Figure 4: Microenvironmental CRISPR against EDN3b abrogates phenotype switching.

(a) Control wild-type fish of the AB strain that was injected with Cas9 protein alone. Higher magnification views of the melanocytes from this fish is shown below, indicating these are fully mature, pigmented melanocytes. (b) An AB fish in the F1 generation that had been injected with a CRISPR gRNA against EDN3b, showing a severe loss of melanocytes over the entire body of the zebrafish. Higher magnification views (below) from this fish show a decreased number of pale, misshapen melanocytes. (c) An AB fish in the F1 generation that had been injected with a CRISPR gRNA against ECE2b, showing a melanocyte defect that is highly similar to that seen in the EDN3b mutant. (d) Schema for testing the effects of microenvironmental EDN3b on melanoma growth and differentiation. Equal numbers of ZMEL1-GFP cells were transplanted subcutaneously either in a WT recipient, EDN3b mutant recipient or the ECE2b mutant recipient, who differ only in the loss of function EDN3b in the microenvironment. (e) Tumour growth in the WT recipient shows large pigmented tumours in multiple subcutaneous sites, consistent with phenotype switching to a more differentiated/proliferative state. (f,g) Tumour growth in the EDN3b or ECE2b recipients is impaired, with smaller tumours that are markedly less pigmented, consistent with reduced phenotypic switching due to loss of endothelin-3 signalling from the microenvironment. (h) Quantification of tumour area in WT versus EDN3b versus ECE2b backgrounds at 14 days post transplant demonstrates a significant decrease in tumour size in the CRISPR mutant (*=P<0.05, WT versus EDN3b and WT versus ECE2b, ANOVA, n=15 WT, n=24 EDN3b, n=19 ECE2b). (i) Measurement of mitf-GFP pixel intensity in WT versus EDN3b versus ECE2b recipients shows a significant decrease in overall GFP+ intensity in the ECE2b mutant (*=P<0.05, WT versus ECE2b). (j) Kaplan–Meier survival curves of WT versus EDN3b−/− recipient fish, showing a significantly longer survival time in the EDN3b and ECE2b recipients (+, P=0.0035, logrank test). Error bars are s.e.m. with the numbers of animals indicated above.