Fig. 3. Pax2^-/-; Pax6^+/- eyes show transdifferentiation of the RPE into a second, inverted NR and reduced Mitf expression. Hematoxylin-Eosin (A,B,D,E,G,H) and immunohistochemical staining of serial, 6 µm sections using an antibody against the retinal ganglion cell marker Brn3b (C,F,I) and the RPE determinant Mitf (J-P). At E18.5, the wild-type eye differentiated to laminated NR, RPE and lens (A,B). The Pax2^-/-; Pax6^+/- eye did not form a lens, but the NR was much more expanded and folded at the expense of RPE development (D; detailed view in E). By contrast, at the same stage the Pax2^-/- mutant eye displays a fully developed optic cup but the NR and the RPE are expanded towards the optic stalk (G,H). The expression of Brn3b in retinal ganglion cells marked the innermost layer of the NR in wild-type (arrow, C) and Pax2^-/- NR (I). In the Pax2^-/-; Pax6^+/- eye, Brn3b expression (arrows) is confined to the inner layer of the inner NR (F: 1. nr) and to the outermost cell layer in the outer NR (F: 2. nr), indicating that the second NR is inverted compared to the inner NR. (J-P) At E9.5, Mitf expression is detected in the wild-type (J) and Pax2^-/-; Pax6^+/- OVs (M), predominantly in the proximal region. At E10.5, Mitf activity is confined to the outer layer of the optic cup in wild type (K, ol), but starts to be downregulated in the folded distal inner layer regions of Pax2^-/-; Pax6^+/- eyes (N, il). Mitf is still expressed the forming RPE in the wild-type E11.5 eye (L), whereas in the Pax2^-/-; Pax6^+/- optic cup, it is only found in regions between the newly formed, folded, NR regions and in the proximal RPE remnant (O, arrows). In the lens-CRE/Pax6^flx/flx eye, Mitf-positive RPE surrounds the folded NRs, similar to wild-type RPE (P).