Abstract
Purpose: :
Laminins and netrins are extracellular matrix (ECM) molecules with roles in: basement membrane assembly; cell adhesion, proliferation and migration; and neural guidance. Here we study the role of laminins and netrins in corneal development and its innervation.
Methods: :
Laminin and netrin expression were studied by immunofluorescence (IF). Corneal organization and innervation were assayed using IF, confocal and electron microscopy (EM) in specimens from wild-type, Lamb2-/-, Lamc3-/- and Ntn4-/- mice. Innervation was examined and quantified in whole-mounts of P20 cornea.
Results: :
Netrin4 IF was present throughout the cornea from limbus to central cornea; it was present in Bowman’s membrane, the stroma and Descemet’s membrane (DM). Laminin β2 IF had a similar distribution, whereas γ3 IF was concentrated in the limbus. Although corneal development was grossly normal in all genotypes, several microscopic alterations were observed. Corneal keratinization was seen (by EM) in P15-16 corneas from all three mutant lines. Moreover, there was a disorganization of the corneal ECM in several of the Lamb2-/- mouse; the most important change was an increase in the ratio of the thickness of DM to the endothelium. Cell proliferation was also measured using phospho-histone H3 IF. While a 3-fold increase in epithelial proliferation was found in the netrin4 null animal, more modest increases were seen in the epithelium of the β2 null animal. An increased density of endothelial cells was seen in the β2 null animal. We next studied corneal innervation; a developmental series of corneas from each genotype was assayed using the nerve-specific antibody TuJ-1, which targets class III β-tubulin. Results from IF microscopy comparing corneas from P20 wild-type and Lamb2-/-, Lamc3-/- and Ntn4-/- mice demonstrate profound reorganization in mutant mice of nerve density and patterning in the central cornea and in axon fasciculation in the pericorneal ring.
Conclusions: :
We show that netrin-4, and the β2 and γ3 chains of laminins, are expressed in corneal ECM. The deletion of these molecules affects not only the proliferation of epithelial cells but also the patterning of innervation. Whether these conditions are linked, or separable phenotypes, is the subject of on-going investigation.
Keywords: cornea: basic science • extracellular matrix • cornea: epithelium