Single-cell analysis of circadian dynamics in tissue explants

Abstract

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.

FIGURE 1:

A Per1-YFP reporter allows for single-cell quantification of circadian rhythms in a mouse organ explant system. (A, B) Bright-field and fluorescence images of bone from the calvarium (A) and tendon from tail (B). (C, D) Images and quantification of three bone (C) and three tendon (D) cells. Cells were imaged every 30 min. Frames every 8 h. Quantification of YFP intensity over time shows an oscillatory pattern with a periodicity of ∼24 h.

FIGURE 2:

Long-term time-lapse imaging and automated segmentation and analysis allow for large-scale acquisition of single-cell circadian data in tissues. (A, B) Heat map and representative line plots showing Per1-YFP oscillations in calvarial cells (A) and tendon (B) drawn from three (A) or two (B) mice. (C–F) Distributions of peak-to-peak intervals and frequencies for calvarial cells (C, D) and tendon cells (E, F). (G) Average autocorrelation of Per1-YFP signal in three mice shows near-identical period and decay rate (N = 343, 129, and 221). (H) Comparison of calvarial (N = 693) and tail tendon (N = 108) signals shows similar average autocorrelation. (I) Scatter plots showing that the length of each individual period is independent of the preceding period (p = 0.78, 0.31 by Student's t test).

FIGURE 3:

Circadian signals in tissues show an initial synchrony that decays over time. (A) Period, measured by peak–peak interval, was calculated for the first and last circadian periods. Note that both measurements have comparable median values, but the last period has a wider variation. (B) The phase distribution of cells from the calvarial bone is plotted after 1, 2, 3, 4, and 5 d (for both A and B, N = 334 cells).

FIGURE 4:

The amplitude of Per1-YFP is constrained within a cell but varies across the population and is independent of the period. (A) Distribution of Per1-YFP amplitude across cells in the calvarial bone. Note that the average intensity of YFP across the population does not change over the duration of the experiment (inset). Error bars represent SD. (B) The coefficient of variation of Per1-YFP amplitude was computed across cells and within single cells across periods. The distributions are significantly different (Kolmogorov–Smirnov test, p = 10⁻⁵⁴). (C) No correlation is observed between Per1-YFP amplitude and the period of the subsequent oscillation (for A–C, N = 254 cells).