Ocular phenotype of Otx1^−/−; Otx2^+/− neonatal mice. Frontal paraffin sections of wild-type (A,B) and Otx1^−/−; Otx2^+/− (C,D) neonatal mice were stained with Cresyl Violet to asses morphology. Images in A and C were taken from sections at equivalent axial levels. B and D are high magnification views of part of A and C, respectively. Note that in mutants both eyes show very similar alterations. The lens is rotated about 90° dorso-nasally, NR is folded toward the lens and fibres exit the eye in aberrant positions (arrows in B,D points to fibre tracts). RPE is totally absent but for a patch of pigmented tissue (arrowhead in D). The majority of the RPE has differentiated as a NR (open arrows in D). lv, lens vesicle; nr, neural retina; on, optic nerve; pe, retinal pigment epithelium; sc, sclera; T, tongue. Bar: A,C, 900 μm; B,D, 300 μm.

Comparison of Otx2, Pax2, Pax6 and Six3 expression domains in the OV of Otx-deficient mice. Dark-field images of transverse paraffin sections of the OV of wild-type and Otx mutant embryos hybridised with radioactive probes for Otx2, Pax2, Pax6 and Six3, as indicated. Note how in all genotypes OV have evaginated normally and Pax6 and Six3 expression is similar in all genotypes. In contrast, expression of Pax2 and of the remaining Otx2 allele is extended respectively ventrally and dorsally in both Otx1^+/−; Otx2^+/− (C, H) and Otx1^−/−; Otx2^+/− (D-E, I-J) as compared to wild-type (A, F) or Otx1^−/− embryos (B, G). Arrows in A-J indicate the limits of expression in the OV. d, dorsal; v, ventral. Bar, 200 μm.

Localisation of RPE markers in Otx-deficient mice. Consecutive sections through E9.5 OV (A-F) or E12.5 OC (G-O) of wild-type (A,D,G,J,M), Otx1^+/−; Otx2^+/− (B,E,H,K,N), and Otx1^−/−; Otx2^+/− mice (C,F,I,L,O) were immunostained with an antiserum against OTX2 (A-C,G-I) and MITF (D-F) or hybridised with digoxigenin-labelled probes for Mitf (J-L) and tyrosinase (Tyr; M-O). Note how in mutant mice the abnormal distribution of MITF extends through the OV (D-F) overlapping with the ventrally diffused expression of OTX2 (A-C). Note the abnormal folding of the OC in Otx1^+/−; Otx2^+/− and Otx1^−/−; Otx2^+/−mutants (J compared with K,L). The outer layer of the OC is thicker (inset in G-I) and lacks pigmentation and expression of Mitf (K,L) and tyrosinase (Tyr; N,O). Note that in mutant mice small patches of RPE co-express OTX2, Mitf and tyrosinase (arrows in H,I,K,L,N,O). enr, ectopic neural retina; lv, lens vesicle; nr, neural retina; ol, outer layer; os, optic stalk; ov, optic vesicle; pe, retina pigment epithelium; pnr, presumptive neural retina. Bar: 200 μm (A-F), 300 μm (G-O).

Gene patterning in the OC of E12.5 Otx-deficient mice. The expression domains of Six3 (A,B), Pax6 (C,D) and Pax2 (E,F) in wild type (A,C,E,G) and Otx1^−/−; Otx2^+/− (B,D,F,H) mice are illustrated with dark-field views of frontal paraffin sections hybridised with radioactive probes (A-F). Corresponding cytoarchitecture is showed with DIC images in G and H. Note the ‘ectopic’ expression of Pax6 and Six3 in the outer layer of the OC (B,D) and the extension of the Pax2 domain (F) in Otx1^−/−; Otx2^+/− embryos. enr, ectopic neural retina; lv, lens vesicle; nr, neural retina; os, optic stalk; pe, retina pigment epithelium. Bar, 200 μm.

PAX2 localisation in the developing eye. Frontal sections of wild-type (A-D) and Otx1^−/−; Otx2^+/−+/− (E-F) mice at E10.5 (A,C,E) and E12.5 (B,D,F) were immunostained with antiserum against PAX2. Otx2 expression was localised in wild-type embryos (A,B) with a digoxigenin-labelled probe. Note that Otx2 and PAX2 colocalise only in a few cells at the boundary of the two expression domains (inset in A, B). In Otx1^−/−; Otx2^+/− mice PAX2 expression is extended into the neural NR (compare C and E) and the RPE (D,F) territories. lv, lens vesicle; nr, neural retina; os, optic stalk. Bar: 150 μm (A,C,E), and 200 μm (B,D,F).

Lens development abnormalities in Otx-deficient mice. Adjacent sections through E10.5 (A-C) and E12.5 OC (D-I) of wild-type (A,D,G), Otx1^+/−; Otx2^+/− (B,E,H), and Otx1^−/−; Otx2^+/− (C,F,I) mice were immunostained with a polyclonal antiserum against α-A-crystallin (A-F) or hybridised with digoxigenin-labelled probe for Prox1 (G-I). Note that lens development is delayed and the overall size of the lens is reduced in Otx1^−/−; Otx2^+/− embryos (C,F,I). Lack of RPE differentiation is evident in Otx1^+/−; Otx2^+/− mice even in the presence of an apparently normal lens (arrowheads in E,H). L, lens; lp, lens placode; nr, neural retina; ol, outer layer. Bar: 200 μm (A-C), 300 μm (D-I).

Improperly patterned RPE differentiates into a NR-like structure in Otx1^−/−; Otx2^+/− mice. Paraffin sections at equivalent axial levels through the eye of E17.5 wild type (A,D,G) and Otx1^−/−; Otx2^+/− mice (B,C,E,F,H,I). C,F and I are high power views of the ectopic neural retinas illustrated in B,E and H, respectively. Sections were immunostained with antiserum against Tuj1 (A-C), Ph-H3 (D-F), and Islet1 (G-I). Note the presence of Tuj1- and Islet1-positive differentiated neurones not only in the vitreal surface of the NR of both wild-type and mutant embryos but also on the sclera side of the ‘ectopic’ NR (arrowheads in C and I). Mitotic Ph-H3-labeled nuclei are located at the vitreal surface of the ‘ectopic’ retina (arrowheads in F). egc, ectopic ganglion cells; enr, ectopic neural retina; gcl, ganglion cell layer; L, lens; nr, neural retina; pe, retinal pigment epithelium; sc, sclera. Bar: 400 μm but C,F,I, 250 μm.

Neural retina development in Otx1^−/−; Otx2^+/− mice. Adjacent paraffin sections of E18.5 wild-type (A,D,G) and Otx1^−/−; Otx2^+/− (B,E,H) retinas were immunostained with antiserum against Ph-H3 (A,B), antibodies against Islet1 (G,H) or stained with Cresyl Violet to determine the amount of apoptosis in the sections (D,E). Note that the number of mitotic (arrows in B compared with A) and apoptotic (arrowheads in E compared with D) cells is greatly increased while those of differentiated cells (compare arrowheads in G and H) is decreased in mutant retinas. Statistical analysis of the number of mitotic, apoptotic and Islet1-positive cells is presented in C,F and I, respectively (n=4). Note that in all cases there is significant difference between control and Otx-deficient mice. enr, ectopic neural retina; gcl, ganglion cell layer; nr, neural retina; pe, retinal pigment epithelium. Bar, 100 μm.