Figures and data
Evolutionarily conserved localization of pcdh15 and cdhr1 in photoreceptors predicts interactions linking outer segments and calyceal processes.
A-A”) Confocal microscopy of wildtype 5dpf retinal cryosections probed with cdhr1a antibody (green), Peanut germ agglutinin (PNA - magenta) to label red-green cones, and pcdh15b (red). White boxes indicate the location of the inset enlargement. White arrowheads highlight the linear localization of cdhr1a along cone OSs (A) and pcdh15b outlining the calyceal process (A’). Merge of all three signals highlights the proximity between cdhr1a and pcdh15b (A”). B=blue cones, RG=red/green cones, UV=UV cones. Scale bar = 5μm. B-B”) Confocal microscopy of wildtype 5dpf retinal cryosections probed with cdhr1a antibody (green), pcdh15b (red) and wheat germ agglutinin (WGA -teal) to label rods. White boxes indicate the location of the inset enlargement. White arrowheads highlight the linear localization of cdhr1a along rod OSs (B) and pcdh15b outlining the calyceal process (B’). Merge of all three signals highlights the proximity between cdhr1a and pcdh15b (B”). Scale bar = 10μm. C-E) Structured illumination microscopy (SIM) of 5 dpf wildtype zebrafish retinal cryosections probed with C) cdhr1a (green) and pcdh15b (red), D) pcdh15b (red) and actin (white) and E) pcdh15b (red) and actin (white). White boxes represent the inset enlargement. White arrowheads highlight the juxtaposition or overlap between the cdhr1a, pcdh15b and actin signals. Scale bar = 2.5μm. F-H) Structured illumination microscopy (SIM) of wildtype zebrafish whole mount 5 dpf retina in transverse orientation (OS outlined in magenta) probed with F) cdhr1a (green) and pcdh15b (red), G) cdhr1a (green) and actin (white) and H) pcdh15b (red) and actin (white). White boxes represent the inset enlargement. Scale bar = 2.5μm. I) Immuno-gold-TEM detection of cdhr1a (green) and pcdh15b (red) in 5 dpf wildtype retinal sections. The CP is outlined in red and the OS in blue. Scale bar = 500nm J) Diagrammatic model of the connection between the OS discs and CPs in both rod and cone cells mediated by the interaction between OS bound cdhr1a and CP bound pcdh15b.
Evolutionarily conserved localization of pcdh15 and cdhr1 in photoreceptors predicts interactions linking outer segments and calyceal processes.
Structured illumination microscopy (SIM) of wildtype xenopus (A), mallard duck (B), macaque (C), human (D), mouse (E), rat (F), spiny mouse (G) and gerbil (H), retinal sections probed for cdhr1 (green), pcdh15 (red) and PNA (magenta) to label cone outer segments. White boxes represent the inset enlargements. White arrowheads highlight the juxtaposition of the cdhr1a and pchd15b signals in each species. Scale bar = 5μm.
Physical interactions between cdhr1a and pchd15b can facilitate cell-cell adhesion.
A) Immunoprecipitation of cdhr1a-FLAG and pcdh15b-MYC performed in HEK293 cell lysates. Pull down of cdhr1a-FLAG using anti-FLAG antibody beads also pulls down pcdh15b-MYC, while pull down of pcdh15b-MYC using anti-MYC antibody beads also pulls down cdhr1a-FLAG. B) Diagrammatic representation of the K562 cell assay for assessing homophilic or heterophilic interactions between cadherins. C) Brightfield microscopy analysis of K562 cell aggregation after transfection with either cdhr1a or pcdh15b or after co-culture of the two transfected populations. Magenta boxes indicate regions enlarged in the insets. D) Quantification of the total number of aggregates observed in a single field of view. E) Quantification of the number of average number of cells in each observed aggregate.
Construction and confirmation of the cdhr1a-/- line.
A) Exon/intron diagram of cdhr1a. The intron 6-exon 6-intron 7 junction is highlighted and the approximate location of the crRNA (black arrows) is depicted. B) Genomic nucleotide sequence from the cdhr1aΔ173 line highlighting the sequence location of the 173 bp deletion compared to WT sequence. C) Amino acid sequence alignment between wildtype and the cdhr1aΔ173 line. Alignment indicates that the 173bp deletion leads to a frameshift and an immediate premature stop codon at AA146. D) Diagrammatic representation of the domain structure of WT cdhr1a protein vs the cdhr1a Δ173 allele. EC = cadherin domain, TD = transmembrane domain, CD = cytoplasmic domain. E) Confocal microscopy of 5 dpf retinal cryosections from WT and cdhr1a-/-retinas stained with anti-cdhr1a antibody (green), PNA (red-green cones. - magenta) and WGA (blue cones -teal). Cdhr1a signal is detected along photoreceptor outer segments in the wildtype and completely missing in cdhr1a-/-. ROS = rod outer segment, RIS = rod inner segment. Scale bar = 5μm.
Loss of cdhr1a function leads to cone outer segment degeneration.
