Abstract
The vertebrate body displays a segmental organization that is most conspicuous in the periodic organization of the vertebral column and peripheral nerves. This metameric organization is first implemented when somites, which contain the precursors of skeletal muscles and vertebrae, are rhythmically generated from the presomitic mesoderm. Somites then become subdivided into anterior and posterior compartments that are essential for vertebral formation and segmental patterning of the peripheral nervous system1,2,3,4. How this key somitic subdivision is established remains poorly understood. Here we introduce three-dimensional culture systems of human pluripotent stem cells called somitoids and segmentoids, which recapitulate the formation of somite-like structures with anteroposterior identity. We identify a key function of the segmentation clock in converting temporal rhythmicity into the spatial regularity of anterior and posterior somitic compartments. We show that an initial ‘salt and pepper’ expression of the segmentation gene MESP2 in the newly formed segment is transformed into compartments of anterior and posterior identity through an active cell-sorting mechanism. Our research demonstrates that the major patterning modules that are involved in somitogenesis, including the clock and wavefront, anteroposterior polarity patterning and somite epithelialization, can be dissociated and operate independently in our in vitro systems. Together, we define a framework for the symmetry-breaking process that initiates somite polarity patterning. Our work provides a platform for decoding general principles of somitogenesis and advancing knowledge of human development.
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Data availability
scRNA-seq data have been deposited to the NCBI Gene Expression Omnibus (GEO; GSE195467). Bulk RNA-seq data have been deposited to the GEO (GSE220634). The Homo sapiens (human) genome assembly (GRCh38) is from NCBI Assembly (GCF_000001405.26). The mouse embryo scRNA-seq data are from the GEO (GSE114186). The RNA-seq data of iPS cells are from the GEO (GSE164874). Source data are provided with this paper.
Code availability
Codes for scRNA-seq analyses can be accessed at GitHub (https://github.com/PourquieLab/Miao_Djeffal_2022.git). Codes for quantitative image analyses are available at GitHub (https://github.com/desimonea/MiaoSomitogenesis2022).
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Acknowledgements
We thank S. G. Tattikota from the N. Perrimon laboratory for help with scRNA-seq experiments; the members of the Biopolymers Facility at Harvard Medical School for providing 10x Genomics Chromium Controller instrument access and sequencing consultation; the staff at the NeuroTechnology Studio at Brigham and Women’s Hospital for providing microscope access and consultation on data acquisition and data analysis; the staff at the Harvard Neurobiology Imaging Facility for access to the FV1000 confocal microscope (NINDS P30 Core Centre grant NS072030); and S. Megason for reading the manuscript. Research in the Pourquié laboratory was funded by a grant from the National Institute of Health (5R01HD085121). Y.D. is supported by Fondation pour la Recherche Médicale (FRM) PLP2020100012456.
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Contributions
Y.M. designed, performed and analysed most of the biological experiments. Y.D. analysed scRNA-seq data; A.D.S. developed the codes and performed quantitative image analysis with Z.L. and S.D.T.; K.Z. performed RNA-seq sample preparation and data analysis. J.G.L. and A.S. conducted embryo experiments and analysis with help from L.M.S.S.; Z.L. performed single-cell-tracking image analysis with A.D.S. and S.D.T.; J.R. and O.A.T. contributed to scRNA-seq experiments. A.S., A.M., P.R. and M.D.-C. contributed to data analysis or experiments. Y.M., Y.D. and O.P. wrote the manuscript with input from all of the authors. O.P. supervised the study.
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O.P. is a scientific founder of Anagenesis Biotechnologies. The other authors declare no competing interests.
