Ewing's Sarcoma EWS protein regulates skeletogenesis by modulation of SOX9

Abstract

Ewing sarcoma is the second most common form of bone cancer in adolescents, characterized by the presence of an aberrant chimeric fusion gene EWS/FLI1. Wildtype EWS has been proposed to play a role in splicing and transcription. Currently, how these functions affect early developmental stages is unknown. To elucidate the function of EWS in early development, we analyzed an ewsa zebrafish mutant line originally isolated from an insertional mutagenesis method. We generated a Maternal Zygotic (MZ) ewsa/ewsa line because ewsa/wt and ewsa/ewsa zebrafish appear to be normal and are fertile. Alizarin Red staining revealed that there are skeletal formation defects in the lower jaw with an aberrant angle and position of the dentary and basihyal bones in adult MZ ewsa/ewsa mutants. Alcian blue staining revealed that the MZ ewsa/ewsa mutation leads to craniofacial defects with higher numbers of smaller cells with disorganized polarization compared to wt/wt zebrafish at four days post fertilization (dpf). In addition, there were reduced intervertebral discs and asymmetrical vertebrae leading to curved spines in MZ ewsa/ewsa mutants. MZ ewsa/ewsa mutants display disorganized alignment of Sox9 expressing neural crest cells at 27hpf. Because both craniofacial skeletons and vertebrae arise from Sox9 expressing cells, we hypothesized that EWS interacts with Sox9 and modulates the transcriptional regulation activity of Sox9. Co-immunoprecipitation (IP) experiment revealed that EWS interacts with SOX9. Furthermore, qPCR analysis identified that known SOX9 target genes are either upregulated (ctgfa, ctgfb, col2a1a, col2a1b) or downregulated (sox5, nog1, nog2, bmp4) in MZ ewsa/ewsa mutants compared to wt/wt zebrafish embryos. This is the first evidence for a tissue specific role of EWS in skeletogenesis and suggests a novel mechanism by which Sox9 transcriptional regulation is modulated as it directs endochondral bone and cartilage development.