Biomechanical forces promote embryonic haematopoiesis

Abstract

Biomechanical forces are emerging as critical regulators of embryogenesis, particularly in the developing cardiovascular system. After initiation of the heartbeat in vertebrates, cells lining the ventral aspect of the dorsal aorta, the placental vessels, and the umbilical and vitelline arteries initiate expression of the transcription factor Runx1 (refs 3-5), a master regulator of haematopoiesis, and give rise to haematopoietic cells. It remains unknown whether the biomechanical forces imposed on the vascular wall at this developmental stage act as a determinant of haematopoietic potential. Here, using mouse embryonic stem cells differentiated in vitro, we show that fluid shear stress increases the expression of Runx1 in CD41(+)c-Kit(+) haematopoietic progenitor cells, concomitantly augmenting their haematopoietic colony-forming potential. Moreover, we find that shear stress increases haematopoietic colony-forming potential and expression of haematopoietic markers in the para-aortic splanchnopleura/aorta-gonads-mesonephros of mouse embryos and that abrogation of nitric oxide, a mediator of shear-stress-induced signalling, compromises haematopoietic potential in vitro and in vivo. Collectively, these data reveal a critical role for biomechanical forces in haematopoietic development.

Figure 1. Shear stress induces haematopoietic commitment from ES-derived cells

a, Experimental protocol used to induce haematopoietic differentiation from ES-derived cells in the presence of wall shear stress (WSS). ES cells are differentiated with the embryoid body method for 3.25 days, disaggregated and plated on gelatinized surfaces. Cell monolayers were exposed to shear stress and then collected on day 6 for further analysis b, Real-time Taqman PCR-based gene expression analysis in FACS-sorted CD41⁺c-Kit⁺ embryoid-body-derived haematopoietic precursors. Exposure to WSS induces upregulation of the haematopoietic markers Runx1 (P=0.01), Myb (P=0.03) and Klf2 (P=0.001), n=3. c, Methylcellulose haematopoietic c.f.u. assay. WSS increases the frequency of haematopoietic progenitors in complete M3434 methylcellulose. n=4, analysis of variance (ANOVA) P=0.01. Dox, doxycycline. Inset shows average distribution of haematopoietic colony types. c.f.u.-GEMM, c.f.u. granulocyte-erythroid-myeloid-megakaryocytes; c.f.u.-G/M/GM, c.f.u. granulocytes/macrophages/granulocyte-macrophages; c.f.u.-E, c.f.u. erythroid. Bar graphs represent average±s.e.m. Pictures show representative colonies: c.f.u.-GEMM (top), c.f.u.-G/M/GM (middle), c.f.u.-E (bottom). Scale bar, 200 μmm. *P<0.05, **P<0.01.

Figure 2. Nitric oxide production regulates the expansion of haematopoietic progenitors

a, Methylcellulose haematopoietic c.f.u. assay. Pharmacological inhibition of nitric oxide synthesis with L-NAME reduces by 50% the WSS-mediated increase in haematopoietic c.f.u.; n=3, P=0.04. Inset shows the average distribution of colony types. b, L-NAME does not affect WSS-mediated Runx1 upregulation; n=3. c, In vivo c.f.u. assay. Exposure of developing embryos to L-NAME from E8.5 to E10.5 leads to a reduction in haematopoietic progenitors in the AGM region. D-NAME, n=30; L-NAME, n=32; P=0.0008. Inset shows average distribution of colonies per AGM. Bar graphs represent average±s.e.m. *P<0.05, **P<0.005.

Figure 3. Shear stress induces haematopoiesis in PSp/AGM-embryo-derived cells

a, Methylcellulose haematopoietic c.f.u. assay. WSS increases the frequency of haematopoietic progenitors in two-dimensional primary PSp cultures from E9.5 embryos; n = 4(P = 0.038); bar graphs represent average ± s.e.m. Inset shows average distribution of haematopoietic colony types. b, WSS induces upregulation of the haematopoietic markers Runx1 (P = 0.01) and Klf2 (P = 0.05) in FACS-sorted AGM-derived CD41⁺ haematopoietic progenitors as documented by real-time PCR; n = 3, bar graphs represent average ± s.e.m. c, FACS analysis. WSS induces an increase in CD31⁺ cells in two-dimensional primary AGM cultures; P = 0.005, n = 3, average ± s.d. d, WSS modulates the differentiation of AGM-derived haematopoietic progenitors as shown by an increase in absolute number of cells positive for the erythroid marker Ter119 (P = 0.02) and for the lymphoid marker B220 (P = 0.01); n = 3, bar graphs represent average ± s.e.m. Shear stress induces maturation of erythroid precursors as documented by cell morphology in cytospins, which show pycnotic erythroblasts in WSS-treated samples, and polychromatic erythroblasts in static cultures. Scale bar, 10 μm. *P < 0.05, ** P < 0.01.

Figure 4. Runx1 expression and c.f.u. activity are shear-stress-dependent

a, PSp isolated from E9.25 Ncx1^−/− embryos show reduced gene expression levels of the haematopoietic markers Runx1 (P = 0.02) and Klf2 (P = 0.003) when compared to matched wild-type (WT) or heterozygous (Het) littermate controls; n = 13 Ncx1^−/−, n = 15 controls. b, Shear stress increases the expression of Runx1 in E9.25 Ncx1^−/− PSp cultures to levels comparable to the one observed in littermate controls; real-time quantitative PCR, n = 4, ANOVA, P = 0.03. Post-hoc multiple comparisons are two-tailed t-test with P < 0.02. c, Shear stress induces c.f.u. activity in Ncx1^−/− PSp-derived cells. Average ± s.e.m.; P = 0.01. Inset shows average distribution of haematopoietic colony types at day 14 after replating; n = 6. *P < 0.05, **P < 0.005.