Understanding the mechanisms of cellular differentiation is challenging
because differentiation is initiated by signaling pathways that drive temporally
dynamic processes, which are difficult to analyse in vivo. We establish a new
Tool, Timer
-
of
-
cell
-
kinet
ics
-
and
-
activity (Tocky [toki], time in Japanese). Tocky
uses the Fluorescent Timer protein, which spontaneously shifts its emission
spectrum from blue
-
to
-
red, in combination with computer algorithms to reveal
the dynamics of differentiation in vivo. Using
a transcriptional target of T cell
receptor (TCR)
-
signaling, we establish Nr4a3
-
Tocky to follow downstream
effects of TCR signaling. Nr4a3
-
Tocky reveals the temporal sequence of
events during regulatory T cell (Treg) differentiation and shows that persist
ent
TCR signals occur during Treg generation. Remarkably, antigen
-
specific T
cells at the site of autoimmune inflammation also show persistent TCR
signaling. In addition, by generating Foxp3
-
Tocky, we reveal the in vivo
dynamics of demethylation of the
Fox
p3
gene. Thus, Tocky is a Tool for cell
biologists to address previously inaccessible questions by directly revealing
dynamic processes in vivo.