Fig. 4. Mitf expression and OV regionalization are lost in Pax2^-/-; Pax6^-/- embryos. Immunohistochemical staining of serial, 6 µm cryosections of E9.5 (A-B,E-F,I-J,M-N) and E11.5 (C,D,G,H,K,L,O,P) using antibodies against Pax2 and Pax6 (A-D), Mitf (E-H), Chx10 (I-L) and Otx2 (M-P). (A) Pax2 (green) and Pax6 (red) are expressed throughout the wild-type E9.5 OV. (C) At E11.5, Pax6 expression (green) is found in all compartments of the eye, but Pax2 (red) is mainly restricted to the ventral NR and RPE and the optic stalk. Both Pax2 and Pax6 are absent in Pax2^-/-; Pax6^-/- OVs at both stages (B and D). The E9.5 wild-type OV expresses Mitf predominantly in the proximal regions (E). At E11.5, Mitf expression is confined to the RPE (G). Mitf fails to be expressed in Pax2^-/-; Pax6^-/- OVs at both stages (F,H). The presumptive NR is marked by the Chx10-positive distal domain at E9.5 in both the wild-type (I) and the Pax2^-/-; Pax6^-/- OVs (J), but seems to be expanded dorsally in the mutant OV (J, arrow). At E11.5, Chx10 is confined to the NR region in the wild type (K) and to the distal OV in Pax2^-/-; Pax6^-/- OVs (L). The proximal expression of Otx2 in the wild type at E9.5 (M) appears expanded ventrally in the mutant OV (N, arrow). The Otx2 expression becomes restricted to the RPE domain at E11.5 in wild type (O), but remains co-expressed with the Chx10 positive distal OV in Pax2^-/-; Pax6^-/- mutants (P). lv, lens vesicle; ov, optic vesicle; os, optic stalk; nr, neural retina; rpe, retinal pigmented epithelium; se, surface ectoderm

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