Fig. 1. Assessment of activities of enhancer N-1 and its subfragments. (A) Scheme of assay using electroporation of chicken embryo at stage 4, and subsequent assessment of EGFP fluorescence. (B) Scheme of deletion analysis of the enhancer N-1. The sequences with enhancer activity are indicated in green, whereas those without activity are in purple. Each deletion construct was examined using more than 10 electroporated embryos, which gave identical results. (C) Enhancer activity of various constructs indicated by EGFP expression. (a-e) Stage 5 (a,c) and stage 9 (b,d,e) chicken embryos, 6 and 12 hours after electroporation, respectively, showing enhancer N-1 activity (c,d), compared with bright-field images (a,b) and expression of co-electroporated DsRed1-N1 (e). (f,g) Enhancer activity of trimerized N-1c, 6 and 12 hours after electroporation, respectively, emulating the activity of enhancer N-1 (compare c,d), in comparison with endogenous Sox2 expression of the same embryo at stage 9 (h) detected by in situ hybridization. (i,j) Loss of enhancer activity by deletion of N-1c sequence (N-1c). a to e, f to h, and i and j are data from the same embryos. Arrowheads indicate the node position.