Single-cell analysis of circadian dynamics in tissue explants.
Tracking molecular dynamics in single cells in vivo is instrumental to understanding how cells act and interact in tissues. Current tissue imaging approaches focus on short-term observation and typically nonendogenous or implanted samples. Here we develop an experimental and computational setup that allows for single-cell tracking of a transcriptional reporter over a period of >1 wk in the context of an intact tissue. We focus on the peripheral circadian clock as a model system and measure the circadian signaling of hundreds of cells from two tissues. The circadian clock is an autonomous oscillator whose behavior is well described in isolated cells, but in situ analysis of circadian signaling in single cells of peripheral tissues is as-yet uncharacterized. Our approach allowed us to investigate the oscillatory properties of individual clocks, determine how these properties are maintained among different cells, and assess how they compare to the population rhythm. These experiments, using a wide-field microscope, a previously generated reporter mouse, and custom software to track cells over days, suggest how many signaling pathways might be quantitatively characterized in explant models.
Department of Neurobiology, Harvard Medical School, Boston, MA 02115.
Laura
L
Lande-Diner
Department of Systems Biology, Harvard Medical School, Boston, MA 02115.
Jacob
J
Stewart-Ornstein
Department of Neurobiology, Harvard Medical School, Boston, MA 02115 charles_weitz@hms.harvard.edu galit@hms.harvard.edu.
Charles J
CJ
Weitz
Department of Systems Biology, Harvard Medical School, Boston, MA 02115 charles_weitz@hms.harvard.edu galit@hms.harvard.edu.
Galit
G
Lahav
D000818:Animals; D001842:Bone and Bones / Q000166:cytology / Q000502:physiology; D057906:Circadian Clocks; D002940:Circadian Rhythm / Q000502:physiology*; D051379:Mice; D008822:Mice, Transgenic; D023421:Models, Animal; D009687:Nuclear Proteins / Q000378:metabolism; D056950:Period Circadian Proteins / Q000378:metabolism*; D059828:Plant Cells / Q000502:physiology; D059010:Single-Cell Analysis / Q000379:methods*; D012984:Software; D013710:Tendons / Q000166:cytology / Q000502:physiology; D014157:Transcription Factors / Q000378:metabolism
D009687:Nuclear Proteins; D056950:Period Circadian Proteins; D014157:Transcription Factors