Fig. 2. Pax6 plays a unique role in each of two spatially distinct subsets of RPCs in the Pax6^flox/flox;-Cre OC. The expression of Pax6, VC1.1 (A-H; green and red, respectively), Crx (I-P), BrdU (Q-V) and syntaxin (W,X red) were characterized on adjacent sections by antibody labeling (A-H,Q-X) or in situ hybridization (I-P) in control (Pax6^flox/flox) and mutant (Pax6^flox/flox;-Cre) retinas in the course of eye development. In the Pax6^flox/flox;-Cre OC, Pax6 was eliminated from the peripheral regions (B,D,F,H; the Pax6-deficient domain is flanked with arrowheads). Two spatially distinct populations of Pax6-deficient RPCs were identified (diagram): the Pax6^- cells that are located in the OC periphery upregulate Crx, whereas the Pax6^- cells located towards the central OC do not upregulate Crx. The border between the two Pax6-deficient cell types is indicated with an arrow and a broken line, and their margins are marked with arrowheads (labeled as region 1 or region 2, respectively). (Y) The percentage of the Crx-expressing domain (region 1, white bars) and the Crx^- domain (region 2, gray bars) relative to the total Pax6-deficient area was calculated for E12, E14, E16 OCs (n=4 eyes for all embryonic stages). (Z) Significant reduction in the percentage of BrdU⁺ cells was detected for both Pax6-deficient regions at all stages of development (^**P<0.005 and ^*P<0.05 by Student's t-test; n=4 eyes for all Pax6^flox/flox;-Cre retinas and three eyes for controls). The reduction in the proliferation index was significantly more extensive in region 1 than in region 2 at E14 (P<0.01 by Student's t-test). inl, inner nuclear layer; gcl, ganglion cell layer; le, lens; nr, neuroretina; prp, prospective photoreceptor layer; rpe, retinal pigmented epithelium. Scale bar: 100 µm.