Individual cellular activities fluctuate but are constantly coordinated at the population level via cell–cell coupling. A notable example is the somite segmentation clock, in which the expression of clock genes (such as Hes7) oscillates in synchrony between the cells that comprise the presomitic mesoderm (PSM)1,2. This synchronization depends on the Notch signalling pathway; inhibiting this pathway desynchronizes oscillations, leading to somite fusion3,4,5,6,7. However, how Notch signalling regulates the synchronicity of HES7 oscillations is unknown. Here we establish a live-imaging system using a new fluorescent reporter (Achilles), which we fuse with HES7 to monitor synchronous oscillations in HES7 expression in the mouse PSM at a single-cell resolution. Wild-type cells can rapidly correct for phase fluctuations in HES7 oscillations, whereas the absence of the Notch modulator gene lunatic fringe (Lfng) leads to a loss of synchrony between PSM cells. Furthermore, HES7 oscillations are severely dampened in individual cells of Lfng-null PSM. However, when Lfng-null PSM cells were completely dissociated, the amplitude and periodicity of HES7 oscillations were almost normal, which suggests that LFNG is involved mostly in cell–cell coupling. Mixed cultures of control and Lfng-null PSM cells, and an optogenetic Notch signalling reporter assay, revealed that LFNG delays the signal-sending process of intercellular Notch signalling transmission. These results—together with mathematical modelling—raised the possibility that Lfng-null PSM cells shorten the coupling delay, thereby approaching a condition known as the oscillation or amplitude death of coupled oscillators8. Indeed, a small compound that lengthens the coupling delay partially rescues the amplitude and synchrony of HES7 oscillations in Lfng-null PSM cells. Our study reveals a delay control mechanism of the oscillatory networks involved in somite segmentation, and indicates that intercellular coupling with the correct delay is essential for synchronized oscillation.
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The nucleotide sequence for Achilles cDNA has been deposited in the DDBJ/EMBL/GenBank under the accession number LC381432. Raw data for Achilles and all the other experiments are available on request from A.M. and the corresponding author, respectively. Correspondence and requests for materials should be addressed to A.M. (email@example.com) for Achilles cDNA and R.K. (firstname.lastname@example.org) for other materials.
Image processing and analysis were performed using Fiji (v.1.0) and Matlab (R2018a). Subsequent analysis was performed using custom Matlab scripts. The codes are available upon request from the corresponding author.
Hubaud, A. & Pourquié, O. Signalling dynamics in vertebrate segmentation. Nat. Rev. Mol. Cell Biol. 15, 709–721 (2014).
Oates, A. C., Morelli, L. G. & Ares, S. Patterning embryos with oscillations: structure, function and dynamics of the vertebrate segmentation clock. Development 139, 625–639 (2012).
Jiang, Y.-J. et al. Notch signalling and the synchronization of the somite segmentation clock. Nature 408, 475–479 (2000).
Riedel-Kruse, I. H., Müller, C. & Oates, A. C. Synchrony dynamics during initiation, failure, and rescue of the segmentation clock. Science 317, 1911–1915 (2007).
Mara, A., Schroeder, J., Chalouni, C. & Holley, S. A. Priming, initiation and synchronization of the segmentation clock by deltaD and deltaC. Nat. Cell Biol. 9, 523–530 (2007).
Özbudak, E. M. & Lewis, J. Notch signalling synchronizes the zebrafish segmentation clock but is not needed to create somite boundaries. PLoS Genet. 4, e15 (2008).
Delaune, E. A., François, P., Shih, N. P. & Amacher, S. L. Single-cell-resolution imaging of the impact of Notch signaling and mitosis on segmentation clock dynamics. Dev. Cell 23, 995–1005 (2012).
Ramana Reddy, D. V., Sen, A. & Johnston, G. L. Time delay induced death in coupled limit cycle oscillators. Phys. Rev. Lett. 80, 5109–5112 (1998).
Bessho, Y. et al. Dynamic expression and essential functions of Hes7 in somite segmentation. Genes Dev. 15, 2642–2647 (2001).
Sparrow, D. B., Guillén-Navarro, E., Fatkin, D. & Dunwoodie, S. L. Mutation of Hairy-and-Enhancer-of-Split-7 in humans causes spondylocostal dysostosis. Hum. Mol. Genet. 17, 3761–3766 (2008).
Shimojo, H. et al. Oscillatory control of Delta-like1 in cell interactions regulates dynamic gene expression and tissue morphogenesis. Genes Dev. 30, 102–116 (2016).
Isomura, A., Ogushi, F., Kori, H. & Kageyama, R. Optogenetic perturbation and bioluminescence imaging to analyze cell-to-cell transfer of oscillatory information. Genes Dev. 31, 524–535 (2017).
