Fig. 6: Misexpression of both Lbx1 and Btrc is required to develop a SHFM phenotype.

a Schematics of the Lbx1/Fgf8 locus for CRISPR/Cas9 Inv2 and Inv1 ∆Lbx1 alleles. Yellow ovals highlight Fgf8 AER enhancers. b cHi-C (data are shown as merged signal of n = 3 biological and 1 technical replicates) of homozygous Inv2 from E11.5 mouse limb buds. The subtraction map between wild-type and Inv2 interactions is shown in mirror view. Dashed circles indicate the position of the original wild-type interactions between boundaries, two of them lost upon reshuffling of the boundary between Lbx1 and Fgf8 TADs as a consequence of this inversion. The boundary involved in the inversion is shown as blurry. Black arrowhead highlights the bow tie configuration representative of the inverted regions. The rectangular dashed area highlighted by black asterisks show ectopic interactions compared to wild-type between the Fgf8 AER enhancers region and the one containing now only Lbx1 and not anymore Btrc. White asterisks point out the loss of interactions between Fgf8 and its AER enhancers. c Schematic of Inv2 configuration. Upon this inversion, the Fgf8 AER enhancers are repositioned within a smaller Lbx1 TAD, whereas the Fgf8 TAD is increased in size due to the inversion of the boundary and contains now Btrc, isolated from the Fgf8 AER enhancers. Below is a zoom-in of the locus highlighting the potential ectopic interactions (red dashed arrow) involving the Fgf8 AER enhancers now repositioned within the Lbx1 TAD. d Whole-mount in situ hybridization for Fgf8, Lbx1 and Btrc at E11.5. Expression was checked and confirmed in at least 3 or more homozygous embryos (at least n = 3 biological replicates). e Skeletal analysis of E18.5 limbs stained with alcian blue (cartilage) and alizarin red (bone). Hands and feet from wild-type, Inv2, Inv1 ∆Lbx1 and Fgf8 AER enhancer KI E18.5 embryos. At least n = 3 biological replicates.