Embryonic stromal clones reveal developmental regulators of definitive hematopoietic stem cells

Abstract

Hematopoietic stem cell (HSC) self-renewal and differentiation is regulated by cellular and molecular interactions with the surrounding microenvironment. During ontogeny, the aorta-gonad-mesonephros (AGM) region autonomously generates the first HSCs and serves as the first HSC-supportive microenvironment. Because the molecular identity of the AGM microenvironment is as yet unclear, we examined two closely related AGM stromal clones that differentially support HSCs. Expression analyses identified three putative HSC regulatory factors, beta-NGF (a neurotrophic factor), MIP-1gamma (a C-C chemokine family member) and Bmp4 (a TGF-beta family member). We show here that these three factors, when added to AGM explant cultures, enhance the in vivo repopulating ability of AGM HSCs. The effects of Bmp4 on AGM HSCs were further studied because this factor acts at the mesodermal and primitive erythropoietic stages in the mouse embryo. In this report, we show that enriched E11 AGM HSCs express Bmp receptors and can be inhibited in their activity by gremlin, a Bmp antagonist. Moreover, our results reveal a focal point of Bmp4 expression in the mesenchyme underlying HSC containing aortic clusters at E11. We suggest that Bmp4 plays a relatively late role in the regulation of HSCs as they emerge in the midgestation AGM.

Fig. 1.

Differential expression of candidate HSC regulators by UG26.1B6 and UG26.3B5 stromal cells. Real-time PCR was performed for β-NGF, MIP-1γ, and Bmp4 on three RNA samples from UG26.1B6 (HSC-supportive) and UG26.3B5 (less supportive) stromal clones. Graphs show the relative expression levels of each factor in both cell lines. n = 3. For MIP-1γ and Bmp4, P < 0.001.

Fig. 2.

Expression of candidate regulators and receptors in E11 embryonic tissues and enriched AGM HSCs. RT-PCR analysis of E11 embryonic tissues for expression of β-NGF and NGF receptors (p75 and TrkA), MIP-1γ and receptor Ccr1, Bmp4, and Bmp4 receptors (Alk3, Alk6, and Bmp-RII), and downstream signaling molecules Smad1, 4, and 5 (A); GFP⁺ and GFP⁻ sorted AGM cells from E11 Ly-6A GFP embryos for expression of Alk3, Alk6 and BmprII (B); and for expression of Bmp4 and Ccr1 (C). β-actin expression was the normalization control. Ao, aorta and mesenchyme; UG, urogenital ridges; FL, fetal liver; YS, yolk sac; LB, limb bud.−rt, no reverse transcriptase.

Fig. 3.

Effect of gremlin on the activity and survival of AGM hematopoietic cells. (A–C) E11 AGMs cultured as explants in the presence or absence of gremlin (250 ng/ml) were analyzed after long term HSC repopulation assay (A), CFU-S₁₁ assay (B), and CFU-C assay (C). For HSC repopulation, 0.3 AGM cell equivalents were injected per recipient. For each CFU-S₁₁ condition, 14 mice were injected with 1 AGM equivalent (n = 3, *, P = 0.002). For CFU-C, n = 3. P < 0.01 for CFU-Mix. Data represent the mean ± SD. (D) FACS plots showing 7AAD and Annexin V staining of CD45⁺ cells from AGMs cultured in the absence or presence of gremlin (250 ng/ml). Percentages of cells shown in gated regions. (E) Graphs show absolute numbers of CD45⁺ cells and Annexin V⁺ 7AAD⁺CD45⁺ cells per AGM (n = 3). (F) Four-color staining dot plots showing percentages of viable cells from AGM explants cultured in the presence or absence of gremlin or Bmp4. (Upper) Cells gated within the CD45⁻ population. (Lower) Cells gated within the total population. C, control; G, gremlin.

Fig. 4.

Expression of Bmp4 in the midgestation AGM. Transverse sections through E11 AGM (dorsal at top; ventral at bottom) stained (A) with anti-Bmp4 antibody. (B) Framed region in A showing Bmp4 staining (green) in the mesenchyme underlying the ventral wall of the dorsal aorta (arrowheads). Nuclear DAPI staining is blue. (C) Immunostaining control with fluorescent (red) secondary antibody used in D in combination with anti-Bmp4 antibody (late E10/early E11). (E) In situ hybridization analysis showing Bmp4 mRNA (purple). Expression is restricted to mesenchyme underlying the ventral aspect of the dorsal aorta (arrowheads). (F) Transverse section through E11 Ly-6A GFP aorta stained with anti-Bmp4 antibody. GFP⁺ cells (green) are located in the ventral endothelium of the aorta with Bmp4 protein (red) detected in the mesenchyme underlying GFP⁺ cells (arrowheads). (G) Framed region in E showing Bmp4-expressing cells (arrowheads) underlying a hematopoietic cluster (arrow). (H) Ventral aspect of aorta in F showing Bmp4 in mesenchymal cells (arrowhead) underlying GFP⁺ hematopoietic cells (arrow). Some GFP⁺ cells show punctate Bmp4 signal. cv, cardinal vein; da, dorsal aorta; e, erythrocytes; g, gonad; m, mesonephros; n, notochord.