Figure 2. Morphological requirements of limb enhancers with overlapping activities.

(a, b) CRISPR-deleted enhancers and their distance to the TSS of predicted target genes (Gli3, Shox2). (c) Left: RNA in situ hybridization (ISH) reveals reduced Gli3 expression in anterior hand plates of mm1179/hs1586 double enhancer knockout (DKO) embryos (white arrowhead). Red arrowhead: local expansion of anterior mesenchyme, a hallmark of Gli3 deficiencies. Right: Forelimb skeletons with digits labeled 1 to 5, from anterior to posterior. DKO embryos exhibit duplication of digit 1 (arrowhead). Scale bars, 200 μm. (d) Shortened femur ossification length in hs741/hs1262 double enhancer knockout (DKO) embryos (normalized to tibia ossification length). Box plot indicates median, interquartile values, range and individual biological replicates. ***, P < 0.001 (two-tailed, unpaired t-test). (e, f) Co-localization of Gli3 (mm1179: green, hs1586: red) and Shox2 (hs741: green, hs1262: red) enhancer activities via enhancer-reporter transgenes and immunofluorescence (IF) in forelimb buds of double transgenic embryos. White arrowheads: examples of double positive cells. Empty arrowheads or arrows: cells marked by single enhancers. Nuclei are stained blue. Scale bars, 50 μm. “n”, independent biological replicates with similar results.