Developmental corneal innervation: Regulation by multiple factors and interactions between nerves and specialized corneal epithelial cells

Proper sensory innervation is necessary for the health and maintenance of the cornea, as well as corneal sensation, which protects the cornea from damage. Previous studies have described the architecture of corneal nerves in the adult and the stages of corneal innervation during development. However, little is known about how these stages are regulated so that the cornea is properly innervated. Here, corneal innervation in the chicken embryo was examined to determine the mechanisms that regulate nerve growth into the cornea during development.;The stages of corneal innervation described previously by others were re-examined using more sensitive immunohistochemical techniques to label corneal nerves with the TuJ-1 antibody, which recognizes an isoform of beta-tubulin thought to be neuron specific. These studies determined that (1) the stages of corneal innervation occur considerably earlier than previously described, (2) the squamous apical corneal epithelial cells are also labeled by the TuJ-1 antibody and (3) the corneal nerves contact these TuJ-1+ epithelial cells during corneal innervation. Immunohistochemistry, electron microscopy and immuno-electron microscopy determined that the nerves and the TuJ-1 + cells form synapse-like contacts. Also, the TuJ-1+ apical epithelial cells are mitotically active. These data suggest the TuJ-1 + apical epithelial cells are a specialized population of cells involved in the formation of synapse-like contacts with corneal nerves.;Innervation has been shown to be regulated during development by both positive and negative guidance cues that result in the proper innervation of a tissue. Here, RT-PCR, qRT-PCR and in situ hybridization were used to correlate the expression of known guidance cues with the stages of corneal innervation. The negative guidance cues semaphorin3A and Slit2, along with their receptors neuropilin1 and roundabout2, were expressed in a pattern consistent with a function in regulating the first stage of corneal innervation. Slit2 has also been proposed to have positive effect on the branching of sensory neurons. Immunoneutralization of Slit2 in ovo during the later stages of corneal innervation reduced the branching of nerves within the corneal epithelium. These results suggest that corneal innervation is regulated by multiple factors, and least one of them, Slit2, has multiple functions during the different stages.