Pcdh15 is expressed in the photoreceptors of the zebrafish retina and is reduced in CRISPR-generated pcdh15b mutants. (A) Representative images of the 10 dpf retina stained for Pcdh15 (magenta). Co-label with F-actin stain, phalloidin (green; present in the CPs and synapse) is shown for the central ONL. (B) Close up of a central region in the ONL stained for Pcdh15 (magenta), phalloidin (green) and an overlap of the two. (C,D) Close up of the co-staining for Pcdh15 and phalloidin in the CPs (C) and for Pcdh15 at the IS-OS junction (D). (E) Representative images of SV2 staining (yellow) and calcium channel 1.4v staining (Cacna1fa; magenta) in the photoreceptor synapse and colocalization with Pcdh15 and phalloidin (green) at 10 dpf. (F) Schematic of Pcdh15 expression in the zebrafish photoreceptor cell. Green represents phalloidin in the synapse and CPs; magenta represents Pcdh15 in the CPs, OS base, IS membrane and synapse; blue dots represent calcium channel 1.4v in the synapse. (G) gRNA used to target and mutate the pcdh15b gene. A 17 bp insertion (magenta nucleotides) and a 7 bp deletion (black nucleotides) mutant were identified. (H) Predicted results of the mutations on the protein sequence. (I) RT-qPCR to analyse pcdh15a (left) and pcdh15b (right) mRNA expression, normalized to actin, in wild-type siblings and pcdh15b mutants. (J) Staining for Pcdh15 in wild-type siblings (+/+) and mutants (Δ7/Δ7 and ins17/ins17) at 10 dpf (n=5+ for each genotype). (K) Genotyping of clutches of heterozygous crosses up to adulthood to measure survival [numbers of fish genotyped (n) are indicated]. CD, variable cytoplasmic domain; CP, calyceal process; EC, extracellular cadherin domains; GCL, ganglion cell layer; INL, inner nuclear layer; IS, inner segment; ONL, outer nuclear layer; OS, outer segment; TD, transmembrane domain.
