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A bioelectrical phase transition patterns the first vertebrate heartbeats

Nature volume 622, pages 149–155 (2023)Cite this article

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Abstract

A regular heartbeat is essential to vertebrate life. In the mature heart, this function is driven by an anatomically localized pacemaker. By contrast, pacemaking capability is broadly distributed in the early embryonic heart1,2,3, raising the question of how tissue-scale activity is first established and then maintained during embryonic development. The initial transition of the heart from silent to beating has never been characterized at the timescale of individual electrical events, and the structure in space and time of the early heartbeats remains poorly understood. Using all-optical electrophysiology, we captured the very first heartbeat of a zebrafish and analysed the development of cardiac excitability and conduction around this singular event. The first few beats appeared suddenly, had irregular interbeat intervals, propagated coherently across the primordial heart and emanated from loci that varied between animals and over time. The bioelectrical dynamics were well described by a noisy saddle-node on invariant circle bifurcation with action potential upstroke driven by CaV1.2. Our work shows how gradual and largely asynchronous development of single-cell bioelectrical properties produces a stereotyped and robust tissue-scale transition from quiescence to coordinated beating.

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Fig. 1: Multiplexed time-lapse calcium imaging captures the first heartbeats of zebrafish embryos.
Fig. 2: The heartbeat starts by means of a noisy tissue-wide SNIC bifurcation.
Fig. 3: The first heartbeats comprise voltage and calcium spikes.
Fig. 4: The heart primordium is excitable and electrically coupled before the first heartbeat.
Fig. 5: Initiation of the early heartbeat is set by competition between spontaneously firing cells.

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Data availability

Raw images are available upon request to the corresponding authors. Source data are provided with this paper.

Code availability

Code is available at https://github.com/adamcohenlab/Jia2023FirstHeartbeat.

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Acknowledgements

This work was supported by the Howard Hughes Medical Institute (A.E.C.), a Vannevar Bush Faculty Fellowship (A.E.C.) and the Harvard Medical School John S. Ladue Fellowship in cardiovascular medicine (B.Z.J.). We thank C. E. Burns and C. G. Burns for providing TgBAC(−36Nkx2.5:ZsYellow) transgenic fish; N. Chi for providing Tg(7xTCF-Xla.Siam:nlsmCherry); I. C. Scott for providing the zACNE20 enhancer element; Y. Shen and R. A. Campbell for providing FR-GECO1c plasmid; L. D. Lavis for providing JaneliaFluor 608 dye; H. C. Davis and F. P. Brooks for developing microscope control software; J. Miller-Henderson, K. Hurley and A. R. Murphy for fish husbandry; and members of the Megason laboratory for assistance with fish husbandry.

Author information

Authors and Affiliations

  1. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA

    Bill Z. Jia, Yitong Qi, J. David Wong-Campos & Adam E. Cohen

  2. Department of Systems Biology, Blavatnik Institute, Harvard Medical School, Boston, MA, USA

    Bill Z. Jia & Sean G. Megason

  3. Systems, Synthetic and Quantitative Biology PhD Program, Harvard University, Cambridge, MA, USA

    Bill Z. Jia

  4. Department of Physics, Harvard University, Cambridge, MA, USA

    Adam E. Cohen

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Contributions

A.E.C., B.Z.J. and S.G.M. conceived the project and designed experiments. B.Z.J. performed all experiments, data analysis and numerical modelling. B.Z.J. constructed molecular tools and transgenic fish lines. Y.Q., J.D.W.-C. and B.Z.J. performed optical engineering of microscopes. B.Z.J., A.E.C. and S.G.M. wrote the manuscript with input from all authors. A.E.C. and S.G.M. supervised the project.

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Correspondence to Sean G. Megason or Adam E. Cohen.