A-G) Confocal microscopy of wildtype retinal cryosections probed with anti-prph2 antibody (green). B=blue cones, RG=red/green cones, UV=UV cones. Scale bar = 10μm. A’-G’) Confocal microscopy of cdhr1a-/- retinal cryosections probed with anti-prph2 antibody (green). A-B’ scale bar = 5μm, C-G’ scale bar = 10μm.. H) Quantification of cone OS length at 5, 15, 30, 90, 180, 360 and 720 dpf measured as length of prhp2 signal in wildtype (black dots) and cdhr1a-/- (blue dots). Standard deviation is shown in red. **** = p<0.0001. ANOVA = p<0.0001. I) Line graph depicting the long-term trend of cone OS length changes between wildtype (black) and cdhr1a-/-(blue). J) Line graph depicting changes in the number of cone cells counted in the observation region over time in wildtype (black) compared to cdhr1a (blue). K) Transmission electron microscopy micrographs from a 15dpf wildtype retina. Yellow rectangle represents the enlarged inset. Magenta arrow heads highlight the proper stacking of OS discs. IS= inner segment, OS = outer segment. Scale bar = 500nm. L) Transmission electron microscopy micrographs from a 15dpf cdhr1a-/- retina. Yellow rectangle represents the enlarged inset. Magenta arrow heads highlight the improper and distorted stacking of OS discs. IS= inner segment, OS = outer segment. Scale bar = 500nm.
Loss of cdhr1a function leads to late onset rod outer segment degeneration.
A-B) Confocal microscopy of wildtype retinal cryosections probed with Gnb1 antibody (magenta) and WGA (teal) to identify rod outer segments at 5 and 15 dpf. Scale bar = 10μm. A’-B’) Confocal microscopy of cdhr1a-/- retinal cryosections probed with Gnb1 antibody (magenta) and WGA (teal) to identify rod outer segments at 5 and 15 dpf. Scale bar = 10μm. C-G) Confocal microscopy of wildtype retinal cryosections probed with prph2 antibody (magenta) and WGA (teal) to identify rod outer segments. A-B’ scale bar = 5μm, C-G’ scale bar = 10μm. C’-G’) Confocal microscopy of cdhr1a-/- retinal cryosections probed with prph2 antibody (magenta) and WGA (teal) to identify rod outer segments. Scale bar = 10μm. H) Quantification of rod OS length at 5, 15, 30, 90, 180, 360 and 720 dpf measured as length of WGA signal in wildtype (black dots) and cdhr1a-/- (blue dots). Standard deviation is shown in red. ** = p<0.001, **** = p<0.0001. ANOVA = p<0.0001. I) Line graph depicting the long-term trend of rod OS length changes between wildtype (black) and cdhr1a-/- (blue). J) Line graph depicting changes in the number of rod cells counted in the observation region over time in wildtype (black) compared to cdhr1a-/- (blue).
Calyceal processes in cones are disorganized in the absence of cdhr1a function.
A-G) Confocal microscopy of wildtype retinal cryosections probed with PNA (magenta) and actin-antibodies (white) at various timepoints detecting cone CPs. Typical length of CPs observed is demonstrated by yellow lines. Scale bar = 5μm. A’-G’) Confocal microscopy of cdhr1a-/- retinal cryosections probed with actin-antibodies (white) at various timepoints detecting cone CPs. Typical length of CPs observed is demonstrated in magenta. H-N) Confocal microscopy of wildtype retinal cryosections probed with actin-antibodies (white) and gnb1 (A’) (magenta) at various timepoints detecting rod CPs. Typical length of CPs observed is demonstrated in yellow. H’-N’) Confocal microscopy of cdhr1a-/- retinal cryosections probed with actin-antibodies (white) and gnb1 (F’) (magenta) at various timepoints detecting rod CPs. Typical length of CPs observed is demonstrated by yellow lines. Scale bar = 5μm. ROS = cone outer segment layer. O) Quantification of cone cells CP length based on measurements of actin staining in wildtype (black) compared to cdhr1a-/- (blue) at 5, 15, 30, 90, 180, 360 and 720 dpf. Standard deviation is shown in red. ** = p<0.01, **** = p<0.0001. P) Quantification of rod cells CP length based on measurements of actin staining in wildtype (black) compared to cdhr1a-/-(blue) at 5, 15, 30, 90, 180, 360 and 720 dpf. Standard deviation is shown in red. ns = not significant, *** = p<0.001, **** = p<0.0001. Q) Line graph depicting the long-term trend of cone cells CP length changes between wildtype (black) and cdhr1a-/- (blue) over time. ** = p<0.01, **** = p<0.0001. R) Line graph depicting the long-term trend of rod cells CP length changes between wildtype (black) and cdhr1a-/- (blue) over time. ns = not significant, *** = p<0.001, **** = p<0.0001. S) Confocal microscopy of wildtype (top panels) and cdhr1a-/- (bottom panels) retinal cryosections probed with pcdh15b antibody (red) at various time points. Scale bar = 5μm. COS = cone outer segment layer, ROS = rod outer segment layer.
