Developmental Biology

Cell-autonomous timing drives the vertebrate segmentation clock’s wave pattern

Laurel A. Rohde
Arianne Bercowsky-Rama
Guillaume Valentin
Sundar Ram Naganathan
Ravi A. Desai
Petr Strnad
Daniele Soroldoni
Andrew C. Oates author has email address

Institute of Bioengineering, Swiss Federal Institute of Technology in Lausanne EPFL; Lausanne, CH
Department of Cell and Developmental Biology, University College London; London, UK
Center of PhenoGenomics, Swiss Federal Institute of Technology in Lausanne EPFL; Lausanne, CH
The Francis Crick Institute; London, UK

https://doi.org/10.7554/eLife.93764.1

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Figures and data

Cell-autonomous oscillatory dynamics reproduce those underlying wave pattern in the embryo.
(A) Experimental design: 1) Dissection of posterior-PSM quarter (PSM4) (dashed lines) from a Tg(her1-YFP;mesp-ba-mKate2) 15 somite-stage embryo, 2) Dissociation into single cells, 3) Culture at low-density, and 4) Her1-YFP and Mesp-ba-mKate2 imaging over time. (B) Her1-YFP and Mesp-ba-mKate2 in a 15 somite-stage embryo Tg(her1-YFP;mesp-ba-mKate2) embryo (bright-field inset). Arrowheads mark the recently formed somite (S1). Dissection lines surround PSM4. Scale bar 100 µm. (C) One cell per field of view imaged (scale bar 50 µm). Boxed region over time (scale bar 5 µm). Intensity trace shown in A. (D) Representative intensity traces of PSM4 cells in culture. (E) Number of Her1-YFP peaks (*mean ± SD, 4.4 ± 1.6 peaks) per PSM4 cell in culture (N = 11 embryos, n = 174 cells). (F) Density plot of ratios of successive Her1-YFP periods and peak intensity (cycleⁿ⁺¹/cycleⁿ) for PSM4 cells in culture (n = 421 successive cycle ratios, circles in inset). Percent of total ratios per quadrant is indicated. (G,H) PSM4 cells in a Tg(her1-YFP;h2b-mCherry) embryo tracked until somite formation (arrowhead) (N = 2 embryos, n = 128 PSM4 cells). Scale bar 100 µm. Cells contributing to the same somite are identically coloured in the embryo and representative Her1-YFP intensity traces (* peaks). Percentage of tracked PSM4 cells contributing to a given somite is shown. (I) Number of Her1-YFP peaks (*mean ± SD, 3.4 ± 1.0 peaks) per PSM4 cell in the embryo. (J) As described in F with PSM4 cells in the embryo (n = 179 total successive cycles).

Clock arrest and Mesp-ba-mKate2 signal-onset within the forming somite.
(A, B) Her1-YFP in a Tg(her1-YFP;h2b-mCherry) embryo (A) and Mesp-ba-mKate2 in a Tg(me-sp-ba-mKate2a;h2a-GFP) embryo (B). Representative lateral PSM light-sheet slice. Scale bar is 25 µm. Arrowheads at somite boundaries. (C) Cartoon of the formed somite (S1), the forming somite (S0) and the prospective somite (S-1). (D, E) S1 cells backtracked in a Tg(her1-YFP;h2b-mCherry) embryo (n = 233 cells) (D) and a Tg(mesp-ba-mKate2a;h2a-gfp) embryo (n = 190 cells) (E). Kymograph of Her1-YFP last peak (D) and Mesp-ba-mKate2 signal-onset time (E) in cells relative to the rostral-cau-dal somite axis (inset with example traces). Dashed grey line at transitions S-1 to S0 and S0 to S-1.

FGF extends cell-autonomous program duration of PSM4 cells in culture.
(A) A pool of dissociated PSM4 cells was split into a control well and one containing FGF-8b, then cultured (N = 4 experiments with n = 44 PSM4 control cells and n = 54 PSM4 cells with added FGF. (B) Representative Her1-YFP and Mesp-mKate2 trace from PSM4 cell culture with added FGF. (C) Number of Her1-YFP Peaks produced per cell, with pink line at the mean (mean ± SD = 4.0 ± 1.8 peaks in control cells, 6.4 ± 3.5 with FGF. (D) Time of Her1-YFP last peak (min post-dissociation), with pink line at the mean (mean ± SD = 269 ± 116 min post-dissociation in control cells, 568 ± 185 min post dissociation in cells with FGF. (E) Ratio of successive cycle Her1-YFP periods (Period (n+1)/n) and peak intensity (Max Intensity (n+1)/n). Upper right quadrant indicates successive intensity rise and oscillation slowing. n = 51 successive cycle ratios in PSM4 control cells and n = 196 in PSM4 cells cultured with FGF, shown as circles in inset and percent of successive cycles is indicated in each quadrant. (F,G) All Her1-YFP intensity traces were aligned by the first peak, cycles were peak-to-peak, then the period (F) and intensity of peak (I⁺) and trough (I^-) (G) was given for each cycle as a median (circle) with 25^th and 75^th interquartiles (bar).

Intrinsic timer initiates upon TB exit and duration shortens in cell flow.
(A) Experimental design: Tg(her1-YFP;mesp-bb-mKate2) PSM was dissected into different anteroposterior quarters, dissociated, and then cultured. PSM4 was dissected and cultured in parallel from each embryo to serve as an internal reference. (B) Representative intensity traces for the different anteroposterior quarters. (C) Her1-YFP last peak times as median (green line) with inter-quartile box and whiskers. Time given as post-dissociation. Data pooled by cell type (N = 3 embryos, n = 32 PSM2 and 32 PSM4 cells; N = 3 embryos, n = 65 PSM3 and 41 PSM4 cells; N = 3 embryos, n = 38 TB and 59 PSM4 cells). PSM2 last peaks that occurred prior to the start of imaging were set to acquisition start time (*). (D) Correlation of Her1-YFP Last Peak and Mesp-bb-mKate2 signal-onset time. (E) Cells backtracked from posterior PSM (blue, n = 17) and S1 (orange, n = 15) to the TB at 15 somite stage in a representative embryo (7.5 h imaging, Tg(her1-YFP;h2b-mCherry, N = 2). Arrowhead at recently formed somite boundary. Scale bar 100 µm. (F, G) Her1-YFP intensity traces for individual cells (green) and mean intensity (black). Inset zoom of oscillations in the TB. (H) Cell distance from posterior tail tip. (I) Mean Her1-YFP period.

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