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First published online 21 January 2004
doi: 10.1242/dev.00961

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The deafness gene dfna5 is crucial for ugdh expression and HA production in the developing ear in zebrafish

¹ Max-Planck-Institut für Entwicklungsbiologie, Spemannstr. 35, 72076 Tübingen, Germany
² Centre for Developmental Genetics, Department of Biomedical Science, University of Sheffield, Firth Court, Sheffield S10 2TN, UK
³ Oregon Hearing Research Center and Vollum Institute, Oregon Health & Science University, Portland, OR 97201, USA

View larger version (34K): [in a new window]   Fig. 1. Protein alignment of zebrafish, human and mouse Dfna5. Black shading indicates identity and gray shading indicates similarity. The overall identity of zebrafish compared with human and mouse Dfna5 is 33% and 30%, respectively. Similarity of zebrafish compared with human and mouse Dfna5 is 51% and 49%, respectively.

View larger version (13K): [in a new window]   Fig. 2. Target sites of the dfna5 morpholinos and aberrant splicing of dfna5 transcript. (A) Genomic organization of human DFNA5 and zebrafish Dfna5. The exon/intron boundaries of DFNA5 are entirely conserved between human and zebrafish. (a) Individuals with DFNA5 mutations carry an insertion/deletion mutation in intron 7, which leads to skipping of exon 8 (indicated in red). Absence of exon 8 causes a frameshift after amino acid 330, resulting in an aberrant stretch of 41 amino acids followed by a premature stop. (b) The two morpholino antisense oligos designed against Dfna5 mRNA are indicated in red. The splice site antisense oligo ('GT-MO') directed against the donor site of exon 8 leads to skipping of the targeted exon (indicated in red), resembling the human mutation. (B) RT-PCR time course of aberrant splicing of dfna5 mRNA caused by the dfna5 GT-MO. At 28 hpf, wild-type dfna5 transcript is not detectable by RT-PCR, only PCR products lacking exon 8 (indicated with exon 8) or with partial retention of the following intron (arrowhead) are present. Wild-type transcript recovers starting at day 2, but the morpholino-modulated transcripts are still present at day 7.

View larger version (97K): [in a new window]   Fig. 3. Expression of dfna5 in zebrafish embryos using whole-mount in situ hybridization. (A,B) 22 hpf embryo. Lateral (A) and dorsal view of the head region (B), showing expression in the intermediate cell mass (arrowhead in A), the olfactory placodes (arrows), ventral diencephalon and migrating neural crest (arrows in B). (C,D) 48 hpf embryo (C) and 55 hpf embryo (D) with expression in the developing semicircular canals of the ear (arrowheads). (E) 72 hpf embryo indicating low level of expression in the mature projections of the semicircular canals in the ear (arrowheads). (F) Dorsal view of the ear at 48 hpf with expression at the tip of the outgrowing projections of the semicircular canals (arrowheads). Scale bar: 300 µm in A,B; 100 µm in C-E; 40 µm in F.

View larger version (119K): [in a new window]   Fig. 4. Reduction of dfna5 activity leads to abnormal ear and cartilage development. (A-C) Lateral views of wild-type (A), 15 ng dfna5 ATG-MO-injected (B) and 25 ng dfna5 ATGMO-injected (C) embryos, indicating a dose-dependent malformation of the lower jaw (arrows). (D,E) Dorsal view of wild-type (D) and 20 ng dfna5 ATG-MO-injected (E) embryos at day 5, showing malformation of the anterior and posterior column in the morphant ear (arrows). (F,G) Close up of the right ear in D and E, respectively. (G) The anterior column (ac, arrow) of the morphant is malformed, the posterior column (pc, large arrows) is interrupted. (H-J) Lateral view of the ear at day 5 (anterior towards the left, ventral towards the bottom) of wild type (H), 15 ng dfna5 ATG-MO injected (I) and 25 ng dfna5 ATG-MO injected (J) embryos. In morphants, either one or more columns do not fuse (arrows), depending on the injected dose of dfna5 ATGMO. ao, anterior otolith; po, posterior otolith. Scale bar: 300 µm in A-C; 250 µm in D,E; 80 µm in F,G; 125 µm in HJ. ac, anterior column; pc, posterior column.