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Extended data figures and tables
Extended Data Fig. 1 Characterization of the Somitoid model.
a, Time lapse confocal images of H2B-mCherry in a spreading Somitoid. b, Illustration of the design of the HES7/MESP2 double-reporter cell line. c, Left, time-lapse confocal images of HES7 wave; Right, temporal profiles of HES7 reporter in two different regions indicated by the blue and orange boxes. d, Left, box plots of projected areas of all rosettes in individual Somitoids. Right, plot of median rosette area of each Somitoid (n = 20 Somitoids). Red bars indicate median with interquartile range. e, Correlation analysis (n = 20 Somitoids; two-sided) between the entire Somitoid area and median rosette area (left); between the entire Somitoid area and total rosette number (right). f, Shape descriptors of individual rosettes (top) and entire Somitoids (bottom). n = 1,957 rosettes from 20 Somitoids. g, h, Confocal slices from the bottom (z = 0 µm) to the top of a rosette in 120 h Somitoid stained with Laminin (g) and N-Cadherin (h) (n = 2 Somitoids). i, Representative images of a Somitoid cultured on gelatin (n = 5 Somitoids) or laminin (n = 5 Somitoids) coated surface, stained with Laminin. j, 3D reconstruction image of a Somitoid cultured in suspension (left; n = 2 Somitoids) and a confocal section (right), stained with Laminin. k, Principal components analysis using the same RNA sequencing datasets shown in Fig. 1h. l, Confocal images of 120 h PAX3-reporting Somitoids treated with 5 µM Blebbistatin (left) and control (right). In box-and-whiskers plots, the middle hinge corresponds to median, the lower and upper hinges correspond to the first and third quartiles, respectively, and the lower and upper whiskers correspond to the minimum and maximum, respectively. Scale bars represent 500 µm (a, c, i, l), 50 µm (g, h) and 100 µm (j).
Extended Data Fig. 2 Antero-Posterior patterning in Somitoids.
a, Illustration of the design of the HES7/MESP2/UNCX triple-reporter cell line. b, Ratio of mean mCherry or YFP intensities in the centre circle vs in the big circle (n = 8 Somitoids and the bars indicate median). c, Normalized RNA counts of selected polarity genes in cell fractions separated by flow cytometry, as measured by RNA sequencing (n = 3 independent experiments, 96 Somitoids in each n). Cells with top 10% mCherry fluorescence are shown on the left (magenta) and top 10% YFP fluorescence on the right (yellow). All four genes were identified as differentially expressed genes by DESeq2 using the Wald test (two-sided). d, Temporal plot of HES7 reporter (mean±s.d., n = 3 Somitoids) and images of an UNCX and MESP2 reporting Somitoid treated with 50 µM DAPT added at 48 h. e, Wide-field images of PAX3-reporting Somitoids treated with 50 µM DAPT (left) since 48 h and control (right). f, Maximum-z-projection confocal images of UNCX and MESP2 reporting Somitoids treated with 10 µM ROCKi (left) or 5 µM Blebbistatin (right) since 48 h. g, Left, percentage of UNCX-positive cells characterized by flow cytometry in 120 h WT (n = 6 experiments), HES7-null (n = 6 experiments), and MESP2-null (n = 5 experiments) Somitoids; Data are represented as mean±s.d., one-way ANOVA, compared with WT, P = 0.89 (HES7-null); 2.49e-10 (MESP2-null). Right, images of MESP2 and UNCX reporters in HES7-null Somitoids, and UNCX reporter in MESP2-null Somitoids. h, Histograms of flow cytometry analysis of UNCX-YFP in 120 h Somitoids (control, WT, HES7-null, and MESP2-null cell lines) with debris and doublets removed. Control is the parental NCRM1 cell line. Fractions on the right side of the red dotted line in the histograms are defined as YFP-positive. i, Scattered plot (top) and histogram (bottom) of flow cytometry analysis on MESP2-mCherry Somitoids at 72 h with debris and doublets removed. j, Time-lapse images of MESP2 reporter in a Somitoid. k, Time-lapse maximum-z-projection confocal images of H2B-GFP in the same region of a Somitoid as in Fig. 2d. l, Cell tracks of MESP2-high cells overlayed on images of MESP2 reporter. The orange outlines represent the forming MESP2-low regions. m, Spatial auto-correlation (sole MESP2 signal, sole UNCX signal or them combined together) once rosettes are formed (representative example from n = 3 Somitoids). n, Additional example of spatial auto-correlation analysis and abscissa-position of the auto-correlation trough (inset) of MESP2/UNCX double