The Pax5 gene encoding the B-cell-specific activator protein (BSAP) is expressed within the haematopoietic system exclusively in the B-lymphoid lineage, where it is required in vivo for progression beyond the pro-B-cell stage. However, Pax5 is not essential for in vitro propagation of pro-B cells in the presence of interleukin-7 and stromal cells. Here we show that pro-B cells lacking Pax5 are also incapable of in vitro B-cell differentiation unless Pax5 expression is restored by retroviral transduction. Pax5-/- pro-B cells are not restricted in their lineage fate, as stimulation with appropriate cytokines induces them to differentiate into functional macrophages, osteoclasts, dendritic cells, granulocytes and natural killer cells. As expected for a clonogenic haematopoietic progenitor with lymphomyeloid developmental potential, the Pax5-/- pro-B cell expresses genes of different lineage-affiliated programmes, and restoration of Pax5 activity represses this lineage-promiscuous transcription. Pax5 therefore plays an essential role in B-lineage commitment by suppressing alternative lineage choices.

The Pax5 gene encoding the B-cell-specific activator protein (BSAP) is expressed within the haematopoietic system exclusively in the B-lymphoid lineage, where it is required in vivo for progression beyond the pro-B-cell stage. However, Pax5 is not essential for in vitro propagation of pro-B cells in the presence of interleukin-7 and stromal cells. Here we show that pro-B cells lacking Pax5 are also incapable of in vitro B-cell differentiation unless Pax5 expression is restored by retroviral transduction. Pax5 −/− pro-B cells are not restricted in their lineage fate, as stimulation with appropriate cytokines induces them to differentiate into functional macrophages, osteoclasts, dendritic cells, granulocytes and natural killer cells. As expected for a clonogenic haematopoietic progenitor with lymphomyeloid developmental potential, the Pax5 −/− pro-B cell expresses genes of different lineage-affiliated programmes, and restoration of Pax5 activity represses this lineage-promiscuous transcription. Pax5 therefore plays an essential role in B-lineage commitment by suppressing alternative lineage choices.All types of blood cell are generated from a pluripotent haematopoietic stem cell (HSC) through developmentally restricted progenitors which undergo lineage commitment and subsequent differentiation along a single pathway. The lymphoid lineages develop through a common lymphoid progenitor (CLP) which gives rise to natural killer, B and T cells 1 . Early B-cell development can be dissected into different stages according to the rearrangement status of the immunoglobulin heavy-chain (IgH) locus, the expression of stage-specific cell-surface markers and growth factor requirements 2,3 . The earliest B-lineage precursor cells carry the IgH locus still in germline configuration. D H -J H recombination is subsequently initiated in pre-BI cells 3 , which are also known as early pro-B cells (fraction B) 2 . These pro-B cells can be cultured in vitro on stromal cells in the presence of interleukin-7 (IL-7) and express the B-cell surface proteins 5, VpreB, Ig␣ and Ig␤ (refs 3, 4). Completion of a functional V H -DJ H rearrangement results in the expression of the pre-B-cell receptor and subsequent differentiation to small pre-B cells, which are no longer responsive to pro-B-cell growth conditions 5 .The initiation of B-cell development critically depends on two transcription factors; the basic helix-loop-helix proteins encoded by the E2A gene and the early B-cell factor (EBF). In the absence of either protein, B-cell development is aborted at the earliest stage, before D H -J H rearrangement of the IgH gene 6-8 . Moreover, forced expression of E2A and EBF in haematopoietic precursor cells revealed that these regulators cooperatively induce the transcription of several B-lymphoid-specific genes 9,10 . Hence, loss-and gain-offunction experiments have implicated E2A and EBF in the control of B-lineage commitment.A third transcriptional regulator involved in early B-lymphopoiesis is the B-cell-specific activator protein (BSAP), which ...

A central challenge in embryonic stem (ES) cell biology is to understand how to impose direction on primary lineage commitment. In basal culture conditions, the majority of ES cells convert asynchronously into neural cells. However, many cells resist differentiation and others adopt nonneural fates. Mosaic activation of the neural reporter Sox-green fluorescent protein suggests regulation by cell-cell interactions. We detected expression of Notch receptors and ligands in mouse ES cells and investigated the role of this pathway. Genetic manipulation to activate Notch constitutively does not alter the stem cell phenotype. However, upon withdrawal of self-renewal stimuli, differentiation is directed rapidly and exclusively into the neural lineage. Conversely, pharmacological or genetic interference with Notch signalling suppresses the neural fate choice. Notch promotion of neural commitment requires parallel signalling through the fibroblast growth factor receptor. Stromal cells expressing Notch ligand stimulate neural specification of human ES cells, indicating that this is a conserved pathway in pluripotent stem cells. These findings define an unexpected and decisive role for Notch in ES cell fate determination. Limiting activation of endogenous Notch results in heterogeneous lineage commitment. Manipulation of Notch signalling is therefore likely to be a key factor in taking command of ES cell lineage choice.

The transcription factor Pax5 is essential for initiating B cell lineage commitment, but its role in maintaining commitment is unknown. Using conditional Pax5 inactivation in committed pro-B cells, we demonstrate that Pax5 is required not only to initiate its B lymphoid transcription program, but also to maintain it in early B cell development. As a consequence of Pax5 inactivation, previously committed pro-B cells regained the capacity to differentiate into macrophages in vitro and to reconstitute T cell development in vivo in RAG2-/- mice. Hence, Pax5 expression is continuously required to maintain B cell lineage commitment, because its loss converts committed pro-B cells into hematopoietic progenitors with multilineage potential.