Fig. S3

Cloning of rw341 mutant gene

(A) The rw341 mutation is mapped on chromosome 19 and is restricted within the genomic region, flanked by two polymorphic markers, Zv7_1912 14 and Zv7_1912 18 (arrow). In this region, six genes including vps45 are annotated. Information regarding polymorphic markers is provided in Table S1.

(B) Whole mount in situ hybridization of wild-type embryos with the vps45 mRNA probe during development.

(C) Section of wild-type lenses labeled with the vps45 mRNA probe at 24hpf, 3 dpf and 5 dpf. Arrowheads indicate mRNA signals in lens vesicle (24 hpf) and lens epithelium (3 and 5 dpf).

(D) GFP expression in lenses of Tg[foxe3:VPS45-GFP] at 32 and 48 hpf. Right panels indicate higher magnification of lenses. GFP is expressed only in lens epithelium (le) but not in lens fiber core (lf).

(E) GFP expression in lenses of Tg[αAcy:VPS45-GFP] at 5 dpf. Bottom panels indicate higher magnification of the interface between lens epithelium and lens fiber core. GFP is expressed only in lens fiber core (lf) but not in lens epithelium (le).

(F) Embryos injected with mRNA encoding VPS45-GFP and VPS45(Δex11)-GFP. VPS45-GFP expression is maintained, but VPS45(Δex11)-GFP expression disappears at 24 hpf.

(G) Western blotting of 24 hpf wild-type embryos, and wild-type embryos injected with vps45-GFP mRNA and vps45(Δex11)-GFP mRNA with anti-GFP antibody. A band of GFP antibody is detected in vps45-GFP mRNA injected embryos, but disappears in vps45(Δex11)-GFP mRNA injected embryos.