reporting Somitoid over time. o, Temporal plot (mean±95%CI) of mean squared displacement (n = 3,422 tracks from 2 Somitoids). p, Additional example of normalized temporal profiles of MESP2 reporter in individual cells (top; n = 52 cells from one Somitoid), and correlation analysis of MESP2 intensities at 72 h and 84 h (bottom; F-test, one-sided, P = 4.67e-14 after removing 3 outliers identified by calculating Mahalanobis distance, as explained in Methods, marked by magenta cross). Temporal profiles are coloured based on relative MESP2 intensity among tracked cells at 72 h, with higher 50% in magenta and lower 50% in cyan throughout the time window. q, Surrounding MESP2 intensity (Methods) of tracked cells at 72 h and 84 h (n = 98 cells from two Somitoids; unpaired two-tailed t-test). Cells at both time points are grouped based on relative MESP2 intensity at 72 h, with lower 50% on the left (cyan) and higher 50% on the right (magenta). r, Left, temporal profile of MESP2 intensity in cells starting in a correct (orange) or wrong (green) region (Method). Right, end-time-point MESP2 intensity of cells with correct or wrong start. n = 98 cells from two Somitoids; unpaired two-tailed t-test. s, Left, temporal profile of displacement in cells starting in a correct (orange) or wrong (green) region (Method). Right, end-time-point displacement of cells with correct or wrong start. n = 98 cells from two Somitoids; unpaired two-tailed t-test. t, Velocity field (arrows) and the corresponding divergence (heatmap) of Particle Image Velocimetry analysis (left) on an additional Somitoid and regions of positive divergence overlayed on the MESP2 reporter image (right; yellow outlines). u, Summary of MESP2 expression and pattern formation processes in the timeline of the Somitoid differentiation. v, Quantification of UNCX reporter in MESP2-high (n = 8 re-aggregates from 3 experiments) and MESP2-low (n = 6 re-aggregates from 3 experiments) re-aggregates in Fig. 2l-n, paired two-sided t-test. In all box and whisker plots, the centre indicates the median, the upper bound indicates 75th percentile, and the lower bound indicates 25th percentile. The maxima and minima of the whiskers represent the most extreme non-outlier data points. The outliers are defined as data points greater than the upper bound or smaller than the lower bound by more than 1.5 times the interquartile range. Scale bars represent 500 µm (b, d, f, g, j), 200 µm (e, t), and 100 µm (k, l).
Extended Data Fig. 3 Differential gene expression during cell sorting and perturbations.
a, Expression fold change plots of selected adhesion proteins between MESP2-low vs MESP2-high cells at 72 h, or between MESP2-high vs UNCX cells at 120 h (n = 3 independent experiments for each time point, with 96 Somitoids in each n). The genes plotted are differentially expressed cadherin and protocadherin encoding genes from the comparison between MESP2-low and MESP2-high cells at 72 h. The dashed (dark red) lines represent log2 fold change values of −0.58 and 0.58. The error bar represents the estimated standard error for the log fold change from the model (DESeq2) which is represented as the centre of the bar. Genes with fold changes greater than 1.5 (above or below the dash line) and padj < 0.05 (estimated by Deseq2 using two-sided Wald test) are considered to be differentially expressed and coloured in either yellow (upregulated in MESP2 low cells) or magenta (upregulated in MESP2 high cells). Genes in blue colour from the comparison between MESP2-high and UNCX cells at 120 h are non-differentially expressed genes. The exact P values for each gene are shown in Supplementary Table 3. b, Normalized RNA counts of selected genes encoding adhesion proteins in MESP2-high and MESP2-low cell fractions at 72 h (n = 3 independent experiments for each time point, with 96 Somitoids in each n; DESeq2 with two-sided Wald test). Cells with top 10% mCherry fluorescence are shown on the left (magenta) and top 10% YFP fluorescence on the right (yellow). c, Expression fold change plots of selected Ephrin protein encoding genes between MESP2-low vs MESP2-high cells at 72 h, or between MESP2-high vs UNCX cells at 120 h (n = 3 independent experiments for each time point, with 96 Somitoids in each n). The error bar represents the estimated standard error for the log fold change from the model (DESeq2) which is represented as the centre of the bar. Genes with fold changes greater than 1.5 (above or below the dash line) and padj < 0.05 (estimated by Deseq2 using two-sided Wald test) are considered to be differentially expressed and coloured in either yellow (upregulated in MESP2 low cells) or magenta (upregulated in MESP2 high cells). Genes in blue colour from the comparison between MESP2-high and UNCX cells at 120 h are non-differentially expressed genes. The exact P values for each gene are shown in Supplementary Table 3. d, Normalized RNA counts of selected genes encoding Ephrin proteins in MESP2-high and MESP2-low cell fractions at 72 h (n = 3 independent experiments for each time point, with 96 Somitoids in each n; DESeq2 with two-sided Wald test). e, Expression fold change plots of selected cytoskeleton regulating proteins between MESP2-low vs MESP2-high cells at 72 h, or between MESP2-high vs UNCX cells at 120 h (n = 3 independent experiments for each time point, with 96 Somitoids in each n). The error bar represents the estimated standard error for the log fold change from the model (DESeq2) which is represented as the centre of the bar. Genes with fold changes greater than 1.5 (above or below the dash line) and padj < 0.05 (estimated by Deseq2 using two-sided Wald test) are considered to be differentially expressed and coloured in either yellow (upregulated in MESP2 low cells) or magenta (upregulated in MESP2 high cells). Genes in blue colour from the comparison between MESP2-high and UNCX cells at 120 h are non-differentially expressed genes. The exact P values for each gene are shown in Supplementary Table 3. After differential gene expression analysis using Deseq2, differentially expressed genes from the MESP2-high vs MESP2-low comparison (72 h) were used to do KEGG functional analysis. The 42 genes plotted represent those that appear in the KEGG pathway “hsa04810” (Regulation of actin cytoskeleton). f, Kymograph of HES7 and MESP2 reporters obtained from a line scan across the centre of a Somitoid overexpressing Tiam1 induced by Doxycycline addition at 48 h. g, percentage of UNCX-positive cells characterized by flow cytometry in 120 h control and Somitoids overexpressing Tiam1 induced by Doxycycline addition at 48 h. Bars represent median. Unpaired two-tailed t-test n = 6 replica from 2 independent experiments, with 12—18 Somitoids in each replica. h, Left, maximum-z-projection confocal image of a MESP2/UNCX-reporting Somitoid at 120 h, overexpressing Tiam1 induced by Doxycycline addition at 48 h. Right, spatial auto-correlation analysis of MESP2 and UNCX signals (n = 3 Somitoids for each condition). All scale bars represent 500 µm.
Extended Data Fig. 4 Characterization of the Segmentoid model.
a, b, Time-lapse bright field images of the Segmentoid model. A, anterior; P, posterior. c, Number of rosettes in each Segmentoid (n = 40 Segmentoids) with the bar representing median. d, Left, projected areas of rosettes in Segmentoids (n = 345 rosettes from 20 Segmentoids). Red bars indicate median with interquartile range. Right, shape descriptors of rosettes in Segmentoids (n = 345 rosettes from 20 Segmentoids). The middle hinge corresponds to median, the lower and upper hinges correspond to the first and third quartiles, respectively, and the lower and upper whiskers correspond to the minimum and maximum, respectively. e, Representative bright-field and DAPI images of organoids without Matrigel, with 10% Laminin supplemented, and embedded in 1% Matrigel (n>10 Segmentoids for each condition). f, Length of organoids in suspension (n = 3 experiments) or embedded in Matrigel (1%, 5%, n = 3 experiments; 10%, n = 5 experiments). Individual structure lengths in each experiment are plotted on the left. The median lengths of each experiment are plotted on the right with red bars indicating median, ordinary one-way ANOVA, P = 0.26 (1%), 0.0023 (5%), 0.00014 (10%) compared with No Matrigel condition. g, Percentage of structures with more than 1 axis in different conditions with red bars indicating median. Ordinary one-way ANOVA, P>0.999 (1%), = 0.0154 (5%), 0.0205 (10%) compared with No Matrigel condition. h, Left, time-lapse maximum-z-projection confocal images of PAX3-YFP reporter (top) and PAX3-YFP merged with H2B-mCherry (bottom) in a Segmentoid. Right, kymographs of PAX3 reporter (top), H2B (middle), and merged channels (bottom) in the same Segmentoid. Segmentoids are aligned to the posterior tip at each time point. Scale bars represent 200 µm (a, b, h) and 100 µm (e).
