Mesoderm-derived PDGFRA+ cells regulate the emergence of hematopoietic stem cells in the dorsal aorta

PDGFRA+ stromal cells (PSCs) in the AGM have MSC properties

Although the existence of stromal cells in the AGM that support haematopoiesis is known and AGM-derived stromal cell lines have proven to be a powerful tool for the identification of environmental HSC regulators21,31,33, we lack knowledge of the characteristics of these cells and their influence on EHT. It has previously been reported that all bone-marrow MSCs in Nestin-GFP transgenic mice (where expression of green fluorescent protein (GFP) is regulated by Nestin)34 were GFP+ and that ablation of these bone-marrow MSCs resulted in significant loss of LT-HSCs in 12–16-week-old mice30. We therefore used Nestin-GFP transgenic mice to investigate stromal cell populations in the AGM (Fig. 1a).

Fig. 1: The E11.5 AGM has resident long- and short-term repopulating CFU-Fs that can be discriminated by expression of PDGFRA and Nestin-GFP.figure 1

a, Schematic outline of experiments performed using E11.5 Nestin-GFP+ embryos. b, Confocal image of an E11.5 Nestin-GFP+ dorsal aorta stained for PDGFRA. Nestin-GFP− and Nestin-GFP+ PDGFRA+ cells are indicated with yellow and white arrows, respectively. c, Flow cytometry analysis of E11.5 Nestin-GFP+ AGM (n = 3) showing that 1:5.3 CD31−Nestin-GFP+ stromal cells are also PDGFRA+. The percentages of cells in the different quadrants (delineated in blue) are indicated. (i)–(iv) The PDGFRA+Nestin-GFP+ cells were further fractionated into high and low positive subpopulations. d, CFU-F potential of E11.5 CD31−Nestin-GFP+ AGM (n = 5) cells, sorted based on CD31, PDGFRA and Nestin-GFP expression according to the gating strategy shown in c. e, Long-term growth of E11.5 Nestin-GFP+ AGM-derived CFU-Fs based on CD31, Nestin-GFP and PDGFRA expression. f, Single-cell clonal analysis of CD31−PDGFRA+Nestin-GFP− CFU-Fs. The CFU-F colony numbers are representative of n = 4 (primary plating) and n = 11–15 (secondary–quaternary plating). g, In vitro differentiation of CD31−PDGFRA+Nestin-GFP− cells (n = 3); ac-LDL, acetylated apoprotein low-density lipoprotein. h, Confocal microscopy image of an E11.5 Nestin-GFP+ dorsal aorta showing that a subset of PDGFRB+ cells co-express Nestin-GFP (n = 3). PDGFRB+Nestin-GFP+ cells are stained in yellow; PDGFRB+Nestin-GFP− cells are stained in red. i, (i) CFU-Fs in FAC-sorted fractions from E11.5 Nestin-GFP+ AGMs (n = 4). (ii) Pericyte colony-forming potential in FAC-sorted fractions from E11.5 Nestin-GFP+ AGMs (n = 7). j, Z-stack reconstruction of confocal microscopy images showing vessel-like structures lined by Nestin-GFP+ endothelial cells with surrounding PDGFRB+ pericytes. These images were taken from tissues harvested 3 weeks after subcutaneous transplantation of a Matrigel plug loaded with PDGFRA+Nestin-GFP−CD31−PDGFRB− FAC-sorted cells from E11.5 Nestin-GFP+ AGMs (see red arrows in i(i) and (ii)); CD31 staining is shown in white. df,i, Data represent the mean ± s.d. d,i, Data were derived from n = 3 biologically independent experiments. d,e,i, A random-effects Poisson regression was used to compare colony counts (d,i) and a linear mixed model was used to compare the growth curves (e); **P < 0.01, ***P < 0.005. Ao, aortic lumen; DAPI, 4′,6-diamidino-2-phenylindole dihydrochloride. Colony sizes: micro colonies, <2 mm, 2–24 cells; small colonies, 2–4 mm, >25 cells; and large colonies, >4 mm, >100 cells. Precise P values are provided in the source data.

