Figures and data in PAX3-FOXO1 transgenic zebrafish models identify HES3 as a mediator of rhabdomyosarcoma tumorigenesis

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Genevieve C Kendall

Sarah Watson

Lin Xu

Collette A LaVigne

Whitney Murchison

Dinesh Rakheja

Stephen X Skapek

Franck Tirode

Olivier Delattre

James F Amatruda

(2018)
PAX3-FOXO1 transgenic zebrafish models identify HES3 as a mediator of rhabdomyosarcoma tumorigenesis

eLife 7:e33800.

https://doi.org/10.7554/eLife.33800
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Additional files

7 figures, 1 table and 4 additional files

Figures

Figure 1 with 2 supplements

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Zebrafish models of human *PAX3-FOXO1* tumorigenesis.

(A) Zebrafish were injected at the single-cell stage with mosaic GFP2A-tagged human *PAX3-FOXO1* under the control of various promoters. At 24 hr old, embryos were sorted for GFP expression indicating …
https://doi.org/10.7554/eLife.33800.003

Figure 1—figure supplement 1

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Promoter restricted expression of human *PAX3-FOXO1* has different effects on survival in developing zebrafish.

Shown are the survival curves for the following promoters and the total number of zebrafish embryos analyzed for each condition: beta actin (n = 120), mitfa (n = 45), fli1 (n = 81), ubi (n = 160), …
https://doi.org/10.7554/eLife.33800.004

Figure 1—figure supplement 2

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Junctional sequence used to map human *PAX3-FOXO1* fusion RNAseq reads from zebrafish tumors.
https://doi.org/10.7554/eLife.33800.005

Figure 2 with 2 supplements

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*PAX3* and *PAX3-FOXO1* have distinct impacts and tolerance during embryonic development and in adult zebrafish.

(A) Strategy for assessing functional differences of beta-actin-driven *PAX3* and *PAX3-FOXO1* in a vertebrate system. (B) Representative images at 24 hr post-fertilization of Uninjected, mCherry2A-*PAX3*,…
https://doi.org/10.7554/eLife.33800.006

Figure 2—figure supplement 1

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The *tp53^M214K* mutation modifies the CMV-*PAX3FOXO1* phenotype in developing zebrafish.

(A) Schematic of the experimental strategy to assess the impact of a *tp53* mutation on survival, cell tolerance, and apoptosis of mosaic CMV restricted GFP, GFP-*PAX3*, or GFP-*PAX3FOXO1* developmental …
https://doi.org/10.7554/eLife.33800.007

Figure 2—figure supplement 2

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A model for *tp53^M214K* mediation of CMV-*PAX3FOXO1* RMS tumorigenesis.

GFP-tagged *PAX3-FOXO1* was injected at the same concentration into developing wildtype or *tp53^M214K* homozygous mutant zebrafish. The number of GFP positive and TUNEL-positive cells was assessed at 24 …
https://doi.org/10.7554/eLife.33800.008

Figure 3

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*PAX3* and *PAX3-FOXO1* induce distinct gene expression signatures during development, identifying a unique *PAX3-FOXO1* target, *her3*.

Microarray analysis identified differentially expressed gene signatures in 24 hr old zebrafish FACS sorted embryonic cells that expressed either GFP2A-*PAX3FOXO1* or GFP2A-*PAX3*. (A) Gene ontology …
https://doi.org/10.7554/eLife.33800.009

Figure 4 with 2 supplements

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*HES3* inhibits myogenic differentiation in developing zebrafish and supports persistence of *PAX3-FOXO1-*positive cells.

(A) Zebrafish embryos were injected at the single-cell stage with the beta actin promoter driving GFP-Cherry, GFP-*PAX3FOXO1*, mCherry-*HES3*, or combined mCherry-*HES3* and GFP-*PAX3FOXO1*. Shown are …
https://doi.org/10.7554/eLife.33800.010

Figure 4—figure supplement 1

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*HES3* facilitates cellular tolerance of CMV-*PAX3FOXO1* expression in developing zebrafish but does not alleviate the apoptosis phenotype.

