Compensatory regulation of sws2 and rh2 opsin genes by six6b and six7. (A) Schematic drawing of the transgene construct. (B) EGFP expression in the anterior region of six6b-tg (ja70Tg) at 5 dpf. (Scale bar: 200 μm.) (C) EGFP signals detected in retinal photoreceptor layers of 4-dpf six6b-tg larvae (green). Weak EGFP signals were also detected in the inner nuclear layer (also SI Appendix, Fig. S2A), consistent with previous work (12). RH2 and LWS cones were labeled with zpr1 antibody (magenta). Cell nuclei were counterstained with DAPI (blue). (Scale bar: 40 μm.) (D and E) Relative mRNA levels of opsin genes and six6b in the 5-dpf larval eyes [mean ± SEM, n = 4 independent samples analyzed for each genotype; *P < 0.05, Tukey’s multiple comparison test (D), Student’s t test (E)]. n.s., not significant. (F) Expression pattern of cone opsin genes examined by in situ hybridization using the 5-dpf larval eyes. Magnified view of the photoreceptor layer (box surrounded with white lines) is indicated on the left side of each panel. The retinal pigmented epithelium is indicated by asterisks. d, dorsal side; v, ventral side. (Scale bar: 50 μm.) (G) Related to F. Quantification of sws2- or rh2-1/2–positive photoreceptors in the central retina (mean ± SEM; *P < 0.05, Tukey’s multiple comparison test). The numbers of fish used for quantification are indicated in the bar graph.
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