A phenotypic screen in zebrafish identifies a novel small-molecule inducer of ectopic tail formation suggestive of alterations in non-canonical Wnt/PCP signaling

PLoS One. 2013 Dec 11;8(12):e83293. doi: 10.1371/journal.pone.0083293. eCollection 2013.

Abstract

Zebrafish have recently emerged as an attractive model for the in vivo bioassay-guided isolation and characterization of pharmacologically active small molecules of natural origin. We carried out a zebrafish-based phenotypic screen of over 3000 plant-derived secondary metabolite extracts with the goal of identifying novel small-molecule modulators of the BMP and Wnt signaling pathways. One of the bioactive plant extracts identified in this screen - Jasminum gilgianum, an Oleaceae species native to Papua New Guinea - induced ectopic tails during zebrafish embryonic development. As ectopic tail formation occurs when BMP or non-canonical Wnt signaling is inhibited during the tail protrusion process, we suspected a constituent of this extract to act as a modulator of these pathways. A bioassay-guided isolation was carried out on the basis of this zebrafish phenotype, identifying para-coumaric acid methyl ester (pCAME) as the active compound. We then performed an in-depth phenotypic analysis of pCAME-treated zebrafish embryos, including a tissue-specific marker analysis of the secondary tails. We found pCAME to synergize with the BMP-inhibitors dorsomorphin and LDN-193189 in inducing ectopic tails, and causing convergence-extension defects in compound-treated embryos. These results indicate that pCAME may interfere with non-canonical Wnt signaling. Inhibition of Jnk, a downstream target of Wnt/PCP signaling (via morpholino antisense knockdown and pharmacological inhibition with the kinase inhibitor SP600125) phenocopied pCAME-treated embryos. However, immunoblotting experiments revealed pCAME to not directly inhibit Jnk-mediated phosphorylation of c-Jun, suggesting additional targets of SP600125, and/or other pathways, as possibly being involved in the ectopic tail formation activity of pCAME. Further investigation of pCAME's mechanism of action will help determine this compound's pharmacological utility.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Bone Morphogenetic Proteins / antagonists & inhibitors
  • Bone Morphogenetic Proteins / metabolism
  • Coumaric Acids / chemistry
  • Coumaric Acids / pharmacology*
  • Drug Evaluation, Preclinical
  • Embryo, Nonmammalian / embryology*
  • Jasminum / chemistry*
  • Pyrazoles / pharmacology
  • Pyrimidines / pharmacology
  • Tail / embryology*
  • Wnt Signaling Pathway / drug effects*
  • Zebrafish / embryology*
  • Zebrafish Proteins / antagonists & inhibitors
  • Zebrafish Proteins / metabolism

Substances

  • Bone Morphogenetic Proteins
  • Coumaric Acids
  • LDN 193189
  • Pyrazoles
  • Pyrimidines
  • Zebrafish Proteins
  • dorsomorphin
  • 4-coumaric acid methyl ester

Grant support

This research was funded by a grant from the Agency for Innovation by Science and Technology in Flanders (IWT). MLCM was funded by a PhD fellowship from the Vlaamse Interuniversitaire Raad (VLIR) linked to the VLIR-UOS project "Pharmacological Characterization of Medicinal Plants from the South of Ecuador." The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.