Single-cell analysis of early B-lymphocyte development suggests independent regulation of lineage specification and commitment in vivo

Abstract

To better understand the process of B-lymphocyte lineage restriction, we have investigated molecular and functional properties in early Blineage cells from Pax-5-deficient animals crossed to a B-lineage-restricted reporter mouse, allowing us to identify B-lineage-specified progenitors independently of conventional surface markers. Pax-5 deficiency resulted in a dramatic increase in the frequency of specified progenitor B-cells marked by expression of a λ5 (Igll1) promotercontrolled reporter gene. Gene expression analysis of ex vivo isolated progenitor cells revealed that Pax-5 deficiency has a minor impact on B-cell specification. However, single-cell in vitro differentiation analysis of ex vivo isolated cells revealed that specified Blineage progenitors still displayed a high degree of plasticity for development into NK or T lineage cells. In contrast, we were unable to detect any major changes in myeloid lineage potential in specified Pax-5-deficient cells. By comparison of gene expression patterns in ex vivo isolated Pax-5-and Ebf-1-deficient progenitors, it was possible to identify a set of B-cell-restricted genes dependent on Ebf-1 but not Pax-5, supporting the idea that B-cell specification and commitment is controlled by distinct regulatory networks.

Positive intergenic feedback circuitry, involving EBF1 and FOXO1, orchestrates B-cell fate

“…It is well established that the nuclear location and abundance of FOXO proteins are regulated by the PI(3)K-AKT pathway (17). Early events in lymphocyte development are modulated by IL7Ra and FLT3-mediated signaling.…”

“…1). To determine how EBF1 and FOXO1 activities are related to promote B-cell fate, we compared our microarray data with expression signatures generated from EBF1-deficient LY6D + CLPs (17). Both FOXO1 and EBF1 transcript levels increased during the developmental transition from LY6D − to LY6D + CLPs and further increased at the pro-B-cell stage (Fig.…”

Early B-cell Factor 1 Regulates the Expansion of B-cell Progenitors in a Dose-dependent Manner

“…In order to investigate the composition of the early lymphoid restricted compartments further, we investigated the expression of Ly6D within the CLP population (8,37). Ly6D is expressed on a subpopulation of the classical CLP that retain B-lineage potential but display reduced NK-and dendritic cell (DC)-lineage potential in vivo and in vitro as well as a reduced T-cell potential in vivo (8,16). The frequency of Ly6D Ϫ cells was reduced as compared with Wt in mice carrying one inactivated allele of E2a (Fig.…”

“…Bone marrows and spleens were harvested from mice aged 7-13 weeks. Adoptive transfers were performed as described previously (16). Animal procedures were performed with consent from the local ethics committee at Linköping University (Linköping, Sweden).…”

Interaction of CCR4–NOT with EBF1 regulates gene-specific transcription and mRNA stability in B lymphopoiesis

“…This process of differentiation is accompanied by a progressive restriction of lineage potential by which CLPs can still generate all adaptive and innate lymphoid cells but no myeloid or erythroid cells (Kondo et al 1997;Adolfsson et al 2005). In CLPs (consisting of all lymphoid progenitors [ALPs] and B-cell-biased lymphoid progenitors [BLPs]) as well as pre-pro-B cells, multilineage priming of enhancers has been implicated in setting a chromatin state that facilitates the activation of the B-lineage program (Inlay et al 2009;Mercer et al 2011;Zandi et al 2012). B-cell programming requires a complex network of transcription factors (TFs) in which feed-forward loops and synergistic and antagonistic actions allow for a robust implementation of the B-cell program (Zandi et al 2008;Lin et al 2010;Mansson et al 2012;Boller and Grosschedl 2014;Singh et al 2014).…”