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.
