September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Development of functional cell junctions at the corneal limbus in the embryonic chicken
Author Affiliations & Notes
  • James Kenneth Kubilus
    Integrative Physiology and Pathobiology, Tufts University School of Medicine, Boston, Massachusetts, United States
  • Carolina Zapater i Morales
    Integrative Physiology and Pathobiology, Tufts University School of Medicine, Boston, Massachusetts, United States
  • Thomas Linsenmayer
    Integrative Physiology and Pathobiology, Tufts University School of Medicine, Boston, Massachusetts, United States
  • Footnotes
    Commercial Relationships   James Kubilus, None; Carolina Zapater i Morales, None; Thomas Linsenmayer, None
  • Footnotes
    Support  NIH Grant R01EY023569
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 4349. doi:
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      James Kenneth Kubilus, Carolina Zapater i Morales, Thomas Linsenmayer; Development of functional cell junctions at the corneal limbus in the embryonic chicken. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4349.

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

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Abstract

Purpose : It is known that the corneal limbus contains a population of corneal stem cells responsible for maintaining the cornea both during health and disease. However, it is unclear how these stem cells become established in their niche during development. We hypothesize that the developmental expression of cell junction proteins is necessary for the establishment of the stem cells in their niche. It has been shown previously that the gap junction protein, Connexin 43, is expressed in the cornea and sclera, but not in the limbus. Here we examined the developmental expression of connexin 43 in the chicken embryo in relation to other corneal markers and tested the potential communication between cells in the corneal limbus.

Methods : Embryonic chicken anterior eyes from embryonic day 4 (E4) through E17 were dissected and either fixed or frozen fresh for immunohistochemistry with antibodies against junctional proteins connexin 43, afadin and ZO-1. Scanning electron microscopy (SEM) was used to observe morphological changes. Cut loading of anterior eyes from corresponding timepoints with rhodamine-dextran (MW 3000), rhodamine dextran (MW 10000), Lucifer yellow (MW 457) and carboxyfluorescein (MW 376) was used to determine cellular communication.

Results : Previous results using the lipophilic dye DiI, suggested that a barrier to diffusion from the cornea to the sclera began to develop within the limbus at approximately E8. Here, both morphological and molecular changes consistent with this development were observed. Beginning at E8 cells at the presumptive corneal limbus began to change shape and level of adhesion in scanning electron microscopy. Consistent with this, expression of connexin43 decreased in cells of the corneal limbus. Further, cut-loading of the fluorescent dyes with a size small enough to pass through gap junctions (rhodamine-dextran [MW3000], carboxyfluorescein and Lucifer yellow) confirmed the function of gap junctions within the cornea, but also showed that the limbal cells were isolated by their lack of gap junction proteins.

Conclusions : The results suggest that the developmental regulation of cellular junctions at the corneal-scleral limbus may have a functional role in establishing the corneal stem cell niche through changes in cell-cell communication. Further work is needed however, to fully test this hypothesis.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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