(A) Schematic of the strategy to assess the impact of human *HES3* expression on *PAX3-FOXO1* cell tolerance and apoptosis in wildtype developing zebrafish. The CMV promoter was utilized in a mosaic …
https://doi.org/10.7554/eLife.33800.011

Figure 4—figure supplement 2

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A model for *HES3* facilitation of CMV-*PAX3FOXO1* embryonic cellular persistence.

GFP-tagged *PAX3-FOXO1* or GFP-tagged *PAX3-FOXO1* in combination with *HES3* were injected in equimolar amounts into developing wild-type zebrafish. The number of GFP-positive and TUNEL-positive cells …
https://doi.org/10.7554/eLife.33800.012

Figure 5 with 2 supplements

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*HES3* overexpression inhibits myogenic differentiation of mouse muscle myoblasts.

(A) Schematic of muscle development. Specifically noted are key skeletal muscle transcription factors and proteins assessed in B. Muscle satellite cells that self-renew express *Pax3*/Pax7. During …
https://doi.org/10.7554/eLife.33800.013

Figure 5—figure supplement 1

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HES3 overexpression inhibits the myogenic differentiation kinetic in mouse muscle myoblasts.

(A) C2C12 cells were transfected with control or CMV-*HES3*, allowed to become confluent and begin fusion, and then whole cell lysate harvested four days post plating. (B) Western blots to detect …
https://doi.org/10.7554/eLife.33800.014

Figure 5—figure supplement 2

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HES3 does not alter MyoD expression during fusion initiation but inhibits MyoD during terminal differentiation.

(A) C2C12 cells were transfected with CMV-Empty or CMV-HES3, allowed to become confluent, and then whole cell lysate harvested 4 days post plating. Remaining C2C12 conditions were changed to …
https://doi.org/10.7554/eLife.33800.015

Figure 6

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*HES3* overexpression promotes pro-tumorigenic features in *PAX3-FOXO1* RMS patient cells and mouse myoblasts.

(A) Rh30 cells stably express CMV-*HES3* as evaluated by qRT-PCR. SD is derived from technical triplicates. (B) *PAX3-FOXO1* levels were assessed by qRT-PCR on the same samples in A. SD is derived from …
https://doi.org/10.7554/eLife.33800.016

Figure 7

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*HES3* is overexpressed in fusion-positive RMS patient tumors, predicts reduced overall survival, and identifies potential therapeutic targets.

*HES3* expression levels determined in fusion-positive ARMS versus fusion-negative ARMS/ERMS. (A) RNAseq data from Shern et al. (2014) . (B) HuEx data from Triche T, Skapek S; GEO: Accession Number …
https://doi.org/10.7554/eLife.33800.017

