DEV032136 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Cre-recombinase activity in the developing RPE of Tyrp1-Cre mice. Anti-β-galactosidase antibodies label cells in the dorsal optic vesicle (presumptive RPE) of E9.5 (A, arrow, 25 somites), and in the RPE in the optic cup of E11.5 (B, arrow) and E15.5 (C, arrow) Tyrp1-Cre; ROSA26R embryos. β-galactosidase expression is also observed ectopically in the retina (B,C); however, retina-specific disruption of β-catenin does not affect RPE development (Fu et al., 2006). Colocalization of β-galactosidase (D) and Otx2 (E) expression in the RPE of BATgal reporter mice at P10 (arrows). (F) Merge of D and E. Arrowheads point to Otx2-labeled photoreceptor cells in the retina (E,F). (G) Axin2 protein expression in the RPE at E13.5 using a conductin antibody (arrow). The adjacent periocular ectoderm that will give rise to the conjunctiva and eyelid, and the underlying mesenchyme also expresses axin2 (G, arrowhead). (H) Control staining without primary antibody. (I) Whole-mount in situ hybridization of E15.5 eyes showing intense labeling of the central RPE (arrow). Asterisks indicate the optic stalk. Scale bars: 50µm

• Supplemental Figure S2 -

  Fig. S2. β-catenin is effectively deleted in the developing RPE by Tyrp1-Cre resulting in downregulation of Mitf expression in recombined cells as shown by the ROSA26R reporter. (A) At E10.75, β-catenin is expressed in the dorsal RPE of control embryos (arrow). (B,E) β-galactosidase expression in the dorsal RPE of controls (arrows). (C) Merge of A and B. (D) Robust expression of Mitf in the dorsal RPE of controls (arrow). (F) Merge of D and E. (G) No β-catenin expression is observed in the dorsal RPE of Tyrp1-Cre^tg/0;β-catenin^floxdel/FL embryos (region between arrowheads), in contrast to the ventral RPE, which has not undergone transdifferentiation yet (arrow). (H,K) β-galactosidase expression in the dorsal RPE of Tyrp1-Cre^tg/0;β-catenin^floxdel/FL embryos. (I) Merge of G and H. (J) Mitf expression in the dorsal RPE of mutant embryos is reduced (region between arrowheads). (L) Merge of J and K. CON, control; CKO, conditional knockout.

• Supplemental Figure S3 -

  Fig. S3. Optic stalk and optic nerve defects in β-catenin mutant eyes. The optic fissure has closed by E15.5 in control eyes (A), but has not sealed in Tyrp1-Cre^tg/0;β-catenin^floxdel/FL eyes resulting in coloboma (B, arrow). (C,D) We analyzed P0 mutant eyes using Neurofilament-M (green) to detect ganglion cell axons and horizontal cells, and Pou4f2 antibodies (red) to label ganglion cell nuclei. No single optic nerve tract or head was observed in sagittal sections in β-catenin mutant RPE and none of the ganglion cell axons appears to exit the eye successfully (D, arrows), in comparison to controls (C, arrows). Arrowheads point to Neurofilament-M-expressing horizontal cells in the control retina (C), as well as in the retina (D) and transdifferentiated RPE in mutant eyes (open arrowheads in D). Asterisk demarcates the lens. Scale bars: 50µm

• Supplemental Figure S4 -

  Fig. S4. ROSA26R reporter analysis in Tyrp1-Cre^tg/0;β-catenin^floxdel/FL eyes reveals that recombined, β-catenin-negative cells in mutant RPE are progressively excluded. Conditional mutants harboring the ROSA26R reporter at E12.5 (A-D), E13.5 (E,F) and P0 (G,H) were immunolabeled for β-catenin (green, A-H) and β-galactosidase (red, B,D,F,H) to examine the fate of cells in which Cre-mediated recombination has occurred (arrows). At E12.5, β-catenin-positive cells start to populate the dorsal edge of the mutant RPE (A-D), and central regions where the two layers appear to form bridges also contain β-catenin-positive cells (D, arrowhead). By E13.5, bridge formation (arrowhead) and more β-catenin-positive cells are detectable in the mutant RPE (F). At P0, the former RPE consists entirely of β-catenin-positive cells, shown by a severe reduction of β-galactosidase expression (H, arrows).