Fig. 3. Identification of an upstream Eomes retinal enhancer containing Pou4f2-binding sites. (A,B) E14.5 retinal sections from Pou4f2^+/lacZ and Pou4f4^lacZ/lacZ embryos immunostained with anti-Eomes antibody and counterstained with propidium iodide. (C) 20 kb Vista analysis of Eomes comparing mouse, human, dog and rat genomes. Pou4f2-binding sites A and B are indicated by arrowheads. (D) The DNA sequence of Pou4f2 sites A and B. The 2.8 kb retinal enhancer fragment is indicated in red. (E) Transgenic embryos carrying Eo2.8k-HSP68p-LacZ-pA construct stain positively in the retina and limb (arrowheads). (F) Histological retinal section of Eo2.8k-HSP68p-LacZ-pA E14.5 embryos shows lacZ expression in RGCs within the NBL. lacZ-expressing cells co-localized with Pou4f2-expressing cells, detected using anti-Pou4f2 antibody (G). (H-K) GFP expression in Bac Eomes::GFP retinas. GFP fluorescence in retinas from E14.5 (H) and E16.5 (J) embryos. There is intense auto-fluorescence in retina pigment epithelia layer (RPE). Immunostaining using Alexa-555-anti-GFP antibody on retinal sections from E14.5 (I) and E16.5 (K) embryos. Scale bars: 100 µm in B,G,K for A,B, F,G and H-K, respectively.