(A) Confocal section through the tailbud of a Looping zebrafish embryo in dorsal view where the dotted line indicates the anterior limit of tissue removed. Nuclei are shown in red and YFP expression …
Summary table of segmentation clock tissue and cellular oscillatory properties.
Summary of statistics of time series traces recorded and analyzed in vitro in tailbud explants or tailbud cells. Peaks were identified, and the period/amplitude of cycles was determined as described in Materials and methods. A maximum period is defined in the method at 140 min, approximately twice the mean. Serum only cells were from the same cell suspension as those that were then treated with Fgf8b for the experiments 280711 and 250112. Pooled data from N = 2 independent cultures, for a total of n = 52 serum only cells. Pooled data from N = 4 independent cultures, for a total of n = 149 Fgf8b treated cells. To culture fully isolated cells, a cell suspension was serially diluted in wells within a 96-well plate, producing wells with a single tailbud cell. These were also treated with Fgf8b. N = 5 independent 96-well plate experiments, with a total of n = 10 fully isolated cells in these experiments.
Summary table of low-density segmentation clock cell experiments.
Description of in vitro cultured tailbud cell population treated with Fgf8b (n = 547), using multiple donor embryos in each of 4 independent experimental replicates (N = 4), carried out on separate days. Across the 29 fields recorded, we observed cell divisions in both YFP-negative (30, 5% of total cells) and YFP-positive cells (13, 2% of total cells). We found a range in the number of cell divisions from 0 to 5 cells per field, with an average of 1.5 (±1 SD) divisions per field. The categories of disqualification list the first event in a recording that led to disqualification. For example, four divisions in YFP-positive cells occurred after the cell had been disqualified for another reason (movement in and out of field, touching another cell).
Time series data from low-density segmentation clock cells.
XLS file containing all time series data for each of the 147 low-density segmentation clock cells in the presence of Fgfb. The file contains 4 work-sheets corresponding to each of the 4 independent replicates and to the plots in Figure 1—figure supplement 5. In each sheet, each cell is described by 3 neighboring columns: average fluorescence, local background, and background subtracted signal. Cells are also listed by their field of view in the original microscopy files.
A confocal section through the tailbud of a Her1-YFP and Histone 2A-mCherry expressing 8-somite stage Looping zebrafish embryo in both lateral and dorsal orientations. The approximate location of …
(A) An example of peak finding from a representative low-density cell trace, showing the steps used to find and estimate inter-peak intervals and amplitude. Top: raw data (red line) and background …
(A) Montage of brightfield and corresponding YFP images from representative explanted tailbud over ~7 hr recording. Brightfield image is a single z-plane, while YFP signal is shown as an average …
(A) Individual tailbud cells from experiment 280711 in the presence of serum. Black time trace is the raw data after background subtraction, the background level is the black line, the smoothed …
Traces from each independent low-density culture replicate (serum + Fgf8b) are shown in separate panels (#070312: yellow, #012512: green, #251012: red, #280711: blue). Each raw trace (black) was …
(A, D) Graphical representation of the full-length Ntla and Tbx16 proteins with exons depicted in different colors. Blue arrows show the predicted T-box domain from amino acid 35 to 212, and 31 to …
(A) z-stack projection showing the expression patterns of Ntla (green) and Tbx16 (red) protein in a 12-somite stage wild type embryo detected using immunohistochemistry with monoclonal antibodies …
(A) Histogram of the number of peaks observed in each cell in the presence of serum + Fgf8b (blue) compared to those from cells in the presence of serum alone (orange). (B) Histogram of periods from …
(Top row) An example 40x transmitted light field of a single, isolated tailbud cell in a 96-well plate well. Scale bar = 20 μm. The cell’s corresponding fluorescence time series at the right shows …
(A) Representative background-subtracted traces displaying different oscillatory behaviors: persistent oscillations, oscillations that initiate, stop, or start and stop within the recording time of …
(A) Definition of averages Ai* (blue dot) of a quantity A measured in consecutive cycles (red square), and the distance Di (green line) to the identity (grey line), employed in panels (B) and (C). (B…
(A) Numerical simulation of equation (S30), see Supplementary ile 1, with μ = 1, b = 1, ω = 2π/78 min-1, q = 0, . Signal x(t) oscillates with a constant amplitude and a period T = 78 min. There is …
(A) Amplitude is not affected by additive noise. Data points show the median peak amplitude for 1000 stochastic simulations (S30), see Supplementary file 1, for . Error bars are the 68% confidence …
(A) Quality factor workflow for time series analysis for an example persistent oscillator. Sub-panel 1: Background-subtracted intensity over time trace from a single tailbud cell (black) with phase …
Precision and period calculation for persistent segmentation clock oscillators.
Each set of panels shows, successively, the background-subtracted average YFP intensity levels over time from a single persistently oscillating cell in black; the cosine of the phase calculated from the wavelet transformation in blue; and the autocorrelation function in green. The dashed green curve shows the analytical fit of the autocorrelation. Both period and quality factor can be calculated from this procedure (see Supplementary file 1). This is the complete persistent cell data set, a sub-set of the low-density set, from which the plots of period andquality factor QP in Figure 3B and D are generated.
Precision and period calculation for the tissue-level segmentation clock in the zebrafish embryo.
As for data set supplement 3–1, each set of panels shows, successively, the background-subtracted average YFP intensity levels from a region of posterior PSM tissue in a Looping embryo in black; the cosine of the phase calculated from the wavelet transformation in blue; and the autocorrelation function in green. The dashed green curve shows the analytical fit of the autocorrelation. Both period and quality factor can be calculated from this procedure. The original intensity versus time data comes from Soroldoni et al. (2014). This is the complete dataset from time-lapse data of 24 embryos from which the plot of quality factor QEmbryo in Figure 3B is generated.
Precision and period calculation for persistent circadian clock oscillators.
As for data set supplement 3–1, each set of panels shows, successively, the background-subtracted intensity levels from a single persistently oscillating Per2-Lucifcerase-expressing fibroblast over time in black; the cosine of the phase calculated from the wavelet transformation in blue; and the autocorrelation function in green. The dashed green curve shows the analytical fit of the autocorrelation. Both period and quality factor can be calculated from this procedure. The original intensity versus time data comes from Leise et al. (2012). This is the complete fibroblast dataset from which the plot of quality factor QF in Figure 3D is generated.
Time series length is defined in terms of the number of cycles. The plot shows the quality factor from stochastic simulations for two parameter sets A and B that display QA = 4 and QB = 10 when the …
Field of view containing cell in Figure 1B–C, highlighted by the red arrow. This field contains 18 cells in total, with 9 expressing YFP. We observed 5 cell divisions, the highest number in any …
Field of view corresponding to cell in top row of Figure 1—figure supplement 9. Total duration = 6.2 hr; Time interval = 2.14 min, field size = 205 x 205 μm, Scale bar = 50 μm.
Theory supplement including Stuart-Landau theory with slow fluctuations, quantification of noisy oscillations, and numerical methods.