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First published online 17 March 2004
doi: 10.1242/dev.01060

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Wnt signaling enhances FGF2-triggered lens fiber cell differentiation

Department of Ophthalmology and Visual Science, College of Medicine, The Catholic University of Korea, Seoul, Korea

View larger version (28K): [in a new window]   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.

View larger version (47K): [in a new window]   Fig. 2. Increased proliferation of epithelial cells in explants treated with Wnt CM. (A,B) BrdU incorporation assays demonstrated stimulation of the cell cycle in explants treated with Wnt 3a CM. (C,D) Nuclei of explants derived from rat lens capsule revealed by Hoechst 33258 staining. (E) Quantification of BrdU incorporation after a 6-hour labeling period. BrdU-positive nuclei from a total of six explants treated with each factor were counted. The mean values (±s.d.) are shown in the histogram. (n=5) [versus control (Cont), *P<0.005].

View larger version (37K): [in a new window]   Fig. 3. Representative immunolabeling of Ser9 phospho-GSK-3ß in the bow region of cryosections from mouse lenses (A), counterstained with Hoechst dye (B). Strong reactivity for phospho-GSK-3ß in fiber cells near the lens equator (arrow), but not in lens epithelial cells (arrowhead). (C) Western blotting of cell extracts was performed with anti-Ser9 phospo-GSK-3ß and normalized with anti-total GSK-3ß. Ser9 phosphorylation of GSK-3ß was increased in differentiating lens cells. Normalized quantification is shown in the graph. (D) Protein extracts from undifferentiated lens epithelium (LE) and differentiating lens fiber cells of the equatorial zone (FC) were assayed by GSK-3ß immune complex kinase assay. GSK-3ß kinase activity was decreased in the rat lens fiber cells. Each error bar represents the mean±s.d. of three independent experiments; each assay was performed in duplicate. le, lens epithelium; lf, lens fiber.

View larger version (74K): [in a new window]   Fig. 4. Localization of ß-catenin on mouse lenses. ß-catenin immunostained cryosections of the bow region of mouse lenses. (A) ß-catenin antibodies localized to the membrane of lens epithelial and fiber cells. However, nuclear ß-catenin was detected only in differentiating fiber cells near the lens equator (C,D). ß-catenin antibodies did not label the nucleus of central (B) and equatorial (E) epithelial cells. Scale bars: A, 50 µm; B-E, 10 µm. (F) Lysates prepared from the nuclei of undifferentiated lens epithelial cells (lane LE) and differentiating lens fiber cells (lane FC) were separated by 10% SDS-PAGE and analyzed with antibodies against ß-catenin. Western blots showed that nuclear ß-catenin was higher in differentiating lens cells compared with undifferentiated epithelial cells. Antibodies against lamin A/C were used to confirm the purity of the nuclear fraction.

View larger version (124K): [in a new window]   Fig. 5. Wnt CM induces elongation of lens epithelial cells. Explants were pre-stimulated with 50 ng/ml FGF2 or Wnt3a CM for 1 hour, followed by stimulation with control medium, Wnt3a CM, 50 ng/ml FGF for 5 days, or in the presence of 50 µM U0126 or DMSO. Explants were also cultured with vehicle or control medium in the absence of stimulation. Untreated explants or explants cultured in control medium after pre-stimulation with FGF2 showed normal epithelial cell morphology (A,C,E,I). Explants pre-stimulated by Wnt3a and cultured in Wnt3a did not undergo morphological change (B). However, explants that were pre-stimulated by FGF2 and cultured for 5 days in Wnt3a elongated (F) similar to explants cultured continuously with FGF2 (D). In the presence of DMSO (as a control for U0126), explants cultured with FGF2/Wnt CM showed a cell elongation (G). However, cell elongation did not occur in the presence of U0126 (H). Scale bar: 50 µm.

View larger version (44K): [in a new window]   Fig. 6. GSK-3ß is inactivated in lens cells treated with Wnt CM. (A) Explants exposed to FGF2 for 1 hour were washed with 2 M NaCl, and cultured in control medium or Wnt3a CM for 5 days. Explants were immunolabeled by using anti-Ser9 phospo-GSK-3ß (a,b), or Hoechst 33258 to stain nuclei. Ser9 phospho-GSK-3ß accumulated in explants cultured with FGF2/Wnt CM (b), but not in explants cultured with FGF2/Control medium (a). (B) In vitro GSK-3b kinase assays were performed using a GSK-3 substrate and [-32P]. Lysates containing 150 µg of protein prepared from explants cultured with FGF2/Control medium (F/C) or FGF2/Wnt CM (F/W) were immunoprecipitated with anti-GSK-3ß and analyzed using a GSK-3ß immune complex kinase assay. The activity of GSK-3ß was decreased in explants cultured with FGF2/Wnt CM compared with explants cultured with FGF2/Control medium. Values represent the mean±s.d. of three independent experiments and are expressed as a percentage of the activity in FGF2/Control explants.

