Light Sheet Fluorescence Microscopy in Intact Bones Reveals Spatial Limitations for Megakaryocyte Migration in the Murine Bone Marrow

Abstract

Blood platelets are small anucleate cell fragments generated from large, polyploid cells in the bone marrow (BM), the megakaryocytes (MKs). According to the current concept, MK development (megakaryopoiesis) progresses spatially from the endosteal niche in the BM to the sinusoidal vascular niche where terminal maturation of MKs occurs and platelets are released into the blood stream. However, in vivo evidence supporting this concept is limited, mainly because previous studies on megakaryopoiesis were hampered by the need to section bones, potentially resulting in cutting artefacts and an underestimation of MK-to-vessel contacts and MK volumes.We studied megakaryopoiesis by visualizing MKs in their 3D environment. To this end, murine sterna were simultaneously stained for MKs and endothelial cells, fixed, chemically cleared and imaged by Light Sheet Fluorescence Microscopy (LSFM). We achieved 3D-reconstructions of the complete and intact bone with subcellular resolution. Surprisingly, we observed an extremely dense blood vessel network, leaving virtually no space for vessel-distant niches. Moreover, MKs were homogenously distributed throughout the BM and, in contrast to previous findings, more than 70% of MKs were in direct contact with the vessel wall. The limited space between vessels questions the need for MKs to migrate. In order to stimulate the production and potentially the migration of MKs, we removed peripheral blood platelets by injecting an anti-platelet antibody, resulting in accelerated megakaryopoiesis to compensate for the platelet loss. Interestingly, this treatment had only minor effects on MK number and distribution. To further understand the principle and spatial restrictions underlying megakaryopoiesis, we use the segmented objects in the 3D image as biological templates for simulations, which perfectly reflect the physiological prerequisites in the bone marrow.