Fig. 3. Phenocopy of the ntn mutation by a cct3 antisense morpholino oligonucleotide and phenotypic rescue of ntn mutant embryos by the cct3 mRNA injection. Broken lines demarcate the tectum (tec). (A) Dorsal view of wild-type (wt) and mutant (ntn) embryos at 4 dpf. An asterisk indicates the turbid tectum. White and black arrowheads indicate small eyes with protruding lens and poor pectoral fins, respectively, in the mutant embryo. An arrow indicates the reduced jaw. (B) Acridine Orange staining of wild-type (wt) and mutant (ntn) embryos at 36 hpf. An arrowhead indicates significant staining signals in the tectum of an ntn mutant embryo. (C) Confocal optical section images of the tectum of Bodipy-ceramide-stained wild-type (wt) and mutant (ntn) embryos at 46 hpf. An arrow indicates patches of dead cells. (D) Confocal composite images of anti-acetylated tubulin immunostaining of wild-type (wt) and mutant (ntn) embryos at 48 hpf. An arrowhead indicates absence of staining signals in the tectum of the mutant embryo. (E) Confocal composite images of the retinae of anti-acetylated tubulin immunostained wild-type (wt) and mutant (ntn) embryos at 48 hpf. An arrow indicates decreased numbers of RGCs and their axons in the mutant embryo at 48 hpf. (F) Dorsal view of cct3 antisense and control morpholino oligonucleotide-injected embryos at 4 dpf. White and black arrowheads indicate small eyes with protruding lens and poor pectoral fins, respectively, in the antisense-treated embryo. An asterisk indicates the turbid tectum. An arrow indicates the reduced jaw. (G) Acridine Orange staining of cct3 antisense and control morpholino oligonucleotide-injected embryos at 36 hpf. An arrowhead indicates significant staining signals in the tectum of the antisense-treated embryo. (H) Confocal optical section images of the tectum of Bodipy-ceramide-stained cct3 antisense and control morpholino oligonucleotide-injected embryos at 48 hpf. An arrow indicates patches of dead cells. (I) Confocal composite images of anti-acetylated tubulin immunostaining of cct3 antisense and control morpholino oligonucleotide-injected embryos at 48 hpf. An arrowhead indicates the absence of staining signals in the tectum of the antisense-treated embryo. (J) Confocal composite images of the retinae of anti-acetylated tubulin immunostaining of cct3 antisense and control morpholino oligonucleotide-injected embryos at 48 hpf. An arrow indicates decreased numbers of RGCs and their axons in the antisense-treated embryo at 48 hpf. (K) Acridine Orange staining of cct3 mRNA- and mock-injected ntn embryos at 36 hpf. An arrowhead indicates the absence of staining signals in the tectum of the treated ntn embryo. (L) Expression of nAChRß3 gene promoter-driven EGFP in cct3 mRNA- and mock-injected ntn embryos at 48 hpf. An arrowhead indicates the recovery of fluorescent signals in RGCs of a cct3 mRNA-injected ntn embryo. (M) Genotyping of a wild-type sibling (lane 1), cct3 mRNA- (lane 2) and mock-injected (lane 3) ntn embryos using primers flanking the 143 bp deletion in the cct3 gene. Lane M shows 100 bp DNA ladder as size markers. Black and white arrowheads on the right indicate the 221 bp and 78 bp PCR products representing the intact and deleted cct3 genes, respectively. ac, anterior commissure neurons; dlf, dorsal longitudinal fasciculus; p, pituitary gland; rgc, retinal ganglion cells; teg, tegmentum; tg, trigeminal ganglion. Scale bars: 50 µm.