Extended Data Fig. 5 Expression of TBXT and SOX2 in Segmentoids.
Confocal images of immunostaining of TBXT and SOX2 at 24 h (a), 48 h (b), 72 h (c), 96 h (d), and 120 h (e) of the Segmentoid model. Representative maximum-z-projection images are shown from b-e. 48 h, n = 3 Segmentoids; 72 h, n = 11 Segmentoids; 96 h, n = 17 Segmentoids; 120 h, n = 21 Segmentoids, with 7 Segmentoids still showing apparent TBXT and SOX2 double positive pole. A, anterior; P, posterior. Scale bars (a, b) represent 100 µm and 20 µm in corresponding enlarged views; Scale bars (c, d, e) represent 100 µm.
Extended Data Fig. 6 scRNA-seq of the segmentoid model.
a, Proportion of cell types identified with Leiden clustering at different timepoints of the Segmentoid model. b, Stream plots of velocities on the UMAP after correction for differential kinetics recapitulating trajectory of cell types at various timepoints. c, Signature gene expression trends (Log2/Normalized) toward somite as the specific terminal population.
Extended Data Fig. 7 scRNA-seq of the Segmentoid and the Somitoid model.
a, UMAP embedding of cells merged from both models (19,551 cells) coloured with cell types identified with Leiden clustering. b, Dot plot of selected genes in cell type clusters from both models. c, Machine-learning classification of a previous data set of E9.5 mouse embryo. d, e, Classifier analysis on cell types comparing the in vitro models with mouse E9.5. A k-NN classifier trained on clusters of mouse clusters was used to predict identities of the human in vitro models. f, g, Classifier analysis on cell types (f) and time points (g) comparing Somitoids with Segmentoids. h, Mean expression heatmap of selected genes in the three datasets. i, Top, somite sub-cluster highlighting cells expressing TBX18 (left) and UNCX (right); Bottom, number of cells expressing TBX18, UNCX, or both in Segmentoids (left) and Somitoids (right). j, k, l, Dot plots of HOX-family genes expression at various timepoints of the Segmentoid model (j), the Somitoid model (k), and the NMP cells of the Segmentoid (l). The mean expression of each cluster is scaled per gene.
Extended Data Fig. 8 Antero-Posterior patterning and the segmentation clock.
a, b, Time auto-correlation of HES7 (a) and MESP2 (b) reporter oscillations in individual WT Segmentoids. Triangles indicate auto-correlation peaks, which in turn indicate oscillation period. c, Merged maximum-z-projection confocal image of a Segmentoid with UNCX reporter, DAPI, and Phalloidin staining (n>10 Segmentoids). d, Distribution of rosette numbers in each segment along the anterior-posterior axis. A segment is defined as the posterior boundary of the UNCX stripe to that of the next posterior UNCX stripe. The maximal number of rosettes along the AP axis observed was used to represent the entire segment (n = 24 Segmentoids). e, Distribution of rosette numbers in each segment along the medial-lateral axis (left; n = 25 Segmentoids). The data is re-grouped based on relative AP location in the Segmentoids (right). f, Kymographs of reporters for pseudoHES7, UNCX, and MESP2 in the same HES7-null Segmentoid. g, Wide-field images and graphs of reporter intensities from posterior (P) to anterior (A) end along 120 h HES7-null (left) and WT (right) Segmentoids (n>10 Segmentoids for each condition). h, Time-lapse, maximum-z-projection confocal images of MESP2 reporter in a HES7-null Segmentoid. i, Average nematic order of MESP2/UNCX signals in WT and HES7-null Segmentoids as a function of time (mean±s.d; n = 7 WT Segmentoids and n = 6 HES7-null Segmentoids). Statistics was performed with a Wilcoxon rank-sum test (two-sided) and P-value is shown. j, Summary of HES7-null phenotypes in Somitoid and Segmentoid. All scale bars represent 100 µm.
