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UBC Theses and Dissertations

Skeletogenic studies in avian primary limb cultures shed new insights into the functions of syndromic Dishevelled-1 (DVL1) variants Bonaparte, Kywana


The Wingless-related (WNT) signaling pathway participates in many aspects of normal and abnormal skeletal development, but the mechanisms are poorly understood. In order to gain functional insights into the role of WNT signaling in human skeletogenesis, we are studying Robinow Syndrome (RS), a rare skeletal dysplasia caused by pathogenic variants in seven genes in the WNT pathway. The most commonly mutated gene in RS is Dishevelled-1 (DVL1), a signal transduction protein in the WNT pathway. The variants of DVL1 substitute an abnormal peptide for the C-terminus of the protein. Previous research in our lab has tested the function of the RS DVL1 variants compared to wtDVL1 in the chicken embryo model. While the wtDVL1 virus shortened the limb bones, only the variants disrupted the smooth cartilage templates that give rise to the bones. The specific steps of chondrogenesis that are affected by the variants are unknown. Here we used primary cultures derived from undifferentiated chicken limb mesenchymal cells and misexpressed GFP, wtDVL1 or one of three human RS DVL1 variants. In micromass culture, the steps of chondrogenesis are recapitulated over a 12-day culture period. Only one of the mutations, DVL11519ΔT produced a severe phenotype. The area of cartilage produced in DVL11519ΔT infected cultures was significantly lower than all other variants tested. Similarly, histological sections of micromass cultures revealed a striking thinning of the DVL11519ΔT infected culture and less COL2A1 stained matrix. The decreased cartilage was likely due to an earlier decrease in RNA expression of cartilage-specific genes, gIHH and gCOL10A. We examined several mechanisms that may mediate the chondrogenic phenotype of the DVL11519ΔT variant and found that the levels of apoptosis were significantly higher. Additionally, we found increased levels of Matrix Metalloproteinase 13 RNA, which when translated could resorb cartilage matrix. This study found that exogenous expression of human wtDVL1 does not alter chondrogenesis or gene expression in the limb. However, certain RS variants of DVL1 alter chondrogenic differentiation and adversely impact cell survival. Our in vitro approach can provide insight into the pathogenicity of specific gene variants and can be applied to genetic dieases of the skeleton beyond RS.

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