A high‐content screening assay in transgenic zebrafish identifies two novel activators of fgf signaling

Abstract

Zebrafish have become an invaluable vertebrate animal model to interrogate small molecule libraries for modulators of complex biological pathways and phenotypes. We have recently described the implementation of a quantitative, high-content imaging assay in multi-well plates to analyze the effects of small molecules on Fibroblast Growth Factor (FGF) signaling in vivo. Here we have evaluated the ability of the assay to identify compounds that hyperactivate FGF signaling from a test cassette of agents with known biological activities. Using a transgenic zebrafish reporter line for FGF activity, we screened 1040 compounds from an annotated library of known bioactive agents including FDA approved drugs. The assay identified two molecules, 8-hydroxyquinoline sulfate and pyrithione zinc that enhanced FGF signaling in specific areas of the brain. Subsequent studies revealed that both compounds specifically expanded FGF target gene expression. Furthermore, treatment of early stage embryos with either compound resulted in dorsalized phenotypes characteristic of hyperactivation of FGF signaling in early development. Documented activities for both agents included activation of extracellular signal-related kinase (ERK), consistent with FGF hyperactivation. To conclude, we demonstrate the power of automated quantitative high-content imaging to identify small molecule modulators of FGF.

Advances In Zebrafish Chemical Screening Technologies

“…Fluorescent zebrafish have also been utilized in multiple screens for potential cancer therapeutics, including screens for antileukemia drugs [70] and anti-angiogenics [71]; each of these studies employed manual screening techniques that were followed up with verification in murine models. A GFP-based reporter of FGF signaling has also been developed and used in a manual screen [72,73] for compounds that activate this pathway (through inhibition of a negative feedback regulator); this screen was subsequently automated using HCI techniques [74]. …”

15 years of zebrafish chemical screening

“…High-content imaging, for example, is becoming a powerful tool when combined with translucent zebrafish larvae. Using high-content screening researchers have identified butafenacil as a potent inducer of anemia [28], stimulators of pancreatic beta-cell proliferation [29], enhancers of FGF signaling [30], and the novel compound ‘lenaldekar,’ which selectively kills leukemic cells [9]. On the data analysis side, phenotypic ‘barcoding,’ where a complex endpoint such as a behavioral response is divided into discrete numeric units enabling comparisons of the phenotypes produced by thousands of chemicals to one another [31].…”

Development of High-Content Assays for Kidney Progenitor Cell Expansion in Transgenic Zebrafish

“…In addition, optical transparency during development makes zebrafish embryos uniquely suited for image-based analysis and high-content screening. Over the past five years, our group has developed phenotype- and platform-independent methodology for automated imaging of transgenic zebrafish embryos 6 that has proven useful in the discovery and characterization of allosteric dual specific phosphatase inhibitors 7; 8 , fibroblast growth factor pathway activators 9 , and antiangiogenic agents 10; 11 . In this report, we present the development of high-content imaging assays for zebrafish kidney progenitor cell expansion.…”