From these data, it appeared that direct,
Opn5-dependent shortwave light sensitivity of the cornea developed following explantation. One possibility for this would be that
Opn5 expression is upregulated following dissection. We assessed the level of opsin transcript relative to
β-actin in corneas maintained in organotypic culture. A large increase of
Opn5 transcript expression was observed after 2 days in culture (
Fig. 4A). A smaller but significant increase in
Opn4 transcript was also detected relative to the fresh cornea (day 0). Transcript levels were variable but not significantly increased for
Opn3 (
Fig. 4A). Unfortunately, no validated antibodies to OPN5 protein have been described.
11 In order to better characterize the
Opn5 induction in a single cornea over time, an
Opn5Cre; Ai14 (tdTomato) reporter
17 was used to observe
Opn5Cre induction over multiple days ex vivo (3-hour resolution for 48 hours; see
Supplementary Video). We noted two populations of
Ai14-positive cells. A sparse, stationary group of cells in the corneal stroma is present at time of dissection; we refer to these cells as “type 0” as they are present at timepoint 0 relative to explantation. In contrast, we noted marked induction of
Ai14 expression in the epithelium (type 1 cells) starting at about 9 to 12 hours postdissection. These cells appear to be highly motile (see
Supplementary Video). The type 1 cells display a morphology typical of epithelial cells with rounded, flat cell bodies, while the type 0 cells have much larger cell bodies and outreaching dendritic processes similar to dendritic cells (
Figs. 4D, 4E).