There's many a CLP on the path to B

Lymphocyte development is an excellent model system to study cell fate choices and the underlying transcriptional mechanisms. However, the earliest events have been poorly understood, due in part to the rarity of the relevant cellular intermediates. In this issue of Blood, Mansson and colleagues identify several subsets within the CLP and fraction A population (B220⁺ CLP phenotype) with differing lineage potentials.¹  The results indicate that B-lineage restriction initiates at much earlier developmental stages than previously thought and provide insight into the hierarchical transcriptional mechanisms involved in B-lineage commitment.

How common lymphoid progenitors (CLPs) give rise to B lineage–restricted progeny is unclear. The B lineage–specific transcription factor Pax5 is critical for B-cell development, and it was suggested that expression of Pax5 as visualized by up-regulation of its gene target CD19 on progenitors drove B-cell lineage commitment.^2,3  However, CD19⁻ CLPs express transcriptional regulators important for B-cell lineage commitment including Ebf1 and Pax5, but surprisingly still maintain non–B-cell lineage potentials.^3,4  Mansson et al used mice transgenic for reporters of RAG1 and Igll1 (itself a transcriptional target of Pax5) to identify substantial heterogeneity within the CD19⁻ CLP/fraction A pool. The authors identified in this early progenitor compartment some cells that were RAG1^low, other cells that were RAG1^hi but negative for Igll1 (referred to here as RAG1^hi), and finally cells that were RAG1^hi and also positive for Igll1 (referred to here as Igll1⁺). In vitro cell culture established that these newly identified populations were linearly related, with a developmental sequence of RAG1^low to RAG1^hi to Igll1⁺.

Consistent with this proposed relationship, the earliest RAG1^low cells had the broadest set of lineage potentials, maintaining natural killer (NK), B, T, and even a degree of myeloid potential in clonal assays. The potential to generate NK cells and also myeloid cells appeared greatly decreased by the RAG1^hi stage (see figure). This finding is supported by other recent data showing that RAG1 expression in CLP marks diminished NK-cell potential at the population level.⁵  Expression of the cell-surface marker Ly6D correlated with increased expression of the RAG1 reporter and also coincided with loss of NK-cell potential. Other recent work has found that the Ly6D⁺ pool of CLP does not possess efficient T-lineage potential in vivo.⁶  However, the Ly6D⁺ subset of CLP did possess a degree of T-cell potential when assessed in vitro in this study.

This new work suggests a novel hypothesis regarding the molecular mechanisms that underpin B-cell lineage commitment. The authors found that the bulk of NK-cell potential is lost in RAG1^hi cells, where levels of the early B-cell specification factor Ebf1 are high, but Pax5 is relatively low (see figure). This could suggest that NK potential is attenuated through an Ebf1-dependent but Pax5-independent mechanism. It is also interesting to note that the majority of myeloid potential appears lost by the RAG1^hi stage, supporting recent work that proposes Ebf1 may also be involved in suppressing myeloid potential of developing B-cell progenitors in a Pax5-independent manner.⁷  However, T-lineage potential is not lost until the stage where the Pax5-dependent target Igll1 is up-regulated, suggesting that final commitment to the B-cell lineage does require higher levels of Pax5 expression. Further insights may develop from examination of Ebf1- and Pax5-deficient progenitors at these newly defined stages of early B-cell development.

The identification of these populations contained within the CLP/fraction A pool clarifies some confusing aspects of early lymphocyte development. The new work will also allow us to better study the molecular mechanisms underlying B-cell lineage commitment. The demonstration that Ly6D is up-regulated at these earliest described steps of B-lineage restriction allows these early transitions to be traced in normal mice, without the requirement for elegant but onerous analysis of multiple transgenic reporter strains. In a difficult and sometimes perplexing area, this paper together with the other recent work discussed here represents significant progress.