Fig. 5

Notch induces proneuromast (rosette) formation independent of Fgf-MAPK-Shroom3 signaling.

(A–D'') The expression of Fgf pathway members in the primordium is altered by Notch signaling. Fgf ligand (B' and C') and target gene expression (D') is largely downregulated in NICD primordia compared to siblings (B–D). (B'') fgf3 and (D'') pea3 expression is downregulated in mib1^ta52b primordia, but fgf10 expression (C'') is largely expanded. fgfr1 expression remains normal in NICD (A') but is downregulated in the center of the mib1^ta52b primordia (A''). (E–H') Notch induces proneuromast formation independent of Fgf-MAPK signaling. NICD primordia form proneuromasts in the presence of Fgfr1 and MAPK inhibitors (F' and F'' and F''') as well as after the induction of dominant-negative Fgfr1 (H'), while sibling proneuromast formation fails after these manipulations (E'–E''' and G'). (G–H') Embryos were heat-shocked 2 times at 39°C for 20 min with 20 min incubation at a room temperature in between, starting at 26 hpf and fixed 4 hr later. (I–L'') Notch regulates apical Rock2a accumulation independent of Fgf signaling. Contrary to sibling primordia (J'), the Rock2a expression is maintained in the apices of the forming proneuromasts in NICD primordia treated with the Fgfr1 inhibitor SU5402 (L'). (J'' and L'') Fgf downregulation is confirmed by the loss of di-pERK1/2 expression in the primordium. (M–P') Notch regulates Phosphorylated Myosin Regulatory Light Chain (pMRLC) expression independent of Fgf signaling. (N') Sibling primordia lose the pMRLC expression after Fgfr1 inhibitor treatment, but pMRLC expression is maintained in the apices of treated (P') NICD proneuromasts. (Q–V') NICD primordia form proneuromasts (black arrows) in the absence of shroom3 expression (V'). Treatment with the Fgfr1 inhibitor SU5402 depletes pea3 (Q' and R'), her4 (S') and shroom3 (U' and V') expression in control and NICD primordia. (T') her4 expression is maintained in NICD primordia after Fgfr1 inhibition. (W) shroom3 is upregulated in mib1^ta52b primordia, although Fgf signaling is downregulated. (X–Y''') Constitutive activation of Fgf signaling by heat-shock induction of Fgfr1 does not rescue rosette formation in mib1^ta52b primordia (Y''') suggesting that Fgf signaling is not sufficient for rosette formation in the absence of Notch signaling. (X–Y''') Embryos were heat-shocked at 39°C for 40 min, starting at 33 hpf and fixed 4 hr later. Scale bar in all panels is 25 μm.