Tables

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Gene (Homo sapiens)	HES3	NA	NM_001024598
Gene (Homo sapiens)	PAX3-FOXO1	PMID:8275086; PMID:8221646	NM_181457; NM_002015.3	exons 1–7 of NM_181457 fused to exons 2–3 of NM_002015.3
Gene (Mus musculus)	Pax3	NA	NM_008781
Gene (Danio rerio)	her3	NA	NM_131080
Strain, strain background (Danio rerio AB)	AB Wildtype	Zebrafish International Resource Center (ZIRC)	ZIRC:ZL1	https://zebrafish.org/fish/lineAll.php
Strain, strain background (Danio rerio AB/TL)	AB\TL Wildtype	NA		Cross of AB and TL
Strain, strain background (Danio rerio TL)	TL Wildtype	ZIRC	ZIRC:ZL86	https://zebrafish.org/fish/lineAll.php
Strain, strain background (Danio rerio WIK)	WIK Wildtype	ZIRC	ZIRC:ZL84	https://zebrafish.org/fish/lineAll.php
Strain, strain background (Danio rerio tp53^M214K mutant)	tp53^M214K mutant	PMID:15630097; available from ZIRC	ZIRC:ZL1057	https://zebrafish.org/fish/lineAll.php
Genetic reagent	Tol2 transposase mRNA	PMID:16959904		Injected at 50 ng/uL
Cell line (Mus musculus)	C2C12	ATCC	ATCC:CRL-1772	Maintained in DMEM + 10% FBS+1X Antimycotic-Antibiotic
Cell line (Homo sapiens)	Rh30	ATCC	ATCC:CRL-2061	Maintained in RPMI-1640 + 10% FBS+1X Antimycotic-Antibiotic
Transfected construct (Homo sapiens)	CMV-HES3 (MYC and FLAG tagged)	Origene	Origene:RC224630
Transfected construct (NA)	CMV-Empty	this paper		HES3 excised using EcoR1 and Mlu1 and re-ligated
Antibody	MF20 (mouse monoclonal)	Developmental Studies Hybridoma Bank	DSHB:MF20c	1:40 in cells, 1:100 in zebrafish embryo whole mounts, 1:1000 Western blot
Antibody	alpha-Tubulin DM1A (mouse monoclonal)	Cell Signaling	Cell Signaling:3873	1:1000 dilution
Antibody	MyoD 5.8A (mouse monoclonal)	Thermo	Thermo:MA5-12902	1:1000 dilution
Antibody	MYC 71D10 (rabbit monoclonal)	Cell Signaling	Cell Signaling:2278	1:1000 dilution
Antibody	GFP (rabbit polyclonal)	MBL International Corporation	MBL:598	1:1000 dilution
Antibody	GFP-488 (rabbit polyclonal)	Thermo	Thermo:A-21311	1:500 dilution
Antibody	Alexa 488 or 594 secondaries	Thermo		1:500 dilution
Antibody	HRP conjugate secondaries	BioRad		1:20000 dilution
Other	DAPI stain (ProLong Gold Antifade mounting media with DAPI)	Thermo	Thermo:P36931
Recombinant DNA reagent	p5E beta actin	PMID:17937395
Recombinant DNA reagent	p5E cmv	PMID:17937395
Recombinant DNA reagent	p5E mcs	PMID:17937395
Recombinant DNA reagent	p5E ubi	PMID:21138979; available from Addgene	Addgene:27320
Recombinant DNA reagent	p5E unc503	PMID:23444339; available from Addgene	Addgene:64020
Recombinant DNA reagent	p5E fli1a	PMID:17948311; available from Addgene	Addgene:31160
Recombinant DNA reagent	p5E mitfa	James Lister; available from Addgene	Addgene:81234
Recombinant DNA reagent	pmE beta globin splice acceptor	PMID:15239961
Recombinant DNA reagent	pmE GFP2A	PMID:17941043
Recombinant DNA reagent	pmE mCherry2A	PMID:17941043
Recombinant DNA reagent	p3E PAX3-FOXO1	this paper		attb2r/attb3 sites added with primers in Supplementary file 3 by high-fidelity PCR
