Fig. 4. In vivo requirement for the NFY site in CR1. (A) EMSA competition experiments showing the ability of specific mutations to interfere with the binding of YY1 and NF-Y to b4Cwt. On the right is a schematic of the various mutations and reporter constructs. Mutated nucleotides are underlined. The binding characteristics of the probes are summarized on the right (b4Cwt, wild type; b4C-mN+Y, double mutation; b4C-mYY1, YY1 specific mutation; b4C-mNFY1 and b4C-mNFY2, NFY specific mutations; +, binding; +/–, partial binding; –, no binding). (B) Transgenic mouse embryos stained for ß-galactosidase activity showing the expression patterns derived from the mutant NF-Y/YY1 constructs. (a) Lateral and (b) dorsal views of a 12.5dpc embryo carrying construct CHZ-mN+Y. Residual staining was consistently observed in nervous system (open triangles). (c,d) Two different 9.5-10dpc embryos carrying the same construct. Weak somitic expression is visible at the level of So 13/14 (blue triangle in c) or in the most caudal somites (blue arrow in d), as is weak expression in the flank mesoderm (red arrow in d). (e) Lateral and (f) dorsal views of an 11.5dpc embryo carrying construct CHZ-mYY1. Black arrows indicate ectopic neural expression and blue arrowhead the anterior limit of somitic expression. Flank mesoderm staining is unaffected (red arrow). (g) Lateral and (h) dorsal view of a 12.5 dpc embryo carrying construct CHZ-mNFY2. (i) TS at the forelimb level of a similar embryo. (j) Lateral and (k) dorsal views of similar 12.5 dpc embryos carrying construct CHZ-mNFY1. (l) TS at the forelimb level of a similar embryo. drg, dorsal root ganglion; v, ventral root; f, floorplate; sg, sympathetic ganglia. Scale bars: 100 µm.