Extended Data Figure 2. No major differences in expression of predicted target genes in individual enhancer knockouts.

(a) Spatial enhancer activity domains (LacZ, see also Fig. 1b) are compared to mRNA expression domains (by in situ hybridization) of the predicted target gene in embryonic fore- and hindlimbs at E11.5. No significant changes in expression patterns were observed in enhancer knockouts compared to wild-type limbs, except in limbs lacking hs741, where a small subdomain of target gene expression was lost (red arrowhead marks loss of the posterior Shox2 domain in the distal limb). Transcript distribution was reproduced in at least n=3 independent biological replicates. (b) Quantitative real-time PCR using limbs of homozygous null (KO, red dots) and wild-type (Wt, blue dots) embryos at E11.5 reveals lack of significantly downregulated transcript levels of predicted enhancer target genes in 9/10 cases. Box plots indicate median, interquartile values, range and individual biological replicates. Outliers are shown as circled data points. **, P=0.0012, unpaired, two-tailed t-test. n.s., not significant. Scale bars, 100 μm.