Figure 5.

T cell precursors in the thymus begin by expressing PU.1. Data from experiments using thymocytes from progeny of a PU.1-GFP reporter mouse (59) crossed with a Bcl11b-mCherry reporter mouse (K. K. H. Ng, H. Y. Kueh, and M. A. Yui, unpublished), in which activation of the T-cell specific Bc11b gene in DN2 stage identifies cells definitively as T-cell precursors. (A) Gating of immature T-cell precursors to separate ETP, DN2a, DN2b, DN3(a), and DN4 cells. (B) Expression of PU.1-GFP relative to Bcl11b-mCherry in the indicated populations of cells from panel A. Note that the level of GFP from this PU.1 reporter is always low in early T cells, but the pattern of expression perfectly fits the measured PU.1 RNA and protein expression patterns determined by realtime PCR, RNA-seq, and intracellular staining (78, 137). The upregulation of the Bcl11b-mCherry reporter is further used to distinguish the earlier DN2a cells (Bcl11b-mCherry-negative) from the later ones beginning to express Bcl11b (Bcl11b-mCherry positive). (C) Quantitation of PU.1-GFP levels in T-cell precursors as they activate the T-lineage specific reporter Bcl11b-mCherry. Points graphed show Mean Fluorescent Intensities for both markers at the indicated stages, from results in (B). Note that when Bcl11b is first turned on, the cells are still expressing PU.1 comparably to ETP cells. Results are representative of three independent experiments.