Recombinant DNA reagent	p3E Pax3	this paper		attb2r/attb3 sites added with primers in Supplementary file 3 by high-fidelity PCR
Recombinant DNA reagent	p3E HES3	this paper		attb2r/attb3 sites added with primers in Supplementary file 3 by high-fidelity PCR
Recombinant DNA reagent	p3E SV40 late poly A	PMID:17937395
Recombinant DNA reagent	p3E 2A-mCherry	PMID:23462469; available from Addgene	Addgene:26031
Recombinant DNA reagent	pDONRP2R-P3 (3′ donor vector; attP2R-P3 flanking chlor/ccdB cassette)	Invitrogen	Invitrogen:pDONR P2R-P3	Used to generate p3E's from this paper
Recombinant DNA reagent	pDestTol2pA2 destination vector	PMID:17937395
Recombinant DNA reagent	BetaActin-GFP2A-pA	this paper		Generated by Gateway Cloning
Recombinant DNA reagent	BetaActin-mCherry2A-pA	this paper		Generated by Gateway Cloning
Recombinant DNA reagent	BetaActin-GFP2A- 2AmCherry	this paper		Generated by Gateway Cloning
Recombinant DNA reagent	BetaActin-mCherry2A-Pax3	this paper		Generated by Gateway Cloning
Recombinant DNA reagent	BetaActin-GFP2A-Pax3	this paper		Generated by Gateway Cloning
Recombinant DNA reagent	BetaActin-mCherry2A-HES3	this paper		Generated by Gateway Cloning
Recombinant DNA reagent	BetaActin-GFP2A-PAX3FOXO1	this paper		Generated by Gateway Cloning
Recombinant DNA reagent	CMV-GFP2A-pA	this paper		Generated by Gateway Cloning
Recombinant DNA reagent	CMV-mCherry2A-pA	this paper		Generated by Gateway Cloning
Recombinant DNA reagent	CMV-GFP2A-Pax3	this paper		Generated by Gateway Cloning
Recombinant DNA reagent	CMV-mCherry2A-HES3	this paper		Generated by Gateway Cloning
Recombinant DNA reagent	CMV-GFP2A-PAX3FOXO1	this paper		Generated by Gateway Cloning
Recombinant DNA reagent	ubi-GFP2A-PAX3FOXO1	this paper		Generated by Gateway Cloning
Recombinant DNA reagent	mitfa-GFP2A-PAX3FOXO1	this paper		Generated by Gateway Cloning
Recombinant DNA reagent	fli1-GFP2A-PAX3FOXO1	this paper		Generated by Gateway Cloning
Recombinant DNA reagent	unc503-GFP2A-PAX3FOXO1	this paper		Generated by Gateway Cloning
Recombinant DNA reagent	SpliceAcceptor- GFP2A-PAX3FOXO1	this paper		Generated by Gateway Cloning
Sequence-based reagent	GFP Morpholino	Gene Tools	Gene Tools: GFP Morpholino	(5' ACAGCTCCTCGCCC TTGCTCACCAT 3')
Commercial assay or kit	ApopTag Red In Situ Apoptosis Detection Kit	Millipore	Millipore:S7165
Commercial assay or kit	Affymetrix Zebrafish Gene 1.1 ST Array strip	Affymetrix	Affymetrix:901802
Commercial assay or kit (Mus musculus)	M384 Rhabdomy osarcoma 384 well panel	BioRad	BioRad:M384 Rhabdomyosarcoma
Commercial assay or kit (Homo sapiens)	H384 Tumor Metastasis (SAB Target List) 384 well panel	BioRad	BioRad:H384 Tumor Metastasis (SAB Target List)
Chemical compound, drug	Geneticin (G418)	Thermo	Thermo:10131027	Select at 1 mg/mL
Software, algorithm	ImageJ	http://imageJ.nih.gov/ij
Software, algorithm	GraphPad Prism 7.0 c	https://www.graphpad.com
Software, algorithm	Rv3.3.1	https://www.R-project.org

