Differential activation of downstream Gsdf signaling pathway components upon selective GsdfX or GsdfY expression. (A) Medaka fibroblast cells (OLF cell line) were cotransfected with a luciferase reporter construct (UAS-luc) and different combinations of Smad-phosphorylation-dependent transactivating GAL4 constructs (Smad1, 2, 3, 5, or 8 -GAL4). Cells were either stimulated (cotransfection) or not (control) with GsdfX or GsdfY. In the absence of any induced signaling, the fusion proteins Smads-GAL4 remain in the cytoplasm and the luciferase reporter is only activated at a basal level. If activated, the Smads-GAL4 proteins are phosphorylated, translocate into the nucleus, and an increased luciferase expression is recorded. Results are expressed as the relative stimulation of Smad phosphorylation after either GsdfX or GsdfY stimulation compared to control (no stimulation). Irrespective of the Gsdf variants employed, monitoring of Smad1, 2, 3, 5, and 8 phosphorylation states (relative luciferase activity) upon stimulation with either GsdfX (B) or GsdfY (C) revealed that only Smads 2 and 3 were activated in both situations, whereas Smads 1/5/8 always remained unresponsive. See also Supplemental Data S1 for raw data. (n.s.) P > 0.05; (*) P < 0.05; (**) P < 0.01; (***) P < 0.001.