Source data

Confocal imaging of the E11.5 AGM of these mice showed that aortic endothelial and sub-endothelial blood cells as well as blood cells adjacent to the aortic endothelium were Nestin-GFP+ (Extended Data Fig. 1a). Both Nestin-GFP+ and Nestin-GFP− stromal cell fractions in the E11.5 AGM were found to express platelet-derived growth factor receptor alpha (PDGFRA), a tyrosine kinase receptor expressed on the surface of MSCs35 (Fig. 1b,c) and on early embryonic mesodermal cells that contribute to haemogenic endothelium and haematopoietic cells36. These PDGFRA+ cells (Nestin-GFP−, yellow arrows; and Nestin-GFP+, white arrows) were distributed deeper in the aortic parenchyma and surrounded the PDGFRA−Nestin-GFP+ cells, which were more concentrated towards the aortic lumen (Fig. 1b).

To explore the transitional functional properties of PDGFRA and Nestin-GFP-expressing and non-expressing cells in the AGM, we used an in vitro colony-forming unit–fibroblast (CFU-F) assay37. We first assessed the CFU-F potential in E9.5–E13.5 AGMs; we noted that colonies were composed of cells of mesenchymal cell morphology and varied in size38 and that their numbers peaked at E11.5 (Extended Data Fig. 1b(i),(ii)). Freshly isolated fluorescence-activated-cell (FAC)-sorted PDGFRA+Nestin-GFP− and PDGFRA+Nestin-GFP+ populations also produced CFU-Fs of different sizes (Fig. 1d). Although both PDGFRA+Nestin-GFP− and PDGFRA+Nestin-GFP+ cells produced large CFU-F colonies (Fig. 1d), their number was lower in the latter and proportionate to the intensity of PDGFRA and Nestin-GFP expression. Only PDGFRA+Nestin-GFP− cells showed long-term replating capacity (Fig. 1e).

Furthermore, serial replating of single cells from PDGFRA+Nestin-GFP− large CFU-F colonies produced consistent numbers of large colonies of CFU-Fs (Fig. 1f), and these cells could be differentiated in vitro into mesodermal and ectodermal derivatives (Fig. 1g(i)–(viii) and Supplementary Video 1). By contrast, single cells from PDGFRA+Nestin-GFP+ large CFU-F colonies showed limited capacity to generate large colonies of CFU-Fs (Extended Data Fig. 1c) and could only be differentiated into adipocytes, endothelium and smooth muscle (Extended Data Fig. 1d). The differentiation potential observed in bulk PDGFRA+Nestin-GFP− and PDGFRA+Nestin-GFP+ cells was best reflected in cells that formed large CFU-F colonies (Extended Data Fig. 2a,b). Together, these data show that CFU-F potential in the E11.5 AGM resides largely in PDGFRA+ cells and that Nestin expression marks a subpopulation of PDGFRA+ cells with more restricted CFU-F and differentiation potential.

Pericytes are characterized by the expression of platelet-derived growth factor receptor beta (PDGFRB)39,40 and were distributed concentrically in the sub-endothelium of the E11.5 dorsal aorta (Fig. 1h). To investigate the relationship between Nestin-GFP+ cells and pericytes, we fractionated cell populations (FAC-sorted) from E11.5 AGMs of Nestin-GFP transgenic mice based on PDGFRA, Nestin-GFP, CD31 and PDGFRB expression (Extended Data Fig. 2c) and performed assays for formation of CFU-F and pericyte colonies as well as a long-term replating assay (Fig. 1i(i),(ii) and Extended Data Fig. 2d). Among the CD31−PDGFRA+ cells, PDGFRB co-expression was proportionately higher in the Nestin-GFP+ subpopulation than the Nestin-GFP− cells (Extended Data Fig. 2c). Although the latter showed the highest large-CFU-F colony and long-term replating potential, unlike the former cells, they lacked potential to form pericyte colonies (Fig. 1i(i),(ii) and Extended Data Fig. 2d). Interestingly, CFU-F potential in the Nestin-GFP+ fraction was exclusively within the PDGFRB+ subfraction (Fig. 1i(i)). We further assessed the contribution of different PDGFRA+ fractions (Fig. 1i) towards in vivo morphological and functional vascular contents. We purified PDGFRA+ fractions using flow cytometry, mixed those cells with Matrigel and transplanted them subcutaneously into C57BL/6 mice. Only purified CD31−PDGFRA+Nestin-GFP−PDGFRB− CFU-Fs formed vessel-like structures (Fig. 1j, Extended Data Fig. 2e and Supplementary Video 2), the luminal surfaces of which were lined with Nestin-GFP+CD31+ endothelial cells and enveloped by PDGFRB+ pericytes.

