Figure 3. Normally dispensable individual enhancers are required for limb morphology in a sensitized background.

Individual and combined enhancer deletions in the presence of only one copy of the Gli3 (a) or Shox2 (b) target genes and the resulting limb morphology at E18.5. Wedges indicate inferred gene dosage. (a) Skeletal forelimb autopod phenotypes at E18.5 resulting from mm1179 and hs1586 enhancer deletions in the presence of reduced Gli3 dosage. 1–5, normal digits. Red asterisk, extra digits with unclear identity. *s, “split” digit. Black arrowhead, hypoplastic distal phalange. (b) Progressive reduction of femur ossification length (double arrows) due to hs741 and hs1262 enhancer loss in a Shox2 sensitized background. The relative length of the femur ossification, normalized to the tibia ossification length, is shown. For comparison, the bottom panel shows absence of the femur ossification in Shox2 deficient limbs at P0 (red arrowhead, reproduced with permission from authors of ref. ²⁵). “n”, number of independent biological replicates with similar results. Box plots indicate median, interquartile values, range and individual biological replicates. ***, P < 0.001 (two-tailed, unpaired t-test). Scale bars, 500 μm.