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tp53 deficiency causes a wide tumor spectrum and increases embryonal rhabdomyosarcoma metastasis in zebrafish

  1. Myron S Ignatius
  2. Madeline N Hayes
  3. Finola E Moore
  4. Qin Tang
  5. Sara P Garcia
  6. Patrick R Blackburn
  7. Kunal Baxi
  8. Long Wang
  9. Alexander Jin
  10. Ashwin Ramakrishnan
  11. Sophia Reeder
  12. Yidong Chen
  13. Gunnlaugur Petur Nielsen
  14. Eleanor Y Chen
  15. Robert P Hasserjian
  16. Franck Tirode
  17. Stephen C Ekker
  18. David M Langenau  Is a corresponding author
  1. Massachusetts General Hospital Research Institute, Massachusetts
  2. Massachusetts General Hospital Cancer Center, Massachusetts
  3. Harvard Stem Cell Institute, Massachusetts
  4. Greehey Children’s Cancer Research Institute, Texas
  5. Mayo Clinic, United States
  6. University of Washington, United States
  7. Université Claude Bernard Lyon, Cancer Research Center of Lyon, France
Research Article
Cite this article as: eLife 2018;7:e37202 doi: 10.7554/eLife.37202
5 figures, 2 tables and 1 additional file

Figures

Figure 1 with 2 supplements
Homozygous tp53del/del zebrafish spontaneously develop a wide range of tumor types.

(A) tp53 genomic locus and CG1 tp53del/del allele. TALEN arms were designed to target the 5’ and 3’ genomic sequence of tp53 (red). (B–M) CG1 tp53del/del zebrafish develop leukemia (B–D), angiosarcoma (E–G), MPNSTs (H–J), and germ cell tumors (K–M). Whole animal images (B,E,H,K), hematoxylin/eosin (H and E) stained sections (C,D,F,G,I,L,M), and immunohistochemistry for Sox10 (J). Blast-like leukemia cells predominate in the kidney marrow and efface the renal tubules (black arrow, (D). (N) Tumor incidence in CG1 tp53del/del zebrafish (n = 134). (O) Quantitation of tumor types that form in CG1 tp53del/del mutant zebrafish by 55 weeks of life based on histology review (n = 51). (P–S) kRASG12D-induced embryonal rhabdomyosarcoma (ERMS) generated in CG1 tp53del/del zebrafish. Whole animal bright field and GFP-epifluorescence overlap images (P and Q, respectively). H and E stained sections revealed features consistent with human ERMS (R,S). Scale bars equal 12.5 mm in whole animal images and 100 μm in histology images.

https://doi.org/10.7554/eLife.37202.002
Figure 1—figure supplement 1
tp53del/del zebrafish survive at expected Mendelian ratios, lack Tp53 protein expression, and are resistant to irradiation-induced cell death.

(A) Survival of animals by genotype from a heterozygous tp53wt/del in-cross. tp53 homozygous wild-type (wt/wt), heterozygous (wt/del), and homozygous (del/del) mutant fish. (B) Western blot analysis at 24 hr post-fertilization (hpf) whole embryos. Actin is used as a loading control. (C–D) TUNEL staining performed on tp53wt/wt and tp53del/del embryos following gamma-irradiation at 24hpf (16 Gray) and fixation at 30hpf. Whole embryos images are shown for representative animals of each genotype (C). Quantification of TUNEL-positive cells within 1000 micron2 area. Regions where cells were counted are outlined by the white boxes in panel C. p<0.001 by Student’s T-test.

https://doi.org/10.7554/eLife.37202.003
Figure 1—figure supplement 2
Tumor latency in tp53del/del zebrafish.

Designation as assessed by histology review.

https://doi.org/10.7554/eLife.37202.004
Figure 1—figure supplemenrt 2—source data 1

Source data for Figure 1—figure supplement 2.

https://doi.org/10.7554/eLife.37202.005
Figure 2 with 1 supplement
tp53del/del tumors efficiently transplant into syngeneic CG1 strain zebrafish.

