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Periodic inhibition of Erk activity drives sequential somite segmentation

Nature volume 613, pages 153–159 (2023)Cite this article

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Abstract

Sequential segmentation creates modular body plans of diverse metazoan embryos1,2,3,4. Somitogenesis establishes the segmental pattern of the vertebrate body axis. A molecular segmentation clock in the presomitic mesoderm sets the pace of somite formation4. However, how cells are primed to form a segment boundary at a specific location remains unclear. Here we developed precise reporters for the clock and double-phosphorylated Erk (ppErk) gradient in zebrafish. We show that the Her1–Her7 oscillator drives segmental commitment by periodically lowering ppErk, therefore projecting its oscillation onto the ppErk gradient. Pulsatile inhibition of the ppErk gradient can fully substitute for the role of the clock, and kinematic clock waves are dispensable for sequential segmentation. The clock functions upstream of ppErk, which in turn enables neighbouring cells to discretely establish somite boundaries in zebrafish5. Molecularly divergent clocks and morphogen gradients were identified in sequentially segmenting species3,4,6,7,8. Our findings imply that versatile clocks may establish sequential segmentation in diverse species provided that they inhibit gradients.

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Fig. 1: Segmentation clock driven dynamics of the ppErk gradient.
Fig. 2: Global periodic inhibition of Fgf signalling drives sequential segmentation of somites in the absence of the clock.
Fig. 3: Drug-induced somites and the COG model for somitogenesis.
Fig. 4: The segmentation clock discretizes the positional information.

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Data availability

The original microscopy image files are provided at BioStudies under accession number S-BSST895. Source data are provided with this paper.

Code availability

MATLAB codes and FIJI macros are provided at GitHub and Zenodo (https://github.com/mfsimsek/erkactivitysegmentation; https://doi.org/10.5281/zenodo.7098199).

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Acknowledgements

We thank I. Ejikeme, H. Seawall, M. Batie, M. Kofron, the staff at Cincinnati Children’s Imaging Core and Cincinnati Children’s Veterinary Services for technical assistance; S. Knierer for help in generating transgenic clock reporter line; K. Keseroglu, B. Dulal, S. Zimik, C. McDaniel, S. Brown, L. Holland, N. Holland, D. Tautz and A. Sarrazin for discussions; and H. Seawall, R. Kopan, A. Zorn, B. Gebelein, H. Meijer and K. Dale for providing feedback on the manuscript. This work was funded by a US NIH (Eunice Kennedy Shriver National Institute of Child Health and Human Development) grant (R01HD103623) to E.M.Ö.

Author information

Authors and Affiliations

  1. Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA

    M. Fethullah Simsek, Angad Singh Chandel, Didar Saparov, Oriana Q. H. Zinani & Ertuğrul M. Özbudak

  2. Systems Biology and Physiology Graduate Program, University of Cincinnati College of Medicine, Cincinnati, OH, USA

    Angad Singh Chandel

  3. Molecular and Developmental Biology Graduate Program, University of Cincinnati College of Medicine, Cincinnati, OH, USA

    Didar Saparov & Oriana Q. H. Zinani

  4. College of Engineering and Applied Sciences, University of Cincinnati, Cincinnati, OH, USA

    Nicholas Clason

  5. Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, USA

    Ertuğrul M. Özbudak

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Contributions

M.F.S. and E.M.Ö. conceived the project. E.M.Ö. designed and supervised the project. M.F.S. administered the project. M.F.S., D.S., A.S.C. and O.Z. performed experiments. M.F.S., D.S. and A.S.C. analysed the data. A.S.C. generated the transgenic Erk activity reporter fish. M.F.S. and A.S.C. wrote the codes for analysis. M.F.S. and N.C. performed the simulations. M.F.S. and E.M.Ö. wrote the manuscript. M.F.S., A.S.C., O.Z. and E.M.Ö. edited the manuscript.

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Correspondence to M. Fethullah Simsek or Ertuğrul M. Özbudak.

