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. 2016 Feb 13;5:e08438. doi: 10.7554/eLife.08438

Figure 1. Zebrafish segmentation clock cells oscillate autonomously in culture.

(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 in green. Scale bar = 50 μm. Kupffer’s vesicle (Kv), notochord (Nc), presomitic mesoderm (PSM), tailbud (Tb). (B) A representative 40x transmitted light field with dispersed low-density Looping tailbud-derived cells. Individual cells highlighted with black arrowheads; green arrowhead shows cell with green time series in (D). Scale bar = 10 μm. Pie chart: More than half of the in vitro population of Looping tailbud cells (n = 321 out of 547 cells combined from 4 independent culture replicates as described in Materials and methods) expresses the Her1-YFP reporter. Some expressing cells are disqualified because they move out of the field of view (4%), touch other cells by colliding in the field of view (12%) or following division (2%) for a total of 14%, or do not survive until the end of the 10-hr recording (7%). (C) Montage of timelapse images (transmitted light, top; YFP, bottom) of a single tailbud cell (green arrowhead in panel B) over 10-hr recording. Scale bar = 10 μm. (D) YFP signal intensity (arbitrary units) measured by tracking a regions of interest over 3 single tailbud cells (green trace follows cell marked by green arrowhead in B, gray traces are two additional cells from culture). Plotted in 2-min intervals. (E) Plot of Her1-YFP (black) and H2A-mCherry (red) signal intensity over time measured together from a representative cell. (EI). Nuclear YFP signal accumulates and degrades over time, as shown in the overlay of H2A-mcherry signal (red channel) and Her1-YFP signal (green channel) during troughs (297, 372) and peaks (342, 402) in Her1 expression. mCherry signal in the nucleus is relatively constant. Plotted in 2-min intervals. (F) Plot of YFP intensity (a.u.) over time in a fully isolated tailbud cell within a single well of a 96-well plate. Plotted in 2-min intervals.

DOI: http://dx.doi.org/10.7554/eLife.08438.003

Figure 1—source data 1. 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.
DOI: http://dx.doi.org/10.7554/eLife.08438.004
elife-08438-fig1-data1.docx (119.2KB, docx)
DOI: 10.7554/eLife.08438.004
Figure 1—source data 2. 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).
DOI: http://dx.doi.org/10.7554/eLife.08438.005
elife-08438-fig1-data2.docx (91KB, docx)
DOI: 10.7554/eLife.08438.005
Figure 1—source data 3. 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.
DOI: http://dx.doi.org/10.7554/eLife.08438.006
elife-08438-fig1-data3.xlsx (1.6MB, xlsx)
DOI: 10.7554/eLife.08438.006

Figure 1.

Figure 1—figure supplement 1. Her1-YFP-expressing cells in the zebrafish tailbud.

Figure 1—figure supplement 1.

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 the segmentation clock cells removed by surgery to generate the tailbud explants and single cell cultures is shown with the dashed line. Nuclei are shown in red and YFP in green. Scale bar = 50 μm. Kupffer’s vesicle (Kv), notochord (Nc), presomitic mesoderm (PSM), tailbud (Tb), neural progenitors (Np), yolk cell (yolk).
DOI: http://dx.doi.org/10.7554/eLife.08438.007
Figure 1—figure supplement 2. Peak finding in time series to estimate period and amplitude.

Figure 1—figure supplement 2.

(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 (grey line). Middle: peak finding (red triangles) and filtering (blue triangles) from the smoothed signal (thin blue line) of the background subtracted trace (red line). Bottom: resulting peaks (blue dots) and troughs (green dots). (B) Definition of period T as inter-peak interval (orange double arrow), and amplitude A as the average of peak heights relative to the trough (green double arrows).
DOI: http://dx.doi.org/10.7554/eLife.08438.008
Figure 1—figure supplement 3. Persistent oscillations in explanted tailbud.

Figure 1—figure supplement 3.

(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 projection of the entire stack. Oscillations in the tailbud were measured using a region of interest (ROI; black circle) to extract average YFP intensity values over time. We placed this region on the most central “tailbud” area, as we observed “PSM”-like protrusions (black arrowheads) emerging from the explant. These areas, as expected for PSM, showed brighter and increasing YFP expression over time, which then switched off. (B) Intensity over time plot for the ROI shown in A. The tailbud area shows 9 cycles over the recording with no slowing of the period. Peak finding was performed as in Figure 1—figure supplement 2: background subtracted raw data (thick red line) was smoothed (thin blue line) and peaks were identified (red down triangles).
DOI: http://dx.doi.org/10.7554/eLife.08438.009
Figure 1—figure supplement 4. Time series of low-density segmentation clock cells in serum-only culture.

Figure 1—figure supplement 4.

