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First published online 31 March 2004
doi: 10.1242/dev.01085

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Identification of chaperonin CCT subunit as a determinant of retinotectal development by whole-genome subtraction cloning from zebrafish no tectal neuron mutant

Department of Molecular Neurobiology and Pharmacology, Graduate School of Medicine, University of Tokyo, and SORST, Japan Science and Technology Agency, Tokyo 113-0033, Japan

View larger version (38K): [in a new window]   Fig. 1. Whole-genome subtraction between ntn mutant embryos and wild-type siblings by RDA. (A) Analysis of RDA products by agarose gel electrophoresis. Driver and tester amplicons were prepared by digestion of genomic DNA from wild-type and ntn mutant embryos using BglII, EcoRI, HindIII, SpeI and XbaI restriction endonucleases, respectively. Lane M, 100 bp DNA ladder as size markers; lane D, driver amplicons; lane T, tester amplicons; lanes 1-4, RDA products of the first, second, third and fourth rounds of subtraction, respectively. (B) Southern blot hybridization analysis of genomic DNA using RDA product E340 as a probe. Genomic DNAs from a wild-type fish of AB strain (lane 1), wild-type siblings (lane 2) and ntn mutant embryos (lane 3) were digested with EcoRI and hybridized to E340 probe. The sizes of markers in kb are indicated on the left. Black and white arrowheads on the right indicate the 3.1 kb and 0.3 kb EcoRI-digested DNA fragments representing polymorphic alleles, respectively. (C) An example of amplicon Southern blot hybridization analysis. Amplicons were prepared from BglII-digested genomic DNA from pools of five wild-type (lane 1) or ntn mutant (lanes 2-10) embryos. Blots were hybridized to B460 (upper) or B470 (lower) probe. Black and white arrowheads indicate fragments in the amplicons hybridizing to B460 and B470 probes, respectively. (D) Amplicon Southern hybridization mapping of the ntn locus using polymorphic RDA products. Amplicons prepared from 93 pools of five ntn mutant embryos were hybridized to respective RDA products. Black and white boxes indicate the presence and absence of amplicons hybridizing to RDA products, respectively. The numbers of pools containing recombinant(s) are given below. (E) Genetic and physical maps of the zebrafish ntn region on the linkage group 16. Numbers in the genetic map (upper) indicate genetic distances in cM between the markers. Physical map around the ntn locus is shown below with markers. Distances in the map are approximately to scale. Short horizontal lines below the physical map indicate YAC, PAC and BAC clones. BAC clones, 82P21, 72A23 and 105K9, were isolated by screening with C18orf1-like cDNAs as probes. B18S is a marker derived from the Sp6 end of BAC 18M9.

View larger version (63K): [in a new window]   Fig. 2. Identification of an internal deletion in the ntn mutant cct3 gene and expression of the cct3 mRNA in wild-type embryos during development. (A) A 143 bp internal deletion in the cct3 transcript. The zebrafish CCT subunit is schematically shown above; the equatorial, apical and intermediate regions are indicated in boxes. The nucleotide sequences of a putative ATPase motif in the wild-type (wt) and mutant (ntn) cct3 transcripts are shown below. The 143 bp deletion corresponding to the nucleotide residues 1205-1347 of the zebrafish cct3 transcript is in the coding sequence for a putative ATPase motif of CCT. The deletion causes frame-shift and aberrant termination of translation. (B) RT-PCR analysis of the cct3 gene transcripts from pools of 1190 wild-type embryos of the AB strain (AB), 650 wild-type siblings (wt) and 200 mutant (ntn) embryos with (+) or without (–) a reverse transcriptase using primers flanking the 143 bp deletion. 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 intact and deleted transcripts, respectively. (C) PCR analysis of genomic DNA from pools of 100 wild-type embryos of AB strain (AB), 300 wild-type siblings (wt), 100 mutant (ntn) and three individual mutant (N151, N150 and S20) embryos using primers flanking the 143 bp deletion. 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. (D) Whole-mount in situ hybridization analysis of cct3 mRNA at 0.2 hpf (one-cell stage), 6 hpf (shield stage), 9 hpf (90%-epiboly stage), 12 hpf, 24 hpf and 36 hpf. A section through the retina was shown for embryos at 36 hpf. mhb, midbrain-hindbrain boundary; ov, optic vesicle. Scale bars: 100 µm.

View larger version (87K): [in a new window]   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.

View larger version (89K): [in a new window]   Fig. 4. Expression patterns of developmental landmark genes in wild-type (wt, upper panels) and ntn mutant (lower panels) embryos at 30 hpf. Lateral views of embryos stained by in situ hybridization with (A) antisense zash1a probe; (B) antisense zash1b probe; (C, left) antisense hlx1 probe; (D) antisense dlx2 probe; (E) antisense krox20 probe; (F) antisense shh probe; (G) antisense pax2a probe; (H) antisense ntl probe. (I) antisense myod probe. (C, right) Dorsal view of embryo stained by in situ hybridization with antisense hlx1 probe. Arrowheads in D indicate pharyngeal arches; arrowheads in G indicate pronephric ducts. Scale bars: 100 µm in A-D,F; 50 µm in E,G-I.

View larger version (107K): [in a new window]   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.

View larger version (77K): [in a new window]   Fig. 6. Impairment of tectal development in ntn mutant embryos. (A) Confocal optical section images of the tectum of Bodipyceramide stained wild-type (wt) and mutant (ntn) embryos at 28 hpf, 36 hpf and 40 hpf. An arrow indicates cell-free spaces. Arrowheads point large and round cells representing mitotically active cells. (B) TUNEL staining of coronal sections through the tectum of wild-type (wt) and mutant (ntn) embryos at 30 hpf, 36 hpf and 40 hpf. An arrow indicates TUNEL-positive cells. (C) Confocal composite images of immunostaining with anti-acetylated tubulin antibody of wild-type (wt) and mutant (ntn) embryos at 30 hpf, 36 hpf and 40 hpf. An arrow indicates the absence of tectal neuropil. (D) In situ hybridization of brn3b mRNA in wild-type (wt) and mutant (ntn) embryos at 48 hpf. Dorsolateral view. Arrows indicate the tectum. Scale bars: 50 µm in A,C; 100 µm in B,D.