Fig 2: ß2-AR Signaling in the Microenvironment Promotes Megakaryocyte Differentiation through IL-6(A) Circulating platelets in young WT (n = 11), young Adrb2-/- (n = 3), or old Adrb2-/- (n = 3) mice.(B–C?) Frequency of (B, B', and B?) CD41+ myeloid or megakaryocyte progenitors within lin-sca-1+c-kit+ (LSK) cells or (C, C', and C?) CD150+CD41+ megakaryocyte progenitors (MkPs) within lin-c-kit+ (LK) cells in endosteal BM cells of the following mice: (B and C) young WT (B, n = 6; C, n = 3), young Adrb2-/- (B, n = 6; C, n = 8) or old Adrb2-/- (n = 4) mice; (B' and C') lethally irradiated WT recipients of WT (n = 5) or Adrb2-/- (n = 4) BM cells; (B? and C?) lethally irradiated WT (n = 5) or Adrb2-/- (n = 4) recipients of WT BM cells.(D) Scheme of human umbilical-cord-blood-derived CD34+ HSPCs cocultured with MS-5 stromal cells.(E–G) Representative immunofluorescence (E and F) and number (G) of CD61+ (red) human megakaryocytes in cocultures treated with (E) vehicle or (F) ß2-AR agonist (clenbuterol, 10 µM) for 10 days (n = 3). Scale bar, 250 µm. (F') Inset of (F).(H) IL6 concentration in endosteal (e) or non-endosteal (n-e) BM supernatant from adult WT (n = 6), Adrb2-/- (n = 4), or Adrb3-/- (n = 7) mice.(I) Il6 mRNA expression (fold change) in MS-5 stromal cells treated with ß2-AR agonist (clenbuterol, 10 µM), PKA inhibitor (H-89, 5 µM), or vehicle for 2 days (n = 3).(J–M) Quantification (J) and representative immunofluorescence (K–M) of CD41+ (red) CD42+ (green) megakaryocytes (yellow) in adult WT (n = 5), Adrb2-/- (n = 3), or Il6-/- (n = 5) mice. Scale bar, 250 µm.(N) Myeloid or megakaryocyte progenitors (CD41+ LSK cells) in primary BM culture from WT or Il6-/- mice treated with ß2-AR agonist (clenbuterol, 10 µM) or vehicle for 4 days (n = 4). Data are means ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001. (B', B?, C', C?, and G) Unpaired two-tailed t test. (A, B, C, H, I, J, and N) one-way ANOVA and Bonferroni pairwise comparisons.

Fig 3: The erythroid cells of Ctnnb1OE-SEC mice show a block in differentiation at the level of orthochromatic erythroblasts and immature reticulocytes.a Flow cytometry quantification (% of live cells) of HSCs (Lin-Sca-1+Kit+CD150+CD48-), short-term HSCs (ST-HSCs) (Lin-Sca-1+Kit+CD150-CD48-), MPPs (Lin-Sca-1+Kit+CD150-CD48+), GMPs (Lin-Sca-1-Kit+CD34+CD16/32+), CMPs (Lin-Sca-1-Kit+CD34+CD16/32-), and MEPs (Lin-Sca-1-Kit+CD34-CD16/32-) of 3-month-old Ctnnb1WT and Ctnnb1OE-SEC females (n = 6). b Numbers of in vitro CFU assays performed with total bone marrow cells from 3-month-old Ctnnb1WT and Ctnnb1OE-SEC females (CFU-GEMM and CFU-E, n = 4; BFU-E, n = 3). Experiments were performed in triplicates. c Representative image of flow cytometry analysis of erythroid bone marrow cells. Plots of CD44 versus FSC of Ter119+-pre-gated BM cells were subdivided into four distinct populations, PII–PV. d Flow cytometry quantification (% of live cells) of erythroid populations PII–PV from of bone marrow cells of 3-month-old Ctnnb1WT and Ctnnb1OE-SEC females (n = 6). e, f GSEA-KEGG pathway alterations analyzed using MSigDB hallmark gene sets in isolated e PIII and f PIV cells from bone marrow of 5-month-old male Ctnnb1WT and Ctnnb1OE-SEC (n = 5). g GSEA plots of Wnt signaling pathway in PIII (upper panel) and PIV (lower panel) cells. A normalized enrichment scores (NES) was calculated after normalization across all analyzed gene sets. The red curve corresponds to the ES (enrichment score) curve, which is the running sum of the weighted enrichment score. h–k Transplantation of 1200 HSC from CD45.2 Ctnnb1WT and Ctnnb1OE-SEC mice into lethally irradiated CD45.1 C57BL/6J recipients. h Survival curves of mice following transplantation with Ctnnb1WT and Ctnnb1OE-SEC donor HSCs (n = 6 per group). i Monthly Hb measurement of CD45.1 C57BL/6J recipients transplanted with Ctnnb1WT and Ctnnb1OE-SEC donor HSCs (n = 6). j Flow cytometry quantification of HSC, ST-HSC, MPP, GMP, CMP, and MEP of transplanted CD45.1 C57BL/6J recipients at 6 months after transplantation (n = 6). k Flow cytometry quantification of CD45.2 positivity in HSC and MEP in the bone marrow of CD45.1 C57BL/6J recipients transplanted with Ctnnb1OE-SEC and Ctnnb1WT HSC CD45.2 donor at 6 months after transplantation (n = 6). The dots in the graphs represent individual mice. Mean ± s.e.m. is shown for each group of mice in all graphs. Statistical significance was determined using Student’s t-test or Mann–Whitney U test (two-sided). ns, not significant. Source data, precise values of n and employed statistical tests for a, b, d, e, f, g, h, i, j, k are provided as a Source Data file.

