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Fig. 2. The regenerating wound is populated by retinal progenitor cells and is organized similarly to the CMZ.
(A–C) In situ hybridization performed using retinal sections of embryos at 9 days post-resection. Cells filling the regenerating wound express pan-RPC markers Rx1A (A), Pax6 (B), and Sox2 (C). (D, E) Cells filling the regenerating wound are proliferating. Immunolabeling of regenerating retinas at 9 days post-resection with anti-BrdU antibody. The putative RPCs incorporate BrdU and are immunoreactive to the anti-BrdU antibody (E, red bracket). The nasal-dorsal quarter of an uninjured retina lacks proliferating RPCs (D). (F, G) In situ hybridization performed on sections of embryos at 9 days post-resection with riboprobes for Notch1 (F) or NeuroD (G). (H) Double in situ hybridization for Notch1 (blue) and NeuroD (red). Different subsets of the RPCs (red) express Notch1 and NeuroD. Notch is expressed closer to the center of the wound (H; blue brackets) than NeuroD (H; red brackets) confirming that the expression of these two markers begins in different subsets of the RPCs that repopulate the wound. (I, J) The cyclin-dependent kinase inhibitor Xic1 is expressed at the extreme periphery of the regenerating region. (I) In situ hybridization for Xic1 (red brackets) demonstrates expression at the periphery of the regenerating wound and not in the center (blue bracket). (J) Overlay of BrdU incorporation (fluorescent green) and Xic1 in situ hybridization from (I). Proliferating cells are largely in the center of the regenerating wound (blue bracket), with little overlap with cells expressing Xic1 (red brackets). (K) Left — Model of normal CMZ (adapted from Perron et al., 1998). Right — Model of the CMZ formed in the regenerating wound. Scale bar = 50 μm. |
