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Fig. 3
Temporal dynamics of genetic FGF signaling perturbation using fgfr1‐dn‐cargo. A: Schematic showing the time line of treatments for genetic and chemical FGF signaling perturbations and time points of embryo fixation for signaling activity readouts. Red arrows indicate time points and duration of drug (10 µM 4‐OHT and 5 µM SU5402) and heatshock treatments; violet arrows mark time points of embryo fixation after treatment onset (heatshock or SU5402 application). B–F: Representative embryos stained for etv4 mRNA expression via in situ hybridization in Cerulean‐negative single‐transgenic controls (control cargo), ubi:creERT2;fgfr1‐dn‐cargo (cargo), and SU5402‐treated embryos; lateral views, anterior to the top. Number “n” indicates individual embryos stained and analyzed for each condition. B,C: After FGF inhibition was initiated at mid‐somitogenesis (10 ss), etv4 expression in ubi:creERT2;fgfr1‐dn‐cargo and SU5402‐treated embryos was decreased 2 hr and absent 4 hr after treatment onset. D‐F: etv4 expression started recovering 2 hr after SU5402‐treatment was stopped (6 hr after initiation of a 4‐hr pulse) and 8 hr after heatshock in ubi:creERT2;fgfr1‐dn‐cargo double transgenics; 9 hr after treatment onset, etv4 expression recovered to a large extent in genetically and pulse‐treated chemically perturbed embryos (F). G–I: FGF signaling perturbation during gastrulation stages. G: Schematic showing the time line of treatments (as in A). H,I: Representative embryos stained for etv4 mRNA expression with in situ hybridization in Cerulean‐negative single‐transgenic controls (control cargo), Cerulean‐positive ubi:creERT2;fgfr1‐dn‐cargo double transgenics (cargo), untreated wild‐type controls (control SU5402), and SU5402‐treated embryos; lateral views, anterior to the top. Number “n” indicates individual embryos stained and analyzed for each condition. etv4 expression in ubi:creERT2;fgfr1‐dn‐cargo and SU5402‐treated embryos was decreased 3 hr and 4 hr after treatment at shield stage, but never completely lost as at later stages (see also C,D). Note that Cerulean‐CAAX expression could not be observed 1–2 hr post‐heatshock, thus etv4 expression analysis is only shown after 3 hr.