Fig 4: SCF from AECs regulates HSCs. a Representative images of femur from Bmx-CreERT2;iTdtomato;Nestin-GFP mice stained with anti-VE-cadherin and anti-CD31. All the panels show the same field for different channels. Scale bar, 100 µm. b Enlarged image of the field highlighted by the yellow rectangle in (a). All panels show the same field for different channels. The Bmx-CreERT2-labelled cells colocalise with Nestin-GFP-labelled cells. Scale bar, 10 µm. c Image of whole-mount sternal BM from Bmx-CreERT2;iTdtomato injected with anti-Sca-1. Scale bar, 50 µm. d Representative FACS plot of the labelling of AECs and SECs by Bmx-CreERT2. e Quantification of the labelling efficiency of AECs and SECs by Bmx-CreERT2. Data are represented as mean ± SEM. n = 8 for AECs and SECs. f Representative FACS plot of Nestin-GFP+ MSCs labelling by Bmx-CreERT2. g Percentage of HSCs (CD150+ CD48- LSK) in the BM. Data are represented as mean ± SEM. n = 11 for Cre (-) and n = 14 for Cre (+). h Number of HSCs (CD150+ CD48- LSK) in the BM. Data are represented as mean ± SEM. n = 11 for Cre (-) and n = 14 for Cre (+). i Percentage of LSK in the BM. Data are represented as mean ± SEM. n = 11 for Cre (-) and n = 14 for Cre (+). j Number of LSK in the BM. Data are represented as mean ± SEM. n = 11 for Cre (-) and n = 14 for Cre (+). k–n Percentage of donor-derived cells after competitive transplantation. n = 6 mice for Cre (-), and n = 7 for Cre (+). The label of X-axis indicates the time (weeks) after transplantation. g–n Data were analysed with two-tailed t-test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig 5: Lack of ß3-AR and Nitric Oxide Synthase 1 (Nos1) Signaling in the Microenvironment Accelerates Aging(A, A', and A?) Frequency of CD150lo/-CD41- lymphoid-biased HSCs in endosteal BM CD34-LSK cells from the following mice: (A) young WT (n = 7), young Adrb3-/- (n = 7), or old Adrb3-/- (n = 5) mice; (A') lethally irradiated WT recipients of WT (n = 5) or Adrb3-/- (n = 5) BM cells; and (A?) lethally irradiated WT (n = 4) or Adrb3-/- (n = 3) recipients of WT BM cells.(B) Nitrate concentration in BM extracellular fluid (BMECF) of young WT (n = 6) or Adrb3-/- (n = 7) mice.(C) Number of lin-sca-1+c-kit+CD34-CD48-CD150+Vwf-eGFP- lymphoid-biased HSCs in primary BM culture from Vwf-eGFP mice treated with ß3-AR agonist (BRL37344, 10 µM), Nos1 inhibitor (L-VINO, 100 µM), or vehicle for 4 days (n = 8).(D–G) Representative immunofluorescence (D–F) and quantification (G) of c-kit+ (green) CD41+ (red) myeloid or megakaryocyte progenitors (arrowheads) in endosteal BM of WT (n = 10), Adrb3-/- (n = 8), or Nos1-/- (n = 4) mice. Endosteal BM is considered as regions within one-fifth marrow width from the bone surface. Scale bar, 300 µm.(H–K) Representative immunofluorescence (H-J) and quantification (K) of CD41+ (red) megakaryocytes adjacent (arrowheads) or nonadjacent (asterisks) to EMCN+ (green) BM vasculature of WT (n = 9), Adrb3-/- (n = 6), or Nos1-/- (n = 4) mice. Scale bar, 250 µm.(L–N') Representative whole-mount immunofluorescence of CD31 (green) and EMCN (red) in WT (n = 7), Adrb3-/- (n = 5), or Nos1-/- (n = 4) BM. Arrowheads in insets (L'), (M'), and (N') depict CD31hiEMCN- capillaries.(O and P) Quantification of (O) CD31hiEMCNhi transition zone vessels and (P) CD31hiEMCN- capillaries with <6 µm diameter. Scale bar, 250 µm. Data are means ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001. (A' and A?) Unpaired two-tailed t test. (A–C, G, K, O, and P) One-way ANOVA and Bonferroni pairwise comparisons.