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

Evelien Gebruers, María Lorena Cordero-Maldonado, Alexander I Gray, Carol Clements, Alan L Harvey, Ruangelie Edrada-Ebel, Peter A M de Witte, Alexander D Crawford, Camila V Esguerra

Research output: Contribution to journalArticle

16 Citations (Scopus)

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.

LanguageEnglish
Pagese83293
JournalPLOS One
Volume8
Issue number12
DOIs
Publication statusPublished - 2013

Fingerprint

coumaric acids
Zebrafish
Danio rerio
Tail
tail
esters
Molecules
embryo (plant)
Embryonic Structures
Bioassay
Biological Assay
Modulators
Oleaceae
Jasminum
bioassays
Pharmacology
Papua New Guinea
Morpholinos
Phosphorylation
Wnt Signaling Pathway

Keywords

  • phenotypic screen
  • zebrafish
  • novel small-molecule inducer
  • ectopic tail formation
  • non-canonical wnt/pcp
  • signalling
  • bmp signaling
  • embryos
  • notochords
  • phenotypes
  • plant signalling
  • tails
  • wnt signaling cascade

Cite this

Gebruers, Evelien ; Cordero-Maldonado, María Lorena ; Gray, Alexander I ; Clements, Carol ; Harvey, Alan L ; Edrada-Ebel, Ruangelie ; de Witte, Peter A M ; Crawford, Alexander D ; Esguerra, Camila V. / A phenotypic screen in zebrafish identifies a novel small-molecule inducer of ectopic tail formation suggestive of alterations in non-canonical Wnt/PCP signaling. In: PLOS One. 2013 ; Vol. 8, No. 12. pp. e83293.
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A phenotypic screen in zebrafish identifies a novel small-molecule inducer of ectopic tail formation suggestive of alterations in non-canonical Wnt/PCP signaling. / Gebruers, Evelien; Cordero-Maldonado, María Lorena; Gray, Alexander I; Clements, Carol; Harvey, Alan L; Edrada-Ebel, Ruangelie; de Witte, Peter A M; Crawford, Alexander D; Esguerra, Camila V.

In: PLOS One, Vol. 8, No. 12, 2013, p. e83293.

Research output: Contribution to journalArticle

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T1 - A phenotypic screen in zebrafish identifies a novel small-molecule inducer of ectopic tail formation suggestive of alterations in non-canonical Wnt/PCP signaling

AU - Gebruers, Evelien

AU - Cordero-Maldonado, María Lorena

AU - Gray, Alexander I

AU - Clements, Carol

AU - Harvey, Alan L

AU - Edrada-Ebel, Ruangelie

AU - de Witte, Peter A M

AU - Crawford, Alexander D

AU - Esguerra, Camila V

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AB - 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.

KW - phenotypic screen

KW - zebrafish

KW - novel small-molecule inducer

KW - ectopic tail formation

KW - non-canonical wnt/pcp

KW - signalling

KW - bmp signaling

KW - embryos

KW - notochords

KW - phenotypes

KW - plant signalling

KW - tails

KW - wnt signaling cascade

U2 - 10.1371/journal.pone.0083293

DO - 10.1371/journal.pone.0083293

M3 - Article

VL - 8

SP - e83293

JO - PLOS One

T2 - PLOS One

JF - PLOS One

SN - 1932-6203

IS - 12

ER -