cytoskeleton ͉ thymus ͉ migration ͉ colitis ͉ knockout mice L ymphoid progenitors enter the thymus to initiate a complex differentiation process resulting in maturation of T cells (1, 2). The well characterized maturation steps of ␣ T cells in the thymus are also governed by trafficking events and positional cues driven by guidance molecules, including chemokines, adhesion molecules, and extracellular matrix components (3-6). Rearrangement of the actin cytoskeleton is regulated during both T cell receptor signaling and T cell migration (7-9), but much less is known about its requirement during T cell development. The small Rho-family GTPases and their guanine nucleotide exchange factors (GEFs), proteins known to regulate the actin cytoskeleton, are clearly required during thymocyte development (10-12). Although these molecules are also known to regulate cell adhesion and migration, their involvement in thymocyte trafficking has not been thoroughly assessed. The small GTPase effector molecule Wiskott-Aldrich syndrome protein (WASP) has also been demonstrated to be a critical regulator of antigen receptor signaling, actin cytoskeletal rearrangements, and lymphocyte migration (13-22). Although WASP deficiency has been correlated with lower numbers of naïve T cells (23), WASP does not seem to play a critical role in T cell development. Moreover, despite a large body of evidence showing a role for WASP in T cell signaling, its role in thymocyte migration has not been studied. WASP belongs to the WASP family of proteins, including WASP, N-WASP, and WAVE/SCAR molecules 1-3 (24). In particular, N-WASP, which shares 50% homology with WASP, may serve specific and redundant function with WASP in hematopoietic cells. In fact, we have shown that expression of N-WASP in WASP-deficient T cells partly restores CD3-mediated proliferation, implying that WASP and N-WASP might share functions in T cells (25).Here we sought to explore the importance of cytoskeletal regulation for thymocyte development by examining the unique and redundant roles of WASP and N-WASP. Using two complementary approaches, we analyzed T cells devoid of WASP and N-WASP and demonstrated that thymopoiesis cannot proceed in the absence of WASP and N-WASP. Results Deletion of WASP and N-WASP in Lymphocytes Using the RAG-2-Deficient Complementation System Leads to a Block in T Cell Development.We have demonstrated that lymphoid development is normal in WASP knockout (WKO) mice (20). We hypothesized that N-WASP might have some overlapping functions with WASP during lymphopoiesis and sought to investigate the unique and redundant activities for WASP and N-WASP in T cell development and function. To circumvent the embryonic lethality of N-WASP germ-line inactivation in mice (26), we used the RAG2-deficient blastocyst complementation system (27). Blastocysts from RAG-2-deficient mice implanted into foster mothers generate animals that fail to rearrange antigen receptor genes and consequently lack mature B and T cells. Injection of gene-targeted ES cells into RAG-2-...
Hematopoietic stem/progenitor cells (HSPC) transition in location during development1 and circulate in mammals throughout life2, moving into and out of the bloodstream to engage bone marrow (BM) niches in sequential steps of homing, engraftment and retention3–5. We show here that HSPC engraftment of BM in fetal development is dependent upon the guanine nucleotide binding protein stimulatory alpha subunit (Gsα). Adult Gsα−/− HSPCs differentiate and undergo chemotaxis, but also do not home to or engraft in the BM in adult mice and demonstrate marked inability to engage the marrow microvasculature. If deleted after engraftment, Gsα did not lead to lack of retention in the marrow, rather cytokine-induced mobilization into the blood was impaired. Testing whether activation of Gsα affects HSPC, pharmacologic activators enhanced homing and engraftment in vivo. Gsα governs specific aspects of HSPC localization under physiologic conditions in vivo and may be pharmacologically targeted to improve transplantation efficiency.
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