Fig. 9: Requirement for Crb1 isoforms in outer limiting membrane integrity.
a Schematic of Crb1 locus showing genetic lesions underlying mouse mutant alleles. Previously studied alleles: Crb1ex1, a targeted deletion of exon 1 that does not impact Crb1-B; Crb1rd8, a point mutation in exon 9. Alleles generated here: Crb1delB, a CRISPR-mediated deletion of the first Crb1-B exon and its promoter region, leaving Crb1-A intact; Crb1null, a CRISPR-mediated deletion of exons used in all Crb1 isoforms. Also see Supplementary Fig. 7A. b Schematic illustrating location of OLM junctions (red) surrounding photoreceptor inner segments. c Electron micrograph from wild-type mouse. All inner segments make OLM junctions with Müller cells. IS inner segment. Red arrowheads, photoreceptor-glial junctions. Blue arrowheads, glial-glial junctions. d, e OLM phenotype in Crb1 mutants. In wild-type control retina (d), OLM (red arrow) divides outer nuclear layer (ONL) from IS layer. In Crb1 mutants (e), OLM gaps allow nuclei to penetrate IS layer. Arrows demarcate region lacking OLM junctions. Image depicts Crb1delB/null mutant, but is representative of OLM phenotypes in null, delB, and rd8 mutants (Supplementary Fig. 7D-F). f–i Higher power views of OLM gaps in Crb1 mutants. In each allelic combination, inner segments lacking OLM junctions (asterisks) were observed. Red and blue arrowheads as in c. j Quantification of OLM gap frequency. No gaps were observed in wild-type (WT) or Crb1null/+ heterozygote (het) controls. Gap frequency was similar in rd8, null, and delB/null mutants, the latter of which lack Crb1-B but still express Crb1-A. Statistics, one-way ANOVA with Tukey’s post-hoc test. Null, rd8, and delB/null differed significantly from controls (P-values given on graph), but did not differ significantly from each other (rd8 vs. null P = 0.991; rd8 vs. delB/null P = 0.784; null vs. delB/null P = 0.967). Also see Supplementary Fig. 7F and Source Data file. Sample sizes: n = 3 (null/+, rd8; null/null, delB/delB); n = 5 (null/delB); n = 6 (WT). WT and het were pooled for plotting and statistics. Error bars, S.E.M. Scale bars 2 µm (C,-E); 1 µm (F-I). Bar in D applies to E; bar in G applies to F.
