Runx1 is sufficient for blood cell formation from non-hemogenic endothelial cells in vivo only during early embryogenesis

Abstract

Hematopoietic cells differentiate during embryogenesis from a population of endothelial cells called hemogenic endothelium (HE) in a process called the endothelial-to-hematopoietic transition (EHT). The transcription factor Runx1 is required for EHT, but for how long and which endothelial cells are competent to respond to Runx1 are not known. Here, we show that the ability of Runx1 to induce EHT in non-hemogenic endothelial cells depends on the anatomical location of the cell and the developmental age of the conceptus. Ectopic expression of Runx1 in non-hemogenic endothelial cells between embryonic day (E) 7.5 and E8.5 promoted the formation of erythro-myeloid progenitors (EMPs) specifically in the yolk sac, the dorsal aorta and the heart. The increase in EMPs was accompanied by a higher frequency of HE cells able to differentiate into EMPs in vitro Expression of Runx1 just 1 day later (E8.5-E9.5) failed to induce the ectopic formation of EMPs. Therefore, endothelial cells, located in specific sites in the conceptus, have a short developmental window of competency during which they can respond to Runx1 and differentiate into blood cells.

Keywords: Embryo; Hematopoiesis; Hemogenic endothelium; Runx1.

Fig. 1.

Conditional endothelial-specific expression of Runx1 initiated before E9.5 increases phenotypic HSPCs in the yolk sac and embryo. (A) Experimental outline for the initiation of ectopic endothelial-specific expression of Runx1 in utero at embryonic day (E) 7.5, E8.5 or E9.5 by the intraperitoneal (IP) injection of tamoxifen into pregnant mice and the subsequent collection of embryos and yolk sacs for analysis at E10.5. (B) Gross images and confocal z-projections of cR1/cR1 and Cre;cR1/cR1 embryos and yolk sacs. Samples were immunostained for CD31 (i), Runx1 (i,ii) and Kit (i,iii). Arrowheads in the gross images indicate sites of hemorrhaging; arrows in yolk sac images indicate the vitelline artery (VA). Scale bars: 500 µm. (C) Percentage of endothelial cells (CD144⁺ CD31⁺ CD41⁻ CD45⁻ Ter119⁻) expressing Runx1 in the embryo and yolk sac of E10.5 cR1/+ and Cre;cR1/+ littermates after the initiation of Runx1 expression at E7.5, E8.5 or E9.5 (mean±s.d.). Unpaired two-tailed Student's t-test was used for comparison of paired littermates (horizontal lines, two litters per time point). Differences between all samples were determined using one-way ANOVA and Tukey's multiple comparison test (brackets). ***P≤0.001; ****P≤0.0001. (D) Median fluorescence intensity (MFI) of intracellular Runx1 in CD144⁺CD31⁺ cells. cR1=cR/+ and cR1/cR1 combined; all embryos were from dams injected with tamoxifen at E9.5 and harvested at E10.5. Differences between all samples were determined by one-way ANOVA and Tukey's multiple comparison test (brackets). **P≤0.01, ****P≤0.0001. Data from three litters in three separate experiments. (E) Percentage of CD144⁺ Kit⁺ cells in the embryo and yolk sac of E10.5 controls (cR1=cR/+ and cR1/cR1 combined), Cre;cR1/+ and Cre;cR1/cR1 conceptuses after initiation of ectopic Runx1 expression at E7.5, E8.5 or E9.5. Data are from four to six litters per time point (mean±s.d.). One-way ANOVA and Tukey's multiple comparison tests were used. ***P≤0.001, **P≤0.01, *P≤0.05.

Fig. 2.

Conditional endothelial-specific expression of Runx1 initiated at E7.5 increases phenotypic HSPCs in the heart and dorsal aspect of the dorsal aorta (DA). (A) Confocal z-projections of DAs with z-intervals of 2 µm from E10.5 embryos immunostained for CD31 (i), Runx1 (i,ii) and Kit (i,iii). Dotted lines demarcate the ventral (V, right) and dorsal (D, left) sides of the DA used for quantification of data in B. Scale bars: 100 µm. (B) Quantification of hematopoietic cluster cells (CD31⁺ Runx1⁺ Kit⁺) in the dorsal and ventral aspects of the dorsal aortas of E10.5 embryos (mean±s.d.). One-way ANOVA and Tukey's, **P≤0.01, *P≤0.05. (C) Confocal z-projections of hearts in E10.5 embryos immunostained for CD31(i), Runx1 (i,ii) and Kit (i,iii). Arrows indicate ectopic angiogenic sprouts; arrowheads indicate ectopic CD31⁺Runx1⁺Kit⁺ HSPCs in the atrium (A) and ventricle (V). Scale bars: 100 µm. (D) Quantification of the number of erythro-myeloid progenitors (EMPs) per heart (including atrium, ventricles and outflow tract) of E10.5 embryos (mean±s.d.). Data from 6 litters, one-way ANOVA and Tukey's multiple comparison test, ***P≤0.001.

Fig. 3.

Conditional endothelial-specific expression of Runx1 at E7.5, but not later, increases the total number of EMPs but not lymphoid progenitors. (A) Experimental outline for the quantification of EMPs in the yolk sac and embryo of E10.5 conceptuses after the initiation of ectopic Runx1 expression at E7.5, E8.5 or E9.5. (B) Total number of EMPs in the yolk sac and embryo of E10.5 conceptuses (mean±s.d.). Data are from 10, six and seven litters in which Runx1 was induced at E7.5, E8.5 and E9.5, respectively. One-way ANOVA and Tukey's multiple comparison tests, ****P≤0.0001, ***P≤0.001, **P≤0.01, *P≤0.05. (C) Experimental outline for the quantification of lymphoid progenitors. (D) Representative scatter plots of Kit⁺ cells sorted for the limiting dilution analyses of lymphoid progenitors. (E) Total number of lymphoid progenitors within the Kit⁺ population sorted from E10.5 embryos and yolk sacs after the initiation of Runx1 expression at E7.5 (mean±s.d.). Data from two independent experiments of pooled litters. Each dilution had 10 replicates per experiment. Unpaired two-tailed Student's t-test. B progenitors, P≤0.1482; T progenitors, P≤0.6632.

Fig. 4.

Runx1 expression is sufficient to specify embryonic endothelial cells as hemogenic. (A) Illustration of a limiting dilution HE assay. (B) Representative scatter plot for isolation of Kit⁺ and Kit⁻ endothelial cells (CD41⁻ CD45⁻ Ter119⁻ CD31⁺ CD144⁺) from E8.5 embryos and yolk sacs. (C) Frequency of HE cells in the Kit⁺ and Kit⁻ populations of CD41⁻CD45⁻Ter119⁻CD31⁺CD144⁺ endothelial cells (±95% CI). Frequencies are indicated above the floating bars. Data represent four biological replicates for the cR1/+ Kit⁺ yolk sac endothelial population and the cR1/+ Kit⁺ embryonic endothelial population; three biological replicates for the Cre;cR1/+ Kit⁺ yolk sac endothelial population, the cR1/+ Kit⁻ yolk sac endothelial population, the Cre;cR1/+ Kit⁺ embryonic endothelial population and the cR1/+ Kit⁻ embryonic endothelial population; and two biological replicates for the Cre;cR1/+ Kit⁻ yolk sac endothelial population and the Cre;cR1/+ Kit⁻ embryonic endothelial population. Each replicate consisted of pooled cells from superovulated litters of E8.5 conceptuses collected in independent experiments. ***P≤0.001.