The role of uxs1 in chondrocyte organization, morphogenesis, and signaling pathways during skeletogenesis
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The role proteoglycans play in molecular-genetic mechanisms of skeletogenesis is not completely understood. UDP-glucuronic acid decarboxylase 1 (Uxs1) converts UDP-glucuronic acid to UDP-xylose which is used by xylosyltransferase 1 to initiate assembly of a common tetrasaccharide linker critical to the biosynthesis of chondroitin sulfate, dermatan sulfate, and heparan sulfate proteoglycans, all of which are found in abundance within the extracellular matrix of cartilage. In this paper, we present two alleles, hi3357 and mow, that have mutations in uxs1. Zebrafish embryos with uxs1 mutations presented an absence of Alcian staining from cells in the pharyngeal cartilages. Fluorescent confocal microscopy revealed improper chondrocyte organization and morphogenesis in cartilage elements of the neurocranium and pharyngeal skeleton. Additionally, we observed reductions in Alizarin red staining at endochondral and intramembranous ossification centers indicating improper bone development. Whole-mount in situ hybridization experiments revealed uxs1 expression to be ubiquitous in developing embryos until 2 dpf and thereafter localized to the pharyngeal skeleton, suggesting a critical role for uxs1 expression during skeletogenesis. These results suggest that chondrocyte organization and morphogenesis, endochondral ossification, and intramembranous ossification are all dependent upon uxs1 function. In mutants homozygous for uxs1hi3357 and uxs1mow, wheat germ agglutinin staining revealed a reduction in proteoglycans in the extracellular matrix. Moreover, antibodies against heparan sulfate revealed deletion of heparan sulfate proteoglycans in the pharyngeal cartilage elements. The deletion of heparan sulfate proteoglycans in uxs1 mutants suggests that Wnt, Fibroblast growth factor, or Hedgehog signaling cascades may be disrupted as previously shown to occur in the fruit fly Drosophila. These findings reveal an absence of UDP-xylose dependent proteoglycans from the extracellular matrix due to mutations in uxs1. Experiments also showed a deletion of type II collagen from the extracellular matrix of chondrocytes, suggesting a role for Uxs1 or proteoglycans in the secretory or localizing mechanisms. Additionally, expression of col10a1 was absent from endochondral ossification centers and reduced at intramembranous sites, suggesting that uxs1 is necessary for proper reciprocal signaling pathways between perichondrial cells and chondrocytes critical for bone formation. Thus, we conclude that UDP-xylose dependent proteoglycans are absent in uxs1 mutants which is altering the composition of the extracellular matrix and disrupting reciprocal signaling between cells. This study provides new insight into the role proteoglycans play in skeletogenesis and the evolutionarily conserved role of uxs1 across life on earth.