Coupling delay controls synchronized oscillation in the segmentation clock

Abstract

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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Fig. 1: Characterization of Achilles and analysis of oscillations of the Hes7-Achilles reporter in control and Lfng-knockout mice.
Fig. 2: Loss of Lfng affects oscillation period, amplitude and synchronization.
Fig. 3: Loss of Lfng affects timing information in cell–cell signal transmission.
Fig. 4: LFNG in sending cells lengthened the time required for Hes1 response to DLL1.

Data availability

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. (matsushi@brain.riken.jp) for Achilles cDNA and R.K. (rkageyam@infront.kyoto-u.ac.jp) for other materials.

Code availability

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.

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Acknowledgements

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.).

Author information

K.Y.-K. developed Hes7 reporter mice, performed the experiments, analysed the data and wrote the manuscript; M.M. performed chemical library screening; A.I. analysed the data; Y.N. and A.M. developed Achilles, analysed the data and wrote the manuscript; H.K. performed mathematical modelling analysis; R.K. designed and supervised the project and wrote the manuscript.

Correspondence to Ryoichiro Kageyama.

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Extended data figures and tables

Extended Data Fig. 1 Loss of Lfng affects Hes7 oscillation dynamics at a tissue level.

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.

Extended Data Fig. 2 Comparative characterization of Achilles versus Venus.

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.

Extended Data Fig. 3 Schematic structures of fluorescent reporters for HES7.

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.

Extended Data Fig. 4 The Achilles–HES7 fusion protein is functional in segment formation.

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.

Extended Data Fig. 6 Synchronization of HES7 oscillation in tail-bud tissue cultures.

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.

ac, 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.

Extended Data Fig. 9 Mathematical modelling and simulation.

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 information

41586_2019_1882_MOESM3_ESM.mp4

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.

41586_2019_1882_MOESM4_ESM.mp4

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.

Reporting Summary

Supplementary Table

Supplementary Table 1: Chemical library screening with iPSM colonies.

Video 1:

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.

Video 2:

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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