PSCs contribute to haemogenic endothelium and HSCs

If PSCs are a reservoir for endothelial and sub-endothelial cells in the developing aorta, they could also contribute to long-term repopulating HSCs that emerge at E11.5. PDGFRA+ cells, when labelled at E7.5–E8, have previously been shown to contribute to blood cells budding from the endothelial lining of the dorsal aorta in E10.5 AGM as well as B, T and Lin−Kit+Sca-1+ (LSK) cells in the bone marrow of adult mice36. To explore whether cells expressing PDGFRA and CD31 proteins in the AGM were early and late constituents of a differentiation continuum, we evaluated the distribution of CD31 in the E8.5, E9.5, E10.5 and E11.5 AGMs of Pdgfra–nGFP knock-in mice (that is, mice whose Pdgfra-expressing cells retain GFP in their nuclei41; Fig. 2a(i)). Pdgfra–nGFP cells are in proximity with endothelial cells of the paired dorsal aorta at early embryonic time points (Fig. 2a(ii)). At E11.5 (Fig. 2a(iii)), cells furthest from the aortic lumen showed robust Pdgfra–nGFPhigh expression but no NESTIN (NES) protein (layer I). Cells co-expressing both Pdgfra–nGFPhigh and NES (layer II) were interspersed between these cells (layer I) and cells that were Pdgfra–nGFPlow but NES+ (layer III), which also co-expressed the smooth-muscle marker aSMA (Extended Data Fig. 3a). Endothelial cells lining the aortic lumen (layer V) were Pdgfra–nGFP− and expressed CD31 but little or no NES (in contrast to the longer-lasting GFP in nGFP transgenic mice; Fig. 1a). A few cells were NES+ and CD31+ but low in Pdgfra–nGFP (layer IV). It is salient that Pdgfra–nGFP+ and PDGFRA+ cells in the E11.5 AGM were comparable in their CFU-F potential and long-term growth potential (Extended Data Fig. 3b). When GFP+CD31−CD45−PDGFRA+ cells were harvested from the E9.5 AGM of ubiquitous GFP mice and cultured on OP9 cells ex vivo, they contributed robustly to GFP+CD31+CD45+ cells (Extended Data Fig. 3c).

Fig. 2: E9.5 PDGFRA+ cells contribute to haemogenic endothelium and LT-HSCs.figure 2

