Fig. 4

Otx function is required for pinealogenesis. Wild-type zebrafish embryos are shown in A,C,E,G,I and otxH MO-injected embryos in B,D,F,H,J (anterior is leftwards, lateral views are shown in A-H, dorsal views in I,J). Asterisks mark the location of the pineal organ in A-H. (A,B) The pineal organ is morphologically reduced in otxH embryos at 48 hpf. (C,D) Immunohistochemical staining for opsin at 48 hpf; staining is absent from the pineal area in D. (E,F) Double in situ hybridisation for otx5 and shh (both in blue) at 24 hpf. otx5-positive pineal progenitors are absent in F. Arrows indicate the zona limitans intrathalamica; this signalling centre is reduced in F. (G,H) In situ hybridisation for otx5 at the eight-somite stage, including dorsal views of the pineal region shown in the insets. (I,J) In situ hybridisation for flh/noto at bud stage; flh/noto-positive pineal progenitors are absent in J (arrow in J indicates persistent flh/noto staining in notochord). Each marker was tested in two independent experiments, comparing at least 30 control and otxH MO-injected embryos for each marker and stage. Images are representative of all analysed embryos. Scale bar: 50 µm.