FIGURE SUMMARY
Title

The NOTCH1/SNAIL1/MEF2C Pathway Regulates Growth and Self-Renewal in Embryonal Rhabdomyosarcoma

Authors
Ignatius, M.S., Hayes, M.N., Lobbardi, R., Chen, E.Y., McCarthy, K.M., Sreenivas, P., Motala, Z., Durbin, A.D., Molodtsov, A., Reeder, S., Jin, A., Sindiri, S., Beleyea, B.C., Bhere, D., Alexander, M.S., Shah, K., Keller, C., Linardic, C.M., Nielsen, P.G., Malkin, D., Khan, J., Langenau, D.M.
Source
Full text @ Cell Rep.

Notch1 Pathway Activation Increases the Number of myf5+ Progenitor Cells in Zebrafish ERMS

(A and B) ERMS generated in Tg(myf5-GFP) zebrafish expressing (A) kRASG12D or (B) kRASG12D + ICN1.

(C and D) Shown here: (C) tumor incidence and (D) size comparing ERMS at 15 and 30 days of life. Relative tumor size was measured at day 30. ns, not significant.

(E and F) Images showing the difference in size of syngeneic zebrafish engrafted with 1 × 104 bulk tumor cells labeled with rag2-dsRedExpress and imaged at day 45. Tumor boundaries are denoted by dashed lines.

(G) Kaplan-Meijer analysis denoting differences in engraftment rates; n = 17 transplant animals per group from four independent tumors per group (p < 0.0001, log-rank statistic).

(H) Real-time qPCR gene expression performed on sorted dsRedExpress+ ERMS cells arising within individual tumors. p < 0.05, Student’s t test.

(I–P) Primary ERMS arising in Tg(myf5-GFP; mylz2-mCherry) animals. ERMS expressing (I–L) kRASG12D alone and (M–P) kRASG12D + ICN1. (I and M) Whole animal images, (J and N) H&E-stained sections, and (K, O, L, and P) representative flow cytometry. Graphical analysis showing percentages of fluorescent-labeled ERMS subpopulations within individual tumors following FACS. Five independent primary tumors were assessed, and each is denoted by numbers on the x axis. (p = 0.013, Student’s t test).

(Q–V) Transplanted ERMS arising from Tg(myf5-GFP; mylz2-mCherry) tumors. (Q–S) ERMS expressing kRASG12D alone and (T–V) kRASG12D + ICN1. (Q and T) Whole animal images of transplant animals, (R and U) H&E-stained sections, and (S and V) bar graphs showing fluorescent-labeled ERMS subpopulations following FACS. Five independent primary transplanted tumors were engrafted into CG1 fish, and each are denoted by numbers on the x axis. FACS populations for two representative engrafted fish per tumor are shown (p < 0.001, Student’s t test).

Scale bars in (I) and (Q), also pertaining to (M) and (T), 2 mm; scale bar in (J), also pertaining to (N), (R), and (U), 50 μm. See also Figure S1.

Notch1 Pathway Activation Confers Tumor-Propagating Activity to Differentiated ERMS Cells

(A) Schematic of limiting dilution cell transplantation assay used to assess engraftment potential of fluorescently labeled ERMS cell fractions.

(B–D) Engraftment with FACS-sorted myf5-GFP+/mylz2-mCherry-negative cells. (B) Whole animal image, (C) engrafted tumor cells analyzed by FACS, and (D) histology. Sort purity is denoted in the lower left corner of (B).

(E–G) Engraftment with FACS-sorted double-positive myf5-GFP+/mylz2-mCherry+ differentiated cells. (E) Whole animal image, (F) engrafted tumor cells analyzed by FACS, and (G) histology. Sort purity denoted in lower left corner of (E).

(H) Table showing combined analysis of engraftment rates for myf5-GFP+/mylz2-mCherry-negative, double-positive myf5-GFP+/mylz2-mCherry+, myf5-GFP-negative/mylz2-mCherry+, and double-negative cells. Number of tumors analyzed per condition is noted. ND, not determined; CI, confidence interval; inf, infinity.

Scale bar in (B), also pertaining to (E), 2 mm; scale bar in (D), also pertaining to (G), 50 μm.

See also Figure S2, Table S2, and Table S3.

The double positive myf5-GFP+/mylz2-mCherrry+ ICN1-expressing ERMS cells generate tumors when engrafted into syngeneic CG1-strain recipient fish. Related to Figure 2.

(A-E) FACS plots of kRASG12D + ICN1-expressing ERMS (A) and following sorting of purified ERMS cell subfractions. These data show sort purity for experiments in Figure 2 B, E and Table S2. (F-J) FACS plots of tertiary transplanted ERMS (F) and following sorting of purified ERMS cell subfractions (G-J). (K-P) Representative animals serially transplanted with FACS sorted myf5-GFP+/mylz2-mCherry-negative ICN1-expressing ERMS cells (K-M, 1x103 cells engrafted/fish) or myf5-GFP+/mylz2-mCherrry+ cells (N-P, 1x103 cells engrafted/fish). Whole animal fluorescent images of engrafted fish with sort purity of transplanted tumor cells denoted in the lower left panel (K, N). Analysis of engrafted cell subfractions by FACs (L, O) or hematoxylin and eosin stained sections (M, P). (Q-V) Engraftment of highly purified double-positive myf5-GFP+/mylz2-mCherry+ ERMS cells verified acquisition of self-renewal and the ability to dedifferentiate into myf5-GFP+ alone expressing cells. myf5-GFP+/mylz2-mCherry+ ERMS cells were isolated from transplanted fish and enriched following three rounds of FACS (Q-S). Single TPC equivalents were injected into CG1 recipient fish (T). Engrafted tumors were analyzed by FACS (U) and histology (V). Scale bar in K, N, Q and T equals 2mm; M, P, and V equals 50μm.

Acknowledgments
This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ Cell Rep.