Fig. 1

Fig. 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 kRAS^G12D-expressing ERMS (A–C) compared with those that express both kRAS^G12Dand 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 kRAS^G12Dand 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). kRAS^G12D 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.