View larger version (80K): [in a new window]   Fig. 5. Specificity of the ATG morpholino-induced phenotype. (A) Dose dependency of the observed phenotype. (B) Injection of up to 25 ng 4 bp mismatch morpholino does not cause any mutant phenotype. (C,D) Partial rescue of the morphant phenotype by co-injection of dfna5 DNA. Dorsal (C, anterior towards the top) and lateral (D, anterior towards the right) view of one d5 larva injected with 25 ng dfna5 ATG-MO and 70 pg dfna5 pCS2+. Left ear (C) and jaw (D) show malformations typical for injections of relatively high doses of ATG-MO, whereas the right ear is rescued and shows no morphological abnormalities. The anterior and posterior columns are properly fused, which is never observed in larvae injected with 25 ng ATG-MO only. Scale bar: 130 µm in B,C; 200 µm in D.

View larger version (132K): [in a new window]   Fig. 6. Histological analysis of the inner ear epithelial columns in dfna5 morphants. Cross-sections of lateral semicircular canals at day 6 of wild-type (A,C) and dfna5 GT-MO injected (B,D) embryos. (A,B) Toluidine Blue staining of 5 µm sections, indicating a loss of the monolayer of the epithelium (A,B, arrow) and the basal lamina (A,B, arrowheads) in morphants (B) compared with wild type (A). (C,D) Structure of the basal lamina in TEM cross-sections. (C) The wild-type lamina has tightly packed, parallel fibers. (D) dfna5 morphant basal lamina is disrupted with loose, disorganized fibers. Scale bar: 20 µm in A,B; 1 µm in C,D.

View larger version (67K): [in a new window]   Fig. 7. Abnormal jaw development in dfna5 morphants. (A-E) Alcian Blue staining of cartilage. (A,C) Lateral (A) and ventral (C) view of a day 5 wild-type larval head. (A) Meckel's cartilage is indicated with a white arrowhead. In C, the ceratobranchials are indicated with 1-5. (B,D,E) Lateral (B) and ventral (D,E) view of a day 5 morphant larval head. (B) Meckel's cartilage (arrowhead) is malformed and the ceratohyal cartilage is inverted, most probably owing to reduction of the branchial cartilages. (E) Cartilage derived from brachial arches 1-5 are present, but strongly reduced. The ceratobranchial cartilage in morphants is less intensely stained than in wild-type. Scale bar: 125 µm in A-D; 200 µm in E.

View larger version (58K): [in a new window]   Fig. 8. In situ analysis of cartilage differentiation in dfna5 morphants. (A,D) Lateral view of col2a1 expression at 55 hpf. White arrows indicate the otic vesicle; white arrowheads indicate pharyngeal arches. Expression in the morphant otic vesicle (D) is unaffected compared with wild type (A). (B,C,E,F) ugdh expression in wild-type (B,C) and morphant (E,F) 55 hpf embryos. (B,E) Lateral view. (E) Expression in the developing morphant ear columns (indicated on wild type) and pharyngeal arches (white arrowhead) is reduced compared with wild-type (B). (C,F) Ventral view of expression in the developing neurocranium reveals no difference between wild-type and morphant embryos (arrows in C and F). Scale bar: 200 µm. c, column; e, eye; fb, fin bud; nc, neurocranium; ov, otic vesicle; pa, pharyngeal arches.

View larger version (54K): [in a new window]   Fig. 9. Reduction of hyaluronic acid (HA) in the developing semicircular canals in dfna5 morphants (54 hpf larvae). (A,D) Outline of the structures in a dorsolateral view of wild-type (B,C) and dfna5 ATG-MO injected larval ears (E,F). The differential interference contrast (DIC) and corresponding fluorescent images are focussed at the level of the anterior column. An outgrowing protrusion of the anterior column (ac) in the wild-type ear (B) is filled with HA as shown by staining with biotinylated HA-binding protein (C). The lateral (lc) and posterior columns (pc) are out of focus in C. HA is reduced in both protrusions of the anterior column in a dfna5 ATG-MO injected larval ear (E,F). Scale bar: 35 mm.

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