Here we identify and characterize very early stages in B cell development in mouse bone marrow, focusing on those before CD19 expression. summarizes the features of the cell stages we have defined. The earliest stage, MLP, generates all lineages assayed in vivo, including B, T, NK, and myeloid. The next stage, identified by cell surface markers as CLP, gives a lymphoid-restricted repopulation in vivo but has the potential to generate myeloid cells in culture, while half of the cells already contain DHJH rearrangements. The third stage, Fr. A, identified by expression of B220, generates predominantly B cells in vivo but has the potential to produce T cells in culture, while showing an even higher frequency of DHJH rearrangement than CLP. Gene expression profiles are consistent with B lineage activation at the MLP stage, resulting in expression of a large number of B lineage–specific genes at the CLP and Fr. A stages.
Figure 8. Diagram of early stages in B lineage development, indicating extent of DHJH rearrangement, expression of key transcription factors, expression of a RAG-2–GFP reporter transgene, extent of B/T/myeloid repopulating capacity, and lineage plasticity (more ...)
T lineage potential in Fr. A was unexpected. Previously we showed that a fraction of B220+
cells expressing CD43 and CD93 (“Fr. A2”) were B lineage precursors that failed to generate T cells in i.v. and intrathymic assays (7
). Using the present approach, applying greater purification criteria than previously, we still find predominantly B lineage repopulation by a “refined Fr. A” using the standard Ly-5 marked competitive repopulation assay. Although these B220+
Fr. A cells constitute only 0.05–0.1% of total bone marrow cells, they constitute an important decision point in B cell development. However, while behaving as B lineage precursors in some assays, their lineage plasticity is revealed in the HOS-FTOC and DL1-OP9 assays. One explanation for this behavior is their expression of Notch-1, which can be activated in the thymic microenvironment or by DL1 interaction in culture, redirecting their lineage fate. The contrasting i.v. repopulation results likely indicate that Fr. A stage cells only inefficiently home to the thymus or else rapidly progress to the irreversibly B lineage–committed CD19+
stage compared with earlier stages like CLP or MLP.
Myeloid cell generation from the CLP fraction was also surprising. Our analysis shows that although these cells have become considerably more lymphoid specified than MLP, expressing higher levels of TdT and RAG message (and functional RAG protein, as indicated by DHJH rearrangement), they nevertheless retain significant myeloid capacity as revealed in the B/myeloid bipotential assay. As with Fr. A, the i.v. repopulation assay shows a more restricted lineage potential, either because these cells predominantly home to microenvironmental niches that disfavor myeloid development or else rapidly progress to Fr. A in vivo. We can only speculate that this in vitro myeloid potential was overlooked previously due to differences in stromal culture conditions that favored lymphoid progression or else to differences in sensitivity of detection of CD45R/B220 that might have included Fr. A in the CLP population, increasing its apparent lymphoid restriction. Therefore, the subdivision of B220− and B220+ fractions of CD117medCD127+ cells is important because it reveals clear differences in lineage restriction read out in both in vivo and in vitro. That is, in contrast with CLP, most Fr. A cells did not respond to myeloid-inducing signals in stromal cell culture. Thus, Fr. A pre-pro–B cells behave as a strict “common lymphoid progenitor” in terms of their lineage potential.
The finding of B220+
CLP is reminiscent of a previous report from Martin et al. (50
) describing a lymphoid-restricted “CLP2” cell type. However, in their studies, the B220+
CLP-like cells were reported to lack CD117, clearly not the case with Fr. A. Furthermore, the CLP2 cells were described functionally as efficiently homing to the thymus, which we do not observe; Fr. A cells injected i.v. generate far fewer thymocytes compared with classical (i.e., B220−
) CLP stage cells (). The capacity of CLP2 stage cells to home to the thymus led Martin et al. to propose that these cells are a founder population for thymic T cell development. In contrast, we would suggest that Fr. A stage cells are an intermediate between CLP and CD19+
pro–B stage cells, in a B lineage–specified, but not yet committed, state. Recent work from Allman et al. (51
) has identified an early thymic progenitor that appears distinct from CLP2. Clearly, this issue will require further investigation to clarify the relationships among B220+
subsets from bone marrow and thymus in terms of lineage potentials.
A very recent report by Balciunaite et al. (21
) described the presence of a B220+
hematopoietic progenitor with B, T, and myeloid potential in vitro and lymphoid potential in vivo. Although these authors' limiting dilution analysis suggests a precursor with some similarity to ours, their population appears more similar to the B220−
CLP stage we report here; the expression of B220 in our experience greatly reduces myeloid potential (by 10-fold) found in CLPs. Furthermore, the decrease in csfR1, a gene encoding the receptor for colony stimulating factor 1 (a key myeloid growth factor), in the progression from CLP to Fr. A provides a mechanistic explanation for the difference we observe. In fact, we found that B220 expression was a better marker for the loss of myeloid potential than induction of a RAG-2–GFP reporter, sounding a cautionary note on the use of RAG reporters for identifying “early lymphoid progenitors” (17
Both CLP and Fr. A stage cells generated T lineage cells in vitro, revealing T lineage potential, but this does not necessarily mean that either are normal intermediates in a developmental pathway from hematopoietic stem cells to T cells. Although the existence of T cell lines or thymocytes with Ig DH
rearrangement have been reported (52
), there are no T lineage precursors among triple negative (CD3−
) thymocytes with a surface phenotype corresponding to bone marrow CLP (51
), and we detect no cells corresponding to Fr. A in the thymus (not depicted). Our results seem most consistent with a type of developmental model where B lineage “specification” precedes B lineage commitment in bone marrow (53
). In this model, B lineage genes are induced and non–B lineage genes are repressed in progressive stages of hematopoietic development, mediated by a hierarchy of transcription factors, resulting in a B lineage–specified stage, coincident with DH
rearrangement and the high-level expression of a set of early B lineage genes, such as Igα/β, λ5/VpreB, and RAG-1/2. However, absolute irreversible lineage commitment occurs at a later stage, coincident with high-level expression of functional Pax-5 (42
In fact, it appears that B lineage specification initiates even before the CLP stage, as MLP cells express some lymphoid/B lineage genes, including TdT, RAG-2, and Ig-β. We also note that most MLP stage cells show activation of a RAG reporter transgene, similar to the previously described CD117hi
early lymphoid progenitors fraction that lacks CD127 (17
). Nevertheless, we found a robust myeloid lineage engraftment with MLP, suggesting that RAG-2 gene transcriptional activation, along with a significant component of the B lineage program, initiates in a cell fraction that maintains considerable myeloid potential as revealed using the cell engraftment competition assay.
Finally, our microarray analysis illustrates the progressive nature of B cell development. We identify clusters of genes with expression shared between MLP and CLP, between CLP and Fr. A, and between Fr. A and CD19+ pro–B stage cells. Continuing examination of the members of these clusters will help to elucidate more fully the gene program resulting in progressive restriction to B lineage development, along with the key microenvironmental interactions that foster this process.