Abstract
Understanding in vivo mechanisms of cellular differentiation is challenging because the temporal regulation of cellular signalling and differentiation in vivo is heterogeneous and highly dynamic. Here we establish a novel cellular technology, Timer of cell kinetics and activity (Tocky [toki], the Japanese word for time). Tocky uses Fluorescent Timer protein, which spontaneously shifts its emission spectrum from blue-to-red, in combination with computer algorithms to reveal the timing of cellular signalling and differentiation events in vivo. Using a transcriptional target of T cell receptor (TCR)-signalling, we establish Nr4a3-Tocky to analyse the temporal dynamics of in vivo TCR signalling activity. Nr4a3-Tocky determines the temporal order of events driving regulatory T cell (Treg) differentiation and identifies their immediate developmental precursors. Furthermore, by generating Foxp3-Tocky, we for the first time reveal in vivo dynamics of DNA demethylation, showing that Foxp3 transcription precedes demethylation of its enhancer region. Thus, Tocky empowers cellular biologists to ask previously inaccessible questions, revealing time-dependent differentiation mechanisms and developmental states.