Figure 5.
Mk budding in the ABM.(A) Optical section from a 3D z-projection of the adult calvarial BM showing Mks (Pf4-Cre:YFP, gray), bone (SHG signal, blue), and vasculature (Dextran-Cy5, green). Scale bar, 200 µm. (B) Representative examples of observed BM Mk morphologies. Scale bar, 20 µm. (C) Frequency of observed Mk morphologies within the BM. Cumulative of three experiments. (D) Frequency of observed BM Mk behaviors during 4D intravital imaging over 6 h. Cumulative: o = 825; n = 2 independent experiments. (E) Duration of proPlt formation observed from individual Mks in 4D intravital imaging. Cumulative: o = 18; n = 2 independent experiments. (F) 4D intravital time series of a proPlt-forming Mk (blue outline). Scale bar, 20 µm. (G) Representative example of in situ volume fold-change of a proPlt-forming Mk (i), summary of volume change (ii), and summary of volume coefficient of variation (CV) of in situ proPlt-forming Mks (o = 10 Mks, n = 2 independent experiments; iii). (H) Representative example of in situ volume fold-change of a sessile Mk (i), summary of volume change (ii), and summary of volume CV of in situ sessile Mks (o = 183 Mks, n = 2 independent experiments; iii). (Ii) Overview z-projection of sessile calvarial Mks (YFP, gray) associated with the vasculature (visualized using Dextran-Cy5, green). Mk of interest outlined in blue. The direction of the flow of blood is indicated by a red arrow. (Iii) 4D intravital time series (2-s resolution) of a sessile Mk (outlined in blue) releasing a Plts into the peripheral circulation via plasma membrane budding. Yellow arrow indicates location of bud formation. Attachment of the bud before presumptive release was confirmed using optical sections (inset yellow boxes). Scale bar, 10 µm. (J) Observed BM Mk behaviors during high-time-resolution (2-s) intravital imaging. Cumulative, o = 45 Mks; n = 4 independent experiments. (K) Example of the frequency of Plt release events from an individual Mk in vivo. (L) Observed in vivo Plt release/budding rate from individual Mks (o = 37 Mks). Cumulative, n = 4 independent experiments. (M) Comparison of observed membrane bud (o = 129) and circulating Plt (o = 264) diameters. Cumulative, n = 4 independent experiments. P = 0.61, two-tailed unpaired t test.

Mk budding in the ABM. (A) Optical section from a 3D z-projection of the adult calvarial BM showing Mks (Pf4-Cre:YFP, gray), bone (SHG signal, blue), and vasculature (Dextran-Cy5, green). Scale bar, 200 µm. (B) Representative examples of observed BM Mk morphologies. Scale bar, 20 µm. (C) Frequency of observed Mk morphologies within the BM. Cumulative of three experiments. (D) Frequency of observed BM Mk behaviors during 4D intravital imaging over 6 h. Cumulative: o = 825; n = 2 independent experiments. (E) Duration of proPlt formation observed from individual Mks in 4D intravital imaging. Cumulative: o = 18; n = 2 independent experiments. (F) 4D intravital time series of a proPlt-forming Mk (blue outline). Scale bar, 20 µm. (G) Representative example of in situ volume fold-change of a proPlt-forming Mk (i), summary of volume change (ii), and summary of volume coefficient of variation (CV) of in situ proPlt-forming Mks (o = 10 Mks, n = 2 independent experiments; iii). (H) Representative example of in situ volume fold-change of a sessile Mk (i), summary of volume change (ii), and summary of volume CV of in situ sessile Mks (o = 183 Mks, n = 2 independent experiments; iii). (Ii) Overview z-projection of sessile calvarial Mks (YFP, gray) associated with the vasculature (visualized using Dextran-Cy5, green). Mk of interest outlined in blue. The direction of the flow of blood is indicated by a red arrow. (Iii) 4D intravital time series (2-s resolution) of a sessile Mk (outlined in blue) releasing a Plts into the peripheral circulation via plasma membrane budding. Yellow arrow indicates location of bud formation. Attachment of the bud before presumptive release was confirmed using optical sections (inset yellow boxes). Scale bar, 10 µm. (J) Observed BM Mk behaviors during high-time-resolution (2-s) intravital imaging. Cumulative, o = 45 Mks; n = 4 independent experiments. (K) Example of the frequency of Plt release events from an individual Mk in vivo. (L) Observed in vivo Plt release/budding rate from individual Mks (o = 37 Mks). Cumulative, n = 4 independent experiments. (M) Comparison of observed membrane bud (o = 129) and circulating Plt (o = 264) diameters. Cumulative, n = 4 independent experiments. P = 0.61, two-tailed unpaired t test.

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