(A–E) A primary tp53del/del MPNSTs that formed in the eye transplanted orthotopically into the periocular space (A–C) or into the peritoneum of CG1-strain recipient fish (D–E). Intraperitoneal injection (i/p). (F–I) tp53del/del Tg(ubi:GFP)-positive angiosarcoma. Primary tumor-bearing fish (F–G) and transplanted animal (H–I). (J–R) tp53del/del Tg(ubi:GFP)-positive leukemia. Primary leukemia (J–K) and transplanted leukemia shown at 20 days post-transplantation (L–R). Whole kidney marrow was isolated from leukemia-engrafted fish and analyzed by FACS (N–O). (N) Forward and side scatter plot of whole kidney marrow of unlabeled CG1 host animal to assess ubi:GFP-positive tp53del/del leukemia cells following transplantation. (O) Analysis of GFP+ ubi:GFP-positive tp53del/del leukemia cells following FACS. Purity was ≥90%. (P–R) Cytospins and Wright/Giemsa staining of whole kidney marrow cells isolated from wildtype fish (P) compared with FACS sorted GFP+ cells from two representative aggressive NK cell-like leukemias, showing large blastic cells with abundant basophilic, vacuolated cytoplasm (Q–R). (S–V) Embryonal rhabdomyosarcoma arising in tp53del/del fish micro-injected at the one-cell stage with linearized rag2:kRASG12D + rag2:GFP. Primary (S), transplanted (2°) (T), and serially transplanted ERMS (3°) (U,V). Whole animal bright-field images (A,D,F,J) and merged GFP-fluorescence images (G,H,K,L,S–U). Hematoxylin and eosin stained sections of engrafted tumors (B–C,E, I, M,V). Scale bars are 5 mm in whole animal images and 100 μm for histology images.

https://doi.org/10.7554/eLife.37202.006
Figure 2—figure supplement 1
Engraftment of tp53del/del tumors into CG1 recipient zebrafish.

Engraftment was scored at >20 days post transplantation. i.p. intraperitoneal.

https://doi.org/10.7554/eLife.37202.007
Figure 2—figure supplemenrt 1—source data 1

Source data for Figure 2—figure supplement 1.

https://doi.org/10.7554/eLife.37202.008
Figure 3 with 1 supplement
Gene expression analysis of tp53del/del tumors.

(A) Principal component analysis (PCA) of gene expression profiles from whole CG1 syngeneic fish, MPNSTs, a germ cell tumor, and FACS sorted GFP+ leukemia, angiosarcomas, and ERMS. All tumor samples were obtained following engraftment in CG1 syngeneic recipient fish. (B) Heat map of genes differentially expressed with respect to controls identifies molecularly defined tumor groups. (C) Upregulated genes identified within each tumor type are enriched for Molecular Signature Database (MSigDB) signatures consistent with the expected tissue of origin. (D) NK cell leukemias are enriched for gene signatures identified from normal NK and NKL cells in the kidney marrow and NK cells isolated from rag1-/-, tg(lck:GFP) transgenic fish (NK cells*). For each analysis, enrichment is shown for the top 30 lineage-restricted genes identified from single-cell transcriptional profiling of transgenic cells using SMARTseq2 (denoted by asterisks) or unsorted cells using InDrops single-cell RNA sequencing approaches. (E) Heat map highlighting NK lineage genes significantly upregulated in tp53del/del leukemias when compared to all other tumor types analyzed. [log2(fold-change)]. Angiosarcoma (AS) and germ cell tumor (GC).

https://doi.org/10.7554/eLife.37202.009
Figure 3—source data 1

Genes and expression values for heatmap rendering shown in Figure 3B.

https://doi.org/10.7554/eLife.37202.014
Figure 3—source data 2

Top 500 transcripts differentially regulated in each tumor subtype identified by RNA sequencing analysis.

These gene lists were used to assess overlap with the GSEA signature database.

https://doi.org/10.7554/eLife.37202.015
Figure 3—source data 3

Table showing GSEAsig analysis for data rendered in Figure 3C.

https://doi.org/10.7554/eLife.37202.016
Figure 3—source data 4

Differential gene expression for tp53del/del and tp53M214K/M214K MPNST.

Significant overlap was observed for both commonly up-regulated (p=4e-321) and down-regulated (p=5e-182) genes. A fold change of log2(FC) ≥2 was considered differential and statistical significance was assessed as p≤0.05 with a one-sided Fisher’s exact test.

https://doi.org/10.7554/eLife.37202.017
Figure 3—source data 5

Genes used for analysis shown in Figure 3D.

https://doi.org/10.7554/eLife.37202.018
Figure 3—figure supplement 1
Zebrafish cancers share common gene expression with human tumors and confirmation of NK-cell linage derivation for tp53del/del leukemias.

