Macrophage and T-cell fate control: insights from tracking transcription factor dynamics in single cells

Abstract

Hematopoietic stem and progenitor cells employ circuits of regulatory genes to integrate developmental signals and stabilize fate choices. These progenitors show considerable cell-to-cell heterogeneity in their fate choices; thus, to better understand how regulatory genes control fate decisions, we tracked their levels in single progenitors over time using timelapse live-cell imaging. We examined two hematopoietic fate transitions: 1) macrophage differentiation, driven by the up-regulation of the myeloid transcription factor PU.1; and 2) T-cell fate commitment, controlled by the activation of the T-cell specific transcription factor Bcl11b. In our study of macrophage differentiation, we found that cell cycle length acts as critical mediator in the positive feedback circuit controlling differentiation (Kueh et al. 2013). By following PU.1 regulation in single cells containing a knock-in PU.1-GFP reporter, we found that developing macrophages lengthen their cell cycles to promote stable PU.1 accumulation, and that PU.1 itself promotes cell cycle lengthening, completing a positive feedback loop that stabilizes its own expression. Mathematical modeling furthered showed that this cell cycle feedback circuit robustly stabilizes a slow-dividing differentiated state. In our studies on T-cell fate commitment, we found that Notch signaling – the primary driver of development – enhances the frequency of all-or-none Bcl11b gene activation to promote commitment. By analyzing progenitors from mice containing a knock-in Bcl11b-YFP reporter, we found that uncommitted (Bcl11b-DN2A) progenitors can activate Bcl11b transcription and undergo fate commitment in the absence of Notch signaling, and that Notch signaling does not modulate the level of Bcl11b transcription, but instead increases the rate at which progenitors switch Bcl11b to an actively expressing state. These results reveal insights into how signaling pathways activate regulatory gene expression to instruct cell fate.