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Fig. S4 Pu1 is required for myeloid differentiation but not for anterior haemangioblast formation. (A) Recovery of the head endothelial programme is seen in gata5 and gata6 morphants. fli1 expression appears normal at 22 and 30 hpf. (B,C) To assess the requirement for pu1 in the ALM, embryos were injected with pu1 morpholino (mo) and gene expression was analysed at 5 and 10 somites. (B) The loss of pu1 expression itself in the ALM, and the PLM (data not shown), confirmed pu1 mo efficiency (red arrows). gata2, gata4, gata5 and gata6 were all expressed as in the wild-type embryos in the ALM, as was draculin. Ectopic expression of gata1 was not observed at 5 somites in pu1 morphants. (C) Expression of the haemangioblast-associated genes scl, fli1, lmo2 and hhex was unchanged at 10 somites in pu1 morphants, demonstrating that pu1 is not required for anterior haemangioblast formation. However, cmyb, runx1 and ikaros were specifically lost in the ALM of embryos depleted of pu1 (red arrowheads), confirming a requirement for pu1 in establishing the myeloid programme. Anterior-dorsal views. For each gene analysed, n=43-97, the images shown represent the findings for >95% of the embryos.