Figure 1.
The inducible Pdx1 mouse model.
(A) The construct of the Tet-off regulation unit for Pdx1 expression that was integrated into the ROSA26 locus. The tetracycline transactivator gene is expressed under the control of the ROSA26 promoter. In knock-in mice (RTF-Pdx1-EGFP mice) heterozygous for the transgene, the continuous administration of Dox prevents the tTA from binding to the tetO, thereby inactivating the transcription of Pdx1 cDNA. After Dox is withdrawn, tTA binds to the tetO, transcribing the Pdx1 cDNA and EGFP cDNA. (B)–(M) Immunofluorescence analysis of Pdx1 (red) and insulin (green) (B–G) or Pdx1 (red) and cytokeratin 19 (CK) (green) (H–M) in pancreas sections from RTF-Pdx1-EGFP mice treated with Dox or untreated for 17 days. Bars = 100 µm. (N)–(O) Magnified view of the area surrounded by the dotted line in (M). Staining with anti-CK antibody (green) and DAPI (4, 6-diamidino-2-phenylindole) (blue) is shown in (O). “d” and “i” represent a duct and an islet, respectively.
Figure 2.
Quantification of histological changes in the AdV-infected mouse pancreas.
(A)–(D) Sections of pancreas excised 1.5 days after injection were stained with an anti-Isl1 antibody (green), anti-amylase antibody (red), and anti-insulin antibody (blue). Bar = 300 µm. Magnified view of the area surrounded by the dotted line in (A) is shown in (B)–(D). Staining with DAPI (blue) is included in (C) and (D). Bar = 50 µm. (E) Pancreatic section stained with an anti-insulin antibody (brown) and counterstained with hematoxylin. The area surrounded by the dotted black line shows TCs. Bar = 100 µm. (F)–(H) Quantification of histological changes. Mice were divided into eight groups: Group 1, RTF-Pdx1-EGFP mice mock-injected after Dox withdrawal (n = 5); group 2, wild-type mice given AdV-CAT (n = 5); group 3, RTF-Pdx1-EGFP mice given AdV-CAT after Dox withdrawal (n = 4); group 4, RTF-Pdx1-EGFP mice given AdV-Ptf1a after Dox withdrawal (n = 3); group 5, RTF-Pdx1-EGFP mice given AdV-Neurod1 after Dox withdrawal (n = 3); group 6, wild-type mice given AdV-Isl1 (n = 5); group 7, RTF-Pdx1-EGFP mice given AdV-Isl1 in the presence of Dox (n = 5); group 8, RTF-Pdx1-EGFP mice given AdV-Isl1 after Dox withdrawal (n = 6). Six days after AdV-Isl1 injection, the pancreata were removed for immunohistochemical analysis. Three nonconsecutive sections from each pancreas were stained with an anti-insulin antibody followed by hematoxylin counterstaining, and examined by light microscopy. Each section was digitally scanned and the image files were analyzed by NIH image to calculate the percentage of the area that was occupied by TCs, as described in Materials and Methods. The islets and insulin-positive cell clusters consisting of 1–4 cells in the TC area on each section were counted. Percentage of TC area/total section area is shown in (F). The numbers of islets and insulin-positive cell clusters/TC area (mm2) are shown in (G) and (H), respectively. Values are means ± SE (*P<0.05 and **P<0.01 by Student’s t-test).
Figure 3.
Immunofluorescence analysis of transdifferentiation in the AdV-Isl1-infected RTF-Pdx1-EGFP mouse pancreas.
(A)–(D) Pancreas was excised 3 days after injection. Pancreatic sections were stained with anti-Ki67 (A, green) and anti-Isl1 (B, red). Merged view of (A) and (B) is shown in (C). Staining with anti-Isl1 (red) and DAPI (light blue) is shown in (D). Arrows indicate doubly stained cells. Note that most of the Ki67-positive cells were Isl1-positive. Bar = 100 µm. (E)–(G) Sections from AdV-Isl1-infected mouse pancreas, excised 3 days after injection, were stained with anti-CK (E, green) and anti-amylase (F, red). Merged view of (E) and (F) is shown in (G) with DAPI (blue). Arrows indicate CK and amylase double-positive cells. Bars = 50 µm. (H)–(J) Control pancreatic sections from wild-type mice not given an AdV injection were stained with anti-CK (H, green) and anti-amylase (I, red). Merged view of (H) and (I) is shown in (J) with DAPI (blue). Note that the duct was clearly stained with anti-CK but not with anti-amylase. Bars = 50 µm. (K)–(P) Pancreatic sections from AdV-Isl1-infected mice were stained with anti-CK (green), anti-Isl1 (magenta), and anti-amylase (red) with DAPI (blue). Merged image of (K) and (L) is shown in (M). Merged image of (N) and (O) is shown in (P). Arrows indicate CK/Isl1/amylase triple positive cells. Bar = 50 µm. (Q)–(S) Immunofluorescence analysis of the pancreas from mice given AdV-Isl1. Pancreas was excised 6 days after injection. Pancreatic sections were stained with anti-CK (green) and anti-Isl1 (magenta) with DAPI (blue) (Q) or with anti-amylase (red) and anti-Isl1 (magenta) (R). Merged view of (Q) and (R) is shown in (S). Note that most of the Isl1-positive cells in TCs were CK-positive. Bar = 50 µm.
Figure 4.
Immunofluorescence analysis of the pancreas of RTF-Pdx1-EGFP mice given AdV-Isl1.
(A)–(C) Pancreas was excised 1.5 days after AdV injection, and sections were stained with anti-CK (red), anti-phospho-STAT3 (light blue), and anti-Isl1 (green). Merged view of (A) and (B) is shown in (C) with DAPI (blue). Arrows indicate cells stained with both anti-Isl1 and anti-phospho-STAT3 antibodies. Bar = 100 µm. (D)–(F) Expression of MafA in the TC area. Sections from AdV-Isl1-injected RTF-Pdx1-EGFP mouse pancreas, excised 6 days after the injection, were stained with anti-insulin (red), anti-MafA (light blue), and anti-CK (green). Merged view of (D) and DAPI staining (blue) is shown in (E). Another part of the TC area is shown in (F) with DAPI staining. Heterogeneous staining for MafA was seen in the insulin-positive cells in the TC area. Arrows indicate insulin-positive, but MafA-negative cells. (G)–(J) Expression of MafA in the non-TC area of the same pancreatic section as (D–F). Merged view of (G) and DAPI staining is shown in (H). Merged view of (I) and DAPI staining is shown in (J). Almost all the insulin-positive islet cells were positive for MafA. Bars = 50 µm.