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

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

Ewing Sarcoma Eswa Protein Regulates Chondrogenesis of Meckel's Cartilage through Modulation of Sox9 in Zebrafish

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.This manuscript was supported by the Massman Family Ewing Sarcoma Research Fund, the Sarcoma Foundation of America, P20RR016475 / P20GM103418 and P20 RR032682-01. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Genetic dissection of a model complex trait using the Drosophila Synthetic Population Resource

Genetic dissection of complex, polygenic trait variation is a key goal of medical and evolutionary genetics. Attempts to identify genetic variants underlying complex traits have been plagued by low mapping resolution in traditional linkage studies, and an inability to identify variants that cumulatively explain the bulk of standing genetic variation in genome-wide association studies (GWAS). Thus, much of the heritability remains unexplained for most complex traits. Here we describe a novel, freely available resource for the Drosophila community consisting of two sets of recombinant inbred lines (RILs), each derived from an advanced generation cross between a different set of eight highly inbred, completely resequenced founders. The Drosophila Synthetic Population Resource (DSPR) has been designed to combine the high mapping resolution offered by multiple generations of recombination, with the high statistical power afforded by a linkage-based design. Here, we detail the properties of the mapping panel of >1600 genotyped RILs, and provide an empirical demonstration of the utility of the approach by genetically dissecting alcohol dehydrogenase (ADH) enzyme activity. We confirm that a large fraction of the variation in this classic quantitative trait is due to allelic variation at the Adh locus, and additionally identify several previously unknown modest-effect trans-acting QTL (quantitative trait loci). Using a unique property of multiparental linkage mapping designs, for each QTL we highlight a relatively small set of candidate causative variants for follow-up work. The DSPR represents an important step toward the ultimate goal of a complete understanding of the genetics of complex traits in the Drosophila model system.This work was supported by the following NIH R01 grants: RR024862 to S.J.M. and A.D.L., GM085260 to S.J.M., GM085251 to A.D.L., GM078338 to S.S., and GM074244 to K.W.B

Reporting the limits of detection and quantification for environmental DNA assays

Background: Environmental DNA (eDNA) analysis is increasingly being used to detect the presence and relative abundance of rare species, especially invasive or imperiled aquatic species. The rapid progress in the eDNA field has resulted in numerous studies impacting conservation and management actions. However, standardization of eDNA methods and reporting across the field is yet to be fully established, with one area being the calculation and interpretation of assay limit of detection (LOD) and limit of quantification (LOQ). Aims: Here, we propose establishing consistent methods for determining and reporting of LOD and LOQ for single‐species quantitative PCR (qPCR) eDNA studies. Materials & Methods/ Results: We utilize datasets from multiple cooperating laboratories to demonstrate both a discrete threshold approach and a curve‐fitting modeling approach for determining LODs and LOQs for eDNA qPCR assays. We also provide details of an R script developed and applied for the modeling method. Discussion/Conclusions: Ultimately, standardization of how LOD and LOQ are determined, interpreted, and reported for eDNA assays will allow for more informed interpretation of assay results, more meaningful interlaboratory comparisons of experiments, and enhanced capacity for assessing the relative technical quality and performance of different eDNA qPCR assays

Reporting the limits of detection and quantification for environmental DNA assays

Background: Environmental DNA (eDNA) analysis is increasingly being used to detect the presence and relative abundance of rare species, especially invasive or imperiled aquatic species. The rapid progress in the eDNA field has resulted in numerous studies impacting conservation and management actions. However, standardization of eDNA methods and reporting across the field is yet to be fully established, with one area being the calculation and interpretation of assay limit of detection (LOD) and limit of quantification (LOQ). Aims: Here, we propose establishing consistent methods for determining and reporting of LOD and LOQ for single‐species quantitative PCR (qPCR) eDNA studies. Materials & Methods/ Results: We utilize datasets from multiple cooperating laboratories to demonstrate both a discrete threshold approach and a curve‐fitting modeling approach for determining LODs and LOQs for eDNA qPCR assays. We also provide details of an R script developed and applied for the modeling method. Discussion/Conclusions: Ultimately, standardization of how LOD and LOQ are determined, interpreted, and reported for eDNA assays will allow for more informed interpretation of assay results, more meaningful interlaboratory comparisons of experiments, and enhanced capacity for assessing the relative technical quality and performance of different eDNA qPCR assays

Reporting the limits of detection and quantification for environmental DNA assays

Background: Environmental DNA (eDNA) analysis is increasingly being used to detect the presence and relative abundance of rare species, especially invasive or imperiled aquatic species. The rapid progress in the eDNA field has resulted in numerous studies impacting conservation and management actions. However, standardization of eDNA methods and reporting across the field is yet to be fully established, with one area being the calculation and interpretation of assay limit of detection (LOD) and limit of quantification (LOQ). Aims: Here, we propose establishing consistent methods for determining and reporting of LOD and LOQ for single‐species quantitative PCR (qPCR) eDNA studies. Materials & Methods/ Results: We utilize datasets from multiple cooperating laboratories to demonstrate both a discrete threshold approach and a curve‐fitting modeling approach for determining LODs and LOQs for eDNA qPCR assays. We also provide details of an R script developed and applied for the modeling method. Discussion/Conclusions: Ultimately, standardization of how LOD and LOQ are determined, interpreted, and reported for eDNA assays will allow for more informed interpretation of assay results, more meaningful interlaboratory comparisons of experiments, and enhanced capacity for assessing the relative technical quality and performance of different eDNA qPCR assays