2 International Journal of Cell Biology HSC MPP CMP MEP GMP Red blood cells Megakaryocyte Neutrophil Macrophage Pro-B Pre-B B cell CLP Pro-T Pre-T T cell Pu.1 expression Low High Figure 1: Schematic showing the changing expression of PU.1 during haematopoiesis. PU.1 levels, where known, are represented by gradient bars. Gradient bars are not drawn to scale. Diﬀerentiation pathways are denoted by arrows. Abbreviations: HSC, haematopoietic stem cell; MPP, multipotent progenitor; CMP, common myeloid progenitor; CLP, common lymphoid progenitor; MEP, megakaryocyte-erythroid progenitor; GMP, granulocyte-macrophage progenitor. two progenitor pools then further diﬀerentiate to form ma- ture blood cells including megakaryocytes, red blood cells (RBCs), neutrophils and macrophages (all CMP derived), and B and T cells (CLP derived) (Figure 1). Iwasaki et al. showed that ablating PU.1 expression results in a de- crease in HSC numbers by an order of magnitude and that CMPs, CLPs, and CMP progeny GMPs (granulocyte- monocyte progenitors; Figure 1) are all undetectable in PU.1 knockout foetal livers [12]. These haematopoietic defects are cell autonomous as PU.1 HSCs fail to generate granular or myeloid colonies when cultured in vitro [12, 13]. Similarly, in competitive repopulation assays, PU.1 knockout foetal liver HSCs that were injected into lethally irradiated mice were barely detectable in peripheral blood, and the low numbers that were found within the bone marrow (BM) failed to generate monocytes and B and T lymphocytes [12, 14, 15]. Interestingly, the formation of megakaryocyte-erythroid progenitors (MEPs), however, remains intact in PU.1 knockout mice [12]. MEPs were conventionally thought to arise only from CMPs (Figure 1). However, the lack of CMPs and abundance of MEPs in PU.1 mice suggest that MEPs may bypass the CMP stage and develop directly from HSCs, a hypothesis that is supported by several other studies [16–19]. Taken together, these data suggest that PU.1 is crucially important for normal development of HSCs into CMPs and CLPs but is dispensable for production of MEPs. In order to explore the role of PU.1 in haematopoietic lineages further, studies have been performed using reporter systems in which green ﬂuorescent protein (GFP) has been knocked into the PU.1 locus to allow its expression pattern to be tracked throughout haematopoietic diﬀerentiation [6– 8, 12]. While a low level of PU.1 is detected in LT-HSCs (Lin− Sca-1+ c-kit+ CD34−), its expression increases as these cells become multipotent progenitors (MPPs; Lin− Sca-1+ c- kit+ CD34+) and subsequently develop into both CMPs and CLPs [7, 12, 20]. At this stage, the level of PU.1 expression is decisive in permitting progression towards either the myeloid or lymphoid lineage [21, 22]. Culturing PU.1+/− foetal liver progenitor cells (Lin−) in cytokines which favour lymphopoiesis predominantly generates pro-B-cells, while overexpression of PU.1 in these cells dramatically skews com- mitment towards the myeloid lineage and results in a marked increase in macrophage production [2]. Therefore, high PU.1 expression promotes macrophage generation, while a lower level is important for B-cell development. Consistent with