fully differentiated cells, with a similar appearance to control cells in the left panel (20×, scale bar: 50 μm).
- Figure 4. The absence of Fzd9 alters the expression of Myc-related, differentiation and Wnt signaling Gene Sets upon MycER activation.
(A) Heat map of differentially expressed genes determined by microarray analysis performed on a pool of multiple isolated islets (∼20–40) from each of four Fzd9WT/WT;MycER;BclXL mice and four Fzd9KO/KO;MycER;BclXL mice. (B, C) Gene sets related to pancreatic differentiation and (C) Myc targets. (D) qRT-PCR analysis of genes related to Gene Sets shown in (B) (GPC1) and in (C) (POLD2, SRSF7, and RPL34). (E) Immunofluorescence stainings (20×) and quantification of MYC targets MCM5 and PCNA. Total MCM5 intensity and PCNA positive nuclei per islet are shown. (F) Gene sets related to Wnt signaling pathway identified in the Gene Set Enrichment Analysis as differentially expressed when comparing expression profiles. (G) β-catenin detected by immunofluorescence (10×) in pancreatic tissue sections treated with tamoxifen for 3 d and its quantification in the islets. Total β-catenin intensity per islet area is shown. (H) Percentage of Ki67-positive cells in individual islets from Fzd9WT/WT;MycER;BclXL and Fzd9KO/KO;MycER;BclXL after 1 wk of tamoxifen treatment. An additional group of the Fzd9WT/WT;MycER;BclXL mice was pretreated for 2 d with the Wnt inhibitor C59 and then received both C59 + tamoxifen during 1 wk. Data information: in (D, G, H), data are represented as mean ± SD, whereas in (E), as median and interquartile range. (D, E, G) Statistical significance of differences was examined using t test (D), Mann-Whitney U test (E) and Tukey’s test (G). *, ** and *** indicate P-values below 0.05, 0.01 and 0.0001, respectively. (E, G) Scale bars: 50 μm in (E), 100 μm in (G). FDR, false discovery rate; NES, normalized enrichment score.
- Figure S4. Cell cycle, metabolism and apoptosis gene sets appear differentially expressed in the absence of Fzd9.
(A, B, C) Gene sets differentially expressed when MycER is activated in Fzd9WT/WT;MycER;BclXL versus Fzd9KO/KO;MycER;BclXL related to Myc-related programs: (A) cell cycle, (B) metabolism, and (C) cell death. (D) Immunofluorescence staining and quantification of β-Actin in pancreatic islets from Fzd9 wt and knockout mice (related to Fig 4F) (Mann-Whitney U test). (E) qRT-PCR analysis of genes related to WNT-β-catenin pathways (related to Fig 4F).
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