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Fig. 5 Spry4 regulates Krox20 initiator enhancers in the chick embryo. (A-D) Chick embryo neural tubes were electroporated on the left side with HA-tagged wild-type or Y52A dominant-negative zebrafish spry4-expressing vectors (pAdRSV-Spry4WT-HA and pAdRSV-Spry4Y52A-HA), and flat-mounted after in situ hybridisation with a Krox20 probe. No difference was detected between the left (experimental) and right (control) sides. The efficiency of electroporation and spry4 expression were monitored by immunolabelling against the HA tag (B,D). (E-P) Chick embryo neural tubes were co-electroporated with HA-tagged wild-type or Y52A dominant-negative spry4-expressing vectors and constructs carrying chicken Krox20 enhancer elements cA, cB or cC driving the gfp reporter, and subjected to HA (red) and GFP (green) immunostaining (merge in yellow). (Q-S) Quantitative evaluation of relative reporter activity obtained by dividing the GFP signal intensity by the HA signal intensity, both quantified using ImageJ. ns, not significant; *, P<0.0005; **, P<0.0001; Student′s t-test. Errors bars indicate s.e.m.