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 kinase (ERK) 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 that BCI inhibits Dusp6 catalytic activation by ERK2 substrate binding. A temporal role for Dusp6 in restricting cardiac progenitors and controlling heart organ size was uncovered with BCI treatment at varying developmental stages. This study highlights the power of in vivo zebrafish chemical screens to identify novel compounds targeting Dusp6, a component of the FGF signaling pathway that has eluded traditional high-throughput in vitro screens.

Background: Fibroblast Growth Factors (FGFs) represent a large family of secreted proteins that are required for proper development and physiological processes. Mutations in mouse and zebrafish FGFs result in abnormal embryogenesis and lethality. A key to understanding the precise role for these factors is to determine their spatial and temporal activity during embryogenesis.

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.

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