Figure 2. Quantifying the segmentation clock rhythm.

(A) Comparison of measurements obtained from a region of interest (ROI) spanning the entire field (‘global ROI’) with those obtained from ROIs in which central regions of increasing size were excluded. The excluded area is represented in terms of the diameter (in pixels, px) of a circular region at the center. The corresponding timeseries are shown in the top panel and marked with different colors. Bottom panel shows the global ROI and excluded region in a snapshot of signal of LuVeLu, a dynamic reporter of Notch signaling driven from the Lfng promoter (Aulehla et al., 2008), in a 2D-assay at 700 min from the start of the experiment. Henceforth, timeseries is obtained using ‘global ROI’, unless otherwise specified. (B) Detrended timeseries of the segmentation clock (obtained using global ROI) in 2D-assays, which express the LuVeLu reporter, subjected to 170 min periodic pulses (magenta bars) of either 2 µM DAPT (in solid line) or DMSO (for control, in dashed line). Timelapse movie and corresponding timeseries from global ROI are available at https://youtu.be/fRHsHYU_H2Q. (C) Detrended timeseries of Axin2-linker-Achilles reporter in 2D-assays, subjected to 170 min periodic pulses (magenta bars) of either 2 µM DAPT (in solid line) or DMSO (for control, in dashed line). (D) Period of morphological segment boundary formation, LuVeLu oscillation, Axin2-linker-Achilles oscillation, and rhythm of Mesp2-GFP in 2D-assays subjected to 170 min periodic pulses of either 2 µM DAPT (or DMSO for control). Each sample is represented as a dot, the median is denoted as a solid horizontal line. For morphological segment boundary formation, period was determined by taking the time difference between two consecutive segmentation events (for CTRL: 17 segmentation events in six samples from four independent experiments, and for DAPT: 24 segmentation events in seven samples from five independent experiments, with p-value < 0.001) in the brightfield channel. For period quantifications based the reporters, the mean period per sample from 650 to 850 min after start of the experiment was plotted. LuVeLu: (CTRL: n=24 and N=7) and (DAPT: n=34 and N=8) with p-value < 0.001, Axin2-linker-Achilles: (CTRL: n=3 and N=1) and (DAPT: n=5 and N=1) with p-value = 0.107, Mesp2-GFP: (CTRL: n=8 and N=3) and (DAPT: n=9 and N=3) with p-value = 0.01. Data were visualized using PlotsOfData (Postma and Goedhart, 2019). To calculate the p-value, two-tailed test for absolute difference between medians was done via a randomization method using PlotsOfDifferences (Goedhart, 2019). The timeseries and corresponding period evolution during entrainment, obtained from wavelet analysis, are in Figure 2—figure supplement 1A and Figure 2—figure supplement 1B, respectively. Timelapse movies are available at https://youtu.be/edFczx_-9hM and https://youtu.be/tQeBk0_U_Qo, respectively.

Figure 2—figure supplement 1. Different readouts of the segmentation clock.

The rhythms of different dynamic readouts of the entrained segmentation clock match. (A) Detrended timeseries of the segmentation clock in 2D-assays subjected to 170 min periodic pulses of 2 µM DAPT, and expressing either LuVeLu (Aulehla et al., 2008), Axin2-linker-Achilles, or Mesp2-GFP (Morimoto et al., 2006). Periodic pulses are indicated as magenta bars and the timeseries of each sample (for LuVeLu: n=34 and N=8, for Axin2-linker-Achilles: n=5 and N=1, for Mesp2-GFP: n=9 and N=3) is marked with a dashed line. The detrended timeseries for samples expressing LuVeLu is the same as the detrended timeseries for the DAPT condition in Figure 3A. (B) Period evolution during entrainment, obtained from wavelet analysis. The period evolution for each sample and the median of the periods are represented here as a dashed line and a solid line, respectively. The gray shaded area corresponds to the interquartile range. Magenta dashed line marks $T_{zeit}$. The period evolution plot for samples expressing LuVeLu is the same as the period evolution plot for the DAPT condition in Figure 3B. (C) Snapshots of segmenting regions in 2D-assays subjected to 170 min periodic pulses of 2 µM DAPT, and expressing either LuVeLu (Aulehla et al., 2008), Axin2-linker-Achilles, or Mesp2-GFP (Morimoto et al., 2006), at different time points. Segment boundaries are marked with either white arrowheads (brightfield channel) or white dashed lines (reporter channel). Samples are re-oriented so that the top is toward the periphery and the bottom is toward the center of the 2D-assay. Time is indicated as minutes elapsed from the start of the experiment. Scale bar: 50 µm. Timelapse movies of 2D-assays expressing either Axin2-linker-Achilles or Mesp2-GFP subjected to said perturbation are available at https://youtu.be/edFczx_-9hM and https://youtu.be/tQeBk0_U_Qo, respectively.

Figure 2—video 1. E10.5 2D-assay, expressing LuVeLu, subjected to 170 min periodic pulses of 2 µM DAPT (or DMSO for control), with corresponding timeseries obtained using a global region of interest (ROI) spanning the entire field of view, available at https://youtu.be/fRHsHYU_H2Q.

Download video file ^{(26.6MB, mp4)}

Figure 2—video 2. E10.5 2D-assay, expressing Axin2-linker-Achilles, subjected to 170 min periodic pulses of 2 µM DAPT (or DMSO for control) available at https://youtu.be/edFczx_-9hM.

Download video file ^{(52.8MB, mp4)}

Figure 2—video 3. E10.5 2D-assay, expressing Mesp2-GFP, subjected to 170 min periodic pulses of 2 µM DAPT (or DMSO for control) available at https://youtu.be/tQeBk0_U_Qo.

Download video file ^{(54.2MB, mp4)}