By E12.5, nerve bundles begin to appear at distinct locations in the periphery of the eye. To establish position identity of the pioneer axons, we partitioned each eye along the dorsal-ventral and temporal-nasal axes into four quadrants (VN, VT, DN, DT;
Fig. 1A). Similar to E11.5, the majority of the embryos (24/35) have innervation of the upper and lower eyelid region and whisker pad, but not the eye (
Fig. 1A). Nonetheless, some E12.5 embryos (11/35) exhibit an interesting variation in nerve staining along the edge of the eye. In some eyes, a single nerve bundle was present in the DN (
Figs. 1B and
1C, arrowhead), VN (
Fig. 1C, arrowhead) or VT (data not shown) quadrants. In other instances (
Fig. 1D), a nerve bundle was present in each of the two quadrants of the same eye: DN and VN (1/35;
Fig. 1C); VN and VT (1/35); and VT and DN (2/35). The VT quadrant was always innervated in combination with either the VN or DN quadrants. All three quadrants were never innervated in the same eye at E12.5.
Figure 1E shows the number of times each quadrant was innervated. However, the majority of the eyes did not have innervation of any of the four quadrants (24/35). For the eyes that were innervated, the majority of the pioneer nerve bundles were located in the VN quadrant (8/35), followed by the DN quadrant (4/35), then the VT quadrant (3/35). Interestingly, the DT quadrant was never innervated at E12.5 (0/35). A representative cross-section through an innervated quadrant at E12.5 reveals a single nerve bundle in the periocular region projecting toward the adjacent presumptive cornea (
Fig. 1F, arrow). Our results indicate that presumptive corneal nerves arrive in the periphery of the anterior surface of the eye as early as E12.5. The DN, VN, and VT quadrants are innervated before the DT quadrant. A single nerve bundle is located in each innervated quadrant of the eye.