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. 2017 Jan 12;6:e19214. doi: 10.7554/eLife.19214

Figure 1. Transgenic myf5 elevates tumor cell differentiation, increases tumor size, and accelerates time to primary tumor-onset when expressed in myosin-expressing ERMS cells.

(A–F) Primary ERMS developing in myf5:GFP/mylpfa:mCherry AB-strain zebrafish. Transgenic kRASG12D-expressing ERMS (A–C) compared with those that express both kRASG12Dand mylpfa:myf5 (D–F). Animals imaged at 35 dpf (A,D). Hematoxylin and Eosin-stained sections of representative tumors (B,E) and quantification of differentiation within individual tumors (C,F; 1-less differentiated and 3-most differentiated). Asterisk denotes p=0.015 by Chi-square test. (G) Quantitative real-time PCR gene expression performed on bulk ERMS cells, confirming high myf5 expression, increased differentiation, and high expression of TPC associated genes in ERMS that co-express kRASG12Dand mylpfa:myf5 (K+M, N = 5). Endogenous myf5 was assessed using primers specific to the 3’UTR and total myf5 assessed by primers that amplify the coding sequence (cds). cadherin 15 (cdh15) and myogenin (myog). kRASG12D alone expressing ERMS (K, N = 4). Average gene expression with 50% confidence intervals denoted by box. Mean, maximum, and minimum also denoted. (H) Relative tumor size of primary ERMS at 30 days post fertilization (dpf). Box shows 50% confidence interval. Mean, maximum, and minimum denoted. Asterisk denotes p=0.0108, Student’s t-test. (I) Kaplan-Meijer analysis denoting time-to-tumor onset (p<0.001, Log-rank Statistic, N = 494 fish analyzed for K and N = 470 for K+M). Scale bars equal 2 mm (A,D) and 50 μm (B,E). Asterisks in panels G-H denote *p<0.05; **p<0.01; ***p<0.001 by Student’s t-test.

DOI: http://dx.doi.org/10.7554/eLife.19214.002

Figure 1.

Figure 1—figure supplement 1. Fluorescence images of primary ERMS developing in stable transgenic myf5:GFP/mylpfa:mCherry zebrafish.

Figure 1—figure supplement 1.

Images of the same representative rag2:kRASG12D –alone (A–C) and rag2:kRASG12D; mylpfa:myf5 (D–F) zebrafish shown in Figure 1A and D, respectively. (A,D) merged (brightfield, GFP and mCherry) image. (B,E) mCherry image. (C,F) GFP image. Scale bars equal 2 mm.
DOI: http://dx.doi.org/10.7554/eLife.19214.003
Figure 1—figure supplement 2. Histological classification of primary zebrafish ERMS based on differentiation score.

Figure 1—figure supplement 2.

Representative H and E-stained sections of zebrafish ERMS assigned to each differentiation category. Scale bars equal 100 μm.
DOI: http://dx.doi.org/10.7554/eLife.19214.004
Figure 1—figure supplement 3. Analysis of proliferation and apoptosis in zebrafish primary ERMS.

Figure 1—figure supplement 3.

(A) Representative H and E-stained sections and immunohistochemistry for phospho-H3 (pH3) and cleaved caspase-3 (CC3). (B) Quantification of the total number of pH3-positive cells per 400x imaging field. (n=average of 3 fields/tumor section). (C) Quantification of the total number of CC3-positive cells per 400x imaging field (n=average of 3 fields/tumor). Boxes in B–C denote 50% confidence interval and mean, maximum, and minimum shown. kRASG12D[K] (N = 5) and kRASG12D; mylpfa:myf5 [K+M] (N = 11). (D) Quantification of total number of EdU+ cells per area (n=average of 3 fields/tumor. N = 3 tumors per genotype). *p<0.05 or **p<0.01 in comparison to each kRASG12D-alone expressing ERMS (Student’s t-test). Error bars denote +/- STD. Scale bars equal 100 μm (A). Not significant by Student’s t-test (n.s.).
DOI: http://dx.doi.org/10.7554/eLife.19214.005