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Nature thanks Emilia Entcheva, Leon Glass and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data figures and tables

Extended Data Fig. 1 Validations of experimental approaches.

a, Histograms of onset time for experimental replicates. b, Comparison of time of onset for alpha-bungarotoxin (aBTX) mRNA-injected embryos (n = 40), tnnt2a morphants (n = 12), and agarose-encased embryos (n = 3) respectively. c, d, Comparison of frequency (c) and inter-spike interval coefficient of variation (ISI CV, d) as a function of time after the first beat. aBTX mRNA-injected replicates colored corresponding to (a). e, Time of onset for jGCaMP7f-expressing embryos (n = 40) and FR-GECO1c-expressing embryos (n = 6). f, g, Comparison of frequency (f) and ISI CV (g) for jGCaMP7f and FR-GECO1c-expressing embryos. h, Difference in time of onset as a function of distance between all pairwise combinations of embryos in the experiments in (a). There was negligible correlation (r = 0.04, −0.05, −0.18 for each replicate, respectively), confirming absence of chemical or mechanical coupling between embryos. i, Time of onset for embryos +/− CoChR expression and exposed to pulsed blue light for the optogenetic stimulus program in Fig. 4. j, Spontaneous beat rate was unchanged before vs. after optogenetic stimulation protocol. k, Method of extracting voltage signals from Tg(z20m:Voltron1-P2A-jGCaMP7f) heart primordia. l, Probability distributions of Voltron1 -ΔF/F in background (blue) and jGCaMP7f principal component 1 (PC1, red) regions, before (top) and after (bottom) initiation of calcium transients. m, Skewness of Voltron1 -ΔF/F distribution before (silent) and after (active) initiation of calcium transients. Positive skewness indicates presence of upwards spikes. n, Pipeline for performing matched filter analysis (Methods). o, Peak matched filter signal-to-noise ratio. p, Gaussian kernel density estimate of candidate spike SNR distribution for background regions. q, Fraction of candidate spike events with SNR greater than the 95th percentile of p. Candidate spike events were identified by performing peak detection on the jGCaMP7f recordings. a – h, Three replicates of jGCaMP7f and aBTX mRNA-injected embryos at n = 13, n = 9, n = 18 animals respectively, tnnt2a morpholino at n = 12 animals, agarose-encased embryos at n = 3 animals, 1 experiment. FR-GECO1c and aBTX mRNA-injected embryos at n = 6 animals. i – j, n = 6 control animals and n = 21 CoChR-expressing animals over 2 experiments. k–q, 18 recordings with calcium transients detectable by peak finding (“Active”), 22 recordings without (“Silent”), across 36 embryos in one experiment. m, 801 traces of regions of interest (ROIs). o, 10408 spiking events from ROIs. q, 597 putative spikes analyzed (377 in “active” recordings, 220 in “silent” recordings). Boxplots represent interquartile range (IQR; 25th, 50th, 75th percentile) with whiskers representing 1.5×IQR. Statistical test values: (i) p = 0.26, Mann–Whitney-Wilcoxon two-sided test; (j) linear regression y = 0.97x – 0.024, r = 0.953, p = 6.6e-281, Wald Test; (m), active background vs. active PC1 p = 2.1e-6, silent PC1 vs active PC1 p = 1.4e-17, Mann-Whitney-Wilcoxon two-sided test with Benjamini-Hochberg correction. (o), p < 5e-324 (below 64-bit numerical precision) in all comparisons shown, Mann-Whitney-Wilcoxon two-sided test with Benjamini-Hochberg correction.

Source data

Extended Data Fig. 2 Calcium dynamics colocalize with genetic markers of developing cardiomyocytes.

a, Tg(myl7:eGFP) expression in a 20 – 21 hpf embryo. a’, Peak ΔF/F of ubiquitously expressed FR-GECO1c during a spike-triggered average video (Methods). a”, Merge. Scale bars 50 µm. b, Relationship between peak FR-GECO1c ΔF/F and myl7:eGFP intensity for tiled regions of interest (Methods). c, Relationship between peak jGCaMP7f ΔF/F and nkx2.5:ZsYellow intensity for tiled regions of interest. Same embryos as in Fig. 5a–d. b, c, dashed line indicates 50th percentile of fluorophor intensity. d, Distribution of LOI motion speed. e, Relative GFP intensity in Ca2+ active regions and LOIs, compared to image mean. n = 10 embryos, 15 – 45 min post onset. f – f’, Example activation maps of ubiquitously expressed FR-GECO1c in Tg(myl7:eGFP) embryos. GFP in grayscale. Scale bars 50 µm. g, Relative nkx2.5:ZsYellow intensity as a function of distance from the inner edge of the heart cone. A region approximately <50 µm from the inner edge had stronger expression than more distal cells. h, Relative nkx2.5:ZsYellow intensity at the location of the 15 mpo LOI did not change between 15 mpo and 105 mpo, suggesting stable nkx2.5 expression in this region. a, b, e, f, n = 10 embryos over one experiment, 15 – 45 min post onset. c, h, Data from Fig. 5a–d (n = 12 embryos, 3 experiments). g, Data from Extended Data Fig. 4e, f. Boxplots represent interquartile range (IQR; 25th, 50th, 75th percentile) with whiskers representing 1.5×IQR. Statistical test values: (e) mean vs. active, p = 1.953e-3, **, mean vs. LOI, p = 1.953e-3, **; (h), p = 0.32. Two-sided Wilcoxon signed-rank test with Benjamini-Hochberg correction.

