Innervation of the mouse cornea during development

Abstract

Purpose: Dense innervation of the cornea is important for maintaining its homeostasis and transparency. Although corneal nerves have been well studied in adults, little is known about mammalian corneal innervation during development. This study provides a detailed profile of nerves at various stages of mouse cornea development.

Methods: Mouse heads and corneas were collected at various stages of development including embryonic days (E)12.5 to E16.5, postnatal days (P)0, P10, three weeks after birth, and the adult. Corneas were immunostained with an anti-neuron-specific β-tubulin antibody (TUJ1). Fluorescently labeled nerves in whole-mount tissues and sections were imaged and analyzed for their axonal projections during eye development.

Results: The first nerve bundles appear at the periphery of the anterior portion of the eye by E12.5. Initial projection into the stroma occurs at E13.5 without formation of a pericorneal nerve ring. Between E13.5 and E16.5, nerve bundles project directly into the periphery of the presumptive cornea stroma. They branch repeatedly as they extend toward the cornea center and epithelium. Concomitantly, nerve bundles originating from four quadrants of the eye bifurcate into smaller branches that innervate the entire stroma. The first epithelial innervation occurs at E16.5. Epithelial nerves arrange into patterns that project toward the center subsequently forming a swirl at three weeks after birth, which becomes more pronounced in adults.

Conclusions: Nerve bundles that arise from four quadrants of the eye innervate the mouse cornea. The nerve bundles directly innervate the stroma without forming a pericorneal nerve ring. Radial arrangement of epithelial nerves gradually becomes centrally oriented, subsequently forming a swirl pattern.

Figure 1.

Innervation of the anterior ocular region at E12.5. (A) An embryo showing innervation of the upper and lower eyelids, and the whisker pad but no innervation of the eye. To determine the positioning of nerve bundles before cornea innervation, the eye was divided into quadrants along the dorsal-ventral and temporal-nasal axes. (B) Innervation of the DN quadrant (arrowhead). (C) Innervation of the DN and VN quadrants (arrowheads). (D) Venn diagram summarizing the number and overlap of innervated quadrants. (E) Quantification of eye innervation and position of pioneer nerve bundles. (F) Cross-section through an E12.5 eye counterstained with DAPI showing a nerve bundle (arrow) in the periocular region projecting toward the presumptive cornea. Figure 1F was imaged from a similar location as the boxed region in Figure 2D. D, dorsal; N, nasal; V, ventral; T, temporal; DN, dorsal-nasal; VN, ventral-nasal; ll, lower eyelid; ul, upper eyelid; wp, whisker pad; pc, presumptive cornea; pr, periocular region; oc, optic cup. Scale bars: (A–C) 100 μm; (F) 50 μm.

Figure 2.

Projection of nerve bundles toward the presumptive cornea at E13.5. (A–C) Showing projection of nerve bundles from the DN, VN, and VT quadrants toward the presumptive cornea. Some nerve bundles innervate the cornea periphery (asterisks). Innervation of the DT quadrant ranges from (A) none, (B) slight (arrow), compared to other quadrants, and (C) moderate (arrow), compared to other quadrants. (D) Cross-section of an immunostained E13.5 eye counterstained with DAPI showing that presumptive cornea nerves project along the optic cup (arrowheads) toward the anterior region of the eye. (E) Close up of the boxed area in D showing innervation of the mid-stromal region (asterisk) of the presumptive cornea. (F) Quantification of innervated quadrants at E13.5. DN, dorsal-nasal; VN, ventral-nasal; VT, ventral-temporal; DT, dorsal-temporal; oc, optic cup; pc, presumptive cornea. Scale bars: (A–D), 100 μm; (E) 50 μm.

Figure 3.

Innervation of the mouse cornea stroma. Whole-mount staining of (A) E14.5 and (C) E15.5 and (E) E16.5 corneas showing progressive extension and branching of nerves as they innervate the entire cornea stroma. (D) Cornea in (C) imaged from an angle to show that a single major nerve bundle (arrow) from the posterior region of the eye bifurcates into several nerves (asterisks) in the VN quadrant. Cross-sections of (B) E14.5 showing that nerve bundles innervate only the anterior two-thirds of the stroma and project toward the epithelium, which is innervated by (F) E16.5 (arrowheads). Dotted lines demarcate in (E) the boundary of the area covered by nerves from each quadrant, and in (B) and (F) the epithelium/stroma boundary. DN, dorsal-nasal; VN, ventral-nasal; VT, ventral-temporal; DT, dorsal-temporal; oc, optic cup; st, stroma; epi, epithelium; ll, lower eyelid nerves. Scale bars: (A, C, D, E) 100 μm; (B, F) 50 μm.

Figure 4.

Innervation of the mouse corneal epithelium. (A, C, E, G, I) Whole-mount immunostaining of cornea epithelia showing the distribution of epithelial nerves at the corneal apex during different stages of development and in adult. (B, D, F, H, J) Cross-sections of corneas at corresponding time points, showing innervation of the epithelium and the formation of the subbasal plexus (arrowheads). epi, epithelium; st, stroma. Scale bars: (A, C, E, G, I) 50 μm; (B, D, F, H, J) 20 μm.