Platelets are anuclear cytoplasmic fragments essential for blood clotting and wound healing. Despite much speculation, the factors determining their life span in the circulation are unknown. We show here that an intrinsic program for apoptosis controls platelet survival and dictates their life span. Pro-survival Bcl-x(L) constrains the pro-apoptotic activity of Bak to maintain platelet survival, but as Bcl-x(L) degrades, aged platelets are primed for cell death. Genetic ablation or pharmacological inactivation of Bcl-x(L) reduces platelet half-life and causes thrombocytopenia in a dose-dependent manner. Deletion of Bak corrects these defects, and platelets from Bak-deficient mice live longer than normal. Thus, platelets are, by default, genetically programmed to die by apoptosis. The antagonistic balance between Bcl-x(L) and Bak constitutes a molecular clock that determines platelet life span: this represents an important paradigm for cellular homeostasis, and has profound implications for the diagnosis and treatment of disorders that affect platelet number and function.

Genetic screens in lower organisms, particularly those that identify modifiers of preexisting genetic defects, have been used successfully to order components of complex signaling pathways. To date, similar suppressor screens have not been used in vertebrates. To define the molecular pathways regulating platelet production, we have executed a large-scale modifier screen with genetically thrombocytopenic Mpl ؊/؊ mice by using N-ethyl-N-nitrosourea mutagenesis. Here we show that mutations in the c-Myb gene cause a myeloproliferative syndrome and supraphysiological expansion of megakaryocyte and platelet production in the absence of thrombopoietin signaling. This screen demonstrates the utility of large-scale N-ethyl-N-nitrosourea mutagenesis suppressor screens in mice for the simultaneous discovery and in vivo validation of targets for therapeutic discovery in diseases for which mouse models are available.

The colonic crypt is the functional unit of the colon mucosa with a central role in ion and water reabsorption. Under steady-state conditions, the distal colonic crypt harbors a single stem cell at its base that gives rise to highly proliferative progenitor cells that differentiate into columnar, goblet, and endocrine cells. The role of c-Myb in crypt homeostasis has not been elucidated. Here we have studied three genetically distinct hypomorphic c-myb mutant mouse strains, all of which show reduced colonic crypt size. The mutations target the key domains of the transcription factor: the DNA binding, transactivation, and negative regulatory domains. In vivo proliferation and cell cycle marker studies suggest that these mice have a progenitor cell proliferation defect mediated in part by reduced Cyclin E1 expression. To independently assess the extent to which c-myb is required for colonic crypt homeostasis we also generated a novel tissue-specific mouse model to allow the deletion of c-myb in adult colon, and using these mice we show that c-Myb is required for crypt integrity, normal differentiation, and steady-state proliferation.colon ͉ hypomorphs ͉ A33 ͉ stem cells ͉ p27

Thrombopoietin (TPO), acting through its receptor Mpl, has two major physiological roles: ensuring production of sufficient platelets via stimulation of megakaryocyte production and maintaining hematopoietic stem cell (HSC) quiescence. Mpl also controls circulating TPO concentration via receptor-mediated internalization and degradation. Here, we demonstrate that the megakaryocytosis and increased platelet mass in mice with mutations in the Myb or p300 genes causes reduced circulating TPO concentration and TPO starvation of the stem-cell compartment, which is exacerbated because these cells additionally exhibit impaired responsiveness to TPO. HSCs from Myb Plt4/Plt4 mice show altered expression of TPOresponsive genes and, like HSCs from Tpo and Mpl mutant mice, exhibit increased cycling and a decline in the number of HSCs with age. These studies suggest that disorders of platelet number can have profound effects on the HSC compartment via effects on the feedback regulation of circulating TPO concentration.

IntroductionThe development of B cells from hematopoietic stem cells (HSCs) proceeds through several well-defined intermediate stages. HSCs are enriched within the Lineage Ϫ Sca-1 hi c-kit hi (LSK) population in the bone marrow. 1 The LSK population also includes progenitors with strong lymphoid and myeloid potential but limited erythromegakaryocyte potential, termed lymphoid-primed multipotent progenitors (LMPPs). 2 LMPPs can be identified by their high level of Flt3 and display expression of several lymphoid transcripts. 3 Downstream of the LMPPs, the common lymphoid progenitor (CLP) up-regulates expression of interleukin-7 receptor ␣ (IL-7R␣) and depends on signaling through this receptor for further development to B-cell progenitor stages. [4][5][6] Together, signals through Flt3 and IL-7R␣ are required for all B-cell development. 7 Transcription factors act in concert with these cytokine signals to regulate B-cell development. 8 Although factors such as Ebf1, E2a, and Pax5 control B-cell commitment and pro-B cell differentiation, [9][10][11][12] less is understood about the earlier stages of lymphoid specification from HSCs. Pu.1 and Ikaros have been implicated in the development of LMPPs; however, this could reflect roles for these factors in Flt3 expression. 13,14 The process of lymphoid priming appears to depend on Ikaros and E2a, 15,16 but additional transcription factors are probably involved.The transcription factor c-Myb is highly expressed in hematopoietic progenitor cells 17 ; however, its role in lymphopoiesis has remained elusive. Mice bearing a germline deletion of c-Myb die embryonically because of anemia. 18 More recently, viable mutant alleles of c-Myb have been developed, enabling the role of c-Myb in adult hematopoiesis to be examined. Mice with impaired c-Myb activity or expression display reduced numbers of B cells. [19][20][21][22][23] Conditional deletion of c-Myb in pro-B cells ablates further B-cell development, indicating that c-Myb is stringently required in the B-cell lineage. 24 The precise role of c-Myb during B-cell development remains undefined, however, and no critical target genes have been described. In addition, it is unclear whether c-Myb also plays a role within multipotent progenitors to influence B-cell development.Here, we have used a range of alleles to dissect the role of c-Myb during B-cell development. Conditional deletion of c-Myb at the earliest B-cell progenitor stage resulted in a complete lack of B cells, confirming that c-Myb is directly required in the B-cell lineage at the point of lineage commitment. Mice expressing a severely hypomorphic allele of c-Myb throughout hematopoiesis display an even earlier defect in lymphoid development, including impaired lymphoid priming in multipotent progenitor cells. Using these hypomorphic mice, we found that c-Myb is required for normal Il7r expression and IL-7-dependent pro-B cell proliferation. Collectively, these data suggest that c-Myb plays a central role in B-cell development by integrating external signals with t...