In situ hybridization of E8.25-E10.5 embryonic tissues with HoxA3 and Runx1 probes. (A-B) HoxA3 is expressed in aortic endothelial cells, while Runx1 is not. Note that Runx1 is expressed in the yolk sac (black arrowheads) while HoxA3 is not. At E8.5, HoxA3 expression (C) begins to decline in aortic endothelium, while Runx1 (D) is still not detected. Note that the omphalomesenteric artery, oa, is negative for HoxA3 at this time but positive for Runx1. (E-H) In situ hybridization of dissected E9.5 and E10.5 AGMs (shown in left panels). At E9.5, HoxA3 (E) expression is barely detectable in aortic endothelial cells, while Runx1 (F) expression is now observed. At E10.5, HoxA3 (G) expression is completely extinguished in aortic endothelial cells. In contrast, Runx1 (H) expression has increased. a, aorta; g, gut tube; nt, neural tube; oa, omphalomesenteric artery. Stippled lines in E-H outline aorta. Scale bar = 50 μm for lower magnifications and AGM explants, = 10 μm for higher magnification panels.

(A) Representative flow cytometric profiles of EBs at day 6 without doxycycline (No Dox) or with 1 μg/mL doxycycline (+Dox) to induce HoxA3 expression from day 4 to day 6. VE-cadherin (VE-cad)/Flk-1 antibody staining or c-Kit/CD41 and c-Kit/CD45 staining were performed to identify vascular and hematopoietic progenitor populations. (B) Frequencies of cells expressing endothelial surface markers (Flk-1⁺/VE-cadherin⁺, F/V), hematopoietic markers CD41+ and CD45+ cells during EB differentiation in 7 independent experiments (for CD41 p=0.0004 and for CD45, p=0.0031). (C) 50,000 cells from day 6 EBs (induced with 1 μg/mL dox to express HoxA3 continually from EB day 4-6 or not) were plated in methylcellulose with hematopoietic cytokines. n=3. Black bar: no dox treatment, gray bar: dox treatment. Colonies: GEMM (granulocyte/erythrocyte/macrophage/megakaryocyte) GM (granulocyte/macrophage) M (macrophage only) Ery-D (definitive erythroid) p=0.032, Ery-P (primitive erythroid) p=0.0002 Ery-Meg (erythrocyte-megakaryocyte) p=0.0009. (D) Brightfield and fluorescence images showing both endothelial (+Dox) and hematopoietic colonies (No Dox or Dox removal) derived from Flk1⁺/VE-cadherin⁺ (F/V) endothelial progenitors from day 6 EBs. Immunofluorescence for VE-cadherin is shown in adherent cells growing in the presence of doxycycline. Bar 100 μm. (E) Equivalent analysis of cultures derived from day 6 EB c-Kit⁺/CD41⁺ (K/41) hematopoietic progenitors. (F) Representative flow cytometric profile of 100,000 Flk-1/VE-cadherin double positive cells or (G) c-Kit/CD41 double positive cells from day 6 uninduced EBs (left), cultured on OP9 for 5 days, in the presence or absence of 1 μg/mL doxycycline. Dox-induced cells were cultured for an additional 4 days in the absence of dox to test the effect of HoxA3 down-regulation. Hematopoietic surface markers, c-Kit, CD41 and CD45 and endothelial markers Flk-1 and VE-cadherin are plotted. (H) AGM tissue dissected from E10.5 embryos, dissociated and transduced with control ires-GFP or HoxA3-ires-GFP retrovirus, cultured on OP9 for 5 days. Bright field images are shown at left, GFP at right. Both hematopoietic and endothelial colonies that acquired GFP were observed with the control, but GFP segregated with endothelial colonies in the HoxA3-ires-GFP transduced sample, indicating skewing of differentiation towards endothelial by HoxA3. Bar 100 μm. (I) Representative flow cytometric profile of AGM cells co-cultured on OP9, and statistical analysis of 5 independent experiments (histogram CD41 p=0.053 CD45 p=0.02).

