Runx1 is required for the endothelial to haematopoietic cell transition but not thereafter

Abstract

Haematopoietic stem cells (HSCs) are the founder cells of the adult haematopoietic system, and thus knowledge of the molecular program directing their generation during development is important for regenerative haematopoietic strategies. Runx1 is a pivotal transcription factor required for HSC generation in the vascular regions of the mouse conceptus-the aorta, vitelline and umbilical arteries, yolk sac and placenta. It is thought that HSCs emerge from vascular endothelial cells through the formation of intra-arterial clusters and that Runx1 functions during the transition from 'haemogenic endothelium' to HSCs. Here we show by conditional deletion that Runx1 activity in vascular-endothelial-cadherin-positive endothelial cells is indeed essential for intra-arterial cluster, haematopoietic progenitor and HSC formation in mice. In contrast, Runx1 is not required in cells expressing Vav1, one of the first pan-haematopoietic genes expressed in HSCs. Collectively these data show that Runx1 function is essential in endothelial cells for haematopoietic progenitor and HSC formation from the vasculature, but its requirement ends once or before Vav is expressed.

Figure 1. VEC-Cre excision marks endothelium and blood

a. Schematic diagram of the VEC-Cre transgene. The VEC promoter (VEC-pro) and Cre plus polyA tail (Cre-pA) were flanked by insulator sequences from the chicken γ-globin gene. b. Sagittal section of a 5 s R26R-lacZ;VEC-Cre conceptus near the hindgut(hg). Arrows indicate β-gal⁺ (blue) PECAM1⁺ (brown) vitelline vasculature. ys, yolk sac; bi, blood island. The blood island contains β-gal⁺ primitive erythrocytes derived from yolk sac mesoderm in which VEC-Cre was active. c. Whole mount conceptuses showing β-gal activity in the embryonic vasculature and placenta (pl). d. VEC-Cre excision in placenta and umbilical artery endothelium. Left panel is from a 9.5 dpc conceptus. Detail of boxed region in placental labyrinth is to the right. β-gal⁺ cells are blue and PECAM1⁺ cells are brown. On the far right is a transverse section through a 10.0 dpc R26R-lacZ;VEC-Cre conceptus with abundant β-gal⁺ endothelial cells in the placenta (pl) and umbilical artery (u). da, dorsal aorta; ys, yolk sac. e. FACS analysis of cells from the AGM region, vitelline and umbilical arteries of 11.5 dpc R26R-YFP;VEC-Cre conceptuses stained for Sca1, VEC, Ter119, and 7-AAD. Gated populations in scatter plot were analyzed for YFP in histograms to the right. Graph shows percent of YFP⁺ cells in each subpopulation, line indicates mean. Non-hematopoietic, non-endothelial cells (Sca1⁺ VEC⁻) are YFP⁻. Means (± SD) for each population are: Sca1⁺ VEC⁻ (0.7 ± 0.2); Sca1⁺ VEC⁺ (86.4 ± 9.9); Sca1⁻VEC⁺ (77.4 ± 11.1). f. On left is a transverse section through the AGM region of a 10.5 dpc conceptus. Detail on right is from boxed region showing β-gal⁺ endothelial cells (e) and hematopoietic clusters (hc) in the dorsal aorta (da). Note that the sub-aortic mesenchyme (m) is β-gal⁻. fl, fetal liver. g. β-gal⁺ cells in the liver of R26R-lacZ;VEC-Cre fetuses (10.5 dpc). h. Percentage of YFP⁺ fetal liver cells of multiple cell surface phenotypes (gated from Ter119⁻ 7-AAD⁻ cells) from 15.5 dpc R26R-YFP;VEC-Cre conceptuses. Mean ± SD for each population: CD45^hi (85.6 ± 6.3); CD45⁻ VEC⁺ (85.7 ± 8.2); CD31^hi VEC⁺ (94.2 ± 2.8); CD34⁺ c-kit⁺ (86.1 ± 6.4); Sca1⁺ Mac1⁺ (89.9 ± 5.1); Gr1⁺ (73.6 ± 10.3); Mac1⁺ (82.3 ± 7.6); B220⁺ (82.4 ± 7.9); CD3/4/8⁺ (89.4 ± 6.4). i. Percentage of YFP⁺ bone marrow cells (gated from Ter119⁻ 7-AAD⁻ cells) in R26R-YFP;VEC-Cre adult mice. CD45^hi (96.1 ± 3.3); LSK (95.5 ± 4.4); CD34⁺ c-kit⁺ (95.2 ± 4.9); CD41⁺ (95.1 ± 4.9); CD31⁺ (95.4 ± 3.5); Gr1⁺ (95.0 ± 3.2); Mac1⁺ (96.5 ± 3.6); B220⁺ (96.0 ± 4.2); CD3/4/8⁺ (88.4 ± 4.1).