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Extended data figures and tables

Extended Data Fig. 1 The segmentation clock drives oscillatory gradient dynamics in wild-type embryos by lowering ppErk levels.

a, Wild-type/heterozygous siblings (with proper segmentation, right) are collected alongside Df(Chr05:her1,her7)b567/b567 clock-deficient embryos (in which somite segmentation fails, left). b-e, Grouping of a single somite stage into three phases according to PSM sizes of clock mutants (b) and their siblings (d). Average PSM sizes (mean±s.d.) of each group of clock mutants (c, n = 19, 20, and 19 embryos over 3 independent experiments respectively) and their clock-intact siblings (e, n = 19, 21, and 20 embryos over 3 independent experiments respectively) for data presented in Fig. 1c. f, her1 expression was ubiquitously driven for various durations from 10 to 60 min. All embryos were simultaneously fixed right after heatshock at 16 somite stage. Three controls were also fixed: Not heatshocked transgenic siblings, not heatshocked wild-type siblings, and 60 min heatshocked wild-type siblings. g, ppErk gradient quantified along the PSM throughout heatshock experiments (mean±s.e.m.) for data presented in Fig. 1g. h, Grouping of each somite stage into two phases according to PSM sizes (tail grows from dark to light colours) for wild-type embryos of three consecutive somite stages (3 somites, blue, 4 somites, purple, 5 somites, orange). i, Average PSM sizes (mean±s.d., n = 12, 9, and 7 embryos over 2 independent experiments for three consecutive stages respectively) of embryos for each phase. j, ppErk levels, aligned from anterior (lab frame), throughout the PSM for these groupings (n = 24, 17, and 14 embryos over 2 independent experiments for 3, 4, and 5 somite stage respectively, mean±s.e.m). k-m, Gradient amplitudes (red, k) and ppErk levels in cells located at pPSM (x = 175 µm, green, l) and mid-PSM (x = 350 µm, black, m) from data in (j). The amplitude of ppErk gradient (k) oscillates within each somite stage (28% average change, p = 0.0037, 0.0076, and 0.035 respectively for the 3, 4 and 5 somite stages). ppErk levels similarly oscillate in the pPSM cells (l, p = 0.035, 0.034, 0.029 for consecutive stages), and decays with a two-speed trend in the mid-PSM cells (m). mean±s.e.m. Posterior is left.

Source data

Extended Data Fig. 2 Erk-Ktr and Her7-Venus reporters faithfully recapitulate dynamics of Erk activity and segmentation clock.

a, Design of the Erk activity reporter line. Orange domains are peptide sequences from Elk1 protein for Erk binding. Teal domains are phospho-acceptor sites at the end of docking site and in NLS sequence. Amino acid sites taking part in NLS and NES functions are coloured according to their chemistry. b, Erk activity gradient along the PSM calculated from live Erk-Ktr reporter (black, N = 4, n = 6 embryos, mean±s.e.m.) or IHC of ppErk (green, n = 10 embryos, mean±s.e.m.) from 14 somites stage embryos. c, Time course response of Erk activity reporter imaged every 250 s before and after 50 µM SU5402 treatment (n = 11 embryos over 4 independent experiments, mean±s.e.m.). Erk activity is measured within a 61 µm diameter circular ROI in pPSM tissue next to notochord tip. Data is normalized (red dashed line) to average of the 6 frames (~25 min) taken in DMSO before addition of the drug after 0 min. Red solid line is exponential decay fit with 90% C.I. p = 0.0135 between before treatment and 3rd time point into the treatment, Brown-Forsythe and Welch ANOVA tests with Dunnett’s T3 correction for multiple comparisons. d, Design of the Tg(her7:her7-Venus) reporter line. All introns (red) and UTR regions (empty boxes) of her7 locus are retained with exons (orange). mVenus (cyan) is inserted upstream of the stop codon and 3’UTR. e, In-situ hybridization (ISH) against Venus RNA in the her7:her7-Venus reporter line. Scale bar is 200 µm. f, Tail bud aligned (left) kymograph of cell nuclei masked Venus signal observed in live intact embryos from 14 to 24 somite stages (N = 3, n = 3 embryos). g-l, 3-D projection (14 µm, 60 z-slices) smFISH images for exemplary heterozygous (g-i) and homozygous (j-l) transgenic fish showing co-expression of endogenous her1 (red) and her7-Venus (cyan) transcripts in the flat mounted 12 somite stage embryo PSM tissues, overlaid with nuclei marker (grey). Posterior is left. Scale bar is 100 µm.

