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.

Maternal Interferon Regulatory Factor 6 is required for the differentiation of primary superficial epithelia in Danio and Xenopus embryos.

Early in the development of animal embryos, superficial cells of the blastula form a distinct lineage and adopt an epithelial morphology. In different animals, the fate of these primary superficial epithelial (PSE) cells varies, and it is unclear whether pathways governing segregation of blastomeres into the PSE lineage are conserved. Mutations in the gene encoding Interferon Regulatory Factor 6 (IRF6) are associated with syndromic and non-syndromic forms of cleft lip and palate, consistent with a role for Irf6 in development of oral epithelia, and mouse Irf6 targeted null mutant embryos display abnormal differentiation of oral epithelia and skin. In Danio rerio (zebrafish) and Xenopus laevis (African clawed frog) embryos, zygotic irf6 transcripts are present in many epithelial tissues including the presumptive PSE cells and maternal irf6 transcripts are present throughout all cells at the blastula stage. Injection of antisense oligonucleotides with ability to disrupt translation of irf6 transcripts caused little or no effect on development. By contrast, injection of RNA encoding a putative dominant negative Irf6 caused epiboly arrest, loss of gene expression characteristic of the EVL, and rupture of the embryo at late gastrula stage. The dominant negative Irf6 disrupted EVL gene expression in a cell autonomous fashion. These results suggest that Irf6 translated in the oocyte or unfertilized egg suffices for early development. Supporting the importance of maternal Irf6, we show that depletion of maternal irf6 transcripts in X. laevis embryos leads to gastrulation defects and rupture of the superficial epithelium. These experiments reveal a conserved role for maternally-encoded Irf6 in differentiation of a simple epithelium in X. laevis and D. rerio. This epithelium constitutes a novel model tissue in which to explore the Irf6 regulatory pathway.

Expression of the zebrafish Staufen gene in the embryo and adult.

Staufen, a double stranded RNA binding protein, has been shown to be involved in creating and maintaining cellular asymmetry in the Drosophila oocyte, neuroblast, and mammalian neuron. Staufen binds to the 3' UTR of specific mRNAs and acts in their localization and anchoring to various subcellular domains. Staufen's molecular interactions during development have been limited to investigations in Drosophila melanogaster. Since a vertebrate Staufen has not been studied in a developmental system, the aim of this study was to clone and characterize a staufen orthologue gene in the vertebrate developmental model, zebrafish. The zebrafish staufen-like sequence shows a 64% homology to the human staufen with a 81.2% homology in the highly conserved double stranded RNA binding domain (dsRBDs). Staufen maps on the LN54 radiation hybrid panel to linkage group 6, 16.25 cR from Z265 between fb22h06 and fi16e01. Northern blot and in situ hybridization showed that staufen is expressed both maternally and zygotically. Zygotically expressed staufen is localized to the developing nervous system and at 24 h is highly concentrated in the subventricular zone of the developing brain. Maternally expressed staufen is dispersed in the mature oocyte and early embryo. In the adult, staufen is expressed in specific brain nuclei, the testis, neurons and Leydig cells.

Touchtone promotes survival of embryonic melanophores in zebrafish.

An outstanding problem in the study of vertebrate development is the identification of the genes that direct neural crest precursor cells to adopt and maintain specific differentiated cell fates. In an effort to identify such genes, we have carried out a mutagenesis screen in zebrafish and isolated mutants that lack neural crest-derived melanophores. In this manuscript we describe the phenotype of one such mutant, touchtone(b722) (tct), and the map position of the gene it defines. Analysis of expression of dopachrome tautomerase (dct) and microphthalmia (mitfa) suggests that melanophore precursors are specified normally in homozygous tct mutants. However, differentiated melanophores are pale, small, and about half of them have disappeared by 48 h of development, apparently by cell death. We show that melanophores require Tct function cell autonomously. Signals from the receptor tyrosine kinase receptor C-kit are essential for survival of melanophores in zebrafish and mammals. However, differences in the phenotypes of tct and c-kit homozygous mutants, and an absence of interaction between c-kit and tct heterozygotes, suggest that Tct functions independently of the C-kit pathway. Other neural crest derivatives, including other pigment cell types, appear normal in tct mutants. Interestingly, tct mutant embryos undergo a temporary period of near complete paralyzis during the second day of development, although markers of axons of motor and sensory neurons look normal in this period. A fraction of tct(b722) mutants survive to adulthood, but mutant adults are small, indicating a role for Tct in post-larval growth. The tct gene maps to a small interval near a telomere of chromosome 18. Thus, we have identified a zebrafish gene that when mutated produces semi-viable offspring and that may serve as a model of human diseases that have both pigmentation and neurological symptoms.

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-2alpha is expressed in neural crest (NC) cells, its role in development of embryonic melanocytes and other neural crest derivatives is unclear because mouse Ap-2alpha mutants die before melanogenesis. We show that zebrafish embryos injected with morpholino antisense oligonucleotides complementary to ap-2alpha (ap-2alpha 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-2alpha MO injected into heterozygous and homozygous c-kit mutants support the notion that Ap-2alpha works through C-kit and additional target genes to mediate melanophore cell number and migration. In contrast to c-kit mutant embryos, in ap-2alpha MO-injected embryos, melanophores are small and under-pigmented, and unexpectedly, analysis of mosaic embryos suggests Ap-2alpha regulates melanophore differentiation through cell non-autonomous targets. In addition to melanophore phenotypes, we document reduction of other neural crest derivatives in ap-2alpha MO-injected embryos, including jaw cartilage, enteric neurons, and sympathetic neurons. These results reveal that Ap-2alpha regulates multiple steps of melanophore development, and is required for development of other neuronal and non-neuronal neural crest derivatives.