Fig. S2

Fgf signalling affects the relative size of the haemangioblast and cardiac populations during a limited time window. (A) SU5402 inhibition performed at the 5-somite stage, after specification of cardiac and blood/endothelial cells, can no longer affect their identity. No difference in expression of the cardiac marker nkx2.5 or the haemangioblast genes pu.1, scl and etsrp was observed at the 10-somite stage, assayed by in situ hybridization, compared with DMSO siblings. Embryos are flat mounted, anterior to the top. Double staining for krox20 (red) provided a landmark. Note that rhombomere 5 expression is no longer reduced, in accordance with observations by Walshe et al. (Walshe et al., 2002). myoD (blue in nkx2.5-, pu.1- and scl-stained embryos, red in etsrp-stained embryos) was used for staging. (B) The overall heart size was reduced on depletion of Fgf signalling from 3 hpf treatment, as seen in cmlc2::GFP transgenic fish at 48 hpf (not shown) but, when Fgf signalling was inhibited from 5S (5 somite stage) onwards, the development of the atrial chamber was no longer affected. A, atrium; V, ventricle. (C,D) nkx2.5 is not essential for anterior lateral plate mesoderm (ALPM) specification. Morpholino (MO) knock-down of nkx2.5 had no effect on cardiac specification (C) or anterior haemangioblast gene expression (D) at the 7-somite stage. nkx2.5-GFP fusion expression was completely abolished at shield stage, confirming MO efficiency. Scale bars: 100μm.