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J.R. Spence, M. Madhavan, K. Del Rio–Tsonis; Hh and FGF Pathways Merge During Retina Regeneration in the Embryonic Chick . Invest. Ophthalmol. Vis. Sci. 2005;46(13):5335.
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Purpose: The embryonic chick has the ability to regenerate a new retina after it has been completely removed during early embryonic development (until E4.5). This regeneration occurs in two different ways; transdifferentiation of the retina pigmented epithelium, and proliferation of stem/progenitor cells located in the anterior margin of the eye that develops into the ciliary body and ciliary marginal zone (CB/CMZ). We have previously shown that both Shh and FGF can independently stimulate regeneration from the CB/CMZ, and that inhibition of the FGF pathway using an FGF receptor (FGFR) antagonist is sufficient to inhibit regeneration stimulated by both FGF and Shh. Our current studies focus on early signaling events during regeneration. Specifically, we are focusing on the influence Shh has on downstream molecules in the FGF/Map Kinase pathway. Methods: To inhibit different members of the FGF/Mek/Erk pathway, we are using different pharmacological inhibitors. We stimulate regeneration using FGF or Shh in the presence of these inhibitors and assay regeneration after 3 days. In addition we are using an in vitro explant culture system and phosphorylation specific western analysis to detect activation levels of Erk and Ras in the presence of FGF, Shh and inhibitors. Results: We show using our in vitro explant system that p–Erk is up regulated when stimulated by FGF after 1 hour, and this upregulation is more robust after 4 hours. We also show that Erk phosphorylation is dependant on FGF concentration. Interestingly, Shh also elicits a robust phosphorylation of Erk. This data is backed up by in vivo inhibitor analysis, which shows that when Mek is inhibited, regeneration is significantly reduced when stimulated by either Shh or FGF. Conclusions: Our in vitro explant data as well as in vivo inhibitor studies indicate that the Hh signaling pathway interacts with the FGF signaling pathway during retina regeneration from the CB/CMZ. In addition, activation of the FGF pathway is required for regeneration stimulated by Shh alone, indicating that there is significant crosstalk between the two pathways during regeneration.
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