ENH_QPD is a conserved hematopoietic enhancer that acts in synergy with the PLAU and VCL promoters. (A) Views of human ENH_QPD. H3K27ac signal from QPD megakaryocytes (blue, including zoom in view) are compared with published ChIP-seq tracks for hematopoietic transcription factors from cord blood–derived megakaryocytes⁴⁴  (red), expressed as fold change over input. Teal track shows a 46-way vertebrate PhastCons score. (B) Similar views of the orthologous equivalent of ENH_QPD in mouse, showing ChIP-seq tracks for corresponding transcription factors profiled in mouse CD41⁺ hematopoietic precursor cells.⁴⁵  Black squares in panels A and B (bottom) depict ENH_{QPD_CONS}. (C) The Tol2 reporter vector system. (D) An embryo at 24 hpf (top), showing known early-stage zebrafish hematopoietic tissues (adapted from Chen and Zon,⁴⁷  with permission); in situ staining (bottom) for GFP in Tg(ENH_{QPD_CONS}:EGFP). A representative F2 embryo at 24 hpf is shown. Embryos were fixed at 24 hpf in 4% paraformaldehyde and incubated with a digoxigenin-labeled antisense gfp probe. RNA-probe hybrids were detected by an alkaline phosphatase–conjugated antibody (anti-digoxigenin-AP and Fab fragments; 1:5000; Roche, Basel, Switzerland) that catalyzed reaction on a chromogenic substrate (NBT/BCIP; Roche). Stained embryos were cleared in 2:1 benzyl benzoate/benzyl alcohol solution and imaged under an Axio Zoom.V16 Stereoscope (Zeiss, Oberkochen, Germany). PLM, posterior lateral mesoderm; ICM, interior cell mass; PBI, posterior blood island. (E) Confocal microscopy image of the PBI in a representative Tg(gata1:dsRed) x Tg(ENH_{QPD_CONS}:EGFP) double-transgenic embryo. At 24 hpf, embryos were mounted in 1% (w/v) low-melt agarose (Sigma-Aldrich, St. Louis, MO) and imaged under a Nikon A1R Si Point scanning confocal microscope (Nikon, Tokyo, Japan). (F) Relative luciferase activity for minimal (minP), PLAU (pPLAU), or VCL (pVCL) promoter constructs, with or without ENH_{QPD_CONS}, assayed in K562 erythroid leukemia cells. Data points show measured values averaged from 4 technical replicates (separate wells) per construct, for 5 separate transfections. All values were normalized relative to minP in their respective cell type. Statistical analysis was performed using 1-way analysis of variance with the Tukey correction. Asterisks immediately above data points denote significance compared with minP. Error bars show standard deviation of mean. Asterisks denote significance vs minP and other select pairwise comparisons. ***P < .001; n.s., not significant. All constructs and inserted sequences are further described in “Materials and methods” and supplemental Methods.