Ewing Sarcoma Protein Ewsr1 Maintains Mitotic Integrity and Proneural Cell Survival in the Zebrafish Embryo

BACKGROUND:The Ewing sarcoma breakpoint region 1 gene (EWSR1), also known as EWS, is fused to a number of different partner genes as a result of chromosomal translocation in diverse sarcomas. Despite the involvement of EWSR1 in these diverse sarcomas, the in vivo function of wild type EWSR1 remains unclear. PRINCIPAL FINDINGS:We identified two zebrafish EWSR1 orthologues, ewsr1a and ewsr1b, and demonstrate that both genes are expressed maternally, and are expressed ubiquitously throughout zebrafish embryonic development. Morpholino induced knockdown of both zebrafish ewsr1 genes led to mitotic defects with multipolar or otherwise abnormal mitotic spindles starting from the bud stage (10 hour post-fertilization (hpf)). The abnormalities in mitotic spindles were followed by p53-mediated apoptosis in the developing central nervous system (CNS) leading to a reduction in the number of proneural cells, disorganization of neuronal networks, and embryonic lethality by 5 days post-fertilization. siRNA silencing of EWSR1 in Hela cells resulted in mitotic defects accompanied by apoptotic cell death, indicating that the role of EWSR1 is conserved between zebrafish and human. CONCLUSIONS:Ewsr1 maintains mitotic integrity and proneural cell survival in early zebrafish development

<i>brca2</i> and <i>tp53</i> Collaborate in Tumorigenesis in Zebrafish

<div><p>Germline mutations in the tumor suppressor genes <i>BRCA2</i> and <i>TP53</i> significantly influence human cancer risk, and cancers from humans who inherit one mutant allele for <i>BRCA2</i> or <i>TP53</i> often display loss of the wildtype allele. In addition, <i>BRCA2</i>-associated cancers often exhibit mutations in <i>TP53</i>. To determine the relationship between germline heterozygous mutation (haploinsufficiency) and somatic loss of heterozygosity (LOH) for <i>BRCA2</i> and <i>TP53</i> in carcinogenesis, we analyzed zebrafish with heritable mutations in these two genes. Tumor-bearing zebrafish were examined by histology, and normal and neoplastic tissues were collected by laser-capture microdissection for LOH analyses. Zebrafish on a heterozygous <i>tp53^M214K</i> background had a high incidence of malignant tumors. The <i>brca2^Q658X</i> mutation status determined both the incidence of LOH and the malignant tumor phenotype. LOH for <i>tp53</i> occurred in the majority of malignant tumors from <i>brca2</i> wildtype and heterozygous mutant zebrafish, and most of these were malignant peripheral nerve sheath tumors. Malignant tumors in zebrafish with heterozygous mutations in both <i>brca2</i> and <i>tp53</i> frequently displayed LOH for both genes. In contrast, LOH for <i>tp53</i> was uncommon in malignant tumors from <i>brca2</i> homozygotes, and these tumors were primarily undifferentiated sarcomas. Thus, carcinogenesis in zebrafish with combined mutations in <i>tp53</i> and <i>brca2</i> typically requires biallelic mutation or loss of at least one of these genes, and the specific combination of inherited mutations influences the development of LOH and the tumor phenotype. These results provide insight into cancer development associated with heritable <i>BRCA2</i> and <i>TP53</i> mutations.</p></div

Malignant zebrafish tumors frequently develop LOH for <i>brca2</i> and/or <i>tp53</i>.

<p>(<b>A–C</b>), Before (upper panels) and after (lower panels) images of LCM-guided sample collection from an MPNST (A), an MPNST and a nephroblastoma (B), and a normal liver (C). Regions of sample collection are outlined in color. (<b>D</b>) MPNST from a <i>brca2+/m</i>;<i>tp53+/m</i> zebrafish shows loss of the <i>brca2</i> and <i>tp53</i> wildtype alleles. (<b>E</b>) MPNST and nephroblastoma from a <i>brca2+/m</i>;<i>tp53+/m</i> zebrafish show disparate LOH profiles. LOH, loss of heterozygosity; MPNST, malignant peripheral nerve sheath tumor; NB, nephroblastoma.</p

Tumor types developed by <i>brca2</i>+/+;<i>tp53</i>+/<i>m</i>, <i>brca2</i>+/<i>m</i>;<i>tp53</i>+/<i>m</i>, and <i>brca2</i><i>m</i>/<i>m</i>;<i>tp53</i>+/<i>m</i> zebrafish.

<p>Abbreviations: MPNST, malignant peripheral nerve sheath tumor; mo, months.</p>I<p>The total numbers of each tumor type observed by histologic analysis are reported. Some zebrafish developed more than one tumor (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087177#pone-0087177-t001" target="_blank">Table 1</a>).</p

Tumor development <i>in tp53+/m</i> zebrafish is influenced by <i>brca2</i> mutation status.

<p>(<b>A</b>) Age at tumor diagnosis is significantly lower in <i>brca2 m/m</i>;<i>tp53+/m</i> zebrafish compared to <i>brca2</i>+/+;<i>tp53+/m</i> and <i>brca2+/m</i>;<i>tp53+/m</i> zebrafish. (<b>B</b>) The percentage of zebrafish that developed at least one malignant tumor, only benign tumors, or no tumors, in <i>brca2</i>+/+;<i>tp53+/m</i>, <i>brca2+/m</i>;<i>tp53+/m</i>, and <i>brca2 m/m</i>;<i>tp53+/m</i> zebrafish. (<b>C</b>) MPNST from a <i>brca2</i>+/+;<i>tp53+/m</i> zebrafish. (<b>D</b>) Undifferentiated sarcoma from a <i>brca2 m/m</i>;<i>tp53+/m</i> zebrafish. (<b>E</b>) MPNST and nephroblastoma from a <i>brca2+/m</i>;<i>tp53+/m</i> zebrafish. MPNST, malignant peripheral nerve sheath tumor; NB, nephroblastoma. Scale bars, 20 μm (C,D) and 200 μm (E).</p