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Jeremiah Martino, Galina Bachay, Michael Dattilo, Douglas Lazzaro, William Brunken; Role of Netrin-4 and Laminin β2 and γ3 Chains in Corneal Development and Maintenance. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4721.
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© ARVO (1962-2015); The Authors (2016-present)
Laminins and netrins are extracellular matrix (ECM) molecules that regulate cell proliferation, migration and neural guidance. Our previous work has shown that these molecules are expressed in the corneal basement membranes and their expression is altered in human disease. Our goal is to understand the role that these molecules play in corneal development and disease. To this end, we are continuing our characterization of the corneal phenotype in mice lacking the Ntn4, LamB2, or LamC3 genes.
Epithelial cell proliferation and differentiation, corneal innervation, and endothelial cell tight-junction formation were assayed via immunofluorescence (IF) in whole cornea and radial section. Key developmental time-points were assayed in wild-type (WT), LamB2-/-, LamC3-/-, double-knockout LamB2-/-LamC3-/-, and Ntn4-/- mice. Cell proliferation and innervation were quantified in P20 and P33/34 corneas.
Although corneal development was grossly normal in all genotypes, alterations in the proliferative behavior of epithelial cells, patterning of the sub-basal neural plexus and endothelial cell tight-junction formation were observed. Epithelial proliferation, assayed with phospho-histone H3 IF, demonstrated increased proliferation in the LamB2-/-, LamC3-/-, double-knockout LamB2-/-LamC3-/- vs. age-matched WT mice. Keratin12 IF data showed no differences between knockout and WT mice, indicating a normal corneal epithelial differentiation. Corneal innervation, assayed by axon labeling with neurotubulin IF (TuJ-1) was aberrant. Increases in nerve branching and pericorneal ring thickness at the level of the sub-basal plexus was observed in P20 Ntn4-/- mice vs. WT littermates. Neural guidance at gestational day 15 (E15) was not altered in Ntn4-/- and LamC3-/- mice, demonstrating that neural pathfinding is largely intact. Lastly, endothelial cell tight-junction assembly, assessed by ZO-1 and β-catenin IF, was profoundly disrupted in P25 LamB2-/- and P33 LamC3-/- and Ntn4-/- compared to age-matched WT mice.
Netrin-4 and laminin β2 and γ3 chains are important components of corneal basement membranes. Genetic deletion of these molecules disrupts epithelial and endothelial cell behavior as well as the branching pattern of the sub-basal neural plexus. Ongoing studies will determine the role of these molecules in corneal wound healing or post-surgical recovery.
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