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Vijay Kalaskar, James Lauderdale; Identification of Signaling Pathways involved in Retina-Lens Tissue Interactions using Embryos Over-Expressing Pairedless Pax6 (Pax6ΔPD) Gene. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5940. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To identify and evaluate the role of signaling pathways involved in lens cell survival and retinal development using mouse embryos over-expressing Pax6ΔPD in a whole embryo culture system.
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 (WT) embryos were CD1-C57BL/6J. Embryos overexpressing Pax6ΔPD were obtained as described by Kim and Lauderdale (2008). Embryos were evaluated for differential expression for genes involved in different signaling pathways by qRT-PCR and in situ hybridization (ISH). Of the genes altered, we first tested the BMP4 signaling by implanting affi-gel agarose beads treated either with recombinant BMP4 or Noggin proteins in the eye region of embryos at E10.5 cultured in a standardized serum free medium. Contralateral eyes implanted with BSA protein treated beads were used as control. Eye tissues from these embryos cultured for 18 - 20 hours were analyzed for development and differential gene expression using immunohistochemistry, western blot, qRT-PCR and ISH.
Mouse embryos overexpressing Pax6ΔPD exhibited apoptotic degeneration of lens cells and analysis of this ocular tissue revealed up-regulation of BMP4 target genes indicating a possible role for BMP4 signaling in lens cell survival. When tested in a mouse embryo culture by implanting BMP4 treated beads in the ocular region in WT embryos, it did not result in lens cell apoptosis. Similarly, Noggin treated bead implantation in Pax6ΔPD overexpressing mouse embryos did not prevent the apoptotic degeneration of the lens tissue indicating that BMP4 signaling may not be involved in lens cell survival. However, BMP4 beads in these embryos inhibited the pigmentation in the retinal pigmented epithelium (RPE) when implanted at ~30 somite stage (ss) and decreased pigmentation at ~35ss and as well affected the dorsal-ventral (D-V) patterning in the retina. Preliminary analysis revealed a 5-fold increase in SOX2 expression apart from other BMP4 target genes in the BMP4 treated eyes compared to the BSA treated eyes.
BMP4 signaling may not have a role in lens cell survival. However, it affects the pigmentation in the RPE in a stage-dependent manner and has a role in D-V patterning of the retina indicating a potential role for BMP4 in RPE and retinal development.
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