Megakaryocytic lineage tracing by chronic time-lapse 2P-IVM

a, Schematic showing experimental setup of chronic 2-photon intravital microscopy (2P-IVM) of mouse calvarian bone marrow. Lower: Time series of BM in VwfeGFP/+ mice. MK lineage (VWF-eGFP; green); Bone (second harmonic generation). Arrow: Fragmentation of MK results in the release of platelet-like particles. Note that after fragmentation, platelet-like particles exit the BM and new MKs grow in size before undergoing fragmentation.

d, Schematic showing experimental setup of PD. Peripheral blood platelet counts monitored at indicated time points after a single injection (i.p.) of R300 or isotype control; R300: before (n?=?9), 30?min (n?=?3), 0.5d (n?=?6), 1d (n?=?8), 2d (n?=?9), 4d (n?=?9), 8d (n?=?4) mice; isotype: before (n?=?7), 30?min (n?=?3), 0.5d (n?=?4), 1d (n?=?5), 2d (n?=?7), 4d (n?=?4), 8d (n?=?3) Mean?±?SD.e, Morphometric analysis of MKs shows decreased sphericity in response to PD corresponding to an increase of cellular protrusions; Control n?=?63 cells and PD 12?h n?=?51 cells, pooled from 4 mice; unpaired t-test/Welch’ correction, ***: p?=?0.0002; Mean?±?SD. Scale bars = 50 μm. f, Representative 2P-IVM time series of proplatelet formation and MK fragmentation. Single cell tracking of MK volumes over time reveals a significant faster decrease of volume following PD. Control n?=?14 and PD n?=?11 cells, pooled from 3 mice; unpaired t-test; ns: p?=?0.0206; Mean?±?SD. g, Upper: Small (?<?15 μm) VWF-eGFP+ cells (arrows) appear 12?h after PD (2P-IVM). Also see Fig. 1i. Scale bar = 50?µm. Middle: PD triggers an instantaneous proliferation of MKPs peaking 1d following platelet depletion; MKPs (CD41+/CD42–) were counted in whole-mount BMs. n?=?3 mice; one-way ANOVA/Dunnett, **: p?=?0.0023, ****: p?=?0.000009, ***: p?=?0.0008, ns: p?=?0.063 and p?=?0.072; Mean?±?SD. Lower: Proliferation of MKPs (VWF-eGFP+/CD41+/CD42–) was measured after in vivo labelling of BM cells with 5-ethynyl-2′-deoxyuridine (EdU) using FACS. Mean fluorescent intensity of EdU and Frequency of EdU-pve cells significantly increases after PD (12?h); n?=?3 mice; paired t-test; **: p?=?0.0046; *: p?=?0.0115; error bar=SD; Mean?±?SD. h, Left: MKPs lodged within the perivascular niche of the BM grow in volume. Volume increase of single cells was tracked over time. Arrested MKP n?=?27 cells and mobile MKP n?=?6 cells. Notably, the speed of cell growth after PD did not significantly differ from steady state control; Control n?=?54 cells and PD?=?18 cells, pooled from 3 mice, unpaired t-test Welch’s correction ns: p?=?0.7503; Mean?±?SD.

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