The haemangioblast generates haematopoietic cells through a haemogenic endothelium stage

Abstract

It has been proposed that during embryonic development haematopoietic cells arise from a mesodermal progenitor with both endothelial and haematopoietic potential called the haemangioblast. A conflicting theory instead associates the first haematopoietic cells with a phenotypically differentiated endothelial cell that has haematopoietic potential (that is, a haemogenic endothelium). Support for the haemangioblast concept was initially provided by the identification during mouse embryonic stem cell differentiation of a clonal precursor, the blast colony-forming cell (BL-CFC), which gives rise to blast colonies with both endothelial and haematopoietic components. Although recent studies have now provided evidence for the presence of this bipotential precursor in vivo, the precise mechanism for generation of haematopoietic cells from the haemangioblast still remains completely unknown. Here we demonstrate that the haemangioblast generates haematopoietic cells through the formation of a haemogenic endothelium intermediate, providing the first direct link between these two precursor populations. The cell population containing the haemogenic endothelium is transiently generated during BL-CFC development. This cell population is also present in gastrulating mouse embryos and generates haematopoietic cells on further culture. At the molecular level, we demonstrate that the transcription factor Tal1 (also known as Scl; ref. 10) is indispensable for the establishment of this haemogenic endothelium population whereas the core binding factor Runx1 (also known as AML1; ref. 11) is critical for generation of definitive haematopoietic cells from haemogenic endothelium. Together our results merge the two a priori conflicting theories on the origin of haematopoietic development into a single linear developmental process.

Figure 1. Analysis of blast colony development

a) Phase contrast time-lapse pictures of blast colony development. b) FACS analysis of CD41 and Tie2 expression during blast colony development between day 1 and 4. c) CD45 expression of CD41⁺ cells at day 4 of blast colony culture. Line represents isotype control. d) FACS analysis of c-Kit and Tie2 expression during blast colony development. e) Tie2 and c-Kit expression of CD41⁺ (left) and CD41^- (right) cells on day 2 of blast culture. Rectangles indicate Tie2^hic-Kit⁺ population. Numbers represent percentages of respective populations.

Figure 2. Tie2^hic-Kit⁺CD41^- cells can generate haematopoietic progenitors

a) FACS analyses of CD31, Flk-1 and MECA32 expression by Tie2^hic-Kit⁺CD41^- cells. b) Generation of endothelial networks in matrigel by isolated day 2 Tie2^hi c-Kit⁺ CD41^- cells. c) Tie2^hic-Kit⁺CD41^- cells at day 2 of blast development were sorted and cultured. FACS analysis of CD41 and Tie2 expression at T₀ and T₀ + 2 days. d) FACS analysis for the presence of Tie2⁺c-Kit⁺CD41^- cells in gastrulating embryos. e) Immunostaining of gastrulating embryos for Tie2/c-Kit and (f) c-Kit/CD41. Amniotic cavity (ac), decidua (de), amnion (am), allantois (al), exocoelomic cavity (ecc), chorion (ch), and yolk sac blood islands (bi) are indicated. g) FACS analysis of CD45 and c-Kit expression (top) and May-Grunwald Giemsa staining (bottom) of cells generated by isolated E7.75 mouse embryos Tie2⁺c-Kit⁺CD41^- cells co-cultured with OP9 cells. Macrophage (arrow) and mast cells (*) are indicated. Numbers indicate percentages of respective populations.

Figure 3. Runx1 requirement in blast colony development

a) Phase contrast time-lapse photographs of Runx1^-/- blast colony development. b) FACS analysis of CD41 and c-Kit expression during Runx1^-/- blast colony development between day 1 and 4. c) FACS analysis of Tie2 and c-Kit expression during Runx1^+/+ (top) and Runx1^-/- (bottom) cells blast colony development. d) FACS analysis of Tie2/CD41 expression after 2 days of culture of iRunx1 Runx1^-/- Tie2^hic-Kit⁺CD41^- cells in absence or presence of doxycycline (dox) (0.1 μg/ml). e) Dil-Ac-LDL and CD41 expression analysis of iRunx1 Runx1^-/- Tie2^hic-Kit⁺CD41^- cells were evaluated in absence (left) and presence of doxycycline (right). Numbers indicate the percentage of the respective populations. f) Numbers of definitive haematopoietic colonies generated in methylcellulose by iRunx1 Runx1^-/- cells harvested after 2 days of culture with or without doxycycline. Error bars indicate standard deviation of the mean (n=3).

Figure 4. SCL requirement during blast colony development

a) Phase contrast pictures of day 3 blast colony culture from Scl^+/+ and Scl^-/- ES cells. b) FACS analysis of CD41 and c-Kit expression during Scl^-/- blast colony development. Numbers indicate the percentage of the respective populations. c) FACS analysis of Tie2 and c-Kit expression during Scl^-/- blast colony development. d) Model of haemangioblast differentiation toward haematopoiesis in the yolk sac.