Moloney, D. J. et al. Fringe is a glycosyltransferase that modifies Notch. Nature 406, 369–375 (2000).
Evrard, Y. A., Lun, Y., Aulehla, A., Gan, L. & Johnson, R. L. lunatic fringe is an essential mediator of somite segmentation and patterning. Nature 394, 377–381 (1998).
Zhang, N. & Gridley, T. Defects in somite formation in lunatic fringe-deficient mice. Nature 394, 374–377 (1998).
Niwa, Y. et al. Different types of oscillations in Notch and Fgf signaling regulate the spatiotemporal periodicity of somitogenesis. Genes Dev. 25, 1115–1120 (2011).
Okubo, Y. et al. Lfng regulates the synchronized oscillation of the mouse segmentation clock via trans-repression of Notch signalling. Nat. Commun. 3, 1141 (2012).
Nagai, T. et al. A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat. Biotechnol. 20, 87–90 (2002).
Hirata, H. et al. Instability of Hes7 protein is crucial for the somite segmentation clock. Nat. Genet. 36, 750–754 (2004).
Lauschke, V. M., Tsiairis, C. D., François, P. & Aulehla, A. Scaling of embryonic patterning based on phase-gradient encoding. Nature 493, 101–105 (2013).
Pikovsky, A., Rosenblum, A. & Kurths, J. Synchronization (Cambridge Univ. Press, 2001).
Hubaud, A., Regev, I., Mahadevan, L. & Pourquié, O. Excitable dynamics and Yap-dependent mechanical cues drive the segmentation clock. Cell 171, 668–682.e11 (2017).
Harima, Y., Takashima, Y., Ueda, Y., Ohtsuka, T. & Kageyama, R. Accelerating the tempo of the segmentation clock by reducing the number of introns in the Hes7 gene. Cell Reports 3, 1–7 (2013).
Lewis, J. Autoinhibition with transcriptional delay: a simple mechanism for the zebrafish somitogenesis oscillator. Curr. Biol. 13, 1398–1408 (2003).
Morelli, L. G. et al. Delayed coupling theory of vertebrate segmentation. HFSP J. 3, 55–66 (2009).
Morimoto, M., Takahashi, Y., Endo, M. & Saga, Y. The Mesp2 transcription factor establishes segmental borders by suppressing Notch activity. Nature 435, 354–359 (2005).
Huppert, S. S., Ilagan, M. X., De Strooper, B. & Kopan, R. Analysis of Notch function in presomitic mesoderm suggests a γ-secretase-independent role for presenilins in somite differentiation. Dev. Cell 8, 677–688 (2005).
Williams, D. R., Shifley, E. T., Braunreiter, K. M. & Cole, S. E. Disruption of somitogenesis by a novel dominant allele of Lfng suggests important roles for protein processing and secretion. Development 143, 822–830 (2016).
Serth, K., Schuster-Gossler, K., Cordes, R. & Gossler, A. Transcriptional oscillation of lunatic fringe is essential for somitogenesis. Genes Dev. 17, 912–925 (2003).
Matsumiya, M., Tomita, T., Yoshioka-Kobayashi, K., Isomura, A. & Kageyama, R. ES cell-derived presomitic mesoderm-like tissues for analysis of synchronized oscillations in the segmentation clock. Development 145, dev156836 (2018).
Shimizu, Y. et al. Cell-free translation reconstituted with purified components. Nat. Biotechnol. 19, 751–755 (2001).
Takashima, Y., Ohtsuka, T., González, A., Miyachi, H. & Kageyama, R. Intronic delay is essential for oscillatory expression in the segmentation clock. Proc. Natl Acad. Sci. USA 108, 3300–3305 (2011).
Abe, T. et al. Establishment of conditional reporter mouse lines at ROSA26 locus for live cell imaging. Genesis 49, 579–590 (2011).
Tinevez, J. Y. et al. TrackMate: an open and extensible platform for single-particle tracking. Methods 115, 80–90 (2017).
Webb, A. B. et al. Persistence, period and precision of autonomous cellular oscillators from the zebrafish segmentation clock. eLife 5, e08438 (2016).
Isomura, A. & Kageyama, R. An optogenetic method to control and analyze gene expression patterns in cell-to-cell interactions. J. Vis. Exp. 133, e57149 (2018).
We thank C. Vissers for critical reading, R. Sueda for technical help, F. Ishidate for live imaging, H. Miyachi for the generation of transgenic mice and M. Uesugi for providing KY02111. This work was supported by Core Research for Evolutional Science and Technology (JPMJCR12W2 to R.K.), Precursory Research for Embryonic Science and Technology (to A.I.), Grant-in-Aid for Scientific Research on Innovative Areas (Ministry of Education, Culture, Sports, Science, and Technology (MEXT), Japan (16H06480 to R.K.; 18H04734 to A.I.; 15H05876 to H.K.)) and the Brain Mapping by Integrated Neurotechnologies for Disease Studies (Brain/MINDS) (JP19dm0207001 to A.M.).
