Etsrp is necessary and sufficient for the formation of myeloid cells. (A-F) Knockdown of Etsrp results in the nearly complete absence of myeloid cells as analyzed by in situ hybridization. Anterior is to the left. (A,C,E) Control uninjected embryos. (B,D,F) Etsrp morphants, injected with 12 ng to 15 ng of etsrp MO1 and etsrp MO2 in a 1:1 mixture. (A,B) L-plastin (lcp1) expression at 24 hours postfertilization (hpf). Note that lcp1-expressing macrophages are nearly completely absent in etsrp morphants (B). (C,D) mpx expression at 24 hpf. Note that mpx-expressing neutrophils are nearly completely absent in etsrp morphants (D). (E,F) pu.1 expression at the 16-somite stage. Embryos have been flat-mounted with the yolk removed. Note that the anterior myeloid-specific pu.1 expression is severely reduced in panel F, whereas posterior erythroid-specific expression is not significantly affected. (G-J) Etsrp RNA overexpression induces ectopic myeloid cell formation. (G,I) Control uninjected embryos. (H,J) etsrp RNA-overexpressing embryos. (G,H) pu.1 expression at the 16-somite stage, anterior view (G), ventro-lateral view (H). Note the strong expansion of pu.1-expressing cells, some of which are located ectopically (H, ). (I,J) lcp1 expression at 24 hpf. Note the increase in the number of lcp1-expressing macrophages in panel J. (K-N) Etsrp RNA with missing MO-binding sites can restore pu.1 expression in etsrp morphants. Embryos are at the 8-somite stage; (K,L) anterior view; (M,N) anterior-ventral view. (K) Control uninjected embryo; (L) 10 ng etsrp MO2-injected embryo; (M) 100 pg etsrp RNA-injected embryo; (N) embryo coinjected with 10 ng etsrp MO2 and 100 pg etsrp RNA. Images were taken using Axioskop2 and 10×/0.30 NA dry objective (Zeiss) (A-F; I,J) or CV11 stereomicroscope (Zeiss) (G,H,K-N), Axiocam color camera (Zeiss, model 412-312) and Openlab 4.0 software (Improvision). Magnification: 100× (A-D); 60× (E-N).