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Vijay K. Kalaskar, James D. Lauderdale; Evaluation of Embryonic Mouse Eye Development in Culture. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4924.
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To evaluate embryonic mouse eye development under defined culture conditions and utilize this system to identify signaling interactions important in anterior eye development.
Mouse embryos were obtained from our breeding colony with noon on the day of plug discovery designated as embryonic day 0.5 (E0.5). Wild-type embryos were CD1-C57BL/6J. Embryos overexpressing ‘pairedless’ Pax6 (Pax6ΔPD) were obtained as described by Kim and Lauderdale (2008). Embryos at E10.5 were cultured in a standardized serum free culture medium comprised of commercially available stem cell media supplements in an oxygenated rolling culture system. Anterior eye development was assessed using morphological and gene expression criteria, including Pax6, Sox2, Prox1, αA-Crystallins and FoxC1.
Wild-type E10.5 mouse embryos cultured for 24 to 36 hours showed morphological development and tissue specific gene expression in the ocular tissue consistent with early eye development. For example, the developing lens had anterior proliferating epithelial cells and posterior αA-Crystallin positive differentiating lens fiber cells. Pigmentation of the retinal pigmented epithelium and FoxC1 positive migrating mesenchymal cells that contribute to the corneal stroma were evident. To evaluate the applicability of this system, and as an initial step to further investigate the signaling interactions involved, we cultured transgenic embryos that overexpressed the Pax6ΔPD isoform. These embryos in culture exhibited a lens degeneration phenotype that was comparable to that observed in Pax6ΔPD overexpressing embryos developing in utero.
Midgestation stage mouse embryos cultured using serum-free medium in oxygenated rolling culture exhibit morphological and molecular correlates of anterior eye development consistent with those observed in utero. We believe this method will be useful for laboratories needing to utilize whole embryo culture to study signaling interactions important in anterior eye development.
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