(A–C) GSEA analysis comparing zebrafish tp53del/del tumors to human counterparts. (A) Human angiosarcoma (FDR q-value = 0.001), (B) human MPNST (FDR q-value = 0.00433526), and (C) human ERMS (FDR q-value = 0) gene sets are significantly enriched in the corresponding zebrafish tp53del/del tumors. (D) Heat map depicting differential gene expression in zebrafish tp53del/del leukemias compared to whole CG1 control animals. Gene-expression defines tp53del/del leukemias based on lineage signatures previously generated using InDrop and SMARTseq (asterisk) sequencing approaches (Tang et al., 2017). (E) Expression of the top 200 tp53del/del ANKL-like genes assessed in the SMARTseq dataset from Tang et al. (2017). HSC/progenitors were isolated as cd41:GFPlow cells from transgenic zebrafish, T cells from tg(lck:GFP) transgenic zebrafish, NK cells from rag1-/-, tg(lck:GFP) transgenic zebrafish, myeloid cells from tg(mpx:EGFP) transgenic zebrafish, B cells from marrow-derived tg(rag2:GFP) transgenic zebrafish, and HSCs from tg(runx1+23:GFP) transgenic zebrafish. The tp53del/del leukemia gene expression signature was significantly enriched only in the NK cells (log2(TPM+ 1)≥2, p-value=0.015, one-sided binomial test).

https://doi.org/10.7554/eLife.37202.010
Figure 3—figure supplement 1—source data 1

GSEA report and human tumor gene expression signatures used for GSEA comparing tp53del/del angiosarcoma, MPNST and ERMS to their human counterparts.

https://doi.org/10.7554/eLife.37202.011
Figure 3—figure supplement 1—source data 2

Differential gene expression for tp53del/del leukemias with respect to blood cells and kidney cells shown in Figure 3—figure supplement 1D.

Gene identifications correspond to SMARTseq and InDrop single cell sequencing from Tang et al. (2017), as indicated.

https://doi.org/10.7554/eLife.37202.012
Figure 3—figure supplement 1—source data 3

Genes used for analysis shown in Figure 3—figure supplement 1E.

https://doi.org/10.7554/eLife.37202.013
tp53del/del kRASG12D-induced ERMS have increased invasion and metastasis.

(A–F) Whole animal fluorescent images of CG1-strain fish engrafted into the dorsolateral musculature with non-disseminated (A–C) and disseminated ERMS (D–F). Days post transplantation (dpt). White lines demarcate GFP+ tumor area. White arrowheads show site of injection and yellow arrowheads denote metastatic lesions. (G) H and E and (H) GFP immunohistological staining of fish engrafted with metastatic tp53del/del kRASG12D-induced ERMS. (I) Quantification of growth confined to site of injection (green bars) and compared with animals that exhibited local invasion or metastatic ERMS following tumor engraftment until fish were moribund. X-axis identifies 5 tp53wt/wt and 11 tp53del/del ERMS primary tumors that were transplanted into wild-type CG1 syngeneic host zebrafish. p=0.003, one-sided Fisher’s exact test. Scale bars denote 5 mm.

https://doi.org/10.7554/eLife.37202.020
Author response image 1
Venn diagram depicting overlap between up-regulated and down-regulated genes when comparing homozygous mutant tp53M214K/M214Kand tp53del/delMPNST to whole adult zebrafish.

Tables

Table 1
Results from limiting dilution cell transplantation experiments comparing engraftment potential of tp53wt/wt and tp53del/del kRASG12D-induced ERMS.
https://doi.org/10.7554/eLife.37202.019
tp53wt/wt + rag2:kRASG12D ERMS
Cell #Tumor 1Tumor 2Tumor 3Tumor 3
100007 of 75 of 66 of 66 of 6
10002 of 62 of 76 of 81 of 8
1000 of 90 of 81 of 80 of 8
TPC#1 in 28321 in 48101 in 7261 in 7388
1 in 3495 (2291–5333)
tp53del/del + rag2:kRASG12D ERMS
Cell #Tumor 1Tumor 2Tumor 3
100003 of 55 of 66 of 6
10003 of 43 of 50 of 7
1003 of 93 of 71 of 8
TPC#1 in 35461 in 22281 in 3640
1 in 3038 (1739–5307), p=0.647
Key resources table
Reagent type (species)
or resource
DesignationSource or referenceIdentifiersAdditional
information
Genetic reagent
(danio rerio)
CG1Mizgireuv and Revskoy (2006)
Genetic reagent
(danio rerio)
rag2E450fs/E450fsTang et al. (2014)
Antibodyanti-tp53 (zebrafish)Abcamab77813, RRID:
AB_10864112
WB 5 ug/mL
Antibodyanti-ActinSigmaA2066, RRID:
AB_476693
WB 1:200
Antibodyanti-mouse HRPSigmaNA931, RRID:
AB_772210
WB 1:1000
Antibodyanti-rabbit HRPCell Signalling
Technology
7074, RRID:
AB2099233
WB 1:1000
Recombinant
DNA construct
rag2:kRASG12DLangenau et al. (2007)
Recombinant
DNA construct
rag2:GFPLangenau et al. (2007)
Langenau et al. (2008)
Recombinant
DNA construct
ubi:GFPMosimann et al. (2011)
Commercial
assay or kit
In situ Cell Death
Detection Kit, TMR red
Sigma12156792910

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