IL7 Counteraction with Notch Signaling Followed by EBF1 Expression Marks the B-Cell Commitment in CLP Stage

Development of B-cell lineage from hematopoietic Stem cells in bone marrow is a stepwise process associated with a gradual loss of myeloid and T cell potential. This process involves a complex interaction of transcription factors like EBF1 and E2A, and extrinsic signals including IL7. It has been suggested that IL7 plays an inductive role in B-cell commitment through EBF activation in early B-Cell development. Mice deficient in Il-7 signaling show a dramatic reduction in the number of B-cell progenitors and reduced expression of EBF1 in the common lymphoid progenitor (CLP) compartment and ectopic expression of EBF can partially rescue the B-cell phenotype. However, the rather limited ability of EBF1 to rescue the phenotype as well as the powerful function of Il-7 in the expansion of committed cells creates a complex situation with an inherent difficulty to separate instructive and permissive actions of Il-7. Using transgenic mice carrying a reporter gene under the control of the EBF1 dependent Igll1 promoter, we were able to identify a B220⁻CD19⁻ committed B-cell progenitor likely to represent the earliest committed population in the mouse bone marrow. This has opened the possibility to investigate lineage commitment in cells not expressing classical surface markers creating increased possibilities to study lineage choices. In order to investigate the inductive role of Il-7 we crossed the Igll1 reporter mice to Il-7 deficient mice. Analysis of reporter gene expression, gene expression by multiplex single cell PCR as well as functional analysis by in vitro differentiation assays, all supported that the committed lineage negative population was dramatically decreased in the absence of Il-7. These data all support the idea that Il-7 is critical not only for expansion of B-lineage progenitors but also for commitment per se. Investigation of the expression of EBF-1 by Real time and single cell PCR suggested that the expression level of EBF was on an average 50% lower in Il-7 deficient progenitors as compared to wild type cells. This expression level was recapitulated in mice heterozygote for a mutation in the EBF1 gene but since the formation of the early committed cells was not as dramatically effected in these mice, we found a need to look for a further function of Il-7 in its instructive role in B-cell development. This prompted us to investigate a potential function of Il-7 in the modulation of Notch signals known to counteract B-cell commitment and EBF function. This revealed that the addition of Il-7 largely inhibits the Notch response in pro-B cells in vitro. Therefore we suggest that Il-7 is directly involved in B-cell commitment and that this function is achieved by modulation of EBF1 both at the transcriptional and functional level.