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

Robinow syndrome (RS) is a rare skeletal dysplasia characterized by skeletal phenotypes affecting the face and limbs. The majority of autosomal dominant RS patients (DRS) have mutations in Dishevelled‐1 (DVL1) (OMIM#1807000) and Dishevelled‐3 (DVL3) (OMIM#616894). DVL is a cytoplasmic phosphoprotein that transduces WNT(Wingless‐related) signals. Any type of WNT signaling that involves Frizzled receptor binding also requires the binding of DVL to activate signaling. All RS DVL1 variants have frameshift mutations in exon 14 that produce an abnormal peptide that is over 200 amino acids long. . This peptide replaces the entire C‐terminus and removes respective binding partners. It is not possible to predict whether this peptide causes a loss‐ gain‐of‐function or whether there may be novel functions. Previous in vivo studies in our lab showed that the ectopic expression of wtDVL did not affect the overall shape of the cartilage and limbs contained closely packed differentiated chondrocytes. The phenotypes of the variants were very different to wtDVL1 but similar to each other. There were pockets of undifferentiated fibroblasts between areas of differentiated chondrocytes thus shape was very irregular. These phenotypes were not providing clear answers as to which steps of chondrogenesis are affected. Our objective is to misexpress RS h DVL1 variants ( DVL1 1519 D T , DVL1 1529 D G , DVL1 1615 D A ) and wtDVL1 in cells isolated from the limb bud and then assess the effect on different stages of chondrogenesis. High density micromass cultures were created from stage 24 chicken limb mesenchyme, infected with viruses and grown for 4, 6 or 8 days of culture. Cultures were photographed and the proportion of the culture occupied by cartilage was determined. Two‐way ANOVA with Tukey's post‐hoc test for multiple comparisons was carried out. All cultures formed nodules stained with Alcian blue by 4 days of culture. Then differences appeared. The GFP controls continued to differentiate into cartilage and there was a significant increased over the 8 day experimental period. The wtDVL1 started out similar to GFP but then chondrogenesis gradually decreased. By 8‐days there was significantly less cartilage than at 4 days in wtDVL1‐infected cultures. Two of the RS DVL1 variants stimulated chondrogenesis significantly compared to GFP and wtDVL1 by 8 days of culture (1519 and 1529). This indicates RS DVL1 variants caused a gain‐of‐function on chondrogenesis. In contrast, the 1615 variant failed to induce cartilage beyond the amount present at 4 days. This seems like the 1615 variant allowed the initial chondrogenesis to take place but then failed to support further differentiation. This unique phenotype could mean that there are interesting effects on downstream transcription of genes. that may indicate signalling differences for this particular variant. In conclusion DVL1 does not play a role in the initial condensation of cartilage but is regulating differentiation.

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