Source data

Extended Data Fig. 3 Spatiotemporal structure of the transition from silent to beating.

a, Active area of single-cell (SC) transients (74 ± 32 µm2) was much lower than that of the first heartbeats (HB, 7000 ± 1400 µm2). b, Single-cell transients were longer lived (3.4 ± 2.1 s) than the first heartbeats (1.3 ± 0.2 s). FWHM: full-width half-maximum. c, Single-cell transients had a longer plateau phase than the first heartbeats, as quantified by the ratio of peak width at 25% (τ25) and 50% (τ50) of maximum height. d, Single-cell transients were rare (0.2 – 0.7 per minute) compared to the first heartbeats (1 – 9.5 per minute). a – d, Data from 19 single-cell transients and 50 beats across n = 3 embryos over 3 experiments. e – e”, Maximum intensity projection fluorescence images of 21-somite stage Tg(-36nkx2.5:ZsYellow; zACNE20:2xLyn-mCherry) heart cone (5/5 embryos over 1 experiment). e, nkx2.5:ZsYellow. e’, zACNE20:2xLyn-mCherry. e”, Merge. f, Cell area distribution. Mean area 82 µm2 (± 29 µm2 SD). n = 5 embryos, 254 cells, over 1 experiment. Scale bars 50 µm. Mean ± SD for all values in caption. g, jGCaMP7f fluorescence starting from 2 min after the first heartbeat in embryos with spontaneous initiation in half the heart. The traces were averaged over the whole heart, so traces that engaged half the heart had approximately half the apparent amplitude of traces that engaged the whole heart. h, Relative active areas of the traces in (g) compared to representative traces of embryos with full-tissue initiation. Area was averaged in a 2-minute sliding window. i, Pearson correlation between fluorescence dynamics of individual pixels and the mean segmented heartbeat jGCaMP7f activity at different timepoints. The sharp boundary in the 9 min post onset (mpo) image is due to roughly half the heart being engaged in some but not all beats. Same experiments as in Fig. 1. Scale bars 100 µm. j, Amplitude of the first detected calcium transient compared to maximum fluctuation from mean in the previous 2 min. k, Ratio of calcium transient amplitudes (ΔF/F) 60 min post onset (mpo, A60) and 0 mpo (A0). l, Ratio of transient widths (full-width half-maximum) 60 min mpo (τ60) and 0 mpo (τ0). m, Amplitude of calcium transients as a function of time, aligned relative to first beat and normalized relative to initial beats. n, Active area of calcium transients as a function of time, aligned relative to first beat and normalized relative to initial beats (Methods). m,n, Colored lines show representative single-embryo traces. Black line shows median. Shading and dashed lines show interquartile range. o, Calcium spike area increased from 13490 ± 7730 µm2 at 0 mpo to 15840 ± 6400 µm2 at 60 mpo. j – o, n = 39 embryos across 3 experiments, same individuals as Figs. 1–2. Boxplots represent interquartile range (IQR; 25th, 50th, 75th percentile) with whiskers representing 1.5×IQR. Statistical test values: (a) Two-sided Mann-Whitney-Wilcoxon test, p = 6.5e-11, ****; (b) Two-sided Mann-Whitney-Wilcoxon test, p = 1.3e-4, ***; (c) Two-sided Mann-Whitney-Wilcoxon test, p = 0.015, *; (j) Two-sided Wilcoxon signed-rank test, p = 1.23e-7, ****; (o) Two-sided paired t-test, p = 5.4e-5, ****.

Source data

Extended Data Fig. 4 Classification of the bifurcation from silent to beating.

a, Mean frequency and b, inter-spike interval (ISI) coefficient of variation (CV) in simulated Morris-Lecar oscillators as a function of input current I and noise σw. Parameters given in Supplementary Table 2, were selected to drive each of the four types of co-dimension 1 bifurcation. c, Squared error (Supplementary Text) of experimental fits to each bifurcation at different values of σw. d, Spiking regimes across the SNIC bifurcation of the noisy quadratic integrate-and-fire (QIF) model (Supplementary Information Equation 2), shown by \(\dot{\varphi }\) as a function of φ. e, Simulated example traces for each spiking regime.

