Extended Data Figure 7. Generation of Gli3 and Shox2 knockout alleles and characterization of enhancer deletions in a sensitized background.

(a, d) Top: Schematic showing CRISPR/Cas9-mediated deletions used to generate Gli3 and Shox2 loss-of-function alleles. Genotyping primers used to validate targeted deletion events are indicated. Middle: Sanger sequencing confirmation of deletion event, with grey and red dashed lines indicating breakpoints. Right: PCR genotyping examples are shown, and the size of the product specific for the deletion allele is depicted in red (primers listed in Supplementary Table 3). (b) In situ hybridization (ISH) showing the gradual decrease of anterior Gli3 transcript in forelimbs of wild-type, Gli3^Δ/+ and sensitized mm1179, hs1586 double enhancer knockout (DKO/Gli3^Δ) embryos. (c) Quantitative real-time PCR (qPCR) validation of Gli3 mRNA levels in forelimb hand plates from the genotypes shown in panel b. (e) Shox2 expression (ISH) in fore- and hindlimbs of wild-type, Shox2^Δ/+ and sensitized hs741, hs1262 double enhancer knockout (DKO/Shox2^Δ) embryos. Arrowheads point to the domains in enhancer DKO/Shox2^Δ embryos where Shox2 expression is nearly abolished. (f) qPCR revealing significantly downregulated Shox2 mRNA levels in hindlimbs of DKO/Shox2^Δ compared to Shox2^Δ/+ embryos. “n” indicates the number of independent biological replicates with similar results. Bar plots illustrate mean and standard deviation (error bars), with individual biological replicates shown. ***, P < 0.001; *, P < 0.05 (two-tailed, unpaired t-test). n.s., not significant. For ISH, transcript distribution was reproduced in at least n=3 independent biological replicates. Scale bars, 100 μm.