Extended Data Fig. 9 Antero-Posterior patterning and cell sorting.
a, Reporter dynamics in forming segments aligned according to phases of HES7 oscillation (n = 6 segments in 2 Segmentoids). Data are represented as mean±s.d. b, Representative images of MESP2 reporter and H2B-GFP in a segment at the salt and petter stage. c, Left, Time-lapse, maximum-z-projection confocal images of MESP2 reporter in the same Segmentoid in Fig. 4d. Right, temporal profile of MESP2 intensity in the forming segment outlined in cyan. Green solid-line boxes indicate the corresponding time points. d, Representative example of spatial auto-correlation analysis (mean±s.e.m) of MESP2 and UNCX reporters as a function of time in a developing segment (n = 6 segments from 2 Segmentoids). e, Cell tracking examples of MESP2-high cells. Dots of the same colour represent the same cell and the orange outlines indicate the forming segment. f, Movement classification of tracked MESP2-high cells starting in the posterior part of the segment (n = 111 cells from 10 segments in 5 Segmentoids). g, Additional examples of velocity field (arrows) and the corresponding divergence (heatmap) of Particle Image Velocimetry analysis and regions of positive divergence overlayed on the MESP2 reporter image. h, Additional examples of merged kymographs of HES7/UNCX (green) and MESP2 (magenta) in a Segmentoid overexpressing Tiam1 induced by Doxycycline addition at 72 h, as well as HES7 and MESP2 oscillations. i, Wide-field images and graphs of reporter intensities from posterior (P) to anterior (A) end along 120 h Segmentoids, overexpressing Tiam1 induced by Doxycycline addition at 72 h (left; n>10 Segmentoids) or treated with 10 µM ROCKi (right; n>10 Segmentoids). All scale bars represent 100 µm.
Extended Data Fig. 10 Embryos stained with Mesp2 HCR probe.
a, Merged maximum-z-projection confocal image of a mouse embryo stained with Mesp2 HCR probe (cyan) and DAPI (magenta). b, Enlarged view of the region indicated by the dotted-line box in a. c, Additional half-embryo pairs stained with MESP2 HCR probe (red) and DAPI (cyan). d, Schemes for quantification. e, High pixel fractions of the anterior and posterior portions of the MESP2 bands at 0 and 45 min. Paired t-test, two-sided. f, Standard Deviations of pixel values in the anterior/posterior portions of the MESP2 bands at 0 and 45 min. Paired t-test, two-sided. Three out of 10 embryos at Time 0 captured the peak of the broad band phase of MESP2 expression, with the criteria that the expression domain roughly occupied the whole segment and MESP2 total intensity was not significantly increased at 45 min. Scale bars represent 100 µm (a, d) and 20 µm (b); 100 µm and 20 µm in corresponding enlarged views (c).
Supplementary information
Supplementary Table 1
Differential gene expression during somitoid differentiation. A Wald test (two-sided hypothesis testing) was performed to compare gene expression between somitoids (48 h, 66 h and 120 h) and iPS cells. This table shows the list of differentially expressed genes from the comparison (fold change > 1.5 and Padj < 0.05).
Supplementary Table 2
Differential gene expression between MESP2- and UNCX-positive cells in 120 h somitoids. Differentially expressed genes of FACS-sorted top 10% YFP (UNCX) fluorescent cells in 120 h somitoids, compared with the top 10% mCherry (MESP2) cells, as measured using RNA-seq. The Wald test (two-sided) was used and genes with Padj < 0.05 and log2[fold change] > 0.58 (fold change > 1.5) were considered to be differentially expressed. Using the above threshold criteria, 233 genes were discovered, among which 70 were upregulated and 163 were downregulated in YFP cells.
Supplementary Table 3
Differential gene expression between MESP2high and MESP2low cells in 72 h somitoids. Differentially expressed genes of the FACS-sorted bottom 10% mCherry (MESP2) fluorescent cells in 72 h somitoids, compared with the top 10% mCherry (MESP2) cells, as measured using RNA-seq. The Wald test (two-sided) was used and genes with Padj < 0.05 and log2[fold change] > 0.58 (fold change > 1.5) were considered to be differentially expressed. Using the above threshold criteria, a total of 1,981 genes were found to be differentially expressed. Among them, 1,321 genes were upregulated and 660 were downregulated in mCherrylow cells.
