A timer for analyzing temporally dynamic changes in transcription during differentiation in vivo.

David Bending,Paz Prieto Martín,Alina Paduraru,Masahiro Ono,Erik Marzaganov,Marie Laviron,Tessa Crompton,Hitoshi Miyachi,Catherine Ducker,Satsuki Kitano

doi:10.1083/jcb.201711048

Abstract

Understanding the mechanisms of cellular differentiation is challenging because differentiation is initiated by signaling pathways that drive temporally dynamic processes, which are difficult to analyze in vivo. We establish a new tool, Timer of cell kinetics and activity (Tocky; or 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 persistent 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 Foxp3 gene. Thus, Tocky is a tool for cell biologists to address previously inaccessible questions by directly revealing dynamic processes in vivo.

Highlights

It is a central question in cell biology how cellular differentiation progressively occurs through the activities of temporally coordinated molecular mechanisms (Kohwi and Doe, 2013; Kurd and Robey, 2016)
Nr4a3-Timer of cell kinetics and activity (Tocky) reveals the temporal sequence of events during regulatory T cell (Treg) differentiation and shows that persistent T cell receptor (TCR) signals occur during Treg generation
Design of the Tocky system for analyzing the time and frequency domains of signal-triggered activation and differentiation events Given the long half-life of stable fluorescent proteins (FPs) like GFP (56 h; Sacchetti et al, 2001), the dynamics of gene transcription cannot be effectively captured using conventional FP expression as a reporter

Summary

Introduction

It is a central question in cell biology how cellular differentiation progressively occurs through the activities of temporally coordinated molecular mechanisms (Kohwi and Doe, 2013; Kurd and Robey, 2016) It is, challenging to investigate in vivo mechanisms at the single-cell level because individual cells are not synchronized and are heterogeneous, receiving key signaling at different times and frequencies in the body. There is a great need for a new technology to experimentally analyze the passage of time after a key differentiation event, or the time domain, of individual cells in vivo Such a new technology would benefit all areas of cellular biology, but it would be useful for the study of T cells under physiological conditions in vivo, where both the time and frequency of signaling are critical to their differentiation

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