S3 B), suggesting that the loss of cadherin-23 also alters the architecture of photoreceptor cells. lead to several abnormal RT-PCR products (asterisks) because of intron retention, exon skipping, and the use of alternative splice sites. The overall reduction in the amount of the RT-PCR products is probably because of the nonsense-mediated decay of Amadacycline methanesulfonate incorrectly spliced mRNAs. Abnormal transcripts and nonsense-mediated decay were not detected in control larvae microinjected with five-base mismatch control oligonucleotide. Ornithine decarboxylase (morphant larvae have altered mechanoelectrical transduction channels and photosensory deficits. (A) Uptake of FM1C43 dye by the hair cells of cranial and caudal neuromasts (high magnification of a neuromast, with 6C12 sensory hair cells shown). Dye uptake in neuromasts (some are outlined) is much weaker in 3-dpf morphants than it is in controls: 120.6 15.5 m2 in morphants (mean SEM; = 33), versus 471.2 37.5 m2 in controls (= 21; unpaired test, ***, P 0.0001). (B, top left) Representative electroretinogram traces (flash intensity increasing from bottom to top). (bottom left) The time-to-peak values obtained for the a- and b-waves (implicit times) did not differ significantly between controls and morphants. (right) Photoresponse curves (V, V), plotted as a function of flash intensity and fitted with the NakaCRushton function, in morphants and controls. The responses show a significant attenuation in morphants (comparison of fits by the least-squares method: P 0.0001 for both waves), whereas after normalization by the maximum value = 7 controls, 13 morphants). Bars, 20 m. Open in a separate window Figure 3. Protocadherin-15 is located at the calyceal processes of photoreceptor cells in larvae. (A) In 4 dpf larvae, protocadherin-15 (green) is located around the base of the lectin-labeled (white) rod (R) and cone (C) outer segments (OS). Protocadherin-15 colocalizes with F-actin (red) that fill the calyceal processes (CPs). (B) In pre-embedding immunogold electron micrographs, silver-enhanced immunogold particles showed protocadherin-15 to be localized at the CPs surrounding the rod (top) and cone (bottom) OS. Sparse gold particles also decorate the lamellar membrane in cones (asterisks). (C) Protocadherin-15 immunolabeled gold particles (arrowheads) were localized with filaments connecting the CPs to the OS plasma membrane in rods (top) and cones (bottom). Bars: (A) 10 m; (A, SEM image) 5 m; (B) 200 nm; (C) 100 nm. Open in a separate window Figure 4. does not affect retinal morphogenesis. (A) Semithin sections of control and morphant retinas at 4 dpf. The retinal layer shows similar organization in morphant and control retina. However, a loss of alignment and shape alterations of the photoreceptor outer segments (OS) are seen in the morphants (see high magnification of the boxed areas). The outer retina is significantly thinner in the morphants: thickness of the OS, outer nuclear layer (ONL), and outer plexiform layer (OPL) (= 3C4; unpaired test, Rabbit Polyclonal to CLIP1 **, P = 0.005. The ratio of the number of OS profiles (= 5; unpaired test, ****, P = 0.0005). (B) Amadacycline methanesulfonate Cryosections of 4 dpf retinas stained with antibodies against rhodopsin (magenta) and cone opsin (green) show no evidence of opsin mislocalization in the morphant retina, but the shape and organization of both cones and rods are altered. INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Bars, 20 m. Open in a separate window Figure 5. The photoreceptor outer segments are misaligned in morphant larvae. 3D rendering of the confocal stacks obtained from acrylamide-embedded vibratome Amadacycline methanesulfonate sections (150 m thick) stained with fluorescent lectin (top) and from SEM micrographs (bottom), highlighting the orderly arrangement of the subretinal space in 4 dpf control retinas (left). In contrast, age-matched morphant retinas contain photoreceptors with misshapen, curved, and misaligned outer segments (OS; middle). The coinjection into the embryo of cRNAs encoding the protocadherin-15 CD1 and CD3 isoforms with the splice-blocking morpholinos essentially preserved the well-ordered organization and parallel alignment of the OS in the larva (right). IS, inner segment, C, cone, R, rod. Bars, 10 m. Open in a separate window Figure 6. causes distinct morphological alterations to the rod and cone outer segments and the associated calyceal processes (scanning electron microscopy.