Additional files

Supplementary file 1 Total number of zebrafish analyzed and resulting tumors for Beta Actin, CMV, and ubiquitin driven PAX3-FOXO1 in wildtype AB or AB/TL strains or tp53^M214K/M214K homozygous mutants.: https://doi.org/10.7554/eLife.33800.018
Download elife-33800-supp1-v1.xlsx
Supplementary file 2 Total number of zebrafish analyzed and the mean age of screening for mitfa, fli1, unc503, and mcs (splice acceptor) driven PAX3-FOXO1.: https://doi.org/10.7554/eLife.33800.019
Download elife-33800-supp2-v1.xlsx
Supplementary file 3 List of oligonucleotide primer sequences.: https://doi.org/10.7554/eLife.33800.020
Download elife-33800-supp3-v1.xlsx
Transparent reporting form: https://doi.org/10.7554/eLife.33800.021
Download elife-33800-transrepform-v1.docx

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Figures

Zebrafish models of human PAX3-FOXO1 tumorigenesis.

Promoter restricted expression of human PAX3-FOXO1 has different effects on survival in developing zebrafish.

Junctional sequence used to map human PAX3-FOXO1 fusion RNAseq reads from zebrafish tumors.

PAX3 and PAX3-FOXO1 have distinct impacts and tolerance during embryonic development and in adult zebrafish.

The tp53^M214K mutation modifies the CMV-PAX3FOXO1 phenotype in developing zebrafish.

A model for tp53^M214K mediation of CMV-PAX3FOXO1 RMS tumorigenesis.

PAX3 and PAX3-FOXO1 induce distinct gene expression signatures during development, identifying a unique PAX3-FOXO1 target, her3.

HES3 inhibits myogenic differentiation in developing zebrafish and supports persistence of PAX3-FOXO1-positive cells.

HES3 facilitates cellular tolerance of CMV-PAX3FOXO1 expression in developing zebrafish but does not alleviate the apoptosis phenotype.

A model for HES3 facilitation of CMV-PAX3FOXO1 embryonic cellular persistence.

HES3 overexpression inhibits myogenic differentiation of mouse muscle myoblasts.

HES3 overexpression inhibits the myogenic differentiation kinetic in mouse muscle myoblasts.

HES3 does not alter MyoD expression during fusion initiation but inhibits MyoD during terminal differentiation.

HES3 overexpression promotes pro-tumorigenic features in PAX3-FOXO1 RMS patient cells and mouse myoblasts.

HES3 is overexpressed in fusion-positive RMS patient tumors, predicts reduced overall survival, and identifies potential therapeutic targets.

Tables

Additional files

Supplementary file 1

Supplementary file 2

Supplementary file 3

Transparent reporting form

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Zebrafish models of human PAX3-FOXO1 tumorigenesis.

Promoter restricted expression of human PAX3-FOXO1 has different effects on survival in developing zebrafish.

Junctional sequence used to map human PAX3-FOXO1 fusion RNAseq reads from zebrafish tumors.

PAX3 and PAX3-FOXO1 have distinct impacts and tolerance during embryonic development and in adult zebrafish.

The tp53M214K mutation modifies the CMV-PAX3FOXO1 phenotype in developing zebrafish.

A model for tp53M214K mediation of CMV-PAX3FOXO1 RMS tumorigenesis.

PAX3 and PAX3-FOXO1 induce distinct gene expression signatures during development, identifying a unique PAX3-FOXO1 target, her3.

HES3 inhibits myogenic differentiation in developing zebrafish and supports persistence of PAX3-FOXO1-positive cells.

HES3 facilitates cellular tolerance of CMV-PAX3FOXO1 expression in developing zebrafish but does not alleviate the apoptosis phenotype.

A model for HES3 facilitation of CMV-PAX3FOXO1 embryonic cellular persistence.

HES3 overexpression inhibits myogenic differentiation of mouse muscle myoblasts.

HES3 overexpression inhibits the myogenic differentiation kinetic in mouse muscle myoblasts.

HES3 does not alter MyoD expression during fusion initiation but inhibits MyoD during terminal differentiation.

HES3 overexpression promotes pro-tumorigenic features in PAX3-FOXO1 RMS patient cells and mouse myoblasts.

HES3 is overexpressed in fusion-positive RMS patient tumors, predicts reduced overall survival, and identifies potential therapeutic targets.

Supplementary file 1

Supplementary file 2

Supplementary file 3

Transparent reporting form

The tp53^M214K mutation modifies the CMV-PAX3FOXO1 phenotype in developing zebrafish.

A model for tp53^M214K mediation of CMV-PAX3FOXO1 RMS tumorigenesis.