a, (i) Schematic outline of experiments performed using E8.5, E9.5, E10.5 and E11.5 Pdgfra–nGFP embryos. (ii) Confocal microscopy images of E.8.5, E9.5 and E10.5 Pdgfra–nGFP embryos showing the distribution of PDGFRA-expressing cells in relation to the developing aorta. (iii) Spatial distribution of Pdgfra–nGFP-, NESTIN- and CD31-expressing cells in a Pdgfra–nGFP E11.5 AGM. The region outlined in white in the main image (left) has been magnified (right) and different cell populations are labelled: I, Pdgfra–nGFPhighNESTIN−CD31−; II, Pdgfra–nGFPhighNESTIN+CD31−; III, Pdgfra–nGFPlowNESTIN+CD31−; IV, Pdgfra–nGFPlowNESTIN+CD31+; V, Pdgfra–nGFP−NESTIN−CD31+. b, (i) Schematic outline of lineage-tracing experiments using Pdgfra–creERT2; R26R–eYFP embryos. (ii) Confocal image of an E11.5 Pdgfra–creERT2; R26R–eYFP AGM following cre activation at E9.5 showing eYFP+ blood cells (white arrows) and endothelium (orange arrows). (iii) Contribution of donor eYFP+ cells to PDGFRA+ cells, pericytes (PDGFRB+), endothelium (CD31+) and blood cells (CD45+) in the E11.5 Pdgfra–creERT2; R26R–eYFP AGM following cre activation at E9.5. Pdgfra-eYFP; PDGFRA cells in the top left panel are boxed in pink, and the adjacent flow cytometry plot to the right (boxed in pink) shows corresponding CD45; CD31 expression. High and low Pdgfra-eYFP expressing cells in the flow cytometry plots to the left in each of the four panels in (iii) are boxed in red and green, respectively. Correspondingly coloured boxes to the right in each of the four panels show expanded phenotypic profiles for these cells. c, (i) Schematic outline of lineage-tracing experiments using Pdgfra–creERT2; R26R–eYFP embryos; e.e., embryonic equivalent. (ii) Contribution of donor eYFP+ cells to peripheral blood in primary and secondary transplants at 4 months post transplantation (n = 5). The arrow indicates the sample for which the expanded flow cytometry profiles are shown in (iii). (iii) Flow cytometry analysis of the contribution of donor eYFP+ cells to peripheral blood in primary transplant. d, (i) Schematic outline of lineage-tracing experiments using Pdgfra–creERT2; R26R–eYFP embryos. (ii) Confocal image of a Pdgfra–creERT2; R26R–eYFP neonatal long-bone section following cre activation at E9.5, showing eYFP+CD45+ blood cells in the bone marrow. (iii) Contribution of donor eYFP+ cells to peripheral blood (PB), bone marrow (BM), thymus and spleen in primary (6 months post transplantation; left) and secondary (4 months post tranplantation; right) transplants (n = 5). Ao, aortic lumen; NT, neural tube; DAPI, 4′,6-diamidino-2-phenylindole dihydrochloride; FSC-A, forward scatter area. The percentage of cells in the different quadrants in the flow cytometry plots are indicated. Data were derived from biologically independent samples, animals and experiments (n = 5). Data represent the mean ± s.d.

Source data

To formally establish a lineage relationship between PDGFRA+ cells at E9.5 and their progeny, we crossed Pdgfra–creERT242 mice with R26R–enhanced yellow fluorescent protein (eYFP)43 mice to generate Pdgfra–creERT2; R26R–eYFP compound transgenic embryos (Fig. 2b(i)) and induced cre recombination at E9.5 by delivering single injections of tamoxifen to pregnant mothers and harvesting embryos at E11.5. CD31+ endothelial cells in the E9.5 AGM do not express Pdgfra (Extended Data Fig. 3d(i),(ii))44. There was sufficient recombination with 6.4% of limb bud cells expressing eYFP following a single injection of tamoxifen at E9.5 (Extended Data Fig. 3e). Bearing in mind that PDGFRA+ cells labelled at E7.5 and E8 also contribute to the endothelium of the dorsal aorta at E10.5 (ref. 36), Pdgfra–creERT2; R26R–eYFP recombination at E9.5 also resulted in eYFP+ aortic endothelial, sub-endothelial and blood cells in the E11.5 AGM (Fig. 2b(ii),(iii)), marking approximately a third of all CD31+CD45+ cells (Extended Data Fig. 3f(i),(ii)). Only a minority of eYFP+ cells still expressed PDGFRA protein (Fig. 2b(iii) and Extended Data Fig. 3g). The eYFP+PDGFRA+ cells were CD31− and CD45− and had lower eYFP fluorescence than CD31+ or CD45+ cells (Fig. 2b). There were no eYFP+CD31+ endothelial cells in the E11.5 yolk sac, placenta or umbilical and vitelline vessels (Extended Data Fig. 3h).

