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M. K. Call, J. Zhang, Y. Zhang, S. Eleswarpu, C.-Y. Liu, W. W. Kao; Lineage Tracing of Periocular Mesenchymal Cells of Neural Crest Origin in Ocular Surface Tissue Morphogenesis During Development. Invest. Ophthalmol. Vis. Sci. 2010;51(13):351.
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© ARVO (1962-2015); The Authors (2016-present)
To determine the kinetics and the lineage of discrete populations of periocular mesenchymal cells of neural crest origin in ocular surface tissue morphogenesis during mouse embryonic development.
Utilizing tet-on triple-transgenic Kera-rtTA/tet-O-Cre/ROSA26mTmG mice, we labeled discrete populations of cells during various stages of embryonic development (E12.5, E14.5, E16.5, E18.5) by pulse induction of doxycycline to the pregnant dam. Cells expressing keratocan at the time of induction will switch from expressing tomato-red fluorescent protein to an enhanced green fluorescent protein (EGFP), whose expression after recombination is regulated by the ubiquitous Rosa allele and independent of Kera (keratocan gene), allowing these cells to be traced. At P0, mice were analyzed by fluorescence microscopy to identify the fate of each cell population labeled at different stages of gestation. In parallel, transgenic mice were also induced with doxycycline postnatally (P0, P12, P14) to further elucidate the involvement of keratocan expressing cells during later stages of ocular surface morphogenesis. These mice were fed doxycycline chow continuously and examined at P21-P30. All reported research was conducted in compliance with the ARVO statement for the Use of Animals in Ophthalmic and Vision Research.
With pulse induction at E12.5 and E14.5, EGFP-expressing cells were localized to the anterior portion of the corneal stroma and densely throughout the eyelid. Induction at E16.5 or E18.5 also resulted in EGFP-expressing cells localized at the anterior portion of the corneal stroma in addition to the posterior portion (conjunctival side) of the eyelid as well as around the eyelash hair follicles. Postnatal induction resulted in strong expression within the corneal stroma as well as the eyelash hair follicles.
Using the Kera-rtTA/tet-O-Cre/ROSA26mTmG mice, we were able to trace the fate and assess the contribution of discrete waves of periocular mesenchymal cells to the morphogenesis of ocular surface tissues during embryonic development. This research yields useful information for a better understanding of the overall biology of the ocular surface but also provides a unique tool for genetic manipulation of discrete cell populations.
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