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

Averting biodiversity collapse in tropical forest protected areas

The rapid disruption of tropical forests probably imperils global biodiversity more than any other contemporary phenomenon¹⁻³. With deforestation advancing quickly, protected areas are increasingly becoming final refuges for threatened species and natural ecosystem processes. However, many protected areas in the tropics are themselves vulnerable to human encroachment and other environmental stresses⁴⁻⁹. As pressures mount, it is vital to know whether existing reserves can sustain their biodiversity. A critical constraint in addressing this question has been that data describing a broad array of biodiversity groups have been unavailable for a sufficiently large and representative sample of reserves. Here we present a uniquely comprehensive data set on changes over the past 20 to 30 years in 31 functional groups of species and 21 potential drivers of environmental change, for 60 protected areas stratified across the world’s major tropical regions. Our analysis reveals great variation in reserve ‘health’: about half of all reserves have been effective or performed passably, but the rest are experiencing an erosion of biodiversity that is often alarmingly widespread taxonomically and functionally. Habitat disruption, hunting and forest-product exploitation were the strongest predictors of declining reserve health. Crucially, environmental changes immediately outside reserves seemed nearly as important as those inside in determining their ecological fate, with changes inside reserves strongly mirroring those occurring around them. These findings suggest that tropical protected areas are often intimately linked ecologically to their surrounding habitats, and that a failure to stem broad-scale loss and degradation of such habitats could sharply increase the likelihood of serious biodiversity declines.Keywords: Ecology, Environmental scienc

A non-genetic switch triggers alternative telomere lengthening and cellular immortalization in ATRX deficient cells

Alternative Lengthening of Telomeres (ALT) is an aberrant DNA recombination pathway which grants replicative immortality to approximately 10% of all cancers. Despite this high prevalence of ALT in cancer, the mechanism and genetics by which cells activate this pathway remain incompletely understood. A major challenge in dissecting the events that initiate ALT is the extremely low frequency of ALT induction in human cell systems. Guided by the genetic lesions that have been associated with ALT from cancer sequencing studies, we genetically engineered primary human pluripotent stem cells to deterministically induce ALT upon differentiation. Using this genetically defined system, we demonstrate that disruption of the p53 and Rb pathways in combination with ATRX loss-of-function is sufficient to induce all hallmarks of ALT and results in functional immortalization in a cell type-specific manner. We further demonstrate that ALT can be induced in the presence of telomerase, is neither dependent on telomere shortening nor crisis, but is rather driven by continuous telomere instability triggered by the induction of differentiation in ATRX-deficient stem cells

A non-genetic switch triggers alternative telomere lengthening and cellular immortalization in ATRX deficient cells

Alternative Lengthening of Telomeres (ALT) is an aberrant DNA recombination pathway which grants replicative immortality to approximately 10% of all cancers. Despite this high prevalence of ALT in cancer, the mechanism and genetics by which cells activate this pathway remain incompletely understood. A major challenge in dissecting the events that initiate ALT is the extremely low frequency of ALT induction in human cell systems. Guided by the genetic lesions that have been associated with ALT from cancer sequencing studies, we genetically engineered primary human pluripotent stem cells to deterministically induce ALT upon differentiation. Using this genetically defined system, we demonstrate that disruption of the p53 and Rb pathways in combination with ATRX loss-of-function is sufficient to induce all hallmarks of ALT and results in functional immortalization in a cell type-specific manner. We further demonstrate that ALT can be induced in the presence of telomerase, is neither dependent on telomere shortening nor crisis, but is rather driven by continuous telomere instability triggered by the induction of differentiation in ATRX-deficient stem cells

MZ <i>ewsa/ewsa</i> mutants display altered expression levels of Sox9 target genes.

<p>The ratios of the Sox9 target mRNA expression levels between the <i>wt/wt</i> and MZ <i>ewsa/ewsa</i> mutants (27 hpf) are shown in a bar plot (* p<0.05 and ** p<0.01).</p

4 dpf MZ ewsa/ewsa mutants display an aberrant angle of Meckel’s cartilage and palatoquadrate.

<p><b>A.</b> Lateral views (anterior to the left) of <i>wt/wt</i> (left) and MZ <i>ewsa/ewsa</i> (right) and ventral views of adult zebrafish. The calcified bones were visualized by alizarin red staining. <b>B.</b> Lateral views (anterior to the left) of (a) <i>wt/wt</i> and (b) MZ <i>ewsa/ewsa</i> and ventral views of (c) <i>wt/wt</i> and (d) MZ <i>ewsa/ewsa</i> chondrocytes from 4 dpf zebrafish embryos visualized with alcian blue. (e and f) Angle formed by Meckel’s cartilage showing that the palatoquadrate is wider in the MZ <i>ewsa/ewsa</i> mutant than <i>wt/wt</i> at 4 dpf and 7 dpf. bh: basihyal, d: dentary, m: Meckel’s cartilage, pq: palatoquadrate, ch: ceratohyal, cb: ceratobranchial.</p

Prehypertrophic chondrocytes of Meckel’s cartilage in MZ <i>ewsa/ewsa</i> fails to differentiate into hypertrophic chondrocytes.

<p><b>A.</b> Flat mounted Meckel’s cartilage of 4 dpf (left) <i>wt/wt</i> and (right) MZ <i>ewsa/ewsa</i>. <b>B.</b> Number of cells of Meckel’s cartilage of 4 dpf <i>wt/wt</i> and MZ <i>ewsa/ewsa</i> zebrafish (P = 0.0002). <b>C.</b> Lateral views (anterior to the left, dorsal to the top) of 4dpf (Left) <i>wt/wt</i> and (Right) MZ <i>ewsa/ewsa</i> visualized by <i>in situ</i> hybridization using probe for <i>ihha</i> (top panel) and <i>colX</i> (bottom panel). m: Meckel's cartilage. <b>D.</b> Ventral views (anterior to the left) of Meckel's cartilage of (Left) <i>wt/wt</i> and (Right) MZ <i>ewsa/ewsa</i> visualized by immunohistochemistry using (top) anti-IHH antibody (green), and anti-Collagen type II antibody (Red) (4dpf), and (bottom) anti-Collagen X antibody (green) and anti-Collagen type II antibody (Red) (5dpf).</p

MZ <i>ewsa/ewsa</i> mutants display altered expression domains of Sox9 target genes.

<p>27hpf of (Left) <i>wt/wt</i> and (Right) MZ <i>ewsa/ewsa</i> visualized by <i>in situ</i> hybridization using antisense RNA probe for <b>A.</b><i>sox5</i>, <b>B.</b><i>noggin1</i>, <b>C.</b><i>noggin2</i>, <b>D.</b><i>bmp4</i>, <b>E.</b><i>ctgfa</i>, <b>F.</b><i>ctgfb</i>, <b>G.</b><i>col2a1a</i> and <b>H.</b><i>col2a1b</i>. Top panel: low magnification images, middle and bottom panel: high magnification images.</p