The effect of disease‐causing DVL1 mutations on chondrogenesis in chicken limb mesenchyme

Abstract

The Wingless (WNT) signaling pathway is a key regulator of skeletal development. Perturbations in WNT signaling are associated with several skeletal disorders, including Robinow syndrome (RS). RS is a rare skeletal dysplasia characterized by deficiencies in facial and limb skeletogenesis. All identified pathogenic variants of RS occur in genes of the WNT signaling pathway. The majority of autosomal dominant RS patients (DRS) cases are attributed to mutations in Dishevelled‐1 (DVL1) (OMIM#1807000) and Dishevelled‐3 (DVL3) (OMIM#616894), a cytoplasmic phosphoprotein. All RS DVL1variants have frameshift mutation in the penultimate exon that substitutes an abnormal peptide for the C‐terminus of the protein. Previous research in our lab has shown the expression of avian‐ specific retroviruses containing human wild‐type DVL1 and RS DVL1 variants in the limb of the chicken embryo in vivo disrupts cartilage formation. In order to examine the possible role of WNT signaling in the dysregulation of chondrogenesis caused by DVL1, high‐density cultures of chicken limb mesenchyme were treated with DVL1 viruses and either TCS JNK6o, which antagonizes non‐canonical, JNK signaling or XAV939, a canonical pathway antagonist. Cultures were grown for eight‐days stained with Alcian blue for cartilage. The area occupied by cartilage was analyzed using two‐way ANOVA with Tukey’s post‐hoc test (Graphpad Prism v9.3). In the absence of drug treatment, wtDVL1 and RS DVL11519ΔTvariant significantly increased chondrogenesis compared to GFP controls (Fig. 1A,B). Cultures treated with TCS6o (Fig. 1A) did not significantly increase cartilage compared to non‐treated controls culture. The combination of wtDVL1 with TCS6o synergistically increased chondrogenesis. This suggests 1) that endogenous non‐canonical WNT signaling is not required for chondrogenesis, 2) that wtDVL1 has a degree of JNK signaling that when antagonized increases chondrogenesis and 3) that the variants are not interacting with JNK signaling. When canonical signaling was antagonized (Fig. 1B), chondrogenesis was significantly increased for all virus types compared to GFPcontrols. However, the effect of XAV939 on the DVL11519ΔT variant‐infected cultures did not further increase chondrogenesis over the virus alone. This suggest that endogenous canonical WNT signaling represses chondrogenesis and but the variant may have altered ability to respond to a decrease in canonical signaling. Misregulation of canonical and non‐canonical WNT signaling is tied to abnormal chondrogenesis and could be contributing to the shortened limbs observed in RS patients.