Supplementary Video 1
Somitoid bright-field video. Time-lapse bright-field video of a somitoid spreading on a Laminin-521-coated surface. Images were acquired every 24.6 min and the video is played at 12 fps.
Supplementary Video 2
HES7 and MESP2 reporters in a somitoid. Time-lapse confocal video of a somitoid reporting HES7 (green fire blue) and MESP2 (magenta) expression. Images were acquired every 15.78 min and the video is played at 15 fps.
Supplementary Video 3
PseudoHES7 and MESP2 reporters in a HES7-null somitoid. Time-lapse confocal video of a HES7-null somitoid reporting pseudoHES7 (green fire blue) and MESP2 (magenta) expression. Images were acquired every 27 min and the video is played at 12 fps.
Supplementary Video 4
MESP2 and UNCX reporters in a somitoid. Time-lapse confocal video of a somitoid reporting MESP2 (magenta) and UNCX (yellow) expression. Images were acquired every 19.8 min and the video is played at 15 fps.
Supplementary Video 5
MESP2 and H2B reporters in a somitoid. Time-lapse maximum z-projection confocal video of a somitoid reporting MESP2 expression (fire; left) and H2B (grey; right). Images were acquired every 6 min and the video is played at 20 fps.
Supplementary Video 6
Tracking of MESP2high cells in a somitoid. Time-lapse maximum z-projection confocal video of a somitoid reporting the expression of MESP2. The orange outline approximately denotes a future MESP2low cluster. The dots all represent MESP2high cells, which are sorted out of the orange region in time. Images were acquired every 5 min and the video is played at 6 fps.
Supplementary Video 7
Segmentoid bright-field video. Time-lapse bright-field video of segmentoids in 10% Matrigel. Images were acquired every 19.8 min and the video is played at 15 fps.
Supplementary Video 8
HES7, MESP2 and UNCX reporters in segmentoids. Time-lapse maximum z-projection confocal video of segmentoids reporting HES7 (green fire blue), MESP2 (magenta) and UNCX (green fire blue) expression. Images were acquired every 18 min and the video is played at 15 fps.
Supplementary Video 9
PseudoHES7, MESP2 and UNCX reporters in HES7-null segmentoids. Time-lapse maximum z-projection confocal video of HES7-null segmentoids reporting pseudoHES7 (green fire blue), MESP2 (magenta), and UNCX (green fire blue) expression. Images were acquired every 18 min and the video is played at 15 fps.
Supplementary Video 10
HES7, MESP2, and UNCX reporters in segmentoid with higher resolution. Time-lapse maximum-z-projection confocal video of a segmentoid reporting the expression of HES7/UNCX (left), MESP2 (middle) and merged signals (HES7/UNCX, green fire blue; MESP2, magenta). The start of the video was at day 4 of differentiation (96 h) and is denoted as time 0. The cyan box approximately outlines a forming segment. Images were acquired every 6 min and the video is played at 15 fps.
Supplementary Video 11
Tracking of MESP2high cells in a segmentoid. Time-lapse maximum z-projection confocal video of a segmentoid reporting the expression of MESP2. The red outline on the right approximately denotes the same forming segment and the dots all represent MESP2high cells. The yellow, blue, red and cyan cells start in the posterior part of the segment and are sorted to the anterior MESP2high compartment in time. The magenta cells move to the anterior MESP2high compartment of the next segment. Images were acquired every 6 min and the video is played at 6 fps.
Supplementary Video 12
HES7, MESP2 and UNCX reporters in segmentoids with Tiam1 overexpression. Time-lapse maximum z-projection confocal video of HES7/UNCX (green fire blue) and MESP2 (magenta) reporting segmentoids overexpressing Tiam1 induced by Doxycycline addition at 72 h. Video started at 84 hr and images were acquired every 18.6 min. The video is played at 12 fps.
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Miao, Y., Djeffal, Y., De Simone, A. et al. Reconstruction and deconstruction of human somitogenesis in vitro. Nature 614, 500–508 (2023). https://doi.org/10.1038/s41586-022-05655-4
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DOI: https://doi.org/10.1038/s41586-022-05655-4
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