April 2010
Volume 51, Issue 13
ARVO Annual Meeting Abstract  |   April 2010
Molecular Regulation of Corneal Innervation During Chicken Embryonic Development
Author Affiliations & Notes
  • J. K. Kubilus
    Anatomy and Cell Biology, Tufts University, Boston, Massachusetts
  • T. F. Linsenmayer
    Anatomy and Cell Biology, Tufts University, Boston, Massachusetts
  • Footnotes
    Commercial Relationships  J.K. Kubilus, None; T.F. Linsenmayer, None.
  • Footnotes
    Support  NIH Grant EY018889
Investigative Ophthalmology & Visual Science April 2010, Vol.51, 6372. doi:
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      J. K. Kubilus, T. F. Linsenmayer; Molecular Regulation of Corneal Innervation During Chicken Embryonic Development. Invest. Ophthalmol. Vis. Sci. 2010;51(13):6372.

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

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Purpose: : Corneal innervation is necessary for the perception of pain as well as the overall maintenance of the cornea through the release of trophic factors. However the factors that regulate the growth and patterning of nerves within the cornea during development remain unclear. We have now analyzed the expression of two axon guidance cues, Sema3A and Slit2, and their respective receptors, Nrp-1 and Robo2, during corneal development. We also functionally tested the effects of Slit2 on the growth and behavior of the corneal nerves.

Methods: : The spatial and temporal growth of axons into the cornea was determined using immunohistochemistry on embryonic chicken eyes between E6 and E14. Expression was determined by in situ hybridization and qRT-PCR, and in some cases Western blot. Also, to functionally analyze the role(s) of Slit2 in corneal innervation, a blocking antibody was used in ovo, and in 3-dimensional collagen gel co-cultures of corneas and trigeminal ganglia.

Results: : Between E7 and E8, nerves grow into the corneal stroma (CS) centripetally from the peri-corneal nerve ring. At E10 they penetrate Bowman’s layer, and within the basal corneal epithelium (CE) they rapidly branch - forming the sub-basal nerve plexus. They then grow through the CE and terminate at the apical cell layer. Between E7 and E8, a rapid decrease in the expression of Sema3A and Slit2 occurs in the CS, but in the lens these factors remain constant. Also at this time, a slow, progressive decrease begins in Nrp-1 (the Sema3A receptor). However, the Slit2 receptor (Robo2) begins a progressive increase, suggesting a switch in function. Such a switch was confirmed, as at the early stage of innervation, immunoneutralization with a Slit2 blocking antibody increases axon growth but at later stages it decreases branching in the sub-basal nerve plexus.

Conclusions: : At early stages of innervation, both Sema3A and Slit2 act as negative regulators - preventing corneal innervation into the CS until the proper time. Later, however, Slit2 switches to positive regulation - promoting branching of nerves once they enter the CE.

Keywords: cornea: basic science • development • innervation: sensation 

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