Ewing sarcoma is the second most common skeletal (bone and cartilage) cancer in adolescents, and it is characterized by the expression of the aberrant chimeric fusion gene EWS/FLI1. Wild-type EWS has been proposed to play a role in mitosis, splicing and transcription. We have previously shown that EWS/FLI1 interacts with EWS, and it inhibits EWS activity in a dominant manner. Ewing sarcoma is a cancer that specifically develops in skeletal tissues, and although the above data suggests the significance of EWS, its role in chondrogenesis/skeletogenesis is not understood. To elucidate the function of EWS in skeletal development, we generated and analyzed a maternal zygotic (MZ) ewsa/ewsa line because the ewsa/wt and ewsa/ewsa zebrafish appeared to be normal and fertile. Compared with wt/wt, the Meckel’s cartilage of MZ ewsa/ewsa mutants had a higher number of craniofacial prehypertrophic chondrocytes that failed to mature into hypertrophic chondrocytes at 4 days post-fertilization (dpf). Ewsa interacted with Sox9, which is the master transcription factor for chondrogenesis. Sox9 target genes were either upregulated (ctgfa, ctgfb, col2a1a, and col2a1b) or downregulated (sox5, nog1, nog2, and bmp4) in MZ ewsa/ewsa embryos compared with the wt/wt zebrafish embryos. Among these Sox9 target genes, the chromatin immunoprecipitation (ChIP) experiment demonstrated that Ewsa directly binds to ctgfa and ctgfb loci. Consistently, immunohistochemistry showed that the Ctgf protein is upregulated in the Meckel’s cartilage of MZ ewsa/ewsa mutants. Together, we propose that Ewsa promotes the differentiation from prehypertrophic chondrocytes to hypertrophic chondrocytes of Meckel’s cartilage through inhibiting Sox9 binding site of the ctgf gene promoter. Because Ewing sarcoma specifically develops in skeletal tissue that is originating from chondrocytes, this new role of EWS may provide a potential molecular basis of its pathogenesis.
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Ewing's sarcoma is the second most common bone cancer in adolescents and is characterized by the aberrant chimeric fusion gene EWS/FLI1. Wild-type EWS has been proposed to play a role in splicing and transcription, but how these functions affect early development is unknown. To elucidate the function of EWS in early development, we analyzed a mutant ewsa zebrafish line. We generated a maternal zygotic (MZ) ewsa/ewsa line because the ewsa/wt and ewsa/ewsa zebrafish appear to be normal and fertile. The adult MZ ewsa/ewsa mutants display defects in the craniofacial bones (dentary and basihyal). Compared to wt/wt, the MZ ewsa/ewsa mutants have a higher number of craniofacial prehypertrophic chondrocytes, and they fail to mature into hypertrophic chondrocytes possibly due to impaired intercalation at 4 days post-fertilization (dpf). The adult MZ ewsa/ewsa mutants also display curved spines due aberrant differentiation of the nucleus pulposus cells in the intervertebral discs (IVD). These altered differentiation is preceded by increased Collagen type II in the IVD, and Ctgf in the craniofacial chondrocytes in MZ ewsa/ewsa mutants compared to wt/wt at 36 hpf. We further discovered that both Ewsa and Ewsb interact with the master transcriptional factor for chondrogenesis, Sox9, in zebrafish. Consistently, qPCR analysis consistently demonstrated that Sox9 target genes are either upregulated (ctgfa, ctgfb, col2a1a, col2a1b) or downregulated (sox5, nog1, nog2, bmp4) in MZ ewsa/ewsa mutants compared to the wt/wt zebrafish embryos. This study is the first demonstration of Ewsa-dependent regulation of skeletogenesis through modulation of Sox9 target gene expression. Citation Format: Chris Merkes, Timothy K. Turkalo, Nicole Wilder, Hyewon Park, Mizuki Azuma. Ewing's sarcoma Ewsa protein regulates Sox9 during skeletogenesis in zebrafish. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 73. doi:10.1158/1538-7445.AM2014-73
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