Abstract
Purpose::
PDZ domain containing proteins are scaffold molecules that have been shown to play a role in maintaining proper cell-cell adhesion, cell polarity and cell growth in Drosophila . Previous studies from our lab have shown that targeting multiple PDZ proteins by expressing the viral oncoprotein E6 leads to increased proliferation, defects in cell adhesion, polarity, and differentiation. Recently, we have provided some evidence for the individual contribution of PDZ protein Dlg-1 in certain aspects of lens development. The purpose of this study is to understand if PDZ protein Scrib plays a role in the lens development and if Dlg-1 and Scrib interact to regulate this tissue. To address this question, lenses from WT, Scrib +/- , Dlg-1 +/- , and Dlg-1 +/- ; Scrib +/- compound heterozygotes were analyzed.
Methods::
Paraffin sections from E18.5 WT, Dlg-1+/-, Scrib+/-, and Dlg-1+/-; Scrib+/- lenses were stained with propidium iodide (PI) and hematoxylin and eosin (H&E) to assess lens morphology. Sections were also immunostained for BrdU and TUNEL to assess the pattern and levels of DNA synthesis and apoptosis, respectively. The patterns of Scrib, N-cadherin and ZO-1 were analyzed by immunofluorescence and confocal microscopy in order to assess cell-cell adhesion and polarity.
Results::
PI and H&E staining showed disorganization of the fiber cells and nuclei in the Dlg-1+/-, Scrib+/-, and Dlg-1+/-; Scrib+/- lenses. The percentage of BrdU positive cells was higher in all three constructs when compared to WT lenses. However, the epithelium of Dlg-1+/-; Scrib+/- mice showed greater clustering of BrdU positive cells and multilayering. While there was mislocalization of the apical marker ZO-1 in the central epithelium of all three constructs, ectopic localization of ZO-1 along the transition zone was observed only in the Dlg-1+/-; Scrib+/- lenses. While apoptosis throughout the epithelial cells was higher in the Scrib+/- lenses, apoptosis within the fiber cell compartment was higher in all three constructs when compared to the WT lenses. Also, N-cadherin staining appeared disorganized and reduced in the posterior lenses of all three strains.
Conclusions::
These results show that loss of Dlg-1 and/or Scrib in the lens is sufficient to alter proper cell cycle regulation, fiber cell organization and cell-cell adhesion. Furthermore, deficiencies in both Scrib and Dlg-1 enhances the cell cycle regulation defect, and results in apico-basal polarity defects. Taken together, these results suggest that these factors act coordinately in regulating lens cell proliferation and differentiation.
Keywords: proliferation • cell adhesions/cell junctions • transgenics/knock-outs