Levels of Fgfrs are altered in the lenses of <i>Dlg10CRE</i> mice.

<p>(A) RIPA, triton soluble (cytosolic) and triton insoluble (cytoskeletal-associated) extracts from P2 control and <i>Dlg10CRE</i> lenses were subjected to western blot analysis for Fgfr1, Fgfr2, and Fgfr3 and the blots reprobed for Gapdh as a loading control. Representative blots are shown. (B) Quantification of protein levels. Shown are the levels of each Fgfr in extracts from <i>Dlg10CRE</i> lenses relative to levels in the control (control levels set at 1.0). Signal intensities were quantified by phosphorimager analysis, as described in Materials and Methods, and the data subjected to statistical analysis using the two-sided One Sample t-test. At least 3 protein pools were analyzed in triplicate over 1–3 blots. The relative levels of Fgfr2 were reduced in the whole cell extract and cytoskeletal associated fraction compared to controls whereas the levels of Fgfr1 were increased as compared to controls. Error bars = standard deviations. * = FDR<0.05, ** = FDR<0.01.</p

Loss of <i>Dlg-1</i> in the Mouse Lens Impairs Fibroblast Growth Factor Receptor Signaling

<div><p>Coordination of cell proliferation, differentiation and survival is essential for normal development and maintenance of tissues in the adult organism. Growth factor receptor tyrosine kinase signaling pathways and planar cell polarity pathways are two regulators of many developmental processes. We have previously shown through analysis of mice conditionally null in the lens for the planar cell polarity gene (PCP), <i>Dlg-1,</i> that <i>Dlg-1</i> is required for fiber differentiation. Herein, we asked if <i>Dlg-1</i> is a regulator of the Fibroblast growth factor receptor (Fgfr) signaling pathway, which is known to be required for fiber cell differentiation. Western blot analysis of whole fiber cell extracts from control and <i>Dlg-1</i> deficient lenses showed that levels of the Fgfr signaling intermediates pErk, pAkt, and pFrs2α, the Fgfr target, Erm, and the fiber cell specific protein, Mip26, were reduced in the <i>Dlg-1</i> deficient fiber cells. The levels of Fgfr2 were decreased in <i>Dlg-1</i> deficient lenses compared to controls. Conversely, levels of Fgfr1 in <i>Dlg-1</i> deficient lenses were increased compared to controls. The changes in Fgfr levels were found to be specifically in the triton insoluble, cytoskeletal associated fraction of <i>Dlg-1</i> deficient lenses. Immunofluorescent staining of lenses from E13.5 embryos showed that expression levels of pErk were reduced in the transition zone, a region of the lens that exhibits PCP, in the <i>Dlg-1</i> deficient lenses as compared to controls. In control lenses, immunofluorescent staining for Fgfr2 was observed in the epithelium, transition zone and fibers. By E13.5, the intensity of staining for Fgfr2 was reduced in these regions of the <i>Dlg-1</i> deficient lenses. Thus, loss of <i>Dlg-1</i> in the lens impairs Fgfr signaling and leads to altered levels of Fgfrs, suggesting that <i>Dlg-1</i> is a modulator of Fgfr signaling pathway at the level of the receptors and that <i>Dlg-1</i> regulates fiber cell differentiation through its role in PCP.</p></div

Levels of the Fgfr signaling intermediate, pErk, are reduced in embryonic <i>Dlg10CRE</i> lenses.

<p>(A) Paraffin embedded sections of eyes from E13.5 control and <i>Dlg10CRE</i> embryos were subjected to immunofluorescence analysis using an anti-pErk antibody (red) and the nuclei counterstained with To-Pro3 (blue). Representative images of the transition zone are shown. c, cornea; e, lens epithelium; r, retina; tz, transition zone. Bar = 50 µm. (B) Quantification of pErk levels. Shown are the relative levels of pErk in the transition zone of <i>DLG10CRE</i> and control lenses (control levels set at 1.0) Quantification of signal intensities in the transition zone was carried out using ImageJ, as described in Materials and Methods, and the data subjected to statistical analysis using the two-sided One Sample t-test. At least 3 different sections from at least 3 different lenses were evaluated. The relative levels of pErk in the transition zone of the <i>Dlg10CRE</i> lens were reduced as compared to levels in the corresponding region of the control lenses. Error bars = standard deviations. * = FDR<0.01.</p

Dlg-1 and Fgfr2 co-localize on the short sides of the fiber cells in the outer cortex.

