A cells still yielded heterogeneous expression levels of IRF8-EGFP (Supplemental Fig

A cells still yielded heterogeneous expression levels of IRF8-EGFP (Supplemental Fig. progenitors. Also surprisingly, IRF8-EGFP revealed a highly heterogeneous pre-pro-B populace with a fluorescence intensity ranging from background to 4 orders above background. Interestingly, IRF8-EGFP readily distinguishes true B cell-committed (EGFPint) from those that are noncommitted. Moreover, dendritic cell progenitors expressed extremely high levels of IRF8-EGFP. Taken together, the IRF8-EGFP reporter revealed previously unrecognized subsets with distinct developmental potentials in phenotypically well-defined oligopotent progenitors, providing new insights into the dynamic heterogeneity of developing hematopoietic progenitors. Introduction Hematopoietic stem cells (HSCs) constantly differentiate into all blood cell lineages via distinct OSI-906 differentiation programs. Lineage specification and commitment are marked by timely activation of one set of transcription factors associated with downregulation of other set(s) of transcription factors important for alternate cell lineage potential. While early studies led to the proposal that this flow of intermediate cells within each lineage is usually fixed (1, 2), recent evidence suggests otherwise – that oligopotent progenitor differentiation is very plastic, especially when the host is usually stressed, by infection for example. This causes reprogramming of early lymphoid and myeloid progenitors leading to enhanced development of myeloid lineage cells but curbed production of lymphoid lineage cells(3-6). The plasticity of hematopoietic differentiation has long been known and was recently confirmed at single cell level by Naik et al. using a novel cellular barcoding technique (7). The developmental heterogeneity of lineage progenitor cells has led not only to inconsistencies in identifying phenotypes of intermediate OSI-906 stage cells but also to troubles in positioning the newly found precursors in the orderly progression of lineage differentiation pathways. For example, macrophages are thought to derive from myeloid progenitors whereas dendritic cells (DCs) are thought to develop from individual pathways originating from either CLPs or CMPs (1, 8-11). However, it was recently found that macrophage-DC progenitors (MDPs) with a phenotype of CD117+CX3CR1+ can give rise to both macrophages and DCs (12). These findings suggest that most, if not all, well-characterized progenitor populations are KR2_VZVD antibody heterogeneous at the clonal level even though they appear to have a homogeneous phenotype by certain criteria. Most of our current knowledge of how blood cells are made came from studies of transcription factors. One of a series of transcription factors modulating hematopoietic fate determination is usually IRF8, also known as ICSBP (interferon consensus sequence binding protein). IRF8 is usually expressed mostly in cells of the hematopoietic system. Microglial cells with a hematopoietic origin also express IRF8 (13, 14). Functional analyses revealed broad contributions of IRF8 to the regulation of myeloid and lymphoid lineage development. The levels OSI-906 of IRF8 transcripts are low in HSCs, but increased in yet poorly defined CLPs, MPs, and common DC progenitors (CDPs) (15, 16). IRF8 deficiency in mice causes disrupted development of monocytes and macrophages but increased differentiation of neutrophils (17). The numbers of several subtypes of DCs including plasmacytoid DCs (pDCs), CD8+ DCs and CD103+ non-lymphoid tissue DCs are also greatly diminished in mice (15, 18-23). In humans, a loss of function mutation of IRF8 also causes a monocytic and DC immunodeficiency (24). While IRF8 expression is usually upregulated in both myeloid and lymphoid progenitors, as determined by conventional PCR methods on sorted bulk populations, little is known about how IRF8 participates in the distinct transcriptional programs that control lineage specification and commitment. Here, we created an IRF8-EGFP reporter mouse by a knockin of the EGFP sequence into the IRF8 stop codon that results in transcription and translation of an IRF8-EGFP fusion protein under the regulation of endogenous IRF8 regulatory elements. Our data revealed previously unappreciated expression patterns of IRF8 that help to explain the functions of IRF8 in distinct lineages of hematopoietic cells and to better understand the heterogeneity of early progenitors. Materials and Methods Mice IRF8-EGFP fusion protein OSI-906 reporter mice were generated by Ozgene using a B6 germ line targeting strategy illustrated in Fig. 1. Mice were genotyped by PCR analysis of tail.