Extended Data Figure 8. Mechanical stimulation is required for the maintenance of peri-arteriolar, but not peri-sinusoidal, niches for lymphoid progenitors in the bone marrow.

(a) The effect of voluntary running for 4 weeks on femoral cortical bone mineral density (6 mice per treatment analyzed in 3 independent experiments). (b) Voluntary running for 4 weeks did not significantly affect the frequencies of HSCs, MPPs, GMPs, MEPs, CMPs, or CLPs in calvarium bone marrow (5 mice per treatment analyzed in 3 independent experiments). (c-h) Hindlimb unloading (c) for 2 weeks did not significantly affect forelimb cortical bone mineral density (d), cortical thickness (e), the percentage of LepR⁺ cells that were Oln-mTomato⁺ (f), or the frequencies of HSCs, MPPs, GMPs, MEPs, CMPs, or CLPs in humerus bone marrow (g). (h) Hindlimb unloading for 2 weeks did significantly reduce hindlimb (femur) cortical bone mineral density (5 mice per treatment analyzed in 3 independent experiments). (i) All point current amplitude histograms of the electrical recordings in Fig. 4h. The single channel conductance was 15 ± 1 pS from the Gaussian fits to the amplitude histograms. The y-axis shows the number of events. (j, k) Single channel current recordings and corresponding all point current histograms of Piezo1 deficient Oln-mTomato⁺ cells isolated from Oln^mT/iCreER; Piezo1^fl/fl mice. The data were collected at −60 mm Hg applied pressure and at the holding potential of +60 mV, without (j) or with (k) 40 μM Yoda1 in the pipette. (l) Piezo1 transcript levels by qRT-PCR in Oln-Tomato⁺ cells from Oln^mT/iCreER; Piezo1^fl/fl and Oln^mT/+; Piezo1^fl/fl control mice (6 mice per genotype analyzed in 3 independent experiments). (m and n) The effect of Piezo1 deletion in Osteolectin⁺ cells on femoral cortical bone mineral density (m) and the percentage of Osteolectin⁺ cells that incorporated a 48 hour pulse of BrdU (n; 6 mice per genotype analyzed in 3 independent experiments). (o) Location of IL7Rα⁺Lineage⁻ cells in the bone marrow of Oln^mT/iCreER; Piezo1^fl/fl and Oln^mT/+; Piezo1^fl/fl control mice (6 mice per genotype analyzed in 3 independent experiments). (p-s) We treated Col1a1-creER; Piezo1^fl/fl; Oln^mT/+ mice and littermate controls with tamoxifen at 2 months of age and analyzed them 1 month later. Piezo1 deletion in osteoblasts significantly reduced femur bone mineral density (p) and cortical thickness (q) but not the frequencies of Osteolectin⁺ cells (r) or HSCs, MPPs, GMPs, MEPs, or CLPs in the bone marrow (s; 5–6 mice per genotype analyzed in 3 independent experiments). (t-w) We analyzed the femurs of Lepr^cre/+; Piezo1^fl/fl; Oln^mT/+ mice and littermate controls at 4 months of age and found that Piezo1 deletion in LepR⁺ cells did not significantly reduce bone mineral density (t) but did reduce cortical thickness (u). Piezo1 deletion in LepR⁺ cells also significantly reduced the frequencies of Osteolectin⁺ cells (v) and CLPs (w) without affecting the frequencies of HSCs, MPPs, GMPs, MEPs, or CMPs (w) in the bone marrow (5 mice per genotype analyzed in 3 independent experiments). (x-z) Compared to Scf^fl/fl littermate controls, Oln^iCreER/+; Piezo1^fl/fl mice had decreased B (x) and T (y) cell counts in the spleen 5 days after oral Listeria infection and increased bacterial CFUs in the spleen 10 days after infection (z; 11–16 mice per genotype analyzed in 3 independent experiments). All data represent mean ± SD. Statistical significance was assessed using paired t-tests (a, l, m, n, p-r, t-v, z), matched samples two-way ANOVAs followed by Sidak’s (b, d-f, h, s, w-y) or Holm-Sidak’s multiple comparisons adjustment (g), or Cochran-Mantel-Haenszel test (o).