March 2012
Volume 53, Issue 14
ARVO Annual Meeting Abstract  |   March 2012
Investigating Roles for the Robo-Slit Pathway and Extracellular GAGs on Nerve Guidance during Cornea Development
Author Affiliations & Notes
  • Tyler Schwend
    Division of Biology, Kansas State University, Manhattan, Kansas
  • Peter Y. Lwigale
    Biochemistry and Cell Biology-MS140, Rice University, Houston, Texas
  • Gary W. Conrad
    Division of Biology, Kansas State University, Manhattan, Kansas
  • Footnotes
    Commercial Relationships  Tyler Schwend, None; Peter Y. Lwigale, None; Gary W. Conrad, None
  • Footnotes
    Support  NIH Grants R01EY000952, F32EY021708
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 1805. doi:
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      Tyler Schwend, Peter Y. Lwigale, Gary W. Conrad; Investigating Roles for the Robo-Slit Pathway and Extracellular GAGs on Nerve Guidance during Cornea Development. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1805.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: : The cornea becomes densely innervated in a series of highly coordinated events. Nerve growth cones projecting from the trigeminal ganglion (TG) reach the cornea margin at embryonic day (E)5, where they are initially repelled from E5-8, instead encircling the corneal periphery in a nerve ring prior to entering on E9-10, at which time growth cones project exclusively within the anterior stroma en route to the corneal epithelium. Here we evaluated potential roles for the Robo-Slit nerve guidance family and extracellular glycosaminoglycans (GAGs) in nerve guidance during cornea development.

Methods: : Chick corneas, lens and TGs were isolated from E5 - E14. Gene expression for Slit ligands 1-3 and their cognate receptor Robo1 were assessed in eye and TG respectively by RT-PCR and in situ hybridization. GAG expression was examined in corneas by immunostaining. To determine the nature of Robo-Slit regulation on trigeminal neurons in vitro, TG explants (source of neurons) and lens or cornea were co-cultured in collagen gels in the presence of inhibitory Robo-Fc protein. Robo-Slit signaling was transiently perturbed in vivo by implanting Robo-Fc coated beads into the eyefield. To determine the effects of individual GAGs on neuronal outgrowth, TGs were cultured in the presence of soluble or substrate-bound GAGs.

Results: : Slit 1-3 expression in cornea and lens persisted during all stages of cornea innervation examined. Robo1 was developmentally regulated in trigeminal cell bodies, expressed robustly during nerve ring formation, then later declining concurrent with growth cone projection into the cornea. Perturbing Robo-Slit signaling in vivo led to premature cornea innervation. Additionally, in organotypic tissue culture both lens and cornea strongly repelled E7 neurites, except in the presence of inhibitory Robo-Fc protein. In contrast, E10 neurites were not as strongly repelled by cornea, and presence of Robo-Fc protein had no effect. At E9-10, growth cones projecting into the cornea encounter chondroitin sulfate/dermatan sulfate (CS/DS) and keratan sulfate (KS)-GAGs, each present in the cornea at this stage. Soluble and immobilized GAGs inhibit neurite outgrowth from E10 trigeminal neurons in vitro.

Conclusions: : These findings suggest repulsion by the cornea during nerve ring formation is mediated by Robo-Slit signaling. Later, a shift in nerve guidance occurs, in part due to molecular changes in trigeminal neurons, including Robo1 downregulation, allowing nerves to find the Slit-expressing cornea permissive for ingrowth. Subsequently, nerves in the cornea are likely regulated by GAGs present in the stromal ECM, possibly directing the advancement of growth cones toward the epithelium.

Keywords: cornea: basic science • innervation: sensation • cell-cell communication 

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