DEV02685 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Injection of crb2a splice-blocking morpholinos phenocopies the moe mutations. (A) crb2a splicing, measured by RT-PCR, is reduced by injection of splice-blocking morpholino antisense oligonucleotides. Morpholino injection results in the amplification of a 1097 bp fragment. The reactions were also performed with no reverse transcriptase (no RT). (B-D) Head morphology of 30-hour wild-type (B), moe^b476 mutant (C) and crb2a morphant (D) embryos. (E-G) The retinal pigmented epithelium is uniform in wild type (E) and patchy in moe^b781 mutants (F) and crb2a morpholinos-injected embryos (G) at 48 hours. (H-J) The three nuclear layers are clearly defined in the wild-type retina (H), but are not defined in the moe^b781 mutant (I) and the crb2a morphant (J) at 3 days. (K-M) Retinal ganglion cells (labeled using Zn5 antibodies) localize to the innermost part of the retina in the wild-type retina (K), but are disorganized and scattered throughout the retina in moe^b781 mutants (L) and crb2a morphants (M) at 3 days. (N-P) Rod photoreceptors (visualized by genetically-encoded GFP) localize to the outermost part of the retina in the wild-type retina (N), but are disorganized and scattered throughout the retina in moe^b781 mutants (O) and crb2a morphants (P) at 4 days. Eyes are outlined. Scale bars: in D, 500 μm for B-D; in P, 100 μm for E-P.

• Supplemental Figure 2 -

  Fig. S2. Co-localization of Moe and panCrb in the brain and eye at 30 hours. Moe is shown in green (guinea pig anti-Moe) and panCrb in red (rabbit anti-CRB3). Images are merged with corresponding DIC images. Apical surfaces are indicated with arrows. Scale bar: 10 μm.

• Supplemental Figure 3 -

  Fig. S3. Nok (Pals1) interacts directly with zebrafish Crumbs proteins. His-Nok-N binds to membrane-immobilized GST-Crb2a and GST-Crb2b but not to GST alone in a far western probed with anti-His. His-LacZ does not show significant binding to any of the GST fusions. Control loading comprises anti-GST western blot with 1/50^th amount of GST-fusion proteins.

• Supplemental Figure 4 -

  Fig. S4. ZO-1 labeling of the outer limiting membrane in transplanted WT and moe^- GFP⁺ rods at 6 days. ZO-1 is shown in red, GFP in green. Scale bar: 5 μm.

• Movie 1 -

  Movie 1. Three-dimensional reconstruction of WT^tgGFP rods (from Fig. 7G) at 10 days. The cells are labeled with anti-GFP (green) and the outer segment with anti-Rhodopsin (blue).

• Movie 2 -

  Movie 2. Three-dimensional reconstruction of a moe^{b781−/−;tgGFP} rod (from Fig. 7I) at 10 days. The cells are labeled with anti-GFP (green) and with anti-Rhodopsin (blue).

• Movie 3 -

  Movie 3. Three-dimensional reconstruction of a moe^{b781−/−;tgGFP} rod (from Fig. 7J) at 10 days. The cells are labeled with anti-GFP (green) and with anti-Rhodopsin (blue).

• Movie 4 -

  Movie 4. Three-dimensional reconstruction of three moe^{b781−/−;tgGFP} rods (from Fig. 7L) at 10 days. The cells are labeled with anti-GFP (green) and with anti-Rhodopsin (blue).

• Movie 5 -

  Movie 5. Three dimensional reconstruction of moe^{b781−/−;tgGFP} rods (from Fig. 7N) at 10 days. The cells are labeled with anti-GFP (green) and with anti-Rhodopsin (blue).