Lineage Instructive Function of C/EBPα in Multipotent Hematopoietic Progenitor Cells Revealed in a Novel Cebpa-Cre Knock-in Model

Transcription factors control the lineage specification and differentiation of hematopoietic progenitor cells. They are expressed in a cell type-restricted pattern and activate lineage specific genetic programs. Recent studies have demonstrated that expression of GATA-1 or PU.1 in multipotent lin⁻Sca-1⁺c-Kit⁺ (LSK) cells specifies them to develop into myeloerythroid progenitors or lymphomyeloid progenitors, respectively. In contrast, C/EBPα, a transcription factor indispensable for the production of granulocytes and macrophages, is thought to predominantly act at a later stage of hematopoietic commitment, by governing the transition from common myeloid progenitors (CMPs) into granulocytic/monocytic progenitors (GMPs). To study whether C/EBPα may already exert a lineage instructive function at an earlier stage of hematopoietic cell development, i.e., at the level of multipotent LSK cells, we generated a knock-in mouse model expressing Cre recombinase under the regulation of the cebpa promoter and crossed C/EBPα^cre/+ mice with R26 YFP reporter mice. This model faithfully demonstrates high levels of C/EBPα expression in myeloid cells and enabled us to trace cebpa-driven Cre/YFP expression in single LSK cells and their progeny by flow cytometry and colony cultures. On average cebpa-driven YFP expression was found in 17% (range 10–25%) of the total LSK fraction (n=12 mice). Within the CD150⁺CD48⁻ CD34⁻ subset of LSK cells, which contains the most primitive hematopoietic stem cells (HSC), 3–8% of the cells expressed YFP, indicating that cebpa is lowly expressed in bona fide HSC. This low level of expression appears insufficient for lineage determination, since the same levels of YFP expression (1–10%) were found in peripheral T and B cells. Within the CD34⁺ fraction of LSK cells, a population enriched for multipotent progenitors, 19% (range 14%–28%) of the cells expressed YFP. Identical distributions of YFP⁺ cells among the different LSK subsets were found in fetal livers of day 14.5 embryos, suggesting a comparable regulation of cebpa expression in fetal and adult cells. Similar to the reported data for GATA-1 and PU.1, cebpa-expressing LSK cells were predominantly found in the Sca-1^low fraction. When cultured in a multilineage cytokine cocktail, YFP⁺ LSK cells gave predominantly rise to GM colonies (73% of all colonies; range 65–85%), whereas YFP⁻ cells formed multiple types of colonies including mixed, megakaryocytic and erythroid colonies. The predominant outgrowth of YFP⁺ LSK cells to GM lineages was further supported in GM-CSF-supplemented colony assays, which gave rise to cloning efficiencies of 26% for YFP⁺ and 4% for YFP⁻ LSK cells, respectively. In conclusion, our results show that C/EBPα starts to exert its instructive function towards GM cell development already within the LSK population, at the level of the multipotent progenitors. This has important ramifications for our understanding of the role of C/EBPα in early hematopoietic cell fate decisions.