Fig. 3: B6-PRX protein distribution is restricted to peripheral lens fibers, whereas 129-PRX proteins are broadly distributed in both peripheral and interior fibers.

a Frozen cross-sections of 129 wild-type (129-WT) and B6L wild-type (B6L/B6L-WT) lenses from 3-week-old mice were immunostained with FITC-phalloidin to label F-actin (green), anti-periaxin antibody (red), and DAPI for nuclei (blue). B6-PRX proteins are exclusively detected in peripheral differentiating fiber cells, approximately 50 μm from the lens surface, while 129-PRX proteins are present in both peripheral and interior fibers. White asterisks indicate inner lens fibers. Scale bar: 50 μm. The schematic lens illustration shows the locations of the imaged areas in panels a, b and c. The arrow marks the denucleation zone, approximately 150 μm from the lens surface. R indicates the lens radius (~1 mm), and the shadowed area represents the cataract. b Three-dimensional (3D) imaging of lens vibratome cross-sections was performed with triple-labeling with FITC-phalloidin (green), anti-periaxin antibody (red), and DAPI (blue). In the B6L/B6L-Cx46KO lens, B6-PRX proteins are localized at the cell boundaries of peripheral differentiating fibers, approximately 50 μm from the lens surface. The staining signals become punctate in inner fibers and gradually diminish. In contrast, in the 129-Cx46KO lens, 129-PRX proteins are detected at the cell boundaries of both peripheral and interior fibers, with pronounced enrichment at tricellular vertices. White arrowheads indicate elongated protrusions, positive for both F-actin and PRX, which are observed along the long sides of hexagonal-shaped fiber cells specifically in the 129-strain background. Lenses are isolated from 3-week-old mice. Scale bar: 10 µm. c 3D images of anterior-posterior (A/P) lens vibratome sections from 3-week-old B6-Cx46KO, 129-Cx46KO, and B6L/B6L-Cx46KO mice were stained with FITC-phalloidin and rhodamine-WGA and reconstructed from a 100 μm × 100 μm area. B6-Cx46KO mice carry wild-type CP49 (CP49(+/+)), whereas both 129-Cx46KO and B6L/B6L-Cx46KO contain a CP49 gene deletion (CP49(del/del)) from the 129 strain genetic background. The upper panels show 3D reconstructions of F-actin (green) and WGA (red) staining in inner mature fibers, located approximately 300–400 μm from the lens surface within the transitional region, where mature fibers on the right side (toward lens core) display reduced F-actin staining. The lower panels present corresponding 2D images of F-actin and WGA labeling from the same transitional regions shown in the upper panels. Scale bars: 10 μm.