Figure 2

HOX and E-box motifs are critical for limb and tail bud activity of the +1 enhancer in vivo. (a) Nucleotide sequence alignment of the +1 enhancer with conserved HOX (red) and Ebox (blue) motifs marked. Arrows indicate the boundaries of the 5′ and 3′ deletion constructs. (b) Representative transgenic mouse embryos at E12.5 showing whole-mount X-Gal reporter expression driven by wild-type and mutated +1 enhancer constructs, with close-up views of limbs and tail bud regions shown to the right of each whole-mount view. Simultaneous mutation of all six putative HOX binding sites (pP/lacZ/+1–HOX1-6) or homeoboxes located in 5′ region of +1 enhancer (pP/lacZ/+1–HOX1-3) abolished staining in developing limbs and the PZ but did not affect endothelial or tail bud staining. Staining pattern of embryos carrying mutation of homeoboxes located in 3′ region of +1 enhancer (pP/lacZ/+1–HOX4-6) was indistinguishable from wild-type enhancer. Simultaneous mutation of two E-boxes located in 5′ region of +1 enhancer (pP/lacZ/+1-5′–Ebox1-2) caused a significant reduction in tail bud staining but did not affect staining in limbs and endothelium. Simultaneous mutation of three homeoboxes and two E-boxes located in 5′ region of +1 enhancer (pP/lacZ/+1-5′–HOX1-3/Ebox1-2) with only endothelial staining remaining.