<p>Cryogenic sections from control P30 lenses were subjected to immunofluorescent staining using anti-Fgfr2 anti-Dlg-1 antibodies. Staining for Fgfr2 (red) was predominantly localized on the short sides of the fiber cells as was staining for Dlg-1 (green). Overlap (yellow) can be seen in the merged image. Bar = 50 µm.</p

Fgfr2 levels are reduced in the transition zone and fiber cells of <i>Dlg39CRE</i> embryos.

<p>Paraffin embedded sections from day E15.5 control, <i>Dlg10CRE</i> and <i>Dlg39CRE</i> embryos were subjected to immunofluorescence analysis using an anti-FGFR2 antibody (red) and the nuclei counterstained with To-Pro3 (blue). Representative images of the epithelium and transition zone of each genotype are shown. The intensity of staining in the epithelium from the <i>Dlg39CRE</i> embryo is similar to that in control whereas intensity in the epithelium of <i>Dlg10CRE</i> embryos is less than in the control. The intensity of Fgfr2 staining was reduced in the transition zone of both <i>Dlg10CRE</i> and <i>Dlg39CRE</i> lenses as compared to controls, indicating that the effect is fiber cell autonomous. At least 3 different sections from at least 3 different lenses were evaluated. c, cornea; e, lens epithelium; f, lens fiber cells; r, retina; tz, transition zone. Bar = 50 µm.</p

Levels of Fgfr signaling intermediates are reduced in both <i>Dlg10CRE</i> and <i>Dlg39CRE</i> fiber cells.

<p>(A) RIPA lysates from P2 control, <i>Dlg10CRE</i>, and <i>Dlg39CRE</i> fiber cells were immunoblotted for the pErk and pFrs2α and the blots reprobed for Gapdh as a loading control. Representative blots are shown. (B) Quantification of protein levels. Shown are the levels of the indicated proteins in extracts from <i>Dlg10CRE</i> fiber cells relative to levels in the controls (control levels set at 1.0). Signal intensities were quantified by phosphorimager analysis, as described in Materials and Methods, and the data subjected to statistical analysis using the two-sided One Sample t-test. At least 3 protein pools were blotted in triplicate over 1–3 blots. The relative levels of Fgfr signaling intermediates were reduced in the fiber cells of <i>Dlg39CRE</i> mice as well as <i>Dlg10CRE</i> mice compared to controls, indicating that the effect was fiber cell autonomous. Error bars = standard deviations. ** = FDR<0.01.</p

Components of the Fgfr signaling pathway are reduced in <i>Dlg10CRE</i> fiber cells.

<p>(A) RIPA lysates from P2 control and <i>Dlg10CRE</i> fiber cells were immunoblotted for the indicated proteins and the blots reprobed for Gapdh as a loading control. Representative blots are shown. (B) Quantification of protein levels. Shown are the levels of the indicated proteins in extracts from <i>Dlg10CRE</i> fiber cells relative to levels in the controls (control levels set a 1.0). Signal intensities were quantified by phosphorimager analysis, as described in Materials and Methods, and the data subjected to statistical analysis using the two-sided One Sample t-test. At least 3 protein pools were blotted in triplicate over 1–3 blots. The relative levels of Fgfr signaling intermediates and target as well as the fiber cell specific protein, Mip26, were all reduced in <i>Dlg10CRE</i> lens fibers as compared to control. Error bars = standard deviation. * = FDR<0.05, ** = FDR<0.01.</p

Loss of Dlg-1 protein following cre-mediated excision of <i>Dlg-1</i> sequences.

<p>Paraffin embedded sections of eyes from control, <i>Dlg10CRE</i> and <i>Dlg39CRE</i> day E12.5, E13.5, and E14.5 embryos were subjected to immunoflourescent staining for Dlg-1 (green). For <i>Dlg10CRE</i> lenses, the intensity of staining throughout the lens was greatly reduced at E12.5 and staining was undetectable at E13.5. For <i>Dlg39CRE</i> lenses, the intensity of staining in the fiber cell compartment was greatly reduced at E13.5 and staining in the fiber cell compartment was undetectable at E14.5. c, cornea; e, lens epithelium; f, lens fiber cells; r, retina. Bar = 50 µm.</p