Retinoic acid treatment during retinal neurogenesis does not result in photoreceptors of mixed phenotype. Control embryos were treated with DMSO (A, C), or 0.3 μM RA (B, D to F) at 36 hpf, and at 60 hpf were processed as 5 μm cryosections for indirect immunofluorescence with rod- and cone-directed markers. Boxed regions in each panel appear at higher magnification in the insets. (A, B) Embryos were stained with a polyclonal anti-red opsin antibody (red color, stains red opsin), and monoclonal antibody 1D1 (green color; specific for rod opsin). Sections from control (A) or from RA-treated (B) embryos showed no evidence of colabeling. (C, D) Embryos were stained with zpr1 (red color, labeling red and green-sensitive cones) and a polyclonal anti-rod opsin antibody (green color, stains rods as well as green cones). In control embryos (C), there are singly- as well as doubly-labeled cells, indicating the likely presence of red cones (red color), green cones (yellow color; arrowheads), and rods (green color, arrows). (D) In embryos treated with RA, there is reduced staining of red cones and green cones and more intense staining for rods. (E, F). In an RA-treated embryo, cells positive for zpr1 (E) and the rod/green cone marker zpr3 (F) are found in ectopic locations (arrowheads). IPL, inner plexiform layer. Bar = 50 μm.