To evaluate whether these eYFP+ cells included LT-HSCs, we again induced cre recombination in Pdgfra–creERT2; R26R–eYFP compound transgenic embryos at E9.5, harvested E11.5 embryos and performed transplantation assays with eYFP+ AGM cells (Fig. 2c(i)). These cells were able to reconstitute haematopoiesis in lethally irradiated mice following primary and secondary transplantation and contributed to multiple blood lineages (Fig. 2c(ii),(iii)). To establish whether Pdgfra–eYFP+ cells populate the bone marrow, Pdgfra–creERT2; R26R–eYFP compound transgenic embryos were matured to term following induction of recombination at E9.5 and delivered by caesarean section (owing to difficulties in parturition; Fig. 2d(i)). eYFP+CD45+ blood cells were present in the bone marrow of the Pdgfra–creERT2; R26R–eYFP compound neonatal mice (Fig. 2d(ii)). These cells were able to reconstitute haematopoiesis in lethally irradiated mice following primary and secondary transplantation (Fig. 2d(iii)) and contributed to multiple blood lineages (Extended Data Fig. 3i,j).

Given the contributions of E9.5 PDGFRA+ cells to structures of the aorta and blood cells that arise therein at E11.5, we predicted that ablation of these cells and their progeny would have a profoundly deleterious impact on the developing aorta and haematopoiesis. To explore this, we crossed Pdgfra–creERT2 mice42 with inducible diphtheria toxin receptor (iDTR) mice45 to generate Pdgfra–creERT2; iDTR embryos (Extended Data Fig. 4a). We conditionally induced expression of diphtheria toxin receptor in E9.5 PDGFRA+ cells through treatment with tamoxifen, followed by ablation of these cells using diphtheria toxin at E10.5 in Pdgfra–creERT2; iDTR embryos (Extended Data Fig. 4a(i)). We then studied the resulting impact on the AGM architecture at E11.5. In whole-mount and tissue sections of compound transgenic embryos, there was severe disruption of normal dorsal aorta development (Extended Data Fig. 4a(ii)). In these embryos, there was concomitant reduction in the number of various cell types: endothelial (CD31+), blood (SCA1+CD45+), perivascular (PDGFRB+) and CFU-Fs (PDGFRA+; Extended Data Fig. 4a(iii)) as well as blood progenitors (Extended Data Fig. 4a(iv)) and CFU-Fs (Extended Data Fig. 4a(v)). These data indicate that the absence of PDGFRA-expressing cells in the developing embryo should have a profoundly deleterious impact on AGM haematopoiesis. Mice carrying a targeted null mutation of Pdgfra show early embryonic lethality46, and PDGFRA signalling has previously been reported to be essential for establishing a microenvironment that supports definitive haematopoiesis47. To directly test whether LT-HSCs were generated in the absence of Pdgfra, we crossed tdTomato/Rosa26; Pdgfra–nGFP knock-in (KI) heterozygote mice to generate Pdgfra KI/KI (null) and KI/+ (heterozygote) embryos with ubiquitous tdTomato expression, and performed colony-forming unit–culture (CFU-C) and transplantation assays with individual E10.5 and E11.5 AGMs from GFP+ KI embryos with retrospective genotyping of yolk sacs (Extended Data Fig. 4b(i)). Consistent with our expectations, Pdgfra-null E10.5 and E11.5 AGMs produced significantly fewer CFU-Cs and no LT-HSCs (Extended Data Fig. 4b(ii),(iii)).

Distinct waves of PSCs serially populate the AGM

To investigate the source of AGM CFU-Fs, we first crossed R26R–eYFP mice with Mesp1–cre (mesoderm) mice48 and harvested embryos for confocal imaging as well as AGM flow cytometry and CFU-F assays (Fig. 3a(i)). Lineage-tracing studies using Mesp1–cre mice have previously shown Mesp1-derived cell contributions to endothelial cells of the dorsal aorta49. At E11.5, CD31+ aortic endothelial cells were Mesp1–eYFP+ and surrounded by a rim of Mesp1–eYFP+CD31− sub-endothelial cells (Fig. 3a(ii), left). Sub-endothelial cells expressing the smooth-muscle marker Calponin were also Mesp1–eYFP+ (Fig. 3a(ii), right)). A survey of E8.5, E9.5 and E10.5 AGMs in Mesp1–eYFP+ embryos showed that Mesp1-derived stromal cells also contributed to the aortic endothelium even at these early time points (Extended Data Fig. 5a). These data collectively show that at the time of HSC emergence, sub-endothelial stromal cells were mesodermal derivatives.