View larger version (102K): [in a new window]   Fig. 7. Wnt CM induces the accumulation of ß-catenin in nuclei. (A) Explants were pre-stimulated by 50 ng/ml FGF2, followed by stimulation with control medium or Wnt3a CM for 5 days. Explants were immunolabeled with anti-ß-catenin or Hoechst 33258 to stain nuclei. ß-catenin accumulated in the nuclei of cells that elongated after treatment with FGF2/Wnt CM (b; arrow), but not in the nuclei of morphologically normal cells cultured with control medium or FGF2/control medium (a). Scale bar: 10 µm. (B) Nuclear extracts were prepared from explants cultured with FGF2/control medium (F/C) or FGF2/Wnt CM (F/W). Western blotting was performed with anti-ß-catenin. Explants displayed an increased level of nuclear ß-catenin in response to FGF2/Wnt CM. Blots probed with an anti-lamin A/C antibody demonstrated equal loading of nuclear proteins.

View larger version (52K): [in a new window]   Fig. 8. Wnt CM induces the accumulation of lens fiber proteins in FGF2-stimulated epithelial cells. Explants pre-stimulated by FGF2 for 1 hour were washed and cultured in control medium or medium supplemented with Wnt3a CM and incubated for 5 days. Explants were analyzed by immunolabeling with antibodies against ß-crystallin (A), aquaporin 0 (B) or were stained with Hoechst 33258. Explants cultured in control medium did not accumulate ß-crystallin or aquaporin 0, but these fiber-specific markers accumulated in cells exposed to Wnt CM. Scale bar: 50 µm.

View larger version (63K): [in a new window]   Fig. 9. Wnt CM induces alteration in the protein levels of cadherins and p57Kip2 in epithelial cells pre-treated with FGF2. Explants were cultured for 5 days with control medium (lane F/C) or Wnt3a CM (lane F/W), after pre-stimulation with FGF2 for 1 hour. Lens epithelium (lane LE) and differentiating lens cells of the zone of early elongation at equator (lane FC) were isolated from neonatal rat lens. Cell lysates were separated by 10% SDS-PAGE and probed with antibodies against E-cadherin, N-cadherin, p27Kip1 or p57Kip2. E-cadherin levels were decreased and N-cadherin levels increased after treatment with FGF2/Wnt CM, consistent with the changes seen during fiber cell differentiation (A). p57Kip2 levels increased in explants treated with FGF2/Wnt CM, similar to the increase in differentiating fiber cells. However, p27Kip1 levels were not affected by FGF2/Wnt CM, unlike the expression pattern seen in differentiating fiber cells (B). Actin was used as a loading control.

View larger version (94K): [in a new window]   Fig. 10. Treatment with lithium or Wnt CM leads to the accumulation of ß-crystallin protein without FGF2 pre-stimulation. (A) After treatment with 50 ng/ml FGF2, explants were cultured for 5 days in the presence of 10 mM NaCl (as a control) or 10 mM LiCl and observed by microscopy. Note that cell elongation did not occur in response to FGF2/LiCl. Scale bar: 50 µm. (B) Western blot analysis of explants cultured for 5 days in the presence of FGF2/NaCl and FGF2/LiCl showed that lithium induced a significant induction of Ser9 phospho-GSK-3ß. (C) Explants were pre-stimulated with or without 50 ng/ml FGF2 for 1 hour, and then cultured with 10 mM NaCl, 10 mM LiCl, control medium, or Wnt3a CM for 5 days, or in the presence of 50 µM U0126 or DMSO. Explants were then fixed and labeled with antibodies against ß-crystallin or Hoechst 33258 for nuclei staining. Immunolabeling for ß-crystallin in FGF2/LiCl-treated explants showed that ß-crystallin was accumulated without cell elongation (b). Lithium also induced the accumulation of ß-crystallin in explants cultured without FGF2 pre-stimulation (d). In the presence of U0126, FGF2/Wnt CM-treated explants revealed an accumulation of ß-crystallin without cell elongation (g), whereas the accumulation of ß-crystallin and cell elongation are induced in FGF2/Wnt CM-treated explants (f). In the presence of Wnt CM without FGF2 pre-stimulation, ß-crystallin was also accumulated (i). Scale bar: 10 µm.

View larger version (22K): [in a new window]   Fig. 11. Wnt/ß-catenin activates the transcription of ß-crystallin. (A) Explants were cultured for 5 days with control medium, Wnt3a CM, 10 mM NaCl, or 10 mM LiCl in the absence of stimulation by FGF2. Total RNA was prepared from each explant and ßB2-crystallin expression was assayed by RT-PCR. (B) To analyze the transcriptional activity of ß-crystallin, mouse lens cells were transiently transfected with pßB2-crystallin-luciferase plasmid, and co-transfected with an expression plasmid of GSK-3ß, ICAT or GFP (as a control). Cells were stimulated by Control medium, Wnt CM, 10 mM NaCl, 10 mM LiCl or a co-transfection of ß-catenin and stabilized ß-catenin (S37A). The activity was measured by dual luciferase analysis. Values present the mean±s.d. (n=5) and are expressed as an n-fold increase relative to the control medium.