Dynamic Visualization of Thrombopoiesis Within Bone Marrow
Tobias Junt, Harald Schulze, Zhao Chen, Steffen Massberg, Tobias Goerge, Andreas Krueger, Denisa D. Wagner, Thomas Graf, Joseph E. Italiano Jr., Ramesh A. Shivdasani, Ulrich H. von Andrian

Supporting Online Material

This supplement contains:
Materials and Methods
Figs. S1 to S4
References

This file is in Adobe Acrobat PDF format.

Other Supporting Online Material for this manuscript includes the following:
(available at www.sciencemag.org/cgi/content/full/317/5845/1767/DC1)
Movies S1 to S11

Movie s1
EYFP+ MKs (red) localize close to BM sinusoids in TPO-treated mice. The blood stream is counterstained with FITC-dextran (green). Image represents a stack of 27 sections (z step of 2 &mgr;m), averaged over three frames and a pixel density of 512x512. Rotation +/- 20° from the vertical plane of view. Scale bar corresponds to 50 &mgr;m.

Movie s2
MKs in the BM of TPO-nontreated mice. Movie represents a stack of 36 sections (z step of 1 &mgr;m) at a pixel density of 512x512. At the beginning and the end of the movie, a 3D reconstruction of the field of view is shown. White circle represents intravascular MK protrusion. Scale bar corresponds to 50 &mgr;m.

Movie s3
Irregular shaped MK (red) with presumed proplatelets in the proximity of BM sinusoids, counterstained with FITC-dextran (green). Image respresents a stack of 27 sections (z step of 2 &mgr;m) at a pixel density of 1024x1024. Rotation +/- 20° from the vertical plane of view. Scale bar corresponds to 50 &mgr;m.

Movie s4
MK with an array of fragments nearby (red) in the proximity of BM sinusoids, counterstained with FITC-dextran (green). The fragments may be connected by a filament that is below the resolution of the MP-IVM. Image represents a stack of 32 sections (z step of 2 &mgr;m) at a pixel density of 1024x1024. Rotation +/- 20° from the vertical plane of view. Size bar corresponds to 50 &mgr;m.

Movie s5
Cytoplasmic protrusions from an EYFP+ MK form proplatelets and intermediate swellings. Three movies of the same z-stack (21 sections, z step of 0.5 &mgr;m), pasted together. Pixel density of 512x512, over a total of 90 frames, with a frame rate of 15 s. White numbers in upper right corner are minutes and seconds, scale bar corresponds to 50 &mgr;m.

Movie s6
Movement of proplatelets, intermediate swellings and MK progeny in the interstitium, close to the MK. Blood stream is counterstained with FITC-dextran (green). Z-stack of 12 sections (z step of 2 &mgr;m) at a pixel density of 256x256 over 200 frames, at a frame rate of 7 s. White numbers in upper left corner are minutes and seconds, scale bar corresponds to 50 &mgr;m. Second harmonic signal of the bone is visible in blue.

Movie s7
Elaborate proplatelets extend into the lumen of a BM sinusoid vessel. Scan of 31 sections (z step of 2 &mgr;m), at a pixel density of 512x512, averaged over three frames. White numbers in the upper right corner are depth in micrometers, scale bar corresponds to 50 &mgr;m.

Movie s8
Formation of a pseudopodium by a EYFP+ MK (red) into a BM sinusoid. The blood stream is counterstained with FITC-dextran (green), and second harmonic emission signal of the collagen appears in blue. Z-stack of 12 sections (z step of 4 &mgr;m) at a pixel density of 256x256, over 61 frames, with a frame rate of 15 s. White numbers in the upper left corner are minutes and seconds, scale bar corresponds to 50 &mgr;m. Arrowhead shows formation of MK pseudopodium at the sinusoid vessel.

Movie s9
Large EYFP+ fragment, potentially a whole MK (red) is shed into a BM sinusoid. It partially occludes the blood flow upstream. Blood stream is counterstained with FITCdextran (green). Six movies of the same z-stack (11 sections, z step of 4 &mgr;m) pasted together. Pixel density of 256x256, over a total of 735 frames, with a frame rate of 15 s. White numbers in upper left corner are hours, minutes and seconds, scale bar corresponds to 50 &mgr;m.

Movie s10
Slow shedding of EYFP+ MK fragment (red, arrowhead) into a BM sinusoid. Blood stream is counterstained with FITC-dextran (green). Z-stack of 14 sections (z step of 4 &mgr;m) at a pixel density of 256x256, over 161 frames, with a frame rate of 15 s. White numbers in upper left corner are minutes and seconds, scale bar corresponds to 50 &mgr;m. White arrowhead shows proplatelet shed into the circulation.

Movie s11
Rapid shedding of EYFP+ MK fragment (red, arrowhead) in a BM sinusoid by the force of the blood stream. Blood stream is counterstained with FITC-dextran (green). Z-stack of 14 sections (z step of 4 &mgr;m) at a pixel density of 256x256, over 161 frames, with a frame rate of 15 s. White numbers in upper left corner are minutes and seconds, scale bar corresponds to 50 &mgr;m. White arrowhead shows proplatelet shed into the circulation.

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