In mammals, megakaryocytes (MKs) in the bone marrow (BM) produce blood platelets, required for hemostasis and thrombosis. MKs originate from hematopoietic stem cells and are thought to migrate from an endosteal niche towards the vascular sinusoids during their maturation. Through imaging of MKs in the intact BM, here we show that MKs can be found within the entire BM, without a bias towards bone-distant regions. By combining in vivo two-photon microscopy and in situ light-sheet fluorescence microscopy with computational simulations, we reveal surprisingly slow MK migration, limited intervascular space, and a vessel-biased MK pool. These data challenge the current thrombopoiesis model of MK migration and support a modified model, where MKs at sinusoids are replenished by sinusoidal precursors rather than cells from a distant periostic niche. As MKs do not need to migrate to reach the vessel, therapies to increase MK numbers might be sufficient to raise platelet counts.

RNA duplex formation between U1 snRNA and a splice donor (SD) site can protect pre-mRNA from degradation prior to splicing and initiates formation of the spliceosome. This process was monitored, using sub-genomic HIV-1 expression vectors, by expression analysis of the glycoprotein env, whose formation critically depends on functional SD4. We systematically derived a hydrogen bond model for the complementarity between the free 5¢ end of U1 snRNA and 5¢ splice sites and numerous mutations following transient transfection of HeLa-T4 + cells with 5¢ splice site mutated vectors. The resulting model takes into account number, interdependence and neighborhood relationships of predicted hydrogen bond formation in a region spanning the three most 3¢ base pairs of the exon (±3 to ±1) and the eight most 5¢ base pairs of the intron (+1 to +8). The model is represented by an algorithm classifying U1 snRNA binding sites which can or cannot functionally substitute SD4 with respect to Rev-mediated env expression. In a data set of 5¢ splice site mutations of the human ATM gene we found a signi®cant correlation between the algorithmic classi®cation and exon skipping (P = 0.018, c 2 -test), showing that the applicability of the proposed model reaches far beyond HIV-1 splicing. However, the algorithmic classi®cation must not be taken as an absolute measure of SD usage as it may be modi®ed by upstream sequence elements. Upstream to SD4 we identi®ed a fragment supporting ASF/SF2 binding. Mutating GAR nucleotide repeats within this site decreased the SD4-dependent Rev-mediated env expression, which could be balanced simply by arti®cially increasing the complementarity of SD4.

Key Points• Rac1 and Cdc42 have redundant functions in platelet biogenesis. • Deficiency of Rac1 andCdc42 results in highly abnormal megakaryocyte morphology associated with severely defective tubulin organization.Blood platelets are anuclear cell fragments that are essential for blood clotting. Platelets are produced by bone marrow megakaryocytes (MKs), which extend protrusions, or socalled proplatelets, into bone marrow sinusoids. Proplatelet formation requires a profound reorganization of the MK actin and tubulin cytoskeleton. Rho GTPases, such as RhoA, Rac1, and Cdc42, are important regulators of cytoskeletal rearrangements in platelets; however, the specific roles of these proteins during platelet production have not been established. Using conditional knockout mice, we show here that Rac1 and Cdc42 possess redundant functions in platelet production and function. In contrast to a singledeficiency of either protein, a double-deficiency of Rac1 and Cdc42 in MKs resulted in macrothrombocytopenia, abnormal platelet morphology, and impaired platelet function. Double-deficient bone marrow MKs matured normally in vivo but displayed highly abnormal morphology and uncontrolled fragmentation. Consistently, a lack of Rac1/ Cdc42 virtually abrogated proplatelet formation in vitro. Strikingly, this phenotype was associated with severely defective tubulin organization, whereas actin assembly and structure were barely affected. Together, these results suggest that the combined action of Rac1 and Cdc42 is crucial for platelet production, particularly by regulating microtubule dynamics. (Blood. 2013;122(18):3178-3187)