Hypomorphic and dominant-negative impact of truncated SOX9 dysregulates Hedgehog-Wnt signaling, causing campomelia

Haploinsufficiency for SOX9, the master chondrogenesis transcription factor, can underlie campomelic dysplasia (CD), an autosomal dominant skeletal malformation syndrome, because heterozygous Sox9 null mice recapitulate the bent limb (campomelia) and some other phenotypes associated with CD. However, in vitro cell assays suggest haploinsufficiency may not apply for certain mutations, notably those that truncate the protein, but in these cases in vivo evidence is lacking and underlying mechanisms are unknown. Here, using conditional mouse mutants, we compared the impact of a heterozygous Sox9 null mutation (Sox9^+/-) with the Sox9^+/Y440X CD mutation that truncates the C-terminal transactivation domain but spares the DNA-binding domain. While some Sox9^+/Y440X mice survived, all Sox9^+/- mice died perinatally. However, the skeletal defects were more severe and IHH signaling in developing limb cartilage was significantly enhanced in Sox9^+/Y440X compared with Sox9^+/-. Activating Sox9^Y440X specifically in the chondrocyte-osteoblast lineage caused milder campomelia, and revealed cell- and noncell autonomous mechanisms acting on chondrocyte differentiation and osteogenesis in the perichondrium. Transcriptome analyses of developing Sox9^+/Y440X limbs revealed dysregulated expression of genes for the extracellular matrix, as well as changes consistent with aberrant WNT and HH signaling. SOX9^Y440X failed to interact with β-catenin and was unable to suppress transactivation of Ihh in cell-based assays. We propose enhanced HH signaling in the adjacent perichondrium induces asymmetrically localized excessive perichondrial osteogenesis resulting in campomelia. Our study implicates combined haploinsufficiency/hypomorphic and dominant-negative actions of SOX9^Y440X, cell-autonomous and noncell autonomous mechanisms, and dysregulated WNT and HH signaling, as the cause of human campomelia.