(A) Venn diagram of regulated genes in endothelial (F/V) and hematopoietic (K/41) progenitor cells. Arrow up upregulated genes, arrow down downregulated genes. (B) Clustering of genes upregulated in the c-Kit CD41 double-positive cells upon HoxA3 induction, during EB differentiation in the hematopoietic c-Kit CD41 double positive cells, based on their expression levels in the Flk-1 VE-cadherin double positive cells (F/V) and in the c-Kit CD41 double positive cells (K/41) of uninduced EBs. (C) Clustering of genes downregulated genes in the Flk-1 VE-cadherin double-positive population upon 6-hour HoxA3 induction, in day 6 EB cells, based on their baseline (control) levels in the Flk-1 VE-cadherin double-positive population (F/V). (D) Real time RTPCR measurements of gene expression changes following HoxA3 induction in sorted endothelial (F/V black bars) or hematopoietic (K/41 grey bars) progenitors. n=5 independent experiments.

(A) Bright field (left) and fluorescent (right) images of HoxA3-induced day 6 EB-derived endothelial progenitors (F/V cells) transduced with control GFP vector or Runx1B-iresGFP retroviral vector and cultured on OP9 for 5 days. Scale bar =100 μm. (B) Representative flow cytometric profiles of F/V cells expressing HoxA3 and transduced with retroviral vectors expressing: ires-GFP (HoxA3), Ikaros (HoxA3+Ikaros), PU.1 (HoxA3+PU.1), Runx1-B (HoxA3+Runx1B), Gata1 (HoxA3+Gata1), Gfi1B (HoxA3+Gfi1B). GFP+ gated events are shown. X axis CD41, Y axis c-Kit. (C) Expression levels of hematopoietic marker genes (fold of Gapdh), without HoxA3 induction (No Dox, black bars) or with HoxA3 induction (+Dox grey bars) for control or Runx1B-transduced cells on OP9. n=3 independent experiments. Gfi1B: p=0.0003, Ikaros: p=0.0073, Phemx: p<0.0001, PU.1: p=0.0009. (D) Genes upregulated by HoxA3 clustered according to their expression in control uninduced, HoxA3+Runx1iresGFP, HoxA3+Gata1iresGFP, and HoxA3+GFP sorted cells. Both Runx1 and Gata1 significantly reverse these HoxA3-induced changes, indicated by their clustering together and near to the non-HoxA3 expressing control. Examples are shown on the heat map below. (E) Genes downregulated by HoxA3. Runx1 reverses downregulation of HoxA3-regulated changes more effectively than Gata1, indicated by its clustering closest to control. Among these genes are critical endothelial regulatory factors, and genes involved in adhesion and cell polarity.

(A) In situ hybridization showing Runx1 expression in HoxA3 +/+, +/− and −/− E8.5 embryos. Runx1 expression is absent in the dorsal aortae of HoxA3 +/+ (i) and HoxA3+/− (ii) embryos, but robustly expressed in yolk sac. Runx1 is ectopically expressed in the dorsal aortae of HoxA3^−/− (iii) embryos. Stippled red lines outline dorsal aortae. Penetrance of this phenotype is indicated at lower left. (i′-iii′) Sections of the embryos shown above. Both endothelial and hematopoietic cells are negative for Runx1 in wild-type or heterozygous embryos, while Runx1-expressing cells are found in HoxA3^−/− embryos. Arrows indicate Runx1-expressing endothelial cells. a, aorta; ys, yolk sac. Scale bar = 50 μm for whole mounts, 10 μm for sections. (B) Model for regulation of endothelial hemogenesis by HoxA3 and Runx1. HoxA3 represses a cascade of transcription factors that promote hemogenesis and induces a set of genes that maintain endothelial character. Runx1 is a positive regulator of most of these transcription factors, and a negative regulator of genes essential for endothelial character, thus transient expression of Runx1 erases the endothelial program and initiates the hematopoietic.