Abstract
Purpose: :
Variations in the gene coding for ephrin type-A receptor-2 (EphA2) have been associated with a cataract phenotype in humans and mice. Here we characterize the lens phenotype of Epha2 gene-targeted mice that do not develop obvious cataract.
Methods: :
Commercially available Epha2-targeted mice were characterized by standard PCR-amplification and sequencing techniques. Lenses were analyzed by immuno-blotting, bright-field microscopy, and confocal fluorescence microscopy techniques.
Results: :
DNA/RNA amplification and sequencing confirmed that the Epha2 gene was disrupted in exon-5 resulting in several mutant transcripts consistent with a functional null at the protein level. Lens immunoblot analysis and confocal immunofluorescence microscopy localized Epha2 to plasma-membranes of the anterior epithelium and peripheral cortical fibers of wild-type but not null mice. Bright-field microscopy showed that Epha2-null lenses were smaller than wild-type and, while transparent, displayed a mild central refractive disturbance. Confocal fluorescence imaging of live Epha2-null lenses revealed at least four other features not observed in wild-type: (1) anterior and posterior Y-sutures were offset from the optical axis and composed of 1-5 asymmetric branches, (2) fiber cell nuclei at the lens equator appeared disorganized and were retained with age, (3) isolated fiber cells lacked characteristic undulation along their anterior-posterior axis, and (4) membranous dome-like structures protruded from the basal surface of the anterior epithelium into the surrounding capsule.
Conclusions: :
Homozygous loss of Epha2 affects lens growth and optical quality, but is not necessarily sufficient to cause cataract in mice. Absence of Epha2 is also associated with mis-targeting of fiber cells to the suture regions, abnormal fiber cell shape and denucleation, and aberrant basal protrusions from the anterior epithelium. These data point to a key role for Epha2-dependent cell signaling in normal lens development.
Keywords: microscopy: confocal/tunneling • cell membrane/membrane specializations • differentiation