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First published online 5 January 2005
doi: 10.1242/dev.01591

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Duplicated genes with split functions: independent roles of protocadherin15 orthologues in zebrafish hearing and vision

¹ Max Planck Institut fur Entwicklungsbiologie, Spemannstrasse 35, 72076 Tübingen, Germany
² Brain Research Institute, University of Zurich and Department of Biology, Swiss Federal Institute of Technology Zurich, Winterthurerstr. 190 8057 Zurich, Switzerland
³ Oregon Hearing Research Center and Vollum Institute, Oregon Health and Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97239, USA

View larger version (94K): [in a new window]   Fig. 2. Expression pattern of pcdh15a using in situ hybridization. (A-E) pcdh15a expression in embryos and larvae. At the four-somite stage (A), pcdh15a is expressed in the anterior neuroectoderm and in the somites. At 24 hpf (B), there is expression in the eye, the hatching gland (blue shading under the eye, out of focus) and the first hair cells of the ear at the margins of the otic vesicle (arrowhead and panel C). At 3 dpf (D), expression of pcdh15a is not detectable in the eye, whereas expression is present in the ear (asterisk), the brain and the lateral parts of the epiphysis (arrowheads). At 4 dpf (E), expression is present in the hair cells of the ear (arrows indicate the posterior maculae present in this focal plane) and the neuromasts (arrowheads). (F) Higher magnification view of the pcdh15a-positive hair cells of the anterior macula. (F') Higher magnication view of a trunk neuromast. Scale bar in F': 180 µm for A,B; 50 µm for C; 120 µm for D; 150 µm for E; 30 µm for F; 20 µm for F'.

View larger version (16K): [in a new window]   Fig. 1. Zebrafish have two pcdh15 genes. (A) Cloning of pcdh15a. The orbiterth236b locus maps to chromosome (LG) 13 between the simple sequence length polymorphic (SSLP) markers z26404 and z25253. The critical interval was determined by generating SSLP markers from overlapping BAC and PAC clones and testing for recombination events in larvae identified with the south marker z26404 (indicated by arrowheads pointing to the right) or north marker z25253 (arrowheads to the left). Sequencing of six clones spanning the contig identified exons of the gene pcdh15a. No other gene was present. (B) Schematic diagram of the Pcdh15a protein. The mutations identified in orbiter are indicated below. (C) Comparison of the predicted extracellular and intracellular domains of zebrafish Pcdh15a and Pcdh15b with the human and mouse PCDH15 proteins. Similarity of the extracellular regions is in the upper right half of the table; the intracellular regions are in the lower left half, shaded in grey. Extracellular domains of human, mouse, and zebrafish Pcdh15 are more conserved than the intracellular domain. (D) Phylogeny of human, mouse, and zebrafish Pcdh15 proteins are shown. The scale indicates phylogenetic distance by substitution events.

View larger version (103K): [in a new window]   Fig. 3. Temporal expression pattern of pcdh15b in differentiating photoreceptor cells. (A) At 2.5 dpf, pcdh15b is expressed in the first differentiating photoreceptors at the ventral margin of the eye (arrowhead). (B) At 3 dpf, the whole proximal optic cup is stained. (C) At 5 dpf, expression is only present at the margin of the optic cup, no expression is visible in the proximal eye. (D) Expression in the epiphysis (arrow) and brain at 3 dpf. pcdh15b expression in the eye is specific to photoreceptor cells (E,F; cryosection of a larva stained as in C) Arrows in (E) indicate photoreceptor proliferation zones shown at higher magnification in (F). Scale bar in F, 50 µm for A; 40 µm for B,C,E; 150 µm for D; and 5 µm for F. IS, inner segment; OS, outer segment.