Source data

Extended Data Fig. 5 A noisy quadratic integrate-and-fire model captures the statistics of the first heartbeats.

a, Simulated frequency and ISI CV for different values of noise σw. b, Example fits (top: frequency, bottom: ISI CV) of individual embryo data (blue) to simulation (black) with choices of σw which minimized squared error. c, Mean squared error of fits of simulated spike statistics to data as a function of σw (n = 39 embryos). d, Histograms of experimental and QIF-generated ISIs for the first 20 heartbeats, using different φreset values. Decreasing φreset modelled an increasing refractory period, and corrected the overrepresentation of short ISIs in the model relative to the data. e, Experimental autocorrelation of instantaneous frequency averaged over the first 50 heartbeats. f, Autocorrelation of instantaneous frequency in the QIF model with experimentally fitted σw and varying values of I. g, Autocorrelation of instantaneous frequency in the four bifurcation classes of the Morris-Lecar model, calculated using experimentally fitted σw and varying values of I.

Source data

Extended Data Fig. 6 Expression patterns of zACNE20 and zACNE20-myl7 (z20m) promoters in the early heart.

a–a”, Heart primordium in a 20-somite stage (ss) Tg(-36nkx2.5:ZsYellow;zACNE20:2xLyn_mCherry) embryo (5/5 embryos). b–b”, Heart cone in a 22 ss stage Tg(zACNE20:2xLyn_mCherry; zACNE20-myl7: FRGECO1c-P2A-CoChR-eGFP) embryo (6/6 embryos). c–c”, Heart primordium in an 18 – 19ss Tg(z20m:FRGECO1c-P2A-CoChR-eGFP) embryo stained for myl7 using HCR-FISH (9/9 embryos). d–d”, Heart primordium in an 16 – 18ss Tg(z20m:FRGECO1c-P2A-CoChR-eGFP) embryo immunostained using MF20 antibody (6/6 embryos over 3 experiments). a – d, All images are confocal maximum intensity projections. e, f, z20m produces stronger expression than zACNE20 at the same stage. Widefield images at 20-21 ss. g, example calcium recording and optogenetic stimulus in zACNE20-myl7: FRGECO1c-P2A-CoChR-eGFP at ~21 – 22 hpf (without additional mRNA). All scale bars 50 μm.

Extended Data Fig. 7 Roles of pacemaker ion transporters in initiation of spontaneity and excitability.

a, Automatic segmentation (Methods) located heartbeats in control embryos but not in embryos injected with the cacna1c morpholino. tnnt2a KD, n = 3 embryos; cacna1c KD, n = 10 embryos. All embryos were injected with jGCaMP7f mRNA. Scale bar 500 µm. b, c, The heartbeats became faster and more regular after initiation in tnnt2a-MO embryos. Manually selected ROIs in the cacna1c morphants did not show spontaneous activity. d – d”, Traces of jGCaMP7f fluorescence at 0, 30, 60, and 90 min after onset for embryos injected with morpholinos for d hcn4, d’ ncx1h, d” atp1a1a (Na+,K+-ATPase α-subunit). e, Time of onset for morphants. f, Mean frequency at 30 min post onset. g, Mean frequency at 60 min post onset. h, Inter-spike interval coefficient of variation (ISI CV) at a mean frequency of 0.1 Hz. ncx1h morphants were omitted in c – e because the plateaus prevented unambiguous determination of spike times. e – h, Uninjected, n = 39 embryos; tnnt2a KD, n = 12 embryos; hcn4 KD, n = 11 embryos; ncx1h KD, n = 10 embryos; atp1a1a KD, n = 12 embryos. Uninjected samples collected over 3 experiments, other samples collected over 1 experiment each. e – h, Boxplots represent interquartile range (IQR; 25th, 50th, 75th percentile) with whiskers representing 1.5×IQR. i, Example traces of FR-GECO1c dynamics (red) with pulsed CoChR stimulus (blue) directed to the entire heart of individual embryos. j, Stimulus-triggered average of calcium activity in control (blue) and cacna1c morphants (orange, population mean ± SD). i, j, tnnt2a KD, n = 11 embryos, cacna1c KD, n = 14 embryos. All recordings acquired at 10 Hz. All embryos were zACNE20-myl7:FRGECO1c-P2A-CoChR-eGFP (+/-) injected with FR-GECO1c mRNA. Statistical test values: e – h, Mann-Whitney-Wilcoxon two-sided test with Benjamini-Hochberg correction. (e) uninjected vs. tnnt2a, p = 0.0775; uninjected vs. hcn4, p = 0.11; uninjected vs. atp1a1a, p = 1.23e-3; uninjected vs. ncx1h, p = 0.0847. (f) uninjected vs tnnt2a, p = 0.149; uninjected vs. hcn4, p = 0.228; uninjected vs. atp1a1a, p = 2.11e-5. (g) uninjected vs. tnnt2a, p = 0.096; uninjected vs. hcn4, p = 0.256; uninjected vs. atp1a1a, p = 7.71e-7. (h) uninjected vs tnnt2a, p = 0.0158; uninjected vs. hcn4, p = 0.165; uninjected vs atp1a1a, p = 0.376.

