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Fig. 2 Morphology of AAC 789-, PTK787-, and FK506-treated zebrafish embryos at 48 hpf. Zebrafish embryos staged at 15 somites (17 hpf) were treated with AAC 789 (5 μM), PTK 787 (5 μM), or FK506 (2 μM) for 4, 12, and 24 hr. At the end of each time period, embryos were washed several times with fresh medium to remove the inhibitor drugs and allowed to develop until 48hpf. Treatment for 4 hr with AAC 789 (b), PTK787 (e), or FK506 (h) resulted in toggling of blood within the heart (see Supplementary Movies), compared to control embryos, while no apparent effect on gross morphogenesis was visible. Treatment with PTK 787 for 12 hr induced minor epicardial edema as well as minor retarded craniofacial development (f and k). Treatment with PTK787 for 24 hr resulted in severe epicardial edema (g, arrow), shortened tails, retarded brain growth, and blood toggling in the heart (k). Embryos treated with AAC 789 or FK506 for 12 hr (c, i) and 24 hr (d, j) showed almost the same morphology at 48 hpf as control embryos (a), with the exception of minor epicardial edema in embryos treated for 24 hr with FK506 (j, arrow). k: The number of embryos with defects in each condition and at each time point (n = 160-500) is shown. No embryos in the untreated group showed any regurgitation of blood (n = 178) (not shown).