Generation of Tg (ela3I-CRE; LSL-KRAS^G12D) fish. (A) Schematic diagram of Tg (ela3I-CRE) driver lines and Tg (LSL-KRAS^G12D) reporter lines. Open triangles indicate Tol2 arms. (B) Confocal images of the microdissected pancreas (5 dpf) in Tg (ela3I-CRE; LSL-KRAS^G12D). GFP signal was detected mainly in the membrane of the pancreas and partly in the cytoplasm as a surrogate marker of KRAS^G12D activation. In the control group (w/o ela3I-CRE), GFP signal was not detected. Asterisk (*) indicates the auto fluorescence of the gut. Scale bar: 25 µm.

Early KRAS^G12D-responsive pancreatic progenitors contribute to endocrine as well as exocrine cells. (A) The pancreas from 5 dpf double transgenic Tg (ela3I-CRE; LSL-KRAS^G12D) larvae was stained with an exocrine specific marker, CPA. CPA staining was observed in the apical cytoplasm as well-developed apical secretory granules (yellow arrows). The GFP signal was detected in both the membrane and cytoplasm as a surrogate marker of KRAS^G12D activation (white arrows). The merged confocal image showed that the pancreatic progenitor cells expressing oncogenic KRAS^G12D expressed CPA in apical secretory granules of exocrine pancreas (red arrows). (B) The pancreas from 5 dpf double transgenic Tg (ela3I-CRE; LSL-KRAS^G12D) larvae was stained with an endocrine specific marker, insulin. Insulin staining was shown in the cytoplasm of islet β-cells (yellow arrows). The merged confocal image showed that some of pancreatic progenitor cells expressing oncogenic KRAS^G12D co-expressed the endocrine cell marker, insulin (red arrows). Asterisk (*) indicates the auto fluorescence of the gut. Scale bar: 25 µm.

Identification of pancreatic tumors in Tg (ela3I-CRE; LSL-KRAS^G12D) fish. (A and B) Transcutaneous fluorescence in the abdomen of Tg (ela3I-CRE; LSL-KRAS^G12D) fish (A) and dissected abdominal viscera with a GFP-positive tumor in pancreas (B). (C and D) In the control group (w/o ela3I-CRE), no fluorescence was detected in the abdomen (C) and the dissected abdominal viscera (D). The dotted line indicated the pancreatic region. Scale bar: 2 mm. (E) Quantification of GFP-positive pancreatic tumor frequency in Tg (ela3I-CRE; LSL-KRAS^G12D) fish. A random subset of fish was anesthetized periodically at 3 (n = 10), 6 (n = 10), and 12 months (n = 10).

Histological profiles of the abnormal pancreatic region at 6 and 12 months of age. (A–C) Histological examination showed that acinar structure of pancreas (black arrows, zymogen granules in cytoplasm) is disrupted by expansion of populations of poorly differentiated round cells that resemble islet cells (blue arrows). Boxed areas indicate regions depicted at higher magnification in adjacent images. Scale bars: 50 μm. (D and E) GFP and PCNA staining in the abnormal pancreatic region. GFP and PCNA staining was observed in the poorly differentiated cells. Boxed areas indicate regions depicted at higher magnification in adjacent images. Scale bars: 50 μm. (F) In the control group (w/o ela3I-CRE), no GFP and infrequent PCNA staining was observed in the pancreas. Scale bars: 50 μm. (G) Comparison of PCNA positive cells. Highly frequent PCNA staining was observed in the poorly differentiated cells (w/ ela3I-CRE) (28.12%) compared to that in the control group (w/o ela3I-CRE) (7.27%) (**P < .01, Student’s t-test). Means ± S.D., N = 3. Gastrointestinal tract (G.I. tract).

Immunohistochemical profiles of the abnormal pancreatic region at 6 and 12 months of age. (A–C) Histological profiles in Tg (ela3I-CRE; LSL-KRAS^G12D) fish (A and B). Boxed areas indicate regions depicted at higher magnification in adjacent images. In the control group (w/o ela3I-CRE), normal histology was observed (C). Scale bars: 50 μm. (D–F) Enhanced chromogranin A staining was observed in the KRAS^G12D-induced pancreatic tumor (D and E), whereas infrequent chromogranin A staining was observed in the endocrine region of the pancreas in the control group (w/o ela3I-CRE) (F). Scale bars: 50 μm. (G–I) The chromogranin A-positive tissues were also positive for GFP staining (G and H), whereas no GFP staining was observed in the control group (w/o ela3I-CRE). Scale bars: 50 μm. (J–L) Trichrome staining to identify the regions of fibrosis and sclerosis. The blue collagen fibers (black arrows) demonstrated that the invading carcinoma triggered fibrosis/sclerosis (J and K). In the control group (w/o ela3I-CRE), typical fibrotic changes were observed in the pancreas (L). Scale bars: 50 μm. (M–R) KRAS^G12D-induced pancreatic tumors showed widespread labeling for both phospho-AKT (M and N) and phospho-ERK (P and Q), in contrast to infrequent AKT and ERK phosphorylation in controls (w/o ela3I-CRE) (O and R). Scale bar: 50 µm.

Molecular characterization of pancreatic tumors at 6 and 12 months of age. (A) To further classify the origin of pancreatic tumors, microdissection was performed. As a control, the pancreas region from the age-matched adult fish in the control group (w/o ela3I-CRE) was selected (exocrine cells: black arrows, endocrine cells: yellow arrows, ductal cells: red arrows). A representative example before/after microdissection was shown. Scale bar: 50 µm. (B) Quantitative PCR was performed against the mRNA for insulin, chromogranin A, amylase, and krt18 (**P < .01, Student’s t-test). Means ± S.D., N = 3.