Figure 2. Runx1 is required in VEC⁺ cells for hematopoietic progenitor formation

a. Gross appearance of Runx1^f/f;VEC-Cre fetuses. Note the central nervous system hemorrhaging (white asterisks) and pale fetal liver (fl). VEC-Cre deletion of Runx1 resulted in the same phenotype as germline Runx1 deletion, but the penetrance was lower. b. FACS analysis of Ter119⁻ 7-AAD⁻ cells from 11.5 dpc fetal livers. Error bars in plot on right represent 95% confidence intervals. Significant differences from combined +/+, f/+, and f/f samples are indicated with asterisks (*P = 0.05, **P = 0.01; ANOVA and Dunnett’s multiple comparison test). d. CFU-C assays of yolk sac, combined AGM region, umbilical and vitelline arteries (AGM+U+V), placenta, and fetal liver (represented as numbers per embryo equivalent, error bars indicate 95% confidence intervals). Significant differences from Runx1^f/f or Runx1^f/+ conceptuses are indicated with asterisks (**P = 0.01). Data are averaged from 16 litters, with 10 – 31 animals per genotype. d. PCR genotyping of single colonies picked from CFU-C assays. On top is shown locations of the three primers (colored triangles) used to detect the wildtype (+), floxed (f), and deleted (Δ) Runx1 alleles. LoxP sites are open triangles. The combined number of colonies from 11.5 dpc AGM+V+U and fetal livers of each genotype are indicated below the gel. Since Runx1 haploinsufficiency decreases the number of CFU-Cs ¹⁸, the percentage of Runx1^f/Δ colonies may actually underestimate the excision frequency.

Figure 3. Runx1 is required in VEC⁺ cells for HSC emergence

a. Scheme of transplantations and analyses. Donor derived cells were isolated from transplant recipients and plated in methylcellulose assays, and individual colonies were genotyped. b. Engraftment as assessed by FACS on peripheral blood to detect donor derived (Ly5.1⁻/5.2⁺) cells. Numbers indicate successfully reconstituted recipients (>5% donor-derived cells) per number transplanted. Mice were transplanted with one embryo equivalent of 11.5 dpc fetal cells from each site. Control samples are from Runx1^f/f, Runx1^f/+ and Runx1^f/+;VEC-Cre conceptuses. c. PCR genotyping of single colonies picked from CFU-C assays of donor-derived placenta cells sorted from the bone marrow of transplant recipients. The number of colonies of each genotype is indicated below the gel. d. Confocal images of the caudal region of 10.5 dpc embryos (35–36 s) stained with antibodies to PECAM1 (pink) and c-kit (green). Diagram on left illustrates area analyzed (da, dorsal aorta; v, vitelline artery; u, umbilical artery). Arrow indicates a c-kit⁺ cluster in the ventral aspect of the dorsal aorta. On right is a bar graph representing the number of c-kit⁺ cells in the dorsal aorta from three embryos of each genotype. Error bars represent standard deviation.

Figure 4. Runx1 is not required in Vav⁺ cells for CFU-C or HSC emergence

a. Flow cytometric assay of AGM region, vitelline and umbilical arteries (AGM+V+U) and fetal livers from 11.5 dpc R26R-YFP;Vav-Cre conceptuses demonstrating deletion in blood but not in endothelium. Scatter plot shows 7AAD⁻ Ter119⁻ cells gated and analyzed for CD45 and VEC expression. Histograms to the right represent the percent of YFP positive cells (± SEM) in each of the gated populations. The CD45⁺VEC⁺ population from the AGM+V+U was too small to analyze for YFP expression. Graph on far right in top row shows mean ± SEM for AGM+V+U. Activation of the R26R-YFP allele in both fetal liver endothelium (CD45⁻ VEC⁺) and blood (CD45⁺ VEC⁻ and CD45⁺ VEC⁺) with VEC-Cre is shown at the bottom for comparison. b. Dorsal aorta from the AGM region of a 10.5 dpc R26R-lacZ;Vav-Cre conceptus. Arrow points to an intra-aortic cluster, all of which are β-gal⁻. c. Transverse section through the dorsal aorta of an 11.5 dpc R26R-lacZ;Vav-Cre conceptus. Arrows indicate two β-gal⁺ cells in the subaortic mesenchyme. d. Detail of β-gal⁺ cell in the subaortic mesenchyme from boxed region in c. e. β-gal⁺ cells in the fetal liver (11.5 dpc). f. CFU-C assays from 15.5 dpc fetal livers. Asterisks indicate significant differences from f/+ or f/f animals (P = 0.05, ANOVA and Dunnett’s multiple comparison test). Data are compiled from 3–8 conceptuses of each genotype. Error bars represent 95% confidence intervals. g. PCR genotyping of colonies from CFU-C assays. The gel is from 11.5 dpc AGM+V+U colonies. Horizontal lines above lanes are Runx1^f/f;Vav-Cre colonies, and Runx1^f/+;Vav-Cre colonies are labeled f/+. Asterisks indicate colonies in which both Runx1^f alleles (or in the case of Runx1^f/+;Vav-Cre colonies, one Runx1^f allele) were completely deleted. Numbers below the gel represent total colonies from 11.5 dpc tissues (AGM+V+U, yolk sac, placenta, fetal liver). h. Engraftment of 11.5 dpc tissues (1 ee) as assessed by FACS on peripheral blood to detect donor derived (Ly5.1⁻/5.2⁺) cells. Numbers within bars indicate successfully reconstituted recipients/number transplanted.