JCS01301 Supplemental Data

Files in this Data Supplement:

• Materials and Methods (Adobe PDF) -

  Materials and Methods:

  PCR analysis of Rpe65 genotype at codon 450

  Codon 450 is CTG encoding a leucine in light sensitive vs ATG encoding a methionine in light insensitive mouse strains (Wenzel et al., 2001). We carried out PCR using primers and genomic DNA from mouse-tails, and determined their DNA sequences using an ABI prism 310 sequencer (ABI, Applera, Nieuwekerk a/d IJssel, The Netherlands). Details are available on request. A correlation between codon 450 polymorphism in Rpe65 and light-sensitivity in Crb1^–/– or wild-type retinas was excluded.

  Immunofluorescence and confocal microscopy

  For immunostaining, the following antibodies were used: rabbit pAb’s anti-Crb1 (AK2, AK7, 1:250; 851, Dr Tepass, 1:50), anti-Crb2 (EP13, EP14, 1:5000, P. Rashbass)(A1, 1:200, Dr A. Le Bivic), anti-Crb3 (1:500, Dr Margolis), anti-Mpp4 (AK4, 1:1000), anti-Mupp1 (1:500, Dr Le Bivic), anti-Patj (1:100, Drs Le Bivic or Margolis), anti-Pals1 (1:500, Dr Margolis), anti-GFAP (1:1000, DAKO, Glostrup, Denmark), anti-ZO-1 (1:500, Zymed, San Francisco, CA), mouse mAb’s anti-PKCl (aPKC, cross-reacts with PKCi, 1:250), anti-p120 (1:1000), anti-moesin (1:500), anti-Dlg (1:500), anti-N-cadherin (1:250), anti-b-catenin (1:500), anti-Cdc42GAP (1:500) (Transduction Laboratories, Alphen aan de Rijn, The Netherlands), anti-pan-cadherin (CH-19, 1:500; Sigma), rat mAb anti-CD44 (1:200, Dr van der Neut) and goat pAb’s anti-Par6 (N18, 1:100), anti-Par6 (T20; 1:100), anti-ZO-2 and anti-ZO-3 (1:500, SantaCruz). Secondary antibodies were conjugated with Cy3 (1:600) or Alexa 488 (1:500, Molecular Probes, Leiden, The Netherlands). Cone segments and pedicles were stained with peanut agglutinin-fluorescein (1:100, Vectorlabs, Burlingame, CA) and F-actin was stained by rhodamine-phalloidin (1:500, Molecular Probes) during incubation with the primary antibody. Eye-cups were fixed in 4% paraformaldehyde in 0.1 M phosphate buffered saline, pH 7.4 for 30 minutes. After rinsing in PBS, eyecups were immersed in 5% sucrose in PBS, followed by 30% sucrose in PBS for 3 hours. Tissue was blotted dry, embedded in TissueTek (Sakura Finetek, Zoeterwoude, The Netherlands) and frozen on dry-ice. Cryosections (7 mm) were rehydrated in PB and blocked for 1 hour using 10% goat or donkey serum, 0.4% Triton X-100 and 0.1% BSA in PB. Tissues for anti-Pals1 staining were permeabilized in 10% goat or donkey serum, 1% SDS and 0.1% BSA in PBS for 1 hour. Primary antibodies (see above) were diluted in 0.3% goat or donkey serum, 0.4% Triton X-100 and 0.1% BSA in PB and incubated for 16 h. Secondary antibodies were diluted in 0.1% goat or donkey serum in PB and incubated for 1 h at RT. Sections were then washed in PB, mounted using vectashield (Vectorlabs, Burlingame, CA).

• Supplemental Figure 1 -

  Fig. S1. Elektronmicroscopic image of the region containing the outer limiting membrane in a 3 months old Crb1^–/– retina. Arrow indicates an elektron dense adherens junction, which are similar as the adherens junction in the wild-type mouse. M= apical villi of a Müller glia cell, IS= inner segment of the photoreceptor cell, PRC=  photoreceptor cell body. Bar represents 0.5 µm.

• Supplemental Figure 2 -

  Fig. S2. Electrophysiology of 9 months old Crb1^–/– mice. (A) Scotopic b-wave amplitude vs. log Intensity (VlogI) function. Boxes indicate the 25% to 75% quantile range, whiskers the 5% and 95% quantiles, and the asterisk the median of the Crb1^–/– data. The normal range is delimited by solid lines indicating the 5% and 95% quantile of the control group. There is no sign of impaired retinal function in Crb1^–/– mice. (B) Typical scotopic intensity series in a wild-type (right column) and a Crb1^–/– mutant mouse (left column). Log light intensities (from top to bottom) were -4, -3, -2, -1.5, -1, -0.5, 0, 0.5, 1, 1.5 log cd*s/m². No differences in amplitude or waveform are visible. (C-D) Electrophysiological results in 3 months old light exposed Crb1^–/– mice. Also under these conditions, there is no sign of impaired retinal function in Crb1^–/– mice. No differences in amplitude or waveform are visible. Calibration marks: Vertical 200 µV/div., horizontal 40 ms/div.

  Reference

  Wenzel, A., Reme, C. E., Williams, T. P., Hafezi, F. and Grimm, C. (2001). The Rpe65 Leu450Met variation increases retinal resistance against light-induced degeneration by slowing rhodopsin regeneration. J.Neurosci. 21, 53-58.