Fig. 1: Engineering of the SHFM-associated structural variations at the Lbx1/Fgf8 locus.

a cHi-C (data are shown as merged signal of n = 3 biological and 1 technical replicates) of extended Lbx1/Fgf8 locus generated from wild-type E11.5 mouse limb buds. Lbx1 and Fgf8 are located within distinct TADs (indicated by single dashed lines) separated by boundaries (indicated by red hexagons) in correlation with CTCF binding sites, as indicated in ChIP-seq from E11.5 mouse limb buds⁶¹ below. Loops (dashed circles) indicate interaction between CTCF sites. Interaction plots using virtual 4C from Fgf8 and Lbx1 viewpoints (VP) are shown below. Regions of interactions relative to Lbx1 and Fgf8 within their own TADs are highlighted in yellow, whereas contacts with the boundary region between Lbx1 and Fgf8 TADs and those over such boundary are in violet and pink, respectively. Published Fgf8 enhancers²⁸ are indicated by ovals. Yellow ovals highlight enhancers driving Fgf8 expression in the AER and localized within the introns of Fbxw4, while in orange is the only AER enhancer in close proximity to Fgf8. b Schematic of human SHFM3 related structural variations (SVs). Red and blue lines in the duplications bar represent the different centromeric and telomeric breakpoints, respectively. Breakpoints of the inversion are shown below. c Schematic of the SHFM-associated SVs engineered in mice using CRISPR/Cas9 genome editing tool, particularly one selected tandem duplication (Dup) and the inversion (Inv1). d Skeletal analysis of fore- and hindlimb stained with alcian blue (cartilage) and alizarin red (bone), from wild-type, heterozygous and homozygous Dup and Inv1 18.5 embryos. No particular phenotype was observed in both heterozygous and homozygous Dup and in heterozygous Inv1, whereas fused bones and split digits were detected in homozygous Inv1 forelimbs.