ABSTRACT
Fibroblast growth factors (FGFs) are secreted molecules that activate the RAS/mitogen-activated protein kinase (MAPK) signaling pathway. In zebrafish development, FGF signaling is responsible for establishing dorsal polarity, maintaining the isthmic organizer, and cardiac ventricle formation. Because several ETS factors are known transcriptional mediators of MAPK signaling, we hypothesized that these factors function to mediate FGF signaling processes. In zebrafish, the simultaneous knock-down of three Pea3 ETS proteins, Etv5, Erm, and Pea3, produced phenotypes reminiscent of embryos deficient in FGF signaling. Morphant embryos displayed both cardiac and left/right patterning defects as well as disruption of the isthmic organizer. Furthermore, the expression of FGF target genes was abolished in Pea3 ETS depleted embryos. To understand how FGF signaling and ETS factors control gene expression, transcriptional regulation of dusp6 was studied in mouse and zebrafish. Conserved Pea3 ETS binding sites were identified within the Dusp6 promoter, and reporter assays showed that one of these sites is required for dusp6 induction by FGFs. We further demonstrated the interaction of Pea3 ETS factors with the Dusp6 promoter both in vitro and in vivo. These results revealed the requirement of ETS factors in transducing FGF signals in developmental processes.
Subject(s)
Fibroblast Growth Factors/metabolism , Proto-Oncogene Proteins c-ets/metabolism , Transcription Factors/metabolism , Zebrafish/metabolism , Animals , Binding Sites/genetics , Cell Differentiation/genetics , Fibroblast Growth Factors/genetics , Gene Expression , Gene Expression Regulation , Mice , Mitogen-Activated Protein Kinases/genetics , Mitogen-Activated Protein Kinases/metabolism , Oncogenes , Proto-Oncogene Proteins c-ets/genetics , Signal Transduction/genetics , Signal Transduction/physiology , Transcription Factors/genetics , Zebrafish/geneticsABSTRACT
The dual-specificity phosphatase 6 (Dusp6) functions as a feedback regulator of fibroblast growth factor (FGF) signaling to limit the activity of extracellular signal-regulated kinases (ERKs) 1 and 2. We have identified a small-molecule inhibitor of Dusp6-(E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one (BCI)-using a transgenic zebrafish chemical screen. BCI treatment blocked Dusp6 activity and enhanced FGF target gene expression in zebrafish embryos. Docking simulations predicted an allosteric binding site for BCI within the phosphatase domain. In vitro studies supported a model in which BCI inhibits Dusp6 catalytic activation by ERK2 substrate binding. We used BCI treatment at varying developmental stages to uncover a temporal role for Dusp6 in restricting cardiac progenitors and controlling heart organ size. This study highlights the power of in vivo zebrafish chemical screens to identify new compounds targeting Dusp6, a component of the FGF signaling pathway that has eluded traditional high-throughput in vitro screens.
Subject(s)
Animals, Genetically Modified/metabolism , Cell Lineage , Cyclohexylamines/pharmacology , Dual Specificity Phosphatase 6/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Heart , Indenes/pharmacology , Zebrafish/genetics , Allosteric Site , Animals , Cell Lineage/genetics , Cyclohexylamines/chemical synthesis , Cyclohexylamines/chemistry , Dual Specificity Phosphatase 6/genetics , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Fibroblast Growth Factors/metabolism , Gene Expression Regulation, Developmental/drug effects , Heart/embryology , Indenes/chemical synthesis , Indenes/chemistry , Mitogen-Activated Protein Kinase 1/metabolism , Protein Binding , Small Molecule Libraries , Substrate Specificity , Zebrafish/embryology , Zebrafish/metabolismABSTRACT
The vertebrate mid-hindbrain boundary (MHB) is a crucial morphological structure required for patterning and neural differentiation of the midbrain and anterior hindbrain. We isolated a novel zebrafish mutant, MHB gone (mgo), that exhibited a defective MHB. Expression of engrailed3 in the prospective MHB was absent at the 1-somite stage, suggesting that initiation of the isthmic organizer was disrupted in mgo mutants. Complementation test with mgo and noi, in which the pax2a gene is mutated, infer that the mgo mutant may represent a novel noi allele. However, pronephric, otic vesicle, and commissural axonal defects described in noi mutants were not associated with mgo mutants. Genetic mapping revealed that the mgo mutation is linked to the Pax2a locus, but no mutation was detected in pax2a exons or within intron-exon boundaries. Based on these findings, we propose that the mgo mutation genetically interacts with pax2a required for the initiation of MHB formation.