View larger version (89K): [in a new window]   Fig. 4. orbiter mutants have a splayed hair-bundle phenotype. (A-D) The actin of the mechanosensory hair bundles of the lateral cristae were labeled with Alexa488-Phalloidin, and visualized in transverse sections with a confocal microscope. In wild-type larvae (A), the bundles are conical and intact in comparison to bundles in orbiterth263b (B). A similar phenotype is visible tc256e bundles (C), but many intact bundles are still present. A null allele of mariner/myosin VIIA (D) causes a more severe phenotype than th263b (B). (E,F) Horizontal optical sections of lateral line hair bundles. The unstained insertion point of the kinocilium is located at the caudal or rostral margin of the hair cells. No differences in planar polarity were observed between wild-type or th263b bundles as indicated with arrows (pointed toward each kinocilium) in (E') and (F'). Scale bar: 1 µm.

View larger version (21K): [in a new window]   Fig. 5. Behavioral analysis and ERG recordings of pcdh15b morphants. (A) Optokinetic responses were measured as a function of spatial frequency of a moving square pattern. Larvae injected with 40 ng ATG Mo (filled circles) have reduced contrast sensitivity and visual acuity compared with control injected larvae (filled squares). Larvae injected with 30 ng ATG Mo (open circles) show a slightly reduced visual performance. (B) pcdh15b knock-down results in significantly reduced retinal sensitivity to light under dark- and light-adapting states. The ERG b-wave amplitudes in the dark-adapted (B) and light-adapted state (C) are plotted as a function of relative light intensity. Uninjected larvae (open squares), larvae injected with control MO (closed squares), 40 ng ATG Mo (closed circles) and 16 ng GT Mo (triangles) were analyzed. Averaged data are plotted with error bars showing mean±s.e.m. (D) RT-PCR analysis of aberrant splicing of pcdh15b transcript. The upper gel contains products amplified with primers flanking exon 4. The lower band is the predicted product without exon 4. The uppermost band presumably includes an extra intronic sequence. The lower gel shows a control reaction with elongation factor -1alpha. Lane 1, GT Mo-injected larvae, day 3; lane 2, uninjected larvae, day 3; lane 3, GT Mo-injected larvae, day 4, lane 4, uninjected larvae, day 5 (pool of 5 larvae per lane). Representative examples of ERG records from four different specimens under dark-adapted (E) and light-adapted states (F). Each trace is an average of 3-7 consecutive responses. Stimulus duration was 100 mseconds. Attenuated light intensity was presented as OD (optical density unit).

View larger version (109K): [in a new window]   Fig. 6. Knock-down of pcdh15b function shows a dose-dependent effect on photoreceptor structure. (A-I) Cryosections of the proximal region of day 4 retinas were stained with Alexa568-Phalloidin (red), and DAPI (blue). All layers are present in the ATG Mo-injected larvae (B,C). The outer segments of rod photoreceptors were stained with zpr-1 antibody (A-F, green) or double cone photoreceptors were stained with rhod-1 antibody (G-I, green). Uninjected larvae (A,D,G), larvae injected with 30 ng ATG Mo (B,E,H), and larvae injected with 40 ng ATG Mo (C,F,I) are shown. Double cone receptors are disorganized and broader in appearance (E,F), and rod outer segments appear shorter and clumped together (H,I). Scale bar in I, 20 µm for A-C; and 5 µm for D-I. gc, ganglion cells; hc, horizontal cells; inl, inner nuclear layer; is, inner segments; os, outer segments; pr, photoreceptors; rpe, retinal pigment epithelium.

View larger version (116K): [in a new window]   Fig. 7. Photoreceptor outer segments are tilted and clumped in pcdh15b morphants. Electron micrographs of day 4 (A-C) and day 7 (D-F) retinas of uninjected or 40 ng pcdh15b ATG Mo-injected larvae. The outer segments are slanted sideways and the nuclear layer is more condensed in morphants (B) in comparison to segments in uninjected larvae (A) or control 4 bp mismatch Mo-injected larvae (A'). In pcdh15b morphants, pigments cell melanosomes are rarely present between photoreceptor outer segments (B,D). The phenotype does not recover at day 7 and outer segments are often clustered together (D). (C,F) Higher magnification views of (B) and (E). Scale bar in F: 1 µm for A,B,D,E; 350 nm for C,F.