Abstract
Purpose:
In Drosophila, Dlg-1 is a regulator of cell proliferation, adhesion and polarity. We have previously shown through analysis of mice conditionally null for Dlg-1 in the lens that Dlg-1 is required for fiber differentiation. Interestingly, the levels of pERK1/2, intermediates in the FGFR signaling pathway, appeared to be reduced in the fibers of Dlg-1 mutant mice, suggesting that loss of Dlg-1 impairs FGFR signaling. In this study, we addressed this hypothesis.
Methods:
Mice carrying a conditional Dlg-1 allele were crossed with MLR10cre mice to generate Dlgf/f (control), and Dlgf/f10(mutant) mice in which Dlg-1 is deleted throughout the lens. Protein extracts of fiber cells from postnatal day 2 Dlgf/f and Dlgf/f10 mice were prepared in RIPA buffer and subjected to western blot analyses using antibodies against FGFRs 1, 2, and 3, the FGFR signaling intermediates, pERK1/2, pFRS2α and the FGFR target ERM. Soluble and cytoskeletal associated fractions were prepared by Triton-X100 extraction and immunoblotted for FGFRs. Blots were reprobed for Gapdh as loading controls and levels quantified. To assess the levels of FGFRs and pERK earlier in lens development, paraffin sections of eyes from Dlgf/f and Dlgf/f10 embryos were subjected to immunofluorescence.
Results:
Analysis of RIPA extracts showed that levels of pERK1/2, pFRS2α, FGFR2, and ERM were reduced in the Dlg10 fibers compared to controls. Conversely, levels of FGFR1 were increased while the levels of FGFR3 were similar to controls. The levels of FGFR2 were reduced specifically in the cytoskeletal associated fraction of Dlg10 fibers compared to controls. The intensity of staining for pERK1/2 was reduced in the transition zone and posterior region in E13.5 Dlgf/f10 lenses. In control lenses, staining for FGFR2 was seen in the epithelium and the transition zone, and was concentrated at the apical and basal tips of the fibers and in the posterior. The intensity of staining for FGFR2 in Dlg10 lenses was reduced at E13.5 and became further reduced at later stages.
Conclusions:
Loss of Dlg-1 in the lens impairs FGFR signaling, suggesting that Dlg-1 is a modulator of this pathway. The change in abundance of FGFRs 1 and 2 between controls and Dlg-1 mutant lenses may contribute to the impaired signaling and observed defects in fiber differentiation. Future studies will explore how Dlg-1 and FGFRs interact with each other and with other signaling pathways to modulate lens development.
Keywords: 543 growth factors/growth factor receptors •
500 differentiation •
714 signal transduction