Source data

Extended Data Fig. 3 Single cell tracking of segmentation clock and Erk activity.

a, Single cell tracks of Erk activity in a clock-intact embryo shown together with their average (magenta, mean±s.e.m.) (n = 42 cells). b, c, Average (mean±s.e.m.) clock (b) and Erk activity (c) signal for cell tracks within each clock-intact embryo presented in Fig. 1j, m (n = 47, 58, 35, 36, 22, 22, 42, and 9 cells for embryos #1–8 resp.). d, Single cell tracks of Erk activity in a clock mutant shown together with their average (black, mean±s.e.m.) (n = 48 cells). e, Average (mean±s.e.m.) dynamics of the Erk activity for cell tracks within each clock mutant presented in Fig. 1l (n = 32, 36, 23, 48, 32, 29, and 42 cells for embryos #1–7 resp.).

Source data

Extended Data Fig. 4 ppErk gradient oscillates in the PSM correlated with clock expression.

a-b, Kymographs of IHC data of Her7-Venus (a) and ppErk (b) from 16 somite stage embryos (n = 47) ordered according to their clock phases. c, Same stage embryos can be grouped into three phases according to their clock expression along the PSM: U-domain (Phase I, orange), mid-PSM stripe (Phase II, purple), and aPSM stripe (Phase III, black). d, Representative pictures of Her7-Venus (magenta) and ppErk (green) for three phases of clock expression (N = 2). Scale bar is 100 µm. e-f, Nuclear density profiles of Her7-Venus (e) and ppErk (f) along the PSM for each group at 16 somite stage embryos (U-domain, Phase I, n = 14; mid-PSM, Phase II, n = 15, and aPSM, Phase III, n = 19). p = 0.0326 (Welch’s two-tailed t-test) between U-domain and mid-PSM ppErk peaks. g, Kymographs of Her7-Venus (top) and ppErk (bottom) for three phases of 16 somite stage IHC data (n = 17, 16, and 17 embryos over 2 independent experiments). h-i, Nuclear density profiles of Her7-Venus (h) and ppErk (i) along the PSM for each group at 17 somite stage embryos (U-domain, Phase I, n = 13; mid-PSM, Phase II, n = 14, and aPSM, Phase III, n = 13). p = 0.0148 (Welch’s two-tailed t-test) between U-domain and mid-PSM ppErk peaks. Lines and shaded error bars indicate mean±s.e.m. in (e,f,h,i). Posterior is left. Note that ppErk level is higher when the PSM size is the smallest in Fig. 1c while it is lower in the corresponding phase in this figure. This difference is due to the change of clock phase profiles over different somite stages as shown in46.

Source data

Extended Data Fig. 5 Simulations suggest pulsatile inhibition of ppErk signalling can imitate the endogenous clock.

a, Simulation predictions for ppErk dynamics (plotted as kymographs in lab frame) for global inhibition experiments: Various drug concentrations (outer y-axis) and treatment/washout durations (outer x-axis) are simulated. Clock mutant (absent, b) and clock-intact (kinematic waves, c) simulations are also presented for comparison. Posterior is left.

Extended Data Fig. 6 Pulsatile inhibition of ppErk activity simulates the effect of the clock.