Source data

Extended Data Fig. 8 Connexin-43 is expressed in the cardiac primordium before fusion.

Tg(z20m:FRGECO1c-P2A-CoChR-eGFP) fish were immunostained for connexin-43 and sarcomeric myosin (MF20). Examples of triple-positive cells indicated by dotted circles on the bottom row. Top row: 4/4 embryos over 1 experiment. Middle and bottom rows: 6/6 embryos over 3 experiments (same individuals as Extended Data Fig. 6d). Cyan in overlay: nuclear staining by SYTOX Blue. Scale bars 50 μm (top and middle row), 25 μm (bottom row).

Extended Data Fig. 9 Optogenetic pacing induces overdrive suppression in the heart cone.

a, Activation map of spontaneous activity (spike-triggered average, n = 9 spikes) in Tg(z20m:FRGECO1c-P2A-CoChR-eGFP) embryo. Purple circle indicates region to be stimulated in (b). b, Activation map of evoked activity (spike-triggered average, n = 19 spikes) in the same heart as (a). Scale bars 50 μm. c, Distance between spontaneous LOI and fixed “target region” after repeated pacing at different frequencies at the target region. d, Drift of the spontaneous LOI from its first recorded position was uncorrelated with repeated pacing. e, A target region away from the spontaneous LOI was paced with period Tstim. Heartbeat was characterized by Tspon = period of spontaneous activity in absence of stimulus, and Tresp = period of activity during periodic stimulus. CoChR activation only paced the heart (i.e. Tresp/Tstim ≈ 1) when Tstim <Tspon. At the highest stimulus frequencies, the heart showed a period doubling, i.e. Tresp/Tstim ≈ 2. f, Ratio of number of evoked spikes to number of total spikes (spontaneous + evoked) during the 30-second stimulus period. At a lower pacing interval, evoked spikes comprised a larger fraction of the total. “Evoked spikes” were defined as spikes which occurred within 200 ms of a pulse of blue light. (c – f) Each color represents one fish. g, Distance between the target region and the LOI of each individual spike grouped by timing and whether the spike was evoked. Evoked spikes had LOI significantly closer to target than any other category. Boxplot represents interquartile range (IQR; 25th, 50th, 75th percentile) with whiskers representing 1.5×IQR. (c – g) Number of beats collected per category – “Pre”: 306; “During”: 309; “Stim”: 363; “Post”: 281. n = 6 embryos in one experiment. Statistical test values: Pre. vs Evoked, U = 9.2e4, p = 1.1e-47, ****; Post vs Evoked, U = 1.5e4, p = 2.3e-52, ****; Spon. vs Evoked, U = 8.6e4, p = 2.6e-33, ****. Mann-Whitney Wilcoxon two-sided test with Benjamini-Hochberg correction.

Source data

Extended Data Fig. 10 Robustness of wave geometry in heterogeneous coupled SNIC oscillators.

