Fig. 5. Impairment of retinal development in ntn mutant embryos. (A) Confocal optical section images through the retina of Bodipy-ceramide stained wild-type (wt) and mutant (ntn) embryos at 27 hpf, 36 hpf and 48 hpf. An arrow indicates cell-free spaces. (B) TUNEL staining of retinal sections of wild-type (wt) and mutant (ntn) embryos at 30 hpf, 36 hpf and 48 hpf. (C) Immunostaining with zn5 antibody (green) of retinal sections of wild-type (wt) and mutant (ntn) embryos at 30 hpf, 36 hpf and 48 hpf. Nuclei of retinal cells were counterstained with Sytox (red). (D) Confocal composite images of nAChRß3 gene promoter-driven EGFP signals in wild-type (wt) and mutant (ntn) embryos at 30 hpf, 36 hpf and 48 hpf in coronal view and those at 48 hpf in lateral view. Wild-type embryos have RGCs over the entire retina, whereas ntn mutants have sparse RGCs. Note that ntn mutants show EGFP signals in the trigeminal ganglion and Rohon-Beard sensory neurons and the pituitary gland. dlf, dorsal longitudinal fasciculus; on, optic nerve; p, pituitary gland; rb, Rohon-Beard neurons; tg, trigeminal ganglion. (E) Confocal composite images of immunostaining with anti-acetylated tubulin of retinal sections of wild-type (wt) and mutant (ntn) embryos at 30 hpf, 36 hpf and 48 hpf. Arrows indicate RGC axons. (F) In situ hybridization of ath5 mRNA in wild-type (wt) and mutant (ntn) embryos at 33 hpf. Ventral view. (G) In situ hybridization of brn3b mRNA in wild-type (wt) and mutant (ntn) embryos at 36 hpf and 48 hpf. Ventral view. Arrowheads indicate the RGC layer. Scale bars: 50 µm in A-C; 100 µm in D-G.