a, Sizes of 18th to 28th somites measured from DIC images of 12-pulse experiments (box (median and interquartile range) and whisker (10th – 90th percentile) plot): induced somites in clock mutants treated with SU5402 (red, n = 23 embryos over 3 independent experiments) and wild-type embryos treated with DMSO (black, n = 17 embryos over 2 independent experiments). p = 0.0544 – 0.9929 (n.s.) for all induced somites except the first one (p < 0.0001 for the 18th somite, 2-way ANOVA with Tukey’s test for multiple comparison). b, SU5402 (5 pulses) increased sizes of 17th – 22nd somites in clock-intact embryos (n = 19 for DMSO, n = 26 for SU5402, p = 0.0034 for 18th somite and p < 0.0001 for 19th, 20th, and 21st somites, 2-way ANOVA with Tukey’s test for multiple comparison; box (median and interquartile range) and whisker (10th – 90th percentile) plot). c, Time-lapse images taken every 45 min beginning from the appearance of first induced boundary at T = 140 min with first drug pulse (n = 36 embryos over 2 independent experiments). Induced boundaries are highlighted with green arrows (right). DMSO treated clock mutants (n = 25 embryos over 2 independent experiments) could not induce any boundaries (left). Lateral view. Note that there is a 3-somite delay from first pulse to first affected boundary in (b) and (c). d, Representative xirp2a boundary staining of clock-deficient mutant (top) or clock-intact sibling (bottom) embryos treated with same concentration (30 µM) SU5402 beginning at 12 somite stage for 225 min at 28 °C. Scale bar is 200 µm. e, At the end of 225 min 30 µM SU5402 treatment, clock intact embryos displayed bi-modal distribution of Erk activity (n = 19 embryos in Group I, magenta, and n = 17 embryos in Group II, red over 2 independent experiments). Approximately half of SU5402 treated sibling embryos could not survive to later stages for xirp2 staining, while the other half formed some segments before onset of axis truncation, indicating necessity of Erk activity gradient for survival and segmentation. f, Induced somite sizes for changing concentrations and durations of drug treatments performed at room temperature, for five pulses; measured from xirp2a boundary staining (box (median and interquartile range) and whisker (10th – 90th percentile) plot and outliers as individual dots, n = 59, 88, 96 and 84 induced somites from left to right respectively). p > 0.1998 for n.s. comparisons, p = 0.0007 between 30 µM, 10 min treatment and 40 µM, 6 min treatment and p < 0.0001 for other comparisons (Kruskal-Wallis ANOVA test with Benjamini-Krieger-Yekutieli false discovery rate multiple comparison correction). g, MEK inhibitor drug PD184352 directly targets Erk activity downstream of Fgf receptor. h, Various durations of 5 pulses of PD184352 (600 nM) can induce somite boundaries (scored by xirp2a staining from left to right, n = 58, 36, 44, 52, 54, 44 embryos over 3 independent experiments, in comparison to SU5402 treatment in Fig. 2j; n = 170 embryos). Truncated violin plots with solid median and dashed quartile lines. p = 0.0002 for DMSO vs. 10’-20’ treatment, p < 0.0001 for DMSO vs. other comparisons, and p > 0.3312 for SU5402 vs. PD184352 comparisons (Kruskal-Wallis ANOVA test with Benjamini-Krieger-Yekutieli false discovery rate multiple comparison correction). i, ppErk gradient along the PSM with 10’-40’ PD184352 pulses (n = 24, 24, and 24 embryos over 2 independent experiments, for before pulse, after pulse, and after recovery from the treatment (before next pulse), mean±s.e.m.). Amplitude comparison is shown on the right (10–90th percentile box-whisker plot together with outlier dots, p = 0.0001 for before vs. after pulse, p = 0.8528 for before pulse vs. after washout; Brown-Forsythe ANOVA, unpaired two-tailed test with Welch correction).

Source data

Extended Data Fig. 7 Characterization of somite boundaries following pulsatile drug treatments.

a, xirp2a ISH staining marks somite boundaries of a clock-deficient mutant treated 5× pulses of 30 µM SU5402. A short working distance objective is used to minimize out of plane staining from the other side of embryos. Drug targeted somites are numbered 1–5 from anterior to posterior. b—d, Boundary intactness and deficiency are calculated after light background subtraction and contrast enhancement (b) and local thresholding of the image for binarization (c). Trace LOIs (magenta) running in between the ventral and dorsal ends of somitic tissue are drawn by automated simple neurite tracer algorithm following xirp2a boundary staining (d). Percentage occupancy along the trace LOIs (average signal) identifies intact boundaries and measures deficiency level of non-intact boundaries. e–g, Phosphorylated focal adhesion kinase (pFAK, gem LUT from dark purple to burnt orange) and cell nuclei (cyan) staining of embryos fixed at 23 h after fertilization (4 h after the 5th pulse). Clock-intact siblings treated with 600 nM PD184352 (n = 37 embryos over 2 independent experiments, e), and clock-deficient mutants treated with DMSO (N = 2, n = 13, f) or 600 nM PD184352 (n = 33 embryos over 2 independent experiments, g). Red stars show induced somite boundary epithelization driven by pulsatile treatments. Bottom images are pFAK staining alone shown in grayscale. Scale bars are 100 µm.

