In vivo, actin architectures were distinct from those in culture, and basal cells showed different patterns of staining from apical cells (
n = 12 wild-type eyes and
n = 8
Pax6 +/− corneas;
Figs. 3C–I). F-actin was concentrated at the borders of wild-type basal cells and did not form networks or stress fibers in the cytoplasm (
Fig. 3C). The F-actin scaffold also aligned to the borders of basal
Pax6 +/− cells but often appeared to split or expand to the cytoplasm (
Fig. 3D, arrowheads). Separated cell borders and vesiclelike cytoplasmic structures were also revealed in basal wild-type cells 21 hours after wounding by actin labeling (
n = 14 eyes;
Fig. 3E). In wild-type squamous cells, actin assembled into short, winding fibers that joined into a fine, dense meshwork in the cell body and aligned to the cell-cell borders (
Fig. 3F). F-actin also aligned to the
Pax6 +/− cell-cell borders, but the fibers often split into a chainlike form (
Fig. 3G, arrowheads). Notably, the F-actin meshwork was diminished or replaced by vesicles in the
Pax6 +/− cell bodies (
Fig. 3G). Lamellipodia and filopodia were visible in some corneal epithelial cells at the leading edge of wounded wild-type epithelia (
Fig. 3H, open arrowhead). Stress fibers were seen in some cells (
Figs. 3H,
3I), but most of the cells retained the actin meshwork structure (
Fig. 3H). Intercellular gaps marked by split F-actin signals at the borders of reepithelializing superficial cells were also noticed (
Fig. 3I, arrowheads), but they were less prominent than in
Pax6 +/− corneas, whose superficial layer actin structure was generally more disrupted than in wounded wild-type epithelia. F-actin binds on adherens junctions
36,43,44 ; therefore, the split chain form may confirm the presence of intercellular gaps found in
Pax6 +/− and regenerating wild-type corneal epithelia under transmission electron microscopy (
Figs. 2C–F). Previous analysis of actin localization in
Pax6 +/− corneal epithelia
18 did not report the defects observed in this study, likely because of the differences in protocol-tissue sections previously and flat-mount staining.