Fig. 1. Wnt gene expression in the mouse lens and activation of Wnt signaling by vitreous humor. Total RNA was isolated from E16, P4, P21 and adult rat lenses (A), or from rat (P4) lens central epithelium (CE) and equatorial epithelium, which contains both the proliferating cells and elongating cells (EC) (B). cDNA was prepared by reverse transcription and amplified with primers specific for each Wnt. Each reaction was normalized to ß-actin. Analyses of at least three different RNA preparations from the same tissues provided similar results. (C) Mouse lens cells were transiently transfected with TOPFLASH or FOPFLASH reporter plasmid, and then stimulated with vehicle (lanes 1, 7), aqueous humor (lane 2), vitreous humor (lane 3), control medium (lane 4), 5xWnt3a conditioned medium (lane 5), 10xWnt3a CM (lane 6), 1 ng/ml EGF (lane 8), 50 ng/ml FGF2 (lane 9), 10 ng/ml PDGF (lane 10), or 5 ng/ml TGF-ß(lane 11) for 16 hours. Cell lysates were assayed for luciferase activity. (D) Control medium (lane 1), vitreous humor pre-incubated with control medium (lane 2), vitreous humor pre-incubated with sFRP-1-conditioned medium (lane 3), or sFRP-1-conditioned medium alone (lane 4) were added to mouse lens cells transfected with the TOPFLASH reporter plasmid. Cell lysates were analyzed by luciferase assay.