Fig. 4. Identification of multiple positive-acting elements within the M250 enhancer for motoneuron expression. (A) Point mutations were introduced into nine sites (M1-M9) as indicated. (B-K) GFP expression in HH stage 24 chick embryos with each mutant reporter tested in the context of the M250+NheI:GFP construct (see Fig. 1B,F). (L) E11.5 transgenic mouse embryo generated with the M250 reporter containing M1 and M9 mutations and stained with anti-GFP antibody. (M) Location of oligonucleotides (indicated by rectangles) in the Hb9 promoter used for gel retardation assays. The sequence of these regions is highly conserved between mouse and human. (N) The binding of Isl1/Lhx3 to the M50 DNA segment was challenged by 20-, 100-fold molar excess of the unlabeled probes indicated above the lanes. M50 and M100C competed efficiently for Isl1/Lhx3 DNA binding, while the M3-oligo and 3'CD-oligos failed to do so. This suggests the M3 element is not a binding site for the LIM-HD factors Isl1 and Lhx3.