Loss of pcdh15b function leads to early cone OS defects and mis-localization of cdhr1a.
A) Diagrammatic representation of the CRISPR/Cas9 strategy for generating a pcdh15b loss of function allele. crRNAs were targeted to the flanking sequences of exon 5 and resulted in a heritable deletion of 68bp which resulted in a frameshift (fs) at AA117 and a premature stop codon (*) at AA118. B-B”) Confocal microscopy of retinal cryosections from wildtype, pcdh15b+/-and pcdh15b-/-individuals probed with pcdh15b antibody (red). Scale bar = 10μm.C-D”) Confocal microscopy of retinal cryosections from wildtype, pcdh15b+/- and pcdh15b-/- individuals probed with prhp2 antibody (green) to visualize the cone outer segments (COS) at 5dpf (C-C”) and 10dpf (D-D”). B=blue cones, RG=red/green cones, UV=UV cones. Scale bar = 10μm.E) Quantification of COS length using prph2 signal for each genotype at each time point is depicted on the right. Standard deviation is shown in red. ns = not significant, ** = p<0.01. **** = p>0.0001. ANOVA = p<0.0001. F-G”) Confocal microscopy of retinal cryosections from wildtype, pcdh15b+/- and pcdh15b-/- individuals probed with actin antibody (white) to visualize cone CPs at 5dpf (F-F”) and 10dpf (G-G”). Scale bar = 10μm.H) Quantification of CP length using actin signal for each genotype at each time point is depicted on the right. Standard deviation is shown in red. ns = not significant, ** = p<0.01. **** = p>0.0001. ANOVA = p<0.0001. I-J”) Confocal microscopy of retinal cryosections from wildtype, pcdh15b+/- and pcdh15b-/- individuals probed with cdhr1a antibody (red) at 5dpf (I-I”) and 10dpf (J-J”). White boxes indicate regions enlarged. Scale bar = 10μm.K) Quantification of cdhr1a OS length for each genotype at each time point is depicted on the right. Standard deviation is shown in red. ns = not significant, *** = p<0.001. **** = p>0.0001. ANOVA = p<0.0001.
Cone phenotypes are exacerbated by simultaneous loss of pcdh15b and cdhr1a.
A-D) Confocal microscopy of retinal cryosections from 5 dpf pcdh15b-/-, cdhr1a-/-, cdhr1a-/- ; pcdh15b+/- and cdhr1a-/- ; pcdh15b-/- larva probed with prph2 antibodies (green). COS = cone outer segment. B=blue cones, RG=red/green cones, UV=UV cones. Scale bar = 10μm. E-H) Confocal microscopy of retinal cryosections from 10 dpf pcdh15b-/-, cdhr1a-/-, cdhr1a-/- ; pcdh15b+/- and cdhr1a-/- ; pcdh15b -/- larva probed with prph2 antibodies (green). COS = cone outer segment. B=blue cones, RG=red/green cones, UV=UV cones. Scale bar = 10μm. I) Quantification of cone outer segment length at 5 dpf based on prph2 signal. ns = not significant, **** =p<0.0001. ANOVA = p<0.0001. J) Quantification of cone outer segment length at 10 dpf based on prph2 signal. ns = not significant, ** = p< 0.01****, =p<0.0001. ANOVA = p<0.0001. K-N) Confocal microscopy of retinal cryosections from 5 dpf pcdh15b-/-, cdhr1a-/-, cdhr1a-/- ; pcdh15b+/- and cdhr1a-/- ; pcdh15b -/- larva probed with actin antibodies (white). COS = cone outer segment. Scale bar = 10μm. O-R) Confocal microscopy of retinal cryosections from 10 dpf pcdh15b-/-, cdhr1a-/-, cdhr1a-/- ; pcdh15b+/- and cdhr1a-/- ; pcdh15b-/-larva probed with actin antibodies (white). COS = cone outer segment. Scale bar = 10μm. R) Quantification of cone CP length at 5 dpf based on prph2 signal. ns = not significant, **** =p<0.0001. ANOVA = p<0.0001. T) Quantification of cone CP length at 10 dpf based on prph2 signal. ns = not significant, * = p< 0.05 ** = p< 0.01****, =p<0.0001. ANOVA = p<0.0001.
Expression of USHR genes in the zebrafish retina.
Whole mount in situ hybridization of 5 dpf zebrafish larva for A) cdhr1a, B) pcdh15b, C) pcdh23, D) ush1ga, E) harmonin, F) myo7aa. Ventral images of the embryos are displayed. White arrows indicate expression in the outer nuclear layer of the retina.
Expression of cdhr1a and cdhr1b.
Whole mount in situ hybridization of 5 dpf zebrafish larva for A-C) cdhr1a, D-F) cdhr1b in WT, cdhr1a+/- or cdhr1a-/- individuals. Ventral images of the embryos are displayed. Yellow arrows indicate expression in the outer nuclear layer of the retina. cdhr1a retinal expression is lost in the homozygous mutant. cdhr1b expression is absent from the retina in wildtype and cdhr1a mutants.
