Antiangiogenic, antimigratory and antiinflammatory effects of 2-methoxyestradiol in zebrafish larvae.

2-Methoxyestradiol (2ME), an endogenous metabolite of 17ß-estradiol, has been previously reported to possess antiangiogenic and antitumor properties. Herein, we demonstrate that the effects of this antiangiogenic steroid can be readily assayed in live zebrafish, introducing a convenient and robust new model system as a screening tool for both single cell and collective cell migration assays. Using the in vitro mammalian endothelial cell line EA.hy926, we first show that cell migration and angiogenesis, as estimated by wound assay and tube formation respectively, are antagonized by 2ME. In zebrafish (Danio rerio) larvae, dose-dependent exposure to 2ME diminishes (1) larval angiogenesis, (2) leukocyte recruitment to damaged lateral line neuromasts and (3) retards the lateral line primordium in its migration along the body. Our results indicate that 2ME has an effect on collective cell migration in vivo as well as previously reported anti-tumorigenic activity and suggests that the molecular mechanisms governing cell migration in a variety of contexts are conserved between fish and mammals. Moreover, we exemplify the versatility of the zebrafish larvae for testing diverse physiological processes and screening for antiangiogenic and antimigratory drugs in vivo.

A high-throughput chemically induced inflammation assay in zebrafish.

BACKGROUND: Studies on innate immunity have benefited from the introduction of zebrafish as a model system. Transgenic fish expressing fluorescent proteins in leukocyte populations allow direct, quantitative visualization of an inflammatory response in vivo. It has been proposed that this animal model can be used for high-throughput screens aimed at the identification of novel immunomodulatory lead compounds. However, current assays require invasive manipulation of fish individually, thus preventing high-content screening. RESULTS: Here we show that specific, noninvasive damage to lateral line neuromast cells can induce a robust acute inflammatory response. Exposure of fish larvae to sublethal concentrations of copper sulfate selectively damages the sensory hair cell population inducing infiltration of leukocytes to neuromasts within 20 minutes. Inflammation can be assayed in real time using transgenic fish expressing fluorescent proteins in leukocytes or by histochemical assays in fixed larvae. We demonstrate the usefulness of this method for chemical and genetic screens to detect the effect of immunomodulatory compounds and mutations affecting the leukocyte response. Moreover, we transformed the assay into a high-throughput screening method by using a customized automated imaging and processing system that quantifies the magnitude of the inflammatory reaction. CONCLUSIONS: This approach allows rapid screening of thousands of compounds or mutagenized zebrafish for effects on inflammation and enables the identification of novel players in the regulation of innate immunity and potential lead compounds toward new immunomodulatory therapies. We have called this method the chemically induced inflammation assay, or ChIn assay. See Commentary article: http://www.biomedcentral.com/1741-7007/8/148.