A high-throughput chemically induced inflammation assay in zebrafish

Artículo de publicación ISIBackground: 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.This work was supported by grants to MA from Fondecyt (1070867), FONDAP (15090007), ICM (P06-039F), CORFO-Innova (09MCSS-6705), DFG-Conicyt 075-2009; to CD from UNAB (DI- 01-09/1) and Fondecyt (24090004); to UL from Dopaminet (EU FP7 223744); and to CG by a Marie Curie International Reintegration Grant (EU FP7; PIRG07-GA-2010-267552)

Transcription factor Ap-2alpha is necessary for development of embryonic melanophores, autonomic neurons and pharyngeal skeleton in zebrafish

The genes that control development of embryonic melanocytes are poorly defined. Although transcription factor Ap-2a is expressed in neural crest (NC) cells, its role in development of embryonic melanocytes and other neural crest derivatives is unclear because mouse Ap-2a mutants die before melanogenesis. We show that zebrafish embryos injected with morpholino antisense oligonucleotides complementary to ap-2a (ap-2a MO) complete early morphogenesis normally and have neural crest cells. Expression of c-kit, which encodes the receptor for the Steel ligand, is reduced in these embryos, and, similar to zebrafish c-kit mutant embryos, embryonic melanophores are reduced in number and migration. The effects of ap-2a MO injected into heterozygous and homozygous c-kit mutants support the notion that Ap-2a works through C-kit and additional target genes to mediate melanophore cell number and migration. In contrast to c-kit mutant embryos, in ap-2a MO-injected embryos, melanophores are small and under-pigmented, and unexpectedly, analysis of mosaic embryos suggests Ap-2a regulates melanophore differentiation through cell non-autonomous targets. In addition to melanophore phenotypes, we document reduction of other neural crest derivatives in ap-2a MO-injected embryos, including jaw cartilage, enteric neurons, and sympathetic neurons. These results reveal that Ap-2a regulates multiple steps of melanophore development, and is required for development of other neuronal and nonneuronal neural crest derivatives.This work was supported by NIH grant HD22486 to J.S.E. and a Carver Foundation seed grant to R.A.C. C. d’., and M.A. were supported by grants ICM P99-137-f and Fondecyt 1031003. E.K.O. was supported by Grant T32 DC00040 (Bruce Gantz, PI)

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

Artículo de publicación ISI.2-Methoxyestradiol (2ME), an endogenous metabolite of 17β-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.GIO was supported by the BRMC CTU06. MA was supported by grants from FONDAP (15090007) and ICGEB (CRP/CHI11-01)