Extended Data Fig. 5: The REX element is required and sufficient for long-range activity at the Shh locus.
From: Range extender mediates long-distance enhancer activity
(a) Shh gene expression analysis using mRNA whole mount in situ hybridization in homozygous ZRSHS72+REX/HS72+REX knock-in mouse embryos at E10.5 (left two panels). Arrows point to the Shh expression in the ZPA. The corresponding hindlimb skeletal preparations at E18.5 are shown (third column). The number of embryos that exhibited representative limb phenotype over the total number of embryos with the genotype is indicated. (b) Forelimb (first panel) and hindlimb (second panel) skeletal phenotypes in heterozygous ZRSHS72+REX/wt knock-in embryos at E18.5. (c) Comparative Shh mRNA in situ hybridization analysis in wild type (top row) and homozygous ZRSHS72+REX/HS72+REX knock-in (bottom row) mouse embryos during limb bud development in E11.75 embryos. Black arrows point to areas of Shh expression in the ZPA. Red arrows point to ectopic expression in the anterior portion of the limb bud. * Extra digits. (d) Schematic showing the sequences from the HS72 (chr16:51623658-51623900 and chr16:51624689-51625572; hg38) and MM1492 (chr4:154706480-154712089; mm10) loci that make up the chimeric MM1492 + REX element. The orange bars indicate the expanded region flanking the core HS72 enhancer that contains the REX element. The purple bar marks the MM1492 enhancer sequence. The core HS72 enhancer was swapped out for the MM1492 enhancer to generate the MM1492 + REX construct (bottom). (e) Shh gene expression analysis using mRNA whole mount in situ hybridization in homozygous ZRSMM1492+REX/MM1492+REX knock-in mouse embryos at E10.5. (f) Forelimb and hindlimb skeletal phenotypes of heterozygous ZRSMM1492+REX/wt embryos at P0.
