Ikaros is expressed in early hematopoietic progenitors and is required for lymphoid differentiation. Analysis of Ikaros null populations revealed a lack of defining markers for early fate-restricted progenitors, but it was difficult to discern whether Ikaros was required for formation of these populations, or for expression of these markers. Here we use a GFP reporter based on Ikaros regulatory elements to identify the HSC and separate early progenitors in both wild-type and Ikaros-null mice. The presence of lympho-myeloid progenitors is revealed in Ikaros-null mice, which lack the defining factor Flt3 and are capable of myeloid, but not lymphoid differentiation. In contrast, lack of Ikaros in the common myeloid progenitor results in increased formation of erythro-megakaryocyte at the expense of myeloid progenitors and influences their subsequent differentiation. By this approach, pivotal roles for Ikaros in distinct fate decisions in the early hematopoietic hierarchy are revealed. KeywordsIkaros-reporter; hematopoiesis; progenitors; cell fateThe long-term hematopoietic stem cell (HSC), capable of self-renewal and differentiation into a number of distinct lineages, is responsible for the lifelong generation of all blood and immune cell types 1-3 . Prospective isolation of HSCs and progenitor populations with conventional cell surface markers has identified rare, multipotent cells with defined lineage activities that in turn have been used to infer prevailing models of lineage restriction 4-6 . For example, the isolation of a common myeloid progenitor (CMP) and a common lymphoid progenitor (CLP), considered to be the respective roots of the erytho-myeloid and lymphoid lineages, has lent support to an early and strict separation of the lymphoid from the erythromyeloid pathways.The HSC compartment is operationally defined within the LinSca-1 hi c-Kit hi (LSK) population that constitutes 0.1% of the adult bone marrow (BM) cells and contains both long-term (LT) and short-term (ST) HSC-also known as multipotent progenitors (MPP) 7,8 . Use of additional markers, including CD34 and Flt3, has separated LT-HSCs (Lin − Sca-1 hi cKit hi CD34 − Flt3 neg-lo ) from ST-HSCs (Lin − Sca-1 hi c-Kit hi CD34 + Flt3 neg-lo ) and more short-lived lymphoid-primed progenitors (Lin − Sca-1 hi c-Kit hi CD34 + Flt3 + ) 9-12 .Restricted erythro-myeloid progenitors are present within the more abundant Lin − Sca-1 − cKit hi (LK) population (0.6-1% of the BM) that can be further subdivided into a common myeloid progenitor (CMP, CD34 + FcγR lo ) and its more restricted progeny of megakaryoerythrocyte (MEP, CD34 − FcγR lo ) and granulo-monocyte (GMP, CD34 + FcγR hi ) progenitors. A restricted common lymphoid progenitor (CLP) capable of B, T and natural killer (NK)

Summary Here we investigate the mechanisms that underlie the induction of developmental potential and establishment of cell fate during early hematopoiesis. A cascade of lineage-affiliated gene expression signatures, primed in hematopoietic stem cells (HSC) and differentially propagated in lineage-restricted progenitors, is identified. First evidence is provided for a stochastic sampling of lymphoid, erythroid and myeloid transcripts in HSC and multipotent progenitors (MPP). Multi-lineage priming is subsequently resolved upon lineage restrictions. Nonetheless, an unexpected association of lymphoid and myeloid signatures is detected past a nominal myeloid restriction point and a previously unappreciated lymphoid potential is revealed for this stage in development. New insight is provided into Ikaros' role as a bivalent regulator of multi-lineage priming during early hematopoiesis. Whereas Ikaros is responsible for activation of a cascade of lymphoid signatures in the HSC, at subsequent restriction points it is also involved in the repression of lineage-inappropriate signatures including stem cell-specific genes.

The death-effector domain (DED) is a critical protein interaction domain that recruits caspases into complexes with members of the TNF-receptor superfamily. Apoptosis can also be induced by expressing certain DED-containing proteins without surface receptor cross-linking. Using Green Fluorescent Protein to examine DED-containing proteins in living cells, we show that these proteins cause apoptosis by forming novel cytoplasmic filaments that recruit and activate pro-caspase zymogens. Formation of these filaments, which we term death-effector filaments, was blocked by coexpression of viral antiapoptotic DED-containing proteins, but not by bcl-2 family proteins. Thus, formation of death-effector filaments allows a regulated intracellular assembly of apoptosis-signaling complexes that can initiate or amplify apoptotic stimuli independently of receptors at the plasma membrane.