| dc.description.abstract | Ewing’s sarcoma is the second most common malignant bone cancer found in adolescents, and the genetic hallmark of this disease is the presence of the aberrant chimeric fusion protein EWS/FLI1. This fusion is induced by the t(11; 22) chromosomal translocation of EWS and FLI1, and is the most prominent and common characteristic of Ewing sarcoma tumors found in approximately 85-90% of reported cases. EWS/FLI1 has been shown to directly bind to and inhibit the function of endogenous EWS in a dominant manner. In this study, we seek to increase our understanding of the role of endogenous EWS during development and skeletogenesis to gain insight into the pathogenesis of the disease. Previously, we demonstrated the role of a zebrafish EWS homolog Ewsa by analyzing the phenotype of a maternal zygotic homozygous ewsa/ewsa mutant line (MZ ewsa/ewsa) of zebrafish null for Ewsa protein. Prehypertrophic chondrocytes of Meckel’s cartilage in 4dpf MZ ewsa/ewsa mutants fail to completely differentiate into hypertrophic chondrocytes, followed by structural defects in craniofacial bones (dentary and basihyal bones) at the adult stage. Based on these results, we sought to understand EWS’s involvement in skeletogenesis by asking if Ewsa regulates activity of critical transcription factors involved in chondrocyte development. We have previously shown that Ewsa directs chondrocyte differentiation through modulation of chondrogenesis master transcription factor Sox9. Runx2 is also known to play a critical role in differentiation of both chondrocytes and osteoblasts, so we have addressed whether and how Ewsa regulates Runx2 expression and transcriptional activity. We discovered that Runx2 protein expression dramatically increases in craniofacial chondrocytes of 4-6dpf MZ ewsa/ewsa compared to wt/wt embryos. We have also observed premature mineralization in MZ ewsa/ewsa embryos at 6dpf and 10dpf. MZ ewsa/ewsa fish also display a decrease in expression of collagen10a1, a hypertrophic-specific Runx2 target gene. These data together suggests that Ewsa regulates Runx2 expression and transcriptional activity during chondrogenesis and regulates mineralization of these domains. Additionally, our lab has previously discovered that adult MZ ewsa/ewsa fish display aberrant curved spines. Based on these results we asked how Ewsa is involved in the formation of the axial skeleton. Since Collagen2a1 is a critical component of notochord development as both a precursor to intervertebral disc (IVD) formation and as a component of notochord sheath extracellular matrix (ECM) we have previously asked if Ewsa regulates the collagen2a1a gene. Ewsa interacts with the col2a1a gene and col2a1 is upregulated in the notochord MZ ewsa/ewsa fish which suggests that the notochord sheath ECM is misregulated in mutant fish. Additionally, our analysis also revealed that notochord cells have failed to intercalate 5-10dpf and remain in a single layer. Based on our previous data and the data in this report, we hypothesize that Ewsa regulates axis development through regulation of collagen2a1 expression, which in turn allows for normal notochord sheath ECM distribution, which in turn allows for notochord cell intercalation and later IVD and vertebral body formation. This is the first in vivo evidence for regulation of RUNX2 by EWS during chondrogenesis and axial skeleton development. | |
