Clone 2b8

Embryos were fixed in 4% paraformaldehyde at 4 °C overnight (P-Smad staining) or for 45 mn (BMP4 and Noggin stainings) and embedded in OCT by flash freezing on dry ice. Transverse sections (7 μM) were produced using a CM1900 Crystat (Leica). Sections were permeabilized in PBS/0.1% Triton/1% BSA for 1 h, before blocking for 30 mn in PBS/10% fetal calf heat-inactivated serum. Immunostainings were performed using rabbit anti-P-Smad1,5,8 (1:100, clone D5B10, Cell Signaling Technology), goat anti-BMP4 (1:50, clone N-16, Santa Cruz Biotechnology), goat anti-Noggin (1:100, clone AF 719, R&D systems), rabbit anti-SCF (1:50, ab64677, Abcam), unconjugated rat anti-CD31 or PE-conjugated anti-CD31 (1:50, MEC13.3, Pharmingen), goat anti-CD45 (1:100, clone AF 114, R&D systems) and rat anti-cKit (1:100, clone 2B8, Biolegend), followed by incubation with anti-goat Alexa fluor 488 (1:100, Invitrogen), anti-goat NL577 (1:100, R&D systems), anti-rabbit Alexa fluor 488 (1:100, Invitrogen) and anti-rat Alexa fluor 647 (1:100, Invitrogen). Images were acquired with an inverted confocal microscope (SP8) and processed using FiJi software.

For HPC isolation, lineage-negative cells were sorted from the BM of C57BL/6 mice followed by staining with Sca-1 (FITC, Biolegend, clone D7, dilution 1:100, lot 108106) and c-kit (PE, Biolegend, clone 2B8, dilution 1:100, lot 105808) antibodies. To differentiate HPCs into MDSCs, 2 × 10⁵ Lin^-Sca-1^-C-kit⁺ HPCs were placed in each well of 24-well plates and cultured in SFEM (STEMCELL Technologies) medium containing GM-CSF (10 ng/mL; PeproTech) and IL-6 (10 ng/ml; PeproTech). At different time points during culturing, 5 μM GSK343 (Selleck Chemicals) was added to the culture system and the newly generated CD11b⁺Gr-1⁺ cells were analyzed at 96 h.

We first incubated cells with PE-conjugated anti-mouse antibodies directed against CD11b (clone M1/70), CD19 (clone 6D5), CD90.2 (clone 53-2.1), CD11c (clone N418), CD4 (clone GK1.5), CD8a (clone 53-6.7), CD127 (clone A7R34), CD49b (clone DX5), Ly-6G (clone 1A8), Ly-6C (clone HK1.4), TER-119 (clone TER-119) (all BioLegend). Then cells were stained with antibodies directed against c-kit (BioLegend, clone 2B8), sca-1 (BioLegend, clone D7), CD34 (BD Bioscience, clone RAM34), CD16/32 (BioLegend, clone 93), CD115 (eBioscience, clone AFS98). The term LSK refers to hematopoietic stem and progenitor cells based on their characteristic surface expression pattern (Lineage ^neg, Sca-1⁺, c-Kit⁺) [42 (link)], specified as Lin (CD11b, CD19, CD90.2, CD11c, CD4, CD8a, CD127, CD49b, Ly-6G, Ly-6C, TER-119) ^low, sca-1 ^high, c-kit ^high and was used throughout the manuscript as it best describes the cell population investigated. Granulocyte–macrophage precursors (GMP) were defined as Lin (CD11b, CD19, CD90.2, CD11c, CD4, CD8a, CD127, CD49b, Ly-6G, Ly-6C, TER-119) ^low, c-kit ^high, sca-1 ^low, (CD34/CD16/32) ^high, CD115 ^int/low. Monocyte-dendritic cell precursor (MDP) were defined as Lin (CD11b, CD19, CD90.2, CD11c, CD4, CD8a, CD127, CD49b, Ly-6G, Ly-6C, TER-119) ^low, c-kit ^int/high, sca-1 ^low, (CD34/CD16/32) ^high, CD115 ^high [25 (link)].