Extended Data Fig. 8 Somite segmentation in mutants can be induced within a range of conditions.

a, Simulation of the COG model predicts only a medium dose of inhibitory treatment would result in discretization of positional information, i.e., successfully induce somite boundaries. SFC detection (middle) could extract reliable positional information (right, critical SFC threshold) from an oscillatory ppErk gradient (left) for a certain range of drug concentrations (middle rows). b, Percentage of successfully induced boundaries (# of intact boundaries over # of pulses) is assessed from xirp2a staining by two independent experimenters for various doses of five 10’+30’ PD184352 pulses (DMSO control left, n = 58 embryos; PD184352 from 50 nM up to 20 µM, n = 44, 70, 18, 54, 54, 50, 90, 48, 22, 64 embryos over 3 independent experiments, respectively, truncated violin plot with solid lines indicating quartiles). p = 0.9824, 0.0932, 0.4365, 0.8045, 0.9824, 0.0633, 0.7284, 0.6164, 0.9824, 0.9824 and 0.1502 between experimenters from left to right resp., 2-way ANOVA with Holm-Šídák test for multiple comparisons. c, Number of induced boundaries (data in (b)) for various doses (black, mean±95% C.I.), fitted with a bell-shaped dose response curve (red solid line with 95% C.I.) in logarithmic scale. Vertical dashed line indicates optimal dose for boundary recovery. d, Induced somite sizes for either short (blue, 6’+24’) or long (10’+40’) pulses of 600 nM PD184352 (5×). Somite sizes are measured from xirp2a boundary staining (n = 136 induced somites for long and n = 58 induced somites for short pulses over 2 independent experiments). p < 0.0001, Welch’s two-tailed t-test. Truncated violin plot, median as solid thick line, and scatter plot of all data points. e, IHC quantification of ppErk gradient along the PSM for short PD184352 pulses (n = 25, 23, and 18 embryos over 2 independent experiments, for before pulse, after pulse, and before the next pulse). p = 0.0391 between before pulse and after pulse (Brown-Forsythe ANOVA, unpaired two-tailed test with Welch correction). mean±s.e.m. f, Determination front position (scatter dots with median (red) and interquartile range error bars) in short (left) and long (right) pulse PD184352 treatments, quantified from data in panel (e) and Extended Data Fig. 6i. p = 0.9987 and 0.9939 for before vs. after pulse in short and long pulse treatments respectively. p < 0.0001 for other comparisons (Brown-Forsythe and Welch ANOVA tests with Dunnett’s T3 correction for multiple comparisons). Determination front stalls during drug treatment and shifts during recovery, similar to that in clock-intact embryos in Fig. 4b. g, Design of the experiment for splitting comparable sizes of trunk axes into varying numbers of induced somites with either 8× short or 5× long pulses of MEK inhibition in her1ci301her7hu2526 clock mutants. h, xirp2a staining for successfully induced somites with either small (top, short pulse, n = 50 embryos over 2 independent experiments) or large (bottom, long pulse, n = 59 embryos over 2 independent experiments) sizes in clock mutants. Red stars indicate induced somite boundaries covering same region of the trunk. Posterior is left.

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Extended Data Fig. 9 Kinematic stripes of clock are required for establishment of rostrocaudal polarity in somites and nuclear β-Catenin gradient does not oscillate.

a, Fluorescent in situ hybridization of mespaa with nuclei staining (blue, dorsal view). Clock-intact sibling embryos treated with DMSO (top, n = 14) or SU5402 (second row, n = 9) and clock mutants (bottom rows; n = 14, and n = 13 respectively). Embryos were fixed at the end of 4th drug treatment pulse. b, in situ hybridization of myoD for DMSO (top, n = 15) or SU5402 (middle, n = 22) treated clock mutants as well as DMSO treated clock-intact siblings (bottom, n = 12). Embryos were fixed 2 h after 4th drug pulse. Induced boundary area is shown with red line. c, in situ hybridization of uncx4.1 for DMSO (top, n = 22) or SU5402 (middle, n = 29) treated clock mutants as well as DMSO treated clock-intact siblings (bottom, n = 11). Embryos were fixed 2 h after 5th drug pulse. Induced boundary area is shown with red line. d, Cell nuclei (Hoescht 33682, blue) and immunofluorescence signals from antibodies against β-Catenin (cyan), Her7-Venus (magenta), and ppErk (green) proteins at 16 somite stage (N = 2). e-f, Nuclear density profiles of β-Catenin proteins along the PSM at 16 (e) and 17 (f) somite stage embryos grouped into U-domain (Phase I, n = 13, 16 somite stage; n = 13, 17 somite stage), mid-PSM (Phase II, n = 15, 16 somite stage; n = 14, 17 somite stage), and aPSM (Phase III, n = 19, 16 somite stage; n = 13, 17 somite stage) phases of clock expression. g, Amplitude quantification of nuclear β-Catenin (blue) gradient for three clock phases of 16-17 somite stages (N = 2, n = 26, 29, and 32). p > 0.6594 for all phases (Welch’s two-tailed tests). Lines and shaded error bars indicate mean±s.e.m. All microscopy images except panels (c) and (d) are dorsal views. Images in (c) and (d) are lateral views. Posterior is bottom / left. Black scale bars are 200 µm. White scale bars are 100 µm.