(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 curve (red) was used for peak counting and identification of peaks and troughs (blue circles). Details of smoothing and peak finding are given in Materials and methods and Figure 1—figure supplement 2. (B) As above for experiment 250112. Corresponding cells grown in the presence of serum + Fgf8b from the same tailbud cell suspensions in this figure are found in Figure 1—figure supplement 5.
DOI: http://dx.doi.org/10.7554/eLife.08438.010
Figure 1—figure supplement 5. Time series of low-density segmentation clock cells.

Figure 1—figure supplement 5.

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 smoothed (red) and peaks and troughs were identified (blue circles). This is the complete low-density cell data set from which representative examples shown in Figure 1B–D are chosen. Details of smoothing and peak finding are given in Materials and methods and Figure 1—figure supplement 2.
DOI: http://dx.doi.org/10.7554/eLife.08438.011
Figure 1—figure supplement 6. Characterization of Ntla and Tbx16 antibodies.

Figure 1—figure supplement 6.

(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 213, for Ntla and Tbx16 respectively. The Ntla antibody (clone D18-4, IgG1) was generated using a peptide from amino acid 1 to 261, while the corresponding sequence for the Tbx16 antibody (clone C24-1, IgG2a) spans the region from amino acid 232 to 405 (bracketed in red in both cases). (B, E) Representative examples showing ntla mRNA and protein expression patterns in wild type and in ntla mutant embryos at 90% epiboly. Immunolabeling using the Ntla antibody was followed by in situ hybridization using a ntla riboprobe. The same procedure was used to characterize the Tbx16 antibody except that wild type embryos are compared to tbx16 morpholino-injected embryos and a tbx16 riboprobe was used. Scale bar = 150 μm. (C, F) Immunolabeling of wild type embryos injected at 1-cell-stage with capped mRNAs (Ntla-T2A-mKate2CAAX, Tbx6-T2AmKate2CAAX, Tbx6l-T2A-mKate2CAAX or Tbx16-T2A-mKate2CAAX), fixed at 4 hr post fertilization and imaged at the animal pole where the endogenous genes are not expressed. Both Ntla (C) and Tbx16 (F) antibodies bind only in embryos injected with ntla and tbx16 mRNA respectively, demonstrating antibody specificity. Scale bar = 20 μm.
DOI: http://dx.doi.org/10.7554/eLife.08438.012
Figure 1—figure supplement 7. Expression of tailbud markers in vivo and in low-density cultures of segmentation clock cells.

Figure 1—figure supplement 7.

(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 D18-4 (IgG1) to Ntla and C24-1 (IgG2a) to Tbx16. Scale bar = 120 μm (B-C) Close up view of the boxed area in (A) showing a single confocal section at dorsal (B) and ventral (C) locations in the tailbud. Scale bar = 60 μm. (D) Representative panels showing expression of Ntla (green) and Tbx16 (red) in single cells within low-density tailbud cultures (serum + Fgf8b) after 5 hr in vitro. Nuclei are labeled with DAPI (blue). Cells single-positive for Ntla and Tbx16 are visible, as are cells co-expressing both proteins. Scale bar = 20 μm. (E) Quantification of nuclear fluorescence intensity of experiment in (D) showing populations of Ntla-positive, Tbx16-positive, and Ntla/Tbx16 co-expressing cells.
DOI: http://dx.doi.org/10.7554/eLife.08438.013
Figure 1—figure supplement 8. Analysis of low-density segmentation clock cell cultures.

Figure 1—figure supplement 8.

(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 all measured cycles for low-density cells in the presence of serum + Fgf8b (blue) compared to those from cells in the presence of serum alone (orange). (C) Histogram of amplitudes from all measured cycles for low-density serum + Fgf8b data set. See Figure 1—source data 1 for statistics. Average amplitude (D) and period (E) plotted vs. time intervals for all cycles in the four different serum + Fgf8b low-density experiments (rows). Data is grouped in bins of 100 min, error bars show variance.
DOI: http://dx.doi.org/10.7554/eLife.08438.014
Figure 1—figure supplement 9. Time series of fully isolated segmentation clock cells.

Figure 1—figure supplement 9.

(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 raw YFP intensity over time (black), smoothed signal (red), and automatically detected peaks (green arrowheads). Cells were obtained from N = 5 independent replicates. From 43 YFP-expressing cells, 33 were disqualified due to death, division or movement from imaging field, leaving n = 10 for analysis. (Bottom panels) As above, each plot shows the background-subtracted average YFP intensity levels from a single cell over time (black), smoothed signal (red), and peaks (green arrowheads) for the remaining 9 fully isolated segmentation clock cells. Peak finding was first performed as in Figure supplement 1–2. Due to the higher noise levels in the fully isolated segmentation clock cells, we modified the parameters of the algorithm to be less stringent, and we introduced an additional step of curation to remove the detected peaks that had very low amplitude and were considered spurious.
DOI: http://dx.doi.org/10.7554/eLife.08438.015