n the levels of transcripts corresponding to Hes1 and Lef1, which encode transducers of the canonical 11756401 Notch and Wnt signaling pathways, respectively. Similarly, we observed a dosedependent increase in the number of GS+BrdU+ cells in 
the sections of retinal explants treated with Jag1 and Wnt3A. GS+BrdU+ cells were observed in the inner nuclear layer where Muller cells somas are located and there was a 1.76 fold and 2.9 fold increase in their number at the highest concentration of Jag1 and Wnt3a, respectively, compared to controls. To rule out the possibility that BrdU+ cells were apoptotic, we examined whether 4 August 2010 | Volume 5 | Issue 8 | e12425 Muller Cells and Regeneration or not these cells also expressed Ki67, a nuclear protein expressed by cells that are active in the cell cycle. We observed that Ki67 immunoreactivities were co-localized in BrdU+ cells and there was a dose-dependent increase in the number of BrdU+Ki67+ cells similar to that observed for GS+BrdU+ cells. The effects of Jag1 and Wnt3A on the increase in GS+BrdU+/ BrdU+Ki67+ cells were abrogated in the presence of DAPT, an inhibitor of gamma secretase, an enzyme essential for the cleavage of Notch receptor to initiate intracellular signaling, and FzdCRD, a soluble FZD receptor that neutralizes Wnt ligands by binding them, suggesting the involvement Notch and Wnt pathways in the activation of Muller cells. Next, the neurogenic MedChemExpress SB-366791 potential of Muller cells was examined using retinal explants that were cultured in the presence Jag1 + Wnt3a + BrdU for 4 days. Results from these experiments demonstrated a 11325787 significant increase in the number of GS+BrdU+ cells in the inner nuclear layer of explant sections, compared to controls. However, compared to the effects of Jag1 and Wnt3A alone, we did not observe a synergistic effect of Jag1+Wnt3A treatment on the number of GS+BrdU+ cells. To August 2010 | Volume 5 | Issue 8 | e12425 Muller Cells and Regeneration understand the underlying mechanism by which Jag1+Wnt3a activated Muller cells, we examined the expression of transcripts corresponding to cyclin-dependent kinase activator, inhibitor, and marker of the active phases of the cell cycle . We observed an increase in levels of CyclinD1 and Ki67 transcripts and a decrease in p27kip1 transcript levels, suggesting that an accentuation of CDK activities underlie Jag1+Wnt3A-mediated activation of Muller cells. That these effects involved the canonical Notch and Wnt pathways was demonstrated by an increase in levels of Hes1/Hes5 and Lef1 transcripts, respectively. Next, whether or not the activated Muller cells possessed the phenotype of neural progenitors was investi- gated. Immunohistochemical analysis of Jag1+Wnt3Atreated retinal explants revealed GS+ cells in the inner nuclear layer, co-expressing immunoreactivities corresponding to Pax6. Since GS+Pax6+ cells were absent in the control retinal explants, this suggests that some Muller cells in Jag1+Wnt3A-treated retina acquired the expression of the neural marker Pax6. For an unambiguous determination of neural phenotype of the activated Muller cells, cells were dissociated from treated and control retinas and subjected to a Hoechst dye efflux assay, which enriched activated Muller cells as Side Population cells. The SP phenotype is a universal feature of stem cells, conferred by the expression of Abcg2, an ATP-binding cassette 6 August 2010 | Volume 5 | Issue 8 | e12425 Muller Cells and Regeneration transporter. We observed appr