(a) Schematic for chain of Morris-Lecar oscillators coupled by a current Ic which depends on coupling strength D (left); and spatial distribution of I for different choices of ΔI and σI,x (right). (b) Mean frequency (f) and inter-spike interval coefficient of variation (ISI CV) for a chain of 25 oscillators with D = 0.5, and current gradient ΔI = 0.514. Simulation with σw = 0.618 (compare qualitatively to experiments) marked with dashed black lines. c – e, Simulated wave properties as a function of σw and D. Here I attained its highest value at normalized x = 1.0. c, Normalized mean LOI position. d, Standard deviation of LOI position. e, Normalized wave area. Parameter choice for dashed line in (b) indicated with white “X”. f, Wave geometry as a function of ΔI and σI,x. Black line indicates the line σI,x = 0.5 ΔI. For sufficiently large ΔI, full-tissue wave propagation occurred with stable LOI at one end of the chain. In high variability, i.e. large σI,x, a stable LOI driving full-tissue waves (low position standard deviation, high normalized mean area) can appear at positions that are not the end of the chain. (e, f) <I > = 4.68, σw = 0.03.

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Supplementary information

Supplementary Information

Supplementary Information containing methods (including modelling), tables and refs.

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Supplementary File 1

CAD file for manufacturing of mould for agarose embryo mounting chamber.

Supplementary File 2

Sequence information of in situ hybridization probe-sets, synthesized mRNAs and morpholinos used in this study.

Supplementary Video 1

Parallel measurements of heartbeat in 18 embryos. Embryos expressed jGCaMP7f and fluorescence was recorded at 10 Hz. Recording at roughly 22 hpf. Raw jGCaMP7f intensity in grayscale. ΔF/F in green. Scale bar, 500 μm.

Supplementary Video 2

Parallel all-optical electrophysiology in 15 embryos. Embryos expressed CoChR and FR-GECO1c ubiquitously and red fluorescence was recorded at 10 Hz. Blue light stimuli were targeted to hearts but are indicated as offset blue dots (bright when illuminated) for clarity. Recording at roughly 22 hpf. Brightfield image in grayscale. Scale bar, 500 μm.

Supplementary Video 3

Calcium dynamics before observable mechanical contractions. Brightfield (left) and jGCaMP7f (right) videos taken consecutively at 10 Hz (20–21 hpf). Playback at 2× speed. Scale bar, 50 μm.

Supplementary Video 4

Calcium dynamics with mechanical contractions. Brightfield (left) and jGCaMP7f (right) videos taken consecutively at 10 Hz on the same embryo as Supplementary Video 3, 1 hour later. Playback at 2× speed. Scale bar, 50 μm.

Supplementary Video 5

The first heartbeat at cellular resolution. Two Tg(zACNE20:2x:Lyn-mCherry) (red) embryos expressing jGCaMP7f (cyan) imaged at roughly 3 Hz. Videos are synchronized by the first detected heartbeat. Scale bar, 50 μm.

Supplementary Video 6

Simultaneous calcium and voltage imaging in the zebrafish heart cone. Two Tg(zACNE20-myl7:Voltron1-P2A-jGCaMP7f) embryos at estimated 15 and 105 min after onset, imaged at 20 Hz, spike-triggered average. jGCaMP7f ΔF/F in green, Voltron1-JF608 ΔF/F in magenta. First principal component of jGCaMP7f ΔF/F in grayscale.

Supplementary Video 7

Calcium wave propagation evoked by targeted CoChR activation before spontaneous cardiac activity. Tg(z20m:FRGECO1c-P2A-CoChR-eGFP) embryo recorded at 50 Hz. CoChR-eGFP in grayscale. FR-GECO1c ΔF/F in red. Blue light stimulus in blue. Recording at roughly 19.5 hpf. Scale bar, 50 μm.

Supplementary Video 8

Heart cone rotation and elongation. Tg(zACNE20:2xLyn-mCherry) embryo recorded every 10 min. Recording starts 21–22 hpf. Scale bar, 25 μm.

Supplementary Video 9

Optogenetic hyperpolarization transiently perturbs calcium wave geometry and LOI. Tg(-36nkx2.5:ZsYellow) embryo expressing gtACR2 and FR-GECO1c. Two consecutive 50 Hz recordings with different targets for optogenetic silencing. ZsYellow in grayscale. FR-GECO1c ΔF/F in red. Blue light stimulus in blue. Recording at roughly 21 hpf. Baselines set separately for blue-illuminated intervals (separated by white frames). Scale bar, 50 μm.

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Jia, B.Z., Qi, Y., Wong-Campos, J.D. et al. A bioelectrical phase transition patterns the first vertebrate heartbeats. Nature 622, 149–155 (2023). https://doi.org/10.1038/s41586-023-06561-z

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