Fig. 2

Zebrafish gata2b is expressed in hemogenic endothelium. (A) Expression of gata2b in whole embryos over time. cDNA isolated from 20-30 pooled embryos per timepoint. Error bars indicate s.e.m. of three biological samples. (B-G) Whole-mount in situ hybridization (WISH) of gata2b expression at the indicated stages. Yellow arrowheads indicate expression in the vascular cord region (C) and dorsal aorta (DA) (D,E). Blue arrowheads indicate branchiomotor neurons (D-F). Pink arrowheads indicate expression in the caudal hematopoietic tissue (CHT) (F,G). (H) qPCR of gata2b expression in GFP⁺ cells isolated from fli1a:GFP embryos by FACS. n.d., not detected. GFP⁺ cells sorted from 20-30 pooled embryos per timepoint. Error bars indicate s.e.m. of three technical replicates. (I) Transverse trunk sections showing WISH for gata2b at 18hpf. Top, rare cell detected outside the midline (asterisk). Bottom, representative section with gata2b⁺ cell localized to the midline. (J-M) Transverse sections showing WISH for gata2b (J), runx1 (K), kdrl (L) and gata2a (M) at 25hpf. Pink dashed ovals indicate the DA; orange dashed ovals indicate the posterior cardinal vein (PCV). Yellow arrowhead indicates a primitive erythrocyte. (N) Quantitation of gata2a and gata2b transcripts in kdrl⁺ cmyb and kdrl⁺ cmyb⁺ cells from 36hpf and 48hpf kdrl:mCherry^mem;cmyb:GFP embryos. Cells isolated from 50-100 pooled embryos per sample. Two biological experiments were performed for each timepoint, with one representative shown. Error bars indicate s.e.m. of three technical replicates for each representative experiment.