The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
a, pHes7-UbLuc imaging in wild-type and Lfng-knockout PSM. Spatiotemporal patterns along the anterior–posterior axis are shown. Top is anterior. b, Period of Hes7 oscillations in the anterior and posterior PSM (n = 4 PSM samples). c, Amplitude of Hes7 oscillations (n = 4 PSM samples). Error bars indicate s.e.m. *P < 0.05, unpaired t-test.
a, Absorption (abs) spectra of Achilles (red) and Venus (black). b, Fluorescence images of bacteria that express Achilles and Venus. Bacterial colonies were grown at 37 °C and photographed at 8, 12, and 20 h after transformation. Exactly the same number of competent bacterial cells was used for transformation. Scale bar, 5 mm. c, Time course of fluorescence intensities of transformed E. coli colonies (mean values ± s.e.m. from three experiments). The data were normalized to the final yields extrapolated by curve fitting (broken line). d, Comparison of properties of Achilles and Venus.
a, Venus was inserted between the 5-kb Hes7 promoter and the Hes7 gene to drive expression of the Venus–HES7 fusion protein. b, Achilles was inserted between the 5-kb Hes7 promoter and the Hes7 gene to drive expression of the Achilles–HES7 fusion protein. c, Achilles fused to NLS-hPEST is expressed under the control of the Hes7 promoter. d, Hes7 cDNA without an initiation codon was inserted between the PEST sequence and the Hes7 3′ UTR of the construct shown in c to enable the transcripts to mimic endogenous mRNA stability. e, The Hes7 gene (exons + introns) without an initiation codon was inserted between the PEST sequence and the Hes7 3′ UTR of the construct shown in c. f, Achilles fused to NLS-hCL1-hPEST is expressed under the control of the Hes7 promoter. g, Hes7 cDNA without an initiation codon was inserted between the PEST sequence and the Hes7 3′ UTR of the construct shown in f.
a, Bone and cartilage were stained with Alizarin red and Alcian blue, respectively, at post-natal day (P)0. Achilles–HES7 rescued the abnormal vertebra and rib formation seen in the Hes7-null background. b, Higher magnification of the thoracic-to-lumbar area in Hes7-Achilles transgene+, Hes7-null mouse in a. Scale bars, 5 mm.
Extended Data Fig. 5 Observation of oscillation dynamics at the single-cell level to analyse the phase-coupling mechanism.
a, Live imaging (wide-field) of PSM carrying the Hes7-Achilles reporter at E10.5. b, Spatiotemporal expression pattern of signals from the Hes7-Achilles reporter in the PSM (wide-field). c, A representative cell tracked by Fiji and TrackMate. d, A representative phase quantification. Fluorescence time series from a cell extracted by tracking was converted into phase information using Hilbert transform. e, HES7 oscillation phase, colour-mapped onto the original image. Scale bars, 100 μm.
a, Expression of Hes7-Achilles reporter in wild-type and Lfng-knockout tail-bud tissue cultures. Scale bar, 100 μm. b, Mean intensity of Hes7-Achilles reporter fluorescence in the whole area. c. Examples of time series of Hes7-Achilles reporter intensity from single-cell tracking data. d, e, Average period (d) and amplitude (e) of HES7 oscillations at a single-cell level. More than 30 cells for each genotype (control and two independent reporter lines) were examined. n, number of peak pairs used for quantification. Error bars indicate s.e.m. *P < 0.05, unpaired t-test. f, Distribution of phase in single cells at the timing of peaks, in mean intensity time series in tail-bud cultures. Control and two independent reporter lines were examined. The number of cells examined (n) is indicated. ***P < 0.001, Rayleigh test. g, Kuramoto order parameter calculated using Achilles–HES7oscillation phase quantified in f. Error bars indicate s.e.m. *P < 0.05, unpaired t-test.
