Figure 2(A-A'') Pax2 (red, OD) and TujI (green, axons) IF on E12.5 optic cup cross-sections in wild-type (WT), Heterozygous (HET) and knock-out (KO) embryos showing abnormal ODs (white dotted lines and arrowhead).(B-B'') Pax6 (red, retinal progenitors) and Tuj1 (green) IF indicating ectopic Pax6+ retinal tissue in HET (B') and KO embryos (B", arrowhead).(C-D") High magnification views highlight a morphological displacement of Pax2- (C-C") and Pax6+ (D-D") cells. Arrowheads in (D',D") point to ectopic Tuj1/Pax6+ neurons.(E,E') Pax2 (red) and Tuj1 (green) IF on tangential sections of E13.5 optic cups revealing strong OD reduction in KO embryos (dotted line and arrowhead in E'), compared to WT (surrounded by a white dotted line in E).(F) Histogram quantifying the gradual reduction of Pax2+ OD cells in HET and KO at different ages. data are represented as mean ± SEM; N=3-4 Student t-test (*P<0.05, **P<0.01, ***P<0.001). Scale bars: 50µm.(G-G''') Low (G) and high (G'-G''') magnifications of E15.5 optic cups stained for Pax2 (red) and Tuj1 (green) confirming Pax2+ reduction in HET and KO ODs and severe morphological malformations in KO (arrowheads in G''').(H-I') Tangential (H,H') and sagittal (I,I') sections of the OD in E18.5 WT and KO retinae stained for Brn3a (red; RGCs) and Tuj1 (green; axons) highlighting the malformed OD and disorganized arrangement of RGCs in KO. In (H,H') white dotted line delineate the borders of the OD and in (I,I') the RGC layer; red dotted line surrounds mesodermal cells forming the hyaloid vessel.(J-K') IF on E13.5 optic cup cross-sections in WT and KO embryos showing increased Pax6 (green, retinal progenitors) and decreased Tuj1+ (red, differentiating cells) in the ventral retina of HET and KO embryos. Arrowhead in (J') points to increased thickness and bending of the ventral retina, while arrowheads in (K,K') indicate Tuj1+ axons exiting the OD.(L) Histogram confirming the decreased number of Tuj1+ neurons in the ventral, but not dorsal, retina of Nr2f1 mutants. data are represented as mean ± SEM; N=3-4 Student t-test (*P<0.05, **P<0.01, ***P<0.001). Scale bars: 50µm.(M-N') Double Tuj1/Brn3a IF on E13.5 WT and KO optic cup sections revealing decreased numbers of differentiating RGCs in bending ventral retinae of KO embryos (N').(O,O') Details of P28 WT and HET retinae indicating decreased number of Brn3a+ neurons (arrowheads) in the GCL of HET animals.(P) Graph illustrating the dynamics of RGC differentiation from E13.5 to P28, normalized to WT animals. data are represented as mean ± SEM; N=3-4 Student t-test (*P<0.05, **P<0.01, ***P<0.001). Scale bars: 50µm.Q-Q") Cross-sections of DiI-labelled P0 ONs in WT and KO pups(R,R') Tuj1+ P8 ON fibers in WT and KO pups showing a progressive volume loss(S) Quantification of the surface occupied by Tuj1+ or DiI+ fibers in different genotypes and ages, as indicated. data are represented as mean ± SEM; N=4-5 Student t-test (*P<0.05, **P<0.01, ***P<0.001). Scale bars: 50µm.(T-U') Cleaved-Caspase-3 (green, apoptotic cells) and Brn3a (red, RGCs) IF on transverse sections of E18.5 retinae in WT and KO fetuses. High magnifications (T'-T'") highlight an example of a double-labeled RGC undergoing apoptosis (arrowheads) in the ganglion cell layer (GCL). Apoptotic RGCs are increased in KO animals (arrowheads in U'), compared to WT (U).(V) Histogram quantifying the number of RGC apoptotic cells in WT, HET and KO from E18.5 to P5. Note the significant increase of dying cells at E18.5. Nuclei (blue) were stained with DAPI. data are represented as mean ± SEM; N=3-4 Student t-test (*P<0.05, **P<0.01, ***P<0.001). Scale bars: 50µm.