Fig. 3: Developmental origins of AGM endothelium and CFU-Fs.figure 3

a, (i) Schematic outlining the genetic cross used to harvest Mesp1–cre; R26R–eYFP (Mesp1–eYFP+) embryos at E11.5. (ii) Confocal microscopy images of E11.5 Mesp1–eYFP AGM showing the contribution of Mesp1-derived cells to the endothelium (left; CD31) and smooth muscle (right; Calponin). Insets: magnified views (2-fold) of the region in the white box in the main image. (iii) Percentage of CD31−Mesp1–eYFP+PDGFRA+ cells in AGMs at E11.5, determined by flow cytometry. (iv) Number of CFU-Fs in cell fractions sorted from Mesp1–eYFP+ AGMs (n = 7) at E11.5. b, (i) Schematic outlining the genetic cross used to harvest Wnt1–eYFP embryos at E11.5. (ii) Confocal microscopy images of E11.5 Wnt1–eYFP AGM showing the absence of contribution to endothelium (left; CD31), sub-endothelial smooth muscle (right; Calponin) and sub-endothelial stroma (left and right). Insets: magnified views (2-fold) of the region in the white box in the main image. (iii) Percentage of CD31−Wnt1–eYFP+PDGFRA+ cells in AGMs at E11.5, determined by flow cytometry. (iv) Number of CFU-Fs in cell fractions sorted from Wnt1–eYFP+ AGMs (n = 5) at E11.5. c, (i) Schematic outlining the genetic cross used to harvest Mesp1–eYFP+ embryos at E13.5. (ii) Confocal microscopy images of E13.5 Mesp1–eYFP AGMs showing the contribution of Mesp1-derived cells to the endothelium (left; CD31) but not to smooth muscle (right; Calponin). Insets: magnified views (2-fold) of the region in the white box in the main image. (iii) Percentage of CD31−Mesp1–eYFP+PDGFRA+ cells in AGMs at E13.5, determined by flow cytometry. (iv) Number of CFU-Fs in cell fractions sorted from Mesp1–eYFP+ AGMs (n = 7) at E13.5. d, (i) Schematic outlining the genetic cross used to harvest Wnt1–eYFP embryos at E13.5. (ii) Confocal microscopy images of E13.5 Wnt1–eYFP AGM showing that Wnt1-derived cells do not contribute to the endothelium (left; CD31) but do contribute to smooth muscle (right; Calponin) and sub-endothelial stroma (left and right). Insets: magnified views (2-fold) of the region in the white box in the main image. (iii) Percentage of CD31−Wnt1–eYFP+PDGFRA+ cells in AGMs at E13.5, determined by flow cytometry. (iv) Number of CFU-Fs in cell fractions sorted from Wnt1–eYFP+ AGMs (n = 5) at E13.5. Ao, aortic lumen; DAPI, 4′,6-diamidino-2-phenylindole dihydrochloride; BV421, brilliant violet 421. Colony sizes: micro, <2 mm, 2–24 cells; small, 2–4 mm, >25 cells; and large; >4 mm, >100 cells. CFU-F data were derived from biologically independent experiments (n = 3) using 5–7 embryos per experiment. Data represent the mean ± s.d. The percentage of cells in the different quadrants in the flow cytometry plots are indicated. A random-effects Poisson regression was used to compare colony counts (ad(iv)); ***P < 0.005. The precise P values are provided in the source data.

Source data

Approximately two-thirds of the Mesp1–eYFP+ cells were PDGFRA+ (Fig. 3a(iii)). In E11.5 Pdgfra–nGFP+; Mesp1–DsRed double-transgenic embryos, Pdgfra–nGFP+DsRed+ cells were distributed in the AGM stroma (Extended Data Fig. 5b(i),(ii)). In contrast, Nestin-GFP+ cells in Nestin-GFP+; Mesp1–DsRed double-transgenic embryos were largely restricted to endothelial and sub-endothelial cells in the E11.5 AGM (Extended Data Fig. 5b(iii),(iv)). Whereas Mesp1–eYFP−PDGFRA+ cells from E10.5 AGMs generated significantly lower numbers of CFU-Fs than the Mesp1–eYFP+PDGFRA+ cells (Extended Data Fig. 5c), this difference was not observed in E11.5 AGMs (Fig. 3a(iv)). PDGFRA− cells on the other hand had limited CFU-F capacity at both time points and formed no large colonies. Together, these data show that the aortic endothelium, sub-endothelium and a proportion of CFU-Fs in the E11.5 AGM were derived from Mesp1+ cells but that a comparable number of CFU-Fs were not.