Extended Data Fig. 7 Acute inhibitor or knockdown treatment of tail-bud and dissociated PSM-cell cultures.
a–c, Expression of the Hes7-Achilles reporter in wild-type tail-bud tissue cultures treated with DMSO control (grey bars) or the Notch inhibitor DAPT (red bars). Period (a), amplitude (b) and synchrony (c) of HES7 oscillations were quantified. Error bars indicate s.e.m. *P < 0.05, unpaired t-test. The number of cells examined (n) is indicated. ****P < 0.0001, Rayleigh test. d, Kuramoto order parameter calculated using Achilles–HES7 oscillation phase quantified in c (time (t) = 400–800 min). Error bars indicate s.e.m. *P < 0.05, unpaired t-test. e, f, Expression of Hes7-Achilles reporter wild-type tail-bud tissue cultures treated with scrambled shRNA (shScramble) (grey bars) or two different shRNAs against Lfng (shLfng-1 and shLfng-2) (blue bars). Synchrony (e) and Kuramoto order parameter (f, t = 600–900 min) of HES7 oscillations were quantified. The number of cells examined (n) is indicated. ****P < 0.0001, Rayleigh test (e). Error bars indicate s.e.m. *P < 0.05, unpaired t-test (f). g, h, Expression of Hes7-Achilles reporter in dissociated PSM cell cultures treated with DAPT. Period (g) and amplitude (h) of HES7 oscillations were quantified. Error bars indicate s.e.m.
Extended Data Fig. 8 Mixed cultures of wild-type PSM cells and PSM cells carrying a faster Hes7 oscillator.
Wild-type (period = 126.6 ± 2.0 min) and mutant (In(3)) PSM cells that carry a faster Hes7 oscillator (period = 115.4 ± 1.1 min)23 were mixed as a minority in mutant or wild-type cells at 1:20 ratio, and fluorescence in the minority and majority cells was quantified over time. a, A small ratio (1:20) of In(3) cells were mixed into an In(3) population. b, A small ratio (1:20) of In(3) cells were mixed into a wild-type population. c, A small ratio (1:20) of wild-type cells were mixed into an In(3) population. The distribution of phase difference between the minority cells and their neighbouring cells was calculated at each time point. At least 100 cells were examined for each genotype. ****P < 0.0001, Rayleigh test.
a, System geometry. We consider 6 × 6 cells, forming a hexagonal lattice with nearest-neighbour coupling. b, Schematic of the mathematical model. c. Dynamical equations of the model. d, Time series of Xi(t) for different τ2 values. The dashed line is the average HES7 level (see ‘Mathematical modelling’ in Methods). In the parameter space for in-phase oscillation, τ2 values of longer or shorter than 1.0 result in smaller amplitudes and larger phase differences. e, τ2-dependence of oscillation amplitude (Xamp) and dispersion among cells (Xdis). The oscillation period is also shown.
Extended Data Fig. 10 KY02111 partially rescued the amplitude and synchrony of HES7 oscillations in Lfng-knockout PSM cells.
a, Effect of WNT-signalling-related chemical compounds on DLL1–Notch signalling delay was examined by a sender–receiver assay in C2C12 cells. Representative time series of the Hes1 reporter signal in receiver cells after light induction of Dll1 in the presence of DMSO, KY02111, kenpaullone or norcantharidin are shown. b, Peak timings of the Hes1 reporter after blue-light stimulation. n > 10 measurements for each condition. c, Fold change of amplitude of the Hes1 reporter after blue-light stimulation. n > 10 measurements for each condition. Error bars indicate s.e.m. *P < 0.05, unpaired t-test. d, Quantification of Hes7-Achilles reporter signals in central area (containing posterior PSM identity) of wild-type and Lfng-knockout tail-bud cultures in the presence of 0.1% DMSO (control), KY02111, kenpaullone or norcantharidin. e, Distribution of phase in single cells at the timing of peaks in mean intensity time series, in Lfng-knockout tail-bud cultures in the presence of DMSO (control) or KY02111. The number of cells examined (n) is indicated. ***P < 0.001, ****P < 0.0001, Rayleigh test. f, Average amplitude of HES7 oscillations in Lfng-knockout tail-bud cultures in the presence of DMSO (control) or KY02111. Error bars indicate s.e.m. *P < 0.05, unpaired t-test. g, Kuramoto order parameter calculated using Achilles–HES7 oscillation phase quantified in e. Error bars indicate s.e.m. *P < 0.05, unpaired t-test.
Supplementary Table 1: Chemical library screening with iPSM colonies.
Hes7-Achilles expression in control and Lfng-KO PSM. Confocal time-lapse imaging of WT (left) and Lfng-KO (right) PSM, carrying Hes7-Achilles and ROSA26-H2B-mCherry at E10.5. Stack images of 30 slices with 3-μm interval were projected with max-intensity. Images were taken every 3 min.
Coloured phase maps of Hes7 oscillations in control and Lfng-KO PSM. Hes7 oscillations of WT (left) and Lfng-KO (right) PSM were converted into colour phase map.
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Yoshioka-Kobayashi, K., Matsumiya, M., Niino, Y. et al. Coupling delay controls synchronized oscillation in the segmentation clock. Nature (2020). https://doi.org/10.1038/s41586-019-1882-z
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