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Fig. 5. The misexpression of Crx is a cell-autonomous response to Pax6 loss in RPCs. (A-E) Pax6 and Crx were detected on the same sections from control (A,B) and Pax6^flox/flox;Chx10-Cre (C-E) E14.5 eyes using indirect immunofluorescence analysis (Pax6, red) or fluorescent in situ hybridization (Crx, green). Only in some of the Pax6-deficient cells was misexpression of Crx detected, whereas other cells were negative for both Pax6 and Crx (white arrowhead in C,D). The Pax6^-Crx⁺ cells (D,E) were detected both adjacent to Pax6-expressing cells (white asterisk) or in regions distant from Pax6 expression (red arrowhead). (F-I) At E16.5, the expression of VC1.1 (red) was compared with Pax6 protein (F,H green) or Crx transcripts (G,I green) in control (F,G) and Pax6^flox/flox;Chx10-Cre embryos (H,I). (J) The distribution of Pax6^-Crx⁺ cells along the central-peripheral regions of the OC was quantified. A significant difference in the proportion of Crx-expressing cells in the Pax6-deficient areas between central and peripheral OC was identified (^*P<0.001 by Student's t-test, n=6 eyes). The scheme illustrates the arbitrary division of the OC into central/peripheral regions. co, cornea; le, lens; nnp, non-neuronal progenitors; oc, optic cup; prp, photoreceptor layer; rpe, retinal pigmented epithelium. Scale bar: in A, 200 µm; in B,E, 25 µm; in C, 75 µm; in D,F,G,H,I, 50 µm.

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