Source data

Extended Data Fig. 10 Alternative signal readout models fail to explain segmental commitment whereas COG model fits observed results.

a, Temporal fold change of ppErk signal throughout the PSM, calculated for clock-intact her7-Venus reporter fish (magenta; from Extended Data Fig. 4f, Phase II–III transition) or SU5402 pulse treated clock-deficient mutants (red; from Fig. 2f, Before Pulse – After Pulse transition) at same stage. Determination front position in clock-intact embryos is indicated with cyan arrow. b-c, Slope of ppErk gradient (reported as ppErk signal difference over a cell distance) for three clock phases in her7-Venus reporter embryos (b; from Extended Data Fig. 4f) or SU5402 pulse treated clock-deficient mutants (c; from Fig. 2f). Data is shown as mean±s.e.m. (d) Simulations of COG model with varying drug parameter (light blue, right axis) fit to experimental data for PD184352 dose optimization in Extended Data Fig. 8c (mean±95% C.I.). (e) Experimental doses used in Extended Data Fig. 8c are converted into drug efficacy from the simulation fit in (d). Fit parameters are provided in Supplementary Table 1. (f) ppErk gradient amplitude oscillations driven by varying drug concentrations. Green zone highlights (26%-65%) amplitude oscillations for the doses succeeding to drive somite segmentation in (d). (g) ppErk Amplitude changes from the simulation of short pulse (6’+24’, blue) 600 nM PD184352 treatment (hollow circles, 35% drop) matching with experimental data in Extended Data Fig. 8e (mean±s.e.m., normalized to before pulse). (h) Positional information (critical SFC of ppErk, 22%) kymograph (grey below, white above threshold) for short pulse simulations. Pink stripes highlight 6 min drug treatment pulses. Determination front makes 31.4 µm shifts every 30 min. (i) Determination front dynamics from the simulations overlaid with the experimental data in Extended Data Fig. 8f (blue, median, error bars are interquartile range). (j) ppErk Amplitude changes from the simulation of long pulse (10’+40’, blue) 600 nM PD184352 treatment (hollow circles, 48% drop) matching with experimental data in Extended Data Fig. 6i (mean±s.e.m., normalized to before pulse). (k) Positional information kymograph for long pulse simulations. Pink stripes highlight 10 min drug treatment pulses. Determination front makes 49.8 µm shifts every 50 min. (l) Determination front dynamics from the simulations overlaid with the experimental data in Extended Data Fig. 8f (green, median, error bars are interquartile range).

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Supplementary Methods; Supplementary Discussion 1–3 and Supplementary Table 1.

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Supplementary Video 1

Simultaneous imaging of the clock and Erk activity dynamics in live embryos. A laterally mounted dual-reporter embryo is observed from the 11 somite stage for 210 min at 25.0 ± 0.7 °C in four fluorescence channels: cell membrane (mCherry–CAAX, red, top left), nuclei (H2B–iRFP, cyan, top right), Erk–Ktr–mTagBFP (yellow, middle left), Her7–mVenus (magenta, middle right); and bright-field channel (bottom right). Erk activity reconstructed from single-cell segmentation of Erk–Ktr data is shown at the bottom left. Scale bar, 100 µm. The time interval is 6 min. The video is 6 fps.

Supplementary Video 2

Live tracking of PSM cells from the determination front to somite formation. A laterally mounted Erk–Ktr reporter embryo is observed from the 13 somite stage for 408 min in three fluorescence channels (cell nuclei, H2B–irFP, cyan, top left; cell membrane, mCherry–CAAX; magenta, bottom left). Erk activity is reported on top right and SFC of Erk activity is shown at the bottom right. Four boundaries (cyan, green, yellow and light grey shaded lines) are tracked on membrane channel (bottom left) from determination (arrows, bottom right) until somite formation. Scale bar, 50 µm. The time interval is 6 min. The video is 5 fps.

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Simsek, M.F., Chandel, A.S., Saparov, D. et al. Periodic inhibition of Erk activity drives sequential somite segmentation. Nature 613, 153–159 (2023). https://doi.org/10.1038/s41586-022-05527-x

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