Fig. 1

Generation and characterization of Tg(ifabp:EGFP-krasV12) transgenic zebrafish. (A) Schematic diagram showing the DNA pDs-ifabp:LexPR-Lexop:EGFP-krasV12 construct used for the generation of Tg(ifabp:EGFP-krasV12) transgenic fish. Ds, maize Ds transposon sequence. (B) Intestine-specific expression of EGFP-krasV12 in F1 transgenic fry and wild-type fish, and confocal image of whole mounted kras+ transgenic larva at 8 dpf following induction by 1 μM Mifepristone. The images were obtained at z = 5 μm over a thickness of 50 μm. (C) Western blot analysis of total proteins from WT and kras+ transgenic fish in the gut tissue from three founder lineage (I, II, and III) for the detection of the Kras protein. β-Actin, internal control for equal loading. (D) RT-qPCR analysis of kras RNA expression in the kras+ transgenic F2 whole larvae from founders I and III. N = 20 larvae from each founder (at 2 dpi using 1 μM mifepristone) were used for isolation of proteins and mRNA. (E) Fluorescence micrographs of intestine cross sections from wild-type and kras+ zebrafish larvae showing green fluorescence or phalloidin staining. (F) Quantification of villi by ratio of inner to outer circumferences of intestine at 10 dpi by 2 μM mifepristone. Statistical significance: ***P < .001.
Fig. 3

Phenotypic characterization of intestinal tumorigeneis in kras+ transgenic zebrafish. Wild-type and kras+ fish were treated with 2 or 3 μM mifepristone from 2 mpf, and samples were collected at 2, 4, and 6 wpi for gross observations and histological analyses. (A) Body length. (B) Body weight. (C) Survival curves. (D) Examples of normal, enteritis, hyperplasia, and tubular adenoma from H&E staining sections of intestine. Wild-type or kras+ fish sources of the sections are indicated. (E) Summary of intestinal histological abnormalities observed in wild-type and kras+ transgenic fish. The data were generated as a result of a blinded histological analysis. Numbers of fish in each group are indicated at the top of each bar. The differences among the variables were assessed using Student's t tests or one-way ANOVA. Statistical significance: *P < .05, **P < .01, ***P < .001.
Fig. 4

Immunocytochemical staining of PCNA, p-ErK, p-Akt, and E-cadherin in sections of normal intestine from wild-type fish and tubular adenoma from kras+ transgenic zebrafish. (A and B) Immunostaining of PCNA (A) and quantification of percentages of positive cells (B). (C and D) Immunostaining of p-Erk (C) and quantification of percentages of positive cells (D). (E and F) Immunostaining of p-Art (E) and quantification of percentages of positive cells (F). (G and H) Immunostaining of E-cadherin (G) and quantification of percentages of positive cells (H). Numbers of samples from each group are indicated in the quantification histograms. Statistical significance: ***P < .001.
Fig. 5

Synergistic effect of krasV12 expression and DSS on intestinal tumorigenesis. Four-mpf wild-type and kras+ fish were co-treated with 2 μM mifepristone and 0.0625% DSS for 3 weeks, and samples were collected for gross observations and histological analyses. There were four groups in the experiment: wild type, wild type/DSS, kras+,  and kras+/DSS. (A) Body length. (B) Body weight. (C) Survival curves. (D) Examples of normal, enteritis, hyperplasia, and tubular adenoma from H&E staining sections of intestine. Sources of the sections are indicated. (E) Summary of intestinal histological abnormalities observed in the four experimental groups. The data were generated as a result of a blinded histological analysis (wild type, n = 20; wild type/DSS, n = 20; kras+, n = 20; kras+ with DSS, n = 16). The differences among the variables were assessed using Student's t tests or one-way ANOVA. Statistical significance: *P < .05, **P < .01, and ***P < .001.
Fig. S1

Dosage-dependent induction of EGFP expression in kras+ transgenic larvae.

F2 kras+ transgenic larvae were treated using 0.005, 0.05, 0.5, 2, or 4 μM of mifepristone from 3 dpf to 8 dpf. (A) EGFP expression in the intestine was recorded using a digital camera attached to a microscope. All of the photos were obtained at the same magnification and exposure. (B) Quantification of EGFP fluorescence. n=20/group. Statistical significance: *p<0.05.

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