Cyclopamine treatment determines time window where Hh signaling inhibits fin induction. Treatment with 10 μM cyclopamine between 50% epiboly and the indicated developmental stages, identified the critical time window for Hh where it actively inhibits fin induction. From a dorsal (A,D, I-L) and lateral (E-H, M-P) view recruitment of tbx5 positive cells in the fin field can be slightly rescued when cyclopamine is administered between 50%- and 100% epiboly (J,N). However, tbx5 is more highly expressed when cyclopamine is administered between 50% epiboly and 5- and 10 somite stage (K,L,O,P). The constitutive activation of Hh signaling after removing cyclopamine, inhibits the outgrowth of the fin bud, clearly visible from a lateral view (M-P). Inhibiting Hh signaling in ptc1;ptc2 double mutants from the 18 ss till 40 hpf, rescues a restricted expression of tbx5, which is not, observed when cyclopamine is administered at 24 ss or 24 hpf (Q-T). These data show that Hh signaling inhibits fin induction during late gastrulation and the segmentation stage.
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