The role of Ewing’s sarcoma protein EWS in endochondral ossification and angiogenesis
Lewin, Seth J.
University of Kansas
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Ewing sarcoma is a bone and cartilage cancer affecting children and adolescents. Up to 90% of Ewing sarcoma patients express a chimeric EWS/FLI1 protein which is formed by a chromosomal translocation, and is known to induce aberrant transcription, bind with endogenous EWS and cause mitotic defects similar to knockdown of EWS. In EWS knockout studies, EWS-/- mice produce smaller pups with shorter, brittle bones compared to wild-type littermates suggesting EWS has a role in skeletal and overall development. Since Ewing’s sarcoma develops in the bone, the role of endogenous EWS is of interest as it may provide an insight into the parthenogenesis of this disease. To investigate this we generated a maternal zygotic (MZ) ewsa null mutant zebrafish, produced by insertional mutagenesis. Chondrocyte maturation begins after mesenchymal condensation at about 3 days past fertilization (dpf), then cells undergo morphological changes by 4dpf to become prehypertrophic chondrocytes with most fully differentiated into hypertrophic chondrocytes by 6dpf. Previously, we reported that Ewsa modulates the mRNA expression of target genes of transcription factor Sox9, the master transcriptional regulator of chondrocyte differentiation. Given the regulatory role of Sox9 in chondrogenesis, an important question is whether Ewsa directly regulates the expression level of Sox9 proteins. Thus, we generated antibodies against Sox9a and Sox9b, and performed immunohistochemistry in 3-6dpf of wt/wt and MZ ewsa/ewsa zebrafish mutants. As a result, Sox9a localized in the nucleus, and the expression levels of Sox9a proteins were unchanged among 3-6dpf wt/wt and MZ ewsa/ewsa zebrafish mutants. This suggests that Sox9a protein is not regulated by Ewsa during chondrocyte maturation. One upregulated Sox9 target gene was connective tissue growth factor (ctgf). Ctgf is known to have a critical role in endochondral ossification, furthermore the overexpression of Ctgf regulation results in skeletal defects and reduced hypertrophy. We suspect that Ews regulates Ctgf during endochonral ossification and aim to confirm this in this study. We discovered that Ctgf is overexpressed in craniofacial cartilage of mutant embryos at 4dpf and 6dpf. Also measurements of cartilage structures show evidence of skeletal defects in these embryos as early as 4dpf. This is evidence that the regulation of Ctgf by Ewsa is essential for normal chondrocyte maturation and skeletal development. In addition, a reduction of ctgfa signal in situ and Ctgf in immunohistochemistry assays was observed in the vascular structures in mutant embryos at 3dpf and 6dpf suggesting Ewsa may regulate ctgf expression in vascular tissue. Which is important since vascular invasion into mature chondrocytes is necessary in skeletal development. In summary, we discovered that Ewsa may regulate stage-specific, and tissue specific expression of Ctgf during embryonic development. Understanding the pathway of Ewsa-dependent skeletogenesis may supply a platform for the molecular pathogenesis of Ewing sarcoma.
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