To explore whether the Mesp1–eYFP− cells were derived from Wnt1+ cells, we next crossed R26R–eYFP mice with Wnt1–cre (neural crest) mice50 and harvested embryos for confocal imaging as well as AGM flow cytometry and CFU-F assays (Fig. 3b(i)). In contrast to Mesp1–eYFP+ cells at corresponding embryonic time points (E8.5–E11.5), Wnt1–eYFP+ cells did not contribute to the endothelium or sub-endothelium and were located deeper in the AGM stroma (E11.5; Fig. 3b(ii)) or distant to the ventral surface of the dorsal aorta (E8.5–E10.5; Extended Data Fig. 5d). However, one-third of the Wnt1–eYFP+ cells were PDGFRA+ (Fig. 3b(iii)), and these cells formed significantly fewer CFU-Fs than Mesp1–eYFP+PDGFRA+ cells at E10.5 (compare Extended Data Fig. 5c,e), but their contributions were comparable at E11.5 (compare Fig. 3a(iv),b(iv)). Although only a minority of PDGFRA+ cells were either Mesp1–eYFP+ (approximately 1:12; Fig. 3a(iii)) or Wnt1–eYFP+ (approximately 1:8; Fig. 3b(iii)), these two subfractions collectively accounted for CFU-F potential in the E10.5 and E11.5 AGM.

Unlike MSCs in the E14.5 embryonic trunk, which were reported to be derived from Sox1+ neuroepithelium35, Sox1–eYFP+PDGFRA+ cells were very rare in the E11.5 AGM (2%) and did not contribute to large CFU-Fs (Extended Data Fig. 5f(i)–(iii)).

Therefore, the E11.5 AGM has at least two populations of PDGFRA+ CFU-Fs that have different lineage ancestries (Mesp1-derived, Mesp1der; and Wnt1-derived, Wnt1der) and occupy distinct anatomical locations with respect to the haemogenic endothelium. Furthermore, Mesp1der PSCs showed greater multilineage differentiation capacity than non-Mesp1der PSCs (Extended Data Fig. 5g).

In contrast to the haemogenic E10.5 and E11.5 AGM, the E13.5 AGM is no longer haemogenic18. To evaluate whether CFU-F populations changed during this transition, we crossed R26R–eYFP mice with Mesp1–cre mice and harvested embryos for confocal imaging as well as AGM flow cytometry and CFU-F assays at E13.5 (Fig. 3c(i)). Whereas the dorsal aorta was still lined by Mesp1–eYFP+CD31+ endothelial cells (Fig. 3c(ii), left), Calponin+ sub-endothelial cells were Mesp1–eYFP− (Fig. 3c(ii), right). Furthermore, Mesp1–eYFP+ PSCs, which were relatively abundant at E11.5 (5.4%; Fig. 3a(iii)), were rare at E13.5 (0.2%; Fig. 3c(iii)). Large-CFU-F potential in the E10.5 and E11.5 AGM was seen in both the Mesp1–eYFP+ and eYFP– PDGFRA+ fractions (Fig. 3a(iv) and Extended Data Fig. 5c), but in the absence of Mesp1–eYFP+PDGFRA+ cells at E13.5, they were derived exclusively from Mesp1–eYFP−PDGFRA+ cells (Fig. 3c(iv)).

