Fig. S1

Lens epithelial phenotypes of the rw341 mutant

(A) Retinal ciliary marginal zone (CMZ) of wild-type and rw341 mutant embryos at 5 dpf. Retinal stem cells and progenitor cells, which are located in the retinal CMZ, were swollen in rw341 mutants.

(B) Labeling of wild-type and rw341 mutant lenses with phalloidin (magenta) and a nuclear stain, Sytox-Green (green) at 3, 4, and 5 dpf. Higher-magnification images of squares in upper panels are shown at the bottom of each panel. In rw341 mutants, lens epithelium is monolayered at 3 dpf, but lens epithelial nuclei become swollen and start to pile up (arrowheads) at 4 dpf. Irregularly aggregated cells (arrows) are observed in the posterior region of the lens in rw341 mutants at 5 dpf.

(C) Labeling 5 dpf wild-type and rw341 mutant lenses with anti-ZO1 antibody (green). Upper and lower panels indicate a lens and higher magnification of lens epithelium, respectively. Dot-like ZO1 signals are detected at the apical interface between wild-type lens epithelial cells (open arrowheads), whereas dotted ZO1 signals are irregularly observed in multilayered lens epithelium of rw341 mutants (le).

(D) Labeling of 5 dpf wild-type and rw341 mutant lenses with antibodies against E-cadherin (green). Nuclei were stained with TOPRO3 (white). Upper left and right panels indicate an eye and a lens, respectively. Lower panels indicate higher magnification of the lens epithelium area. Right upper and lower panels indicate only the green channel. E-cadherin is normally localized at the adherens junction between wild-type lens epithelial cells (open arrowheads), but scattered in the multilayered lens epithelium of rw341 mutants (le).

(E) Labeling of 5 dpf wild-type and rw341 mutant lenses with anti-pH3 (green) and anti-PCNA (magenta) antibodies. Nuclei were counter-stained with TOPRO3 (white). Upper left and right panels indicate an eye and a lens, respectively. Lower panels indicate higher magnification of lens epithelium area. pH3-positive lens epithelial cells are few in wild type (open arrowhead) and do not increase in rw341 mutants.

(F) Confocal scanning of 5 dpf wild-type and rw341 mutant lenses with a transgene, Tg[acta2: EGFP] (green). Upper left and right panels indicate an eye and a lens, respectively. Lower panels indicate higher magnification of lens epithelium area. No EGFP expression was observed in either wild-type or rw341 mutant lens epithelium (le). Scale bars: 20 μm.