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Fig. 4 Endoderm-derived cxcl12b is required for LDA formation. (A,B) In situ hybridization at 24 hpf showing cxcl12b expression in tissue underlying the LDA (arrows). (B) Loss of endoderm in sox32 morpholino-injected embryos leads to severe reduction in cxcl12b expression. Dorsal view, anterior is to the left. (C–E) Confocal micrographs of 22-hpf embryo subjected to double-fluorescent whole-mount in situ hybridization, lateral view, anterior is to the left, dorsal is up. (C) fli1a expression, marking the LDA. (D) cxcl12b expression. (E) Overlay; fli1a is red, cxcl12b is green. (F) Influence of loss of endoderm or cxcl12b on circulation. (ctl) Control morpholino-injected; (circ+) embryos with circulation; (circ) embryos without circulation. (G–L) Vascular defects in embryos lacking endoderm or cxcl12b. (G,I,K) Bright-field views at 32 hpf, anterior is to the left, dorsal is up. (H,J,L) In situ hybridization for kdrl at 24 hpf. Black arrows indicate anterior LDA, arrowheads indicate posterior LDA. (G,H) Embryos injected with control morpholino. (I,L) Embryos injected with 15 ng of cxcl12b MO. (K,L) Embryos injected with 2 ng of sox32 morpholino. Arrows mark the anterior LDA, arrowheads mark the posterior LDA.