We then crossed R26R–eYFP mice with Wnt1–cre mice and harvested embryos at E13.5 for confocal imaging as well as AGM flow cytometry and CFU-F assays (Fig. 3d(i)). As observed in E8.5–E11.5 embryos (Fig. 3b(ii), left and Extended Data Fig. 5d), there was no evidence of Wnt1–eYFP+-derived endothelial cells at E13.5 (Fig. 3d(ii), left), but the layer of Mesp1–eYFP+CD31−Calponin+ sub-endothelial cells that were evident at E11.5 had been replaced by Wnt1–eYFP+CD31−Calponin+ cells (Fig. 3d(ii), right). There were equal proportions (8.4%) of Wnt1–eYFP PSCs at E13.5 and E11.5 (compare Fig. 3b(iii),d(iii)). In the absence of Mesp1–eYFP+ PSCs at E13.5, large-CFU-F potential was mostly seen in Wnt1–eYFP+ PSCs (Fig. 3d(iv)).

It is important to note that Mesp1 transcripts were absent in PDGFRA+ (CFU-F), PDGFRB+ (pericytes) and CD31+ (endothelial) cells in the AGM at both E11.5 and E13.5 (Extended Data Fig. 6a). Therefore, Mesp1–eYFP+ cells in the AGM at these time points are Mesp1-derived cells that do not currently express Mesp1. Although Wnt1 transcripts were absent in E13.5 cells, there was variable and low-level Wnt1 expression at E11.5 in PDGFRA+ but not PDGFRB+ or CD31+ cells (Extended Data Fig. 6b).

Together, these data show that at the time of HSC emergence at E11.5 (and E10.5), sub-endothelial stromal cells were mesodermal (that is, Mesp1) derivatives. The loss of Mesp1der cells in the sub-endothelium, along with replacement by Wnt1der cells at E13.5, temporally coincides with the loss of EHT in the dorsal aorta.

Mesp1 der PSCs induce EHT in non-haemogenic endothelium

To determine whether there were EHT-promoting attributes in E10.5 and E11.5 Mesp1der PSCs that were absent in E11.5 and E13.5 Wnt1-derived progenitors, we performed co-aggregate cultures of FAC-sorted Mesp1der and Wnt1der PSCs with endothelial cells from ubiquitous GFP+ (UBC–gfp/BL6)51 mice. The Mesp1der and Wnt1der PSCs were harvested from the AGMs of compound transgenic embryos generated by crossing Mesp1–cre or Wnt1–cre mice with STOCK Tg(CAG-Bgeo-DsRed*MST)1Nagy/J (Z/Red) reporter mice52.

Endothelial cells (UBC–GFP+PDGFRA−PDGFRB−CD31+VE-cadherin(VE-Cad)+CD41−CD45−) from E10.5, E11.5 and E13.5 AGM or 12–16-week-old female adult mice (heart, lung, aorta and inferior vena cava) were co-aggregated with stromal cells (PDGFRA+PDGFRB−CD31−VE-Cad−CD41−CD45−) from E10.5 and E11.5 Mesp1−DSRed+ AGM (Fig. 4a(i) and Extended Data Fig. 6c(i),(ii)). Following 96 h of culture, the co-aggregates were cryosectioned for confocal imaging or used for flow cytometry, CFU-C and transplantation assays to establish progenitor and stem cell potential of emerging blood cells. Confocal microscopy and flow cytometry showed GFP+CD45+ cells in all endothelial and Mesp1der PSC co-aggregates (Fig. 4a(ii) and Extended Data Fig. 6d; E13.5 AGM endothelium and E11.5 Mesp1der PSCs). No DsRed+CD45+ cells were found in any co-aggregate. Co-aggregation of both E10.5 and E11.5 Mesp1der PSCs with E11.5 (haemogenic) or E13.5 (non-haemogenic) AGM or adult heart, lung, aortic or inferior vena cava endothelium resulted in the emergence of UBC–GFP+ CFU-Cs (Fig. 4a(iii)) and endothelial cell-derived (UBC–GFP+) LT-HSCs with robust multilineage haematopoietic reconstitution (Fig. 4a(iv),(v) and Extended Data Fig. 7a,b). There were no PSC-derived (DsRed+) haematopoietic cells in any co-aggregate transplant recipient (Extended Data Fig. 7a(ii)–(iv)). Transplantation of aggregates composed of endothelial cells or PSCs alone did not contribute to haematopoietic cells (Extended Data Fig.

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