Progression from the Common Lymphoid Progenitor to B/Myeloid PreproB and ProB Precursors during B Lymphopoiesis Requires C/EBPα

The C/EBP alpha transcription factor is required for myelopoiesis, with prior observations suggesting additional contributions to B lymphopoiesis. Cebpa expression is evident in common lymphoid progenitor (CLP) and preproB cells but is absent in proB and preB cells. We previously observed that marrow lacking the Cebpa +37 kb enhancer is impaired in producing B cells upon competitive transplantation. Additionally, a Cebpa enhancer/promoter-hCD4 transgene is expressed in B/myeloid CFU. Extending these findings, pan-hematopoietic murine Cebpa enhancer deletion using Mx1-Cre leads to expanded CLP, fewer preproB cells, markedly reduced proB and preB cells, and reduced mature B cells, without affecting T cell numbers. In contrast, enhancer deletion at the proB stage using Mb1-Cre does not impair B cell maturation. Further evaluation of CLP reveals that the Cebpa transgene is expressed almost exclusively in Flt3(+) multipotent CLP versus B cell-restricted Flt3(-) CLP. In vitro, hCD4(+) preproB cells produce both B and myeloid cells, whereas hCD4(-) preproB cells only produce B cells. Additionally, a subset of hCD4(-) preproB cells express high levels of RAG1-GFP, as seen also in proB cells. Global gene expression analysis indicates that hCD4(+) preproB cells express proliferative pathways, whereas B cell development and signal transduction pathways predominate in hCD4(-) preproB cells. Consistent with these changes, Cebpa enhancer-deleted preproB cells downmodulate cell cycle pathways while upregulating B cell signaling pathways. Collectively, these findings indicate that C/EBPa is required for Flt3(+) CLP maturation into preproB cells and then for proliferative Cebpa(int) B/myeloid preproB cells to progress to Cebpa(lo) B cell-restricted preproB cells and finally to Cebpa(neg) proB cells.