Heat shock-inducible Cre/Lox approaches to induce diverse types of tumors and hyperplasia in transgenic zebrafish

Abstract

RAS family members are among the most frequently mutated oncogenes in human cancers. Given the utility of zebrafish in both chemical and genetic screens, developing RAS-induced cancer models will make large-scale screens possible to understand further the molecular mechanisms underlying malignancy. We developed a heat shock-inducible Cre/Lox-mediated transgenic approach in which activated human kRASG12D can be conditionally induced within transgenic animals by heat shock treatment. Specifically, double transgenic fish Tg(B-actin-LoxP-EGFP-LoxP-kRASG12D; hsp70-Cre) developed four types of tumors and hyperplasia after heat shock of whole zebrafish embryos, including rhabdomyosarcoma, myeloproliferative disorder, intestinal hyperplasia, and malignant peripheral nerve sheath tumor. Using ex vivo heat shock and transplantation of whole kidney marrow cells from double transgenic animals, we were able to generate specifically kRASG12D-induced myeloproliferative disorder in recipient fish. This heat shock-inducible recombination approach allowed for the generation of multiple types of RAS-induced tumors and hyperplasia without characterizing tissue-specific promoters. Moreover, these tumors and hyperplasia closely resemble human diseases at both the morphologic and molecular levels.

Fig. 1.

Human kRASG12D transgene expression can be induced by Cre-mediated recombination in stable transgenic B-actin-LoxP-EGFP-LoxP-kRASG12D zebrafish. (A) Diagram of B-actin-LoxP-EGFP-LoxP-kRASG12D transgene. (B–E) Stable transgenic animals (line 25A) at 24 hpf (B and C), and 44 dpf (D and E). (B and D) Bright-field. (C and E) EGFP fluorescence. (F and G) Whole-mount in situ hybridization for kRASG12D performed on 24 hpf double transgenic embryos (LGL-RAS; hsp70-Cre) with heat shock (F) or without (G). Arrowheads denote kRASG12D transcript-expressing cells. (H and I) Whole-mount immunostaining with anti-phospho-ERK1/2 antibody performed on 24 hpf double transgenic embryos with heat shock (H) or without (I). Cells with ERK1/2 activation are denoted by arrows. (J) Percentage of recombination at the genomic DNA in single embryos heat shocked from 4 to 5 hpf and analyzed at 8, 16, and 24 hpf. (K) Number of kRASG12D-expressing cells in single embryos heat shocked from 4 to 5 hpf and analyzed at 8, 12, 16, 20, and 24 hpf. Statistic significance (P < 0.001) is indicated by asterisks. +HS, heat shock; NoHS, non-heat shocked.

Fig. 2.

Early induction of kRASG12D expression leads to juvenile lethality and four types of tumors and hyperplasia in adult transgenic fish. (A) Kaplan–Meier survival curves. Double transgenic embryos were heat shocked at 24 hpf (LGL-RAS; hsp70-Cre +HS) or were not heat shocked (LGL-RAS; hsp70-Cre NoHS). Single transgenic animals were treated with heat shock (hsp70-Cre + HS or LGL-RAS + HS). (B) Tumor spectrum in diseased fish surviving past 25 days of life (n = 25 of 180 in the heat-shocked group and n = 19 of 120 in the non-heat-shocked group). Four types of lesions were observed: RMS, intestinal hyperplasia, MPD, and MPNST.

Fig. 3.

RMS is the most common tumor type in the heat-shocked group. (A) Normal zebrafish side view. (B) Double transgenic fish (46 dpf) with externally visible tumor mass at the tail region. (C and G) Hematoxylin/eosin (H&E)-stained sections of normal muscle (C) and RMS (G). (D–F) RNA in situ hybridization of normal muscle. (H–J) RNA in situ hybridization of RMS muscle. (C–J) Antisense probes are designated in the lower left corners. [Scale bars: 1 cm (A and B) and 50 μm (C–J).]

Fig. 4.

MPD is common in the non-heat-shocked group. (A) Normal zebrafish side view. (B) Double transgenic adult fish (53 dpf) with MPD. (C and G) Hematoxylin/eosin (H&E)-stained sections of normal (C) and MPD kidney (G). (D–F) RNA in situ hybridization of normal kidneys. (H–J) RNA in situ hybridization of MPD kidneys. Antisense probes are designated within each panel. (E) Arrows denote l-plastin-expressing monocytes in normal kidney. (F) Arrowheads denote mpo-expressing granulocytes in normal kidney. (K and M) FACS analysis of whole kidney marrow cells from normal (K) and MPD fish (M). Erythrocytes are shown in red, lymphocytes in blue, granulocytes and monocytes in green, and blood cell precursors in purple. FSC, Forward scatter; SSC, side scatter. (L and N) May–Grunwald–Giemsa-stained cytospins of normal (L) and MPD (N) kidney marrow cells. [Scale bars: 1 cm (A and B), 25 μm (C–J), and 12.5 μm (L and N).]

Fig. 5.

MPD can be induced in transplant animals by ex vivo heat shock and cell transplantation strategy. (A) Procedure diagram. No HS donor, non-heat-shocked double transgenic donors. (B) FACS analysis of the donor cells showing that before ex vivo heat shock, the donor fish had normal blood cell numbers within the kidney marrow. (C) FACS analysis showing that primary transplant animals developed MPD 2 months after transplantation. (D and E) May–Grunwald–Giemsa-stained cytospins of the donor cells (D) and primary transplant (E). (Scale bars: 12.5 μm.)