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?2F, G, and I), markers of CMZ zones 2, 3, and 4 respectively (Perron et al., 1998). None of these markers was expressed in the RPCs at GDC-0449 mouse the center of the wound. Further, NeuroD was absent from a region of the regenerating wound that expressed Notch1 (Fig.?2H). Xic1 was expressed at the periphery of the wound and was largely excluded from proliferating cells at the center of the wound (Fig.?2J). This organization was reminiscent of the organization of the CMZ at the retinal periphery, where Notch is expressed in zones 2�C4 and NeuroD is expressed in zones 3�C4, and Xic1 is primarily expressed in zone 4 (Perron et al., 1998). It is not surprising that there was some overlap between BrdU-positive cells and Xic1-expressing cells, as it has recently been demonstrated that Xic1 is expressed in some proliferating RPCs (Bilitou and Ohnuma, 2010). These results suggest that the RPCs are organized into zones, similar to the endogenous CMZ, at this stage of retinal regeneration (Fig.?2K). To investigate the involvement of Rx in retinal regeneration, we used a MS-275 in vivo transgenic shRNA approach to knock down Rx expression (Pan et al., 2010). Previously, we demonstrated that Rx expression is knocked down 50�C90% in Rx shRNA transgenics but the eye develops with apparently normal morphology through st 41. To address the function of Rx during retinal regeneration, we induced regeneration in Rx shRNA transgenic tadpoles. We found that regeneration is impaired in Rx shRNA transgenics (Figs.?3C and D) as compared to nontransgenic controls (Fig.?3A) and control shRNA transgenics (Fig.?3B). The wound is disorganized at 9?days post-resection (Figs.?3C and D). In some tadpoles the cells repopulating the wound do not appear to be normal RPCs. The cells have a rounder morphology than the typical spindle-shaped RPCs found at the CMZ (Figs.?3C and S1). In some cases, the cells repopulating the wound lack the columnar organization we had previously Quinapyramine observed in the RPCs that repopulate the wound by 9?days post-resection. Others have both defects in RPE reformation and RPC repopulation of the wound. We did not find tadpoles in which only the RPE regeneration at the wound site was defective. On the other hand, in some tadpoles, the RPE is either not completely reformed at the wound site or is disorganized (Figs.?3C and D). To quantify our observations we developed a classification of the regeneration defects we found in Rx shRNA transgenic tadpoles (Table?1). Based on the regeneration defects, regenerating embryos were classified into 3 categories, defined by morphological criteria. Using this classification system we found that 72% (p?
