Key Points• Using state-of-the-art three-dimensional electron microscopy approaches, we show that the onset of the DMS formation is at the megakaryocyte plasma membrane.• A pre-DMS structure is formed in the perinuclear region, through a PM invagination process that resembles cleavage furrow formation.The demarcation membrane system (DMS) in megakaryocytes forms the plasma membrane (PM) of future platelets. Using confocal microscopy, electron tomography, and large volume focused ion beam/scanning electron microscopy (FIB/SEM), we determined the sequential steps of DMS formation. We identified a pre-DMS that initiated at the cell periphery and was precisely located between the nuclear lobes. At all developmental stages, the DMS remained continuous with the cell surface. The number of these connections correlated well with the nuclear lobulation, suggesting a relationship with cleavage furrow formation and abortive cytokinesis. On DMS expansion, Golgi complexes assembled around the pre-DMS, and fusion profiles between trans-golgi network-derived vesicles and the DMS were observed. Brefeldin-A reduced DMS expansion, indicating that the exocytic pathway is essential for DMS biogenesis. Close contacts between the endoplasmic reticulum (ER) and the DMS were detected, suggesting physical interaction between the 2 membrane systems. FIB/SEM revealed that the DMS forms an intertwined tubular membrane network resembling the platelet open canalicular system. We thus propose the following steps in DMS biogenesis:(1) focal membrane assembly at the cell periphery; (2) PM invagination and formation of a perinuclear pre-DMS; (3) expansion through membrane delivery from Golgi complexes; and (4) ER-mediated lipid transfer. (Blood. 2014;123(6):921-930) IntroductionThe maturation of megakaryocytes (MKs) includes the development of a unique and extensive membrane system known as the demarcation membrane system (DMS), which divides the cytoplasm into small platelet territories. One MK is thought to produce an average of 4000 platelets. Although it has been known for many years that the DMS ultimately forms the cell membrane of the future platelets, 1,2 the exact mechanism of the formation of this unique membrane system remains unclear. Early electron microscopy (EM) examination of late stage mature MKs led to the proposal that the DMS demarcates already preformed platelets. 3 Observations by De Bruyn of long MK extensions protruding into the sinusoidal lumen modified this idea and suggested that the DMS divides the MK cytoplasm into intertwined and compacted cylindrical regions. 4 Such a model had already been proposed earlier by Thiery and Bessis, who observed that MKs from bone marrow (BM) explants extended elongated projections they called proplatelets. 5 Studies using cultured MKs led to a more refined model, where the DMS appears to function as a membrane reservoir for the extension of proplatelets, which would then fragment into platelets along their length 6 or at their tips. [7][8][9] More recently, using intravital microscopy...

Summary Background Transgenic mice expressing cre recombinase under the control of the platelet factor 4 (Pf4) promoter, in the context of a 100‐kb bacterial artificial chromosome, have become a valuable tool with which to study genetic modifications in the platelet lineage. However, the specificity of cre expression has recently been questioned, and the time of its onset during megakaryopoiesis remains unknown. Objectives/Methods To characterize the expression of this transgene, we used double‐fluorescent cre reporter mice. Results In the bone marrow, Pf4‐cre‐mediated recombination had occurred in all CD42‐positive megakaryocytes as early as stage I of maturation, and in rare CD42‐negative cells. In circulating blood, all platelets had recombined, along with only a minor fraction of CD45‐positive cells. However, we found that all tissues contained recombined cells of monocyte/macrophage origin. When recombined, these cells might potentially modify the function of the tissues under particular conditions, especially inflammatory conditions, which further increase recombination in immune cells. Unexpectedly, a subset of epithelial cells from the distal colon showed signs of recombination resulting from endogenous Pf4‐cre expression. This is probably the basis of the unexplained colon tumors developed by Apcflox/flox;Pf4‐cre mice, generated in a separate study on the role of Apc in platelet formation. Conclusion Altogether, our results indicate early recombination with full penetrance in megakaryopoiesis, and confirm the value of Pf4‐cre mice for the genetic engineering of megakaryocytes and platelets. However, care must be taken when investigating the role of platelets in processes outside hemostasis, especially when immune cells might be involved.