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Fig. 3 The PPR specifier genes dlx3b/dlx4b in conjunction with flh/noto are required and sufficient for pineal progenitor specification. (A-D) 24 hpf zebrafish embryos injected with the MOs indicated in B-D and stained for the pineal marker otx5 in red (lateral views, anterior is leftwards). There is reduced otx5 staining in B and C, and a complete absence in D. Scale bar: 50 µm. MO injection experiments were performed three times independently, injecting each MO or combination of MOs in parallel into 40-50 embryos from the same parents. Images are representative for each experimental condition. Fifty percent of the embryos injected with dlx3b/4b MOs looked as in C, the other 50% had a range of even weaker otx5 staining. (E,F) Injection of dlx3 and flh mRNAs at the one-cell stage results in ectopic induction of the pineal marker otx5 (blue) after 24 h of incubation (n=21/32; control embryo shown in E; two independent experiments). Inset in F is an image focusing on the ectopically induced otx5-expressing cells that are marked with the arrow in F. Scale bar: 50 µm. (G,H) Lateral views of embryos injected at the one-cell stage with hs:flh/noto and hs:dlx4b, heat-shocked at shield stage and stained by in situ hybridisation for otx5 (red) after 24 h of incubation. There is sparse ectopic induction of otx5 in superficial cells (arrowheads). Scale bars: 50 µm (bar in A applies to A-D; bar in E applies to E-H). (I) Quantification of otx5 induction in cells injected with hs:gfp alone, with hs:flh/noto and hs:dlx4b, or with hs:flh/noto alone (two independent experiments; six embryos were selected for statistical analysis; data are mean±s.d.). P<0.0001 (GFP versus flh+dlx), P<0.0001 (GFP versus flh), P=0.041 (flh+dlx versus flh).