Data availability
All scRNA-seq and tomo-seq data are available at the Gene Expression Omnibus: GSE215983 (SuperSeries composed of two SubSeries (single-cell and tomo-seq files))—the tomo-seq data (GSE215982) and the scRNA-seq data (GSE215981). The human embryo scRNA-seq data used in Extended Data Fig. 10 can be found at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE171892 and https://www.ebi.ac.uk/biostudies/arrayexpress/studies/E-MTAB-9388. For single-cell analysis, we used GRCh38-2020-A (from 10x Genomics) based on hg38. The human genome CRCh38 is available at https://www.ncbi.nlm.nih.gov/datasets/genome/GCF_000001405.26/. Additional data supporting the findings of this study are available in the article and the Supplementary Information. Further information for resources or technical information are available upon reasonable request from the corresponding author. Requests for transcriptomic analysis should be sent to [email protected].
Code availability
The tomo-seq analysis script is available at https://github.com/anna-alemany.
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Acknowledgements
We thank M. Russeau for help and assistance on pluripotent stem cell culture; P. Lacour, F. Luciani, T. Rashed and H. Malek for their help during the project; J. Dasen for providing guinea pig anti-HOXC9 antibody; C. Metin for sharing ARL13B antibody; and L. Goutebroze for providing the Phalloidin probes. We thank H. Wichterle and the Ribes laboratory, in particular V. Ribes and P. Gilardi, as well as F. Causeret for helpful discussions along the project and critical reading of the manuscript. We thank J.-A. Girault and I. Caillé for their comments on the manuscripts. Imaging experiments and iPSC cultures were carried out, respectively, at the Imaging Platform and the Cell and Tissue Engineering Facility of the Institut du Fer à Moulin. This project was supported by two grants from the ATIP/Avenir program (S.N. and J.F.), Laboratoire d’Excellence (LabEx) Biopsy (ANR-10-LABX-73, S.N.), ANR SYMASYM (ANR-18-CE16-0021-03, S.N. and X.M.) as well as the NWO Gravitation Project: BRAINSCAPES: A Roadmap from Neurogenetics to Neurobiology (NWO: 024.004.012, B.v.S. and A.v.O.) and a ZonMW PSIDER grant (GREAT, 40-46800-98-015, A.L. and A.v.O.).
Author information
Authors and Affiliations
Institut du Fer à Moulin, Paris, France
Simona Gribaudo, Rémi Robert, Julien Ferent & Stéphane Nedelec
Inserm, UMR-S 1270, Paris, France
Simona Gribaudo, Rémi Robert, Julien Ferent & Stéphane Nedelec
Sorbonne Université, Science and Engineering Faculty, Paris, France
Simona Gribaudo, Rémi Robert, Julien Ferent & Stéphane Nedelec
Oncode Institute, Utrecht, The Netherlands
Björn van Sambeek, Anna Lyubimova & Alexander van Oudenaarden
Hubrecht Institute-KNAW (Royal Netherlands Academy of Arts and Sciences) and University Medical Center Utrecht, Utrecht, The Netherlands
Björn van Sambeek, Anna Lyubimova & Alexander van Oudenaarden
Université Paris Cité, CNRS, Institut Jacques Monod, Paris, France
Camil Mirdass
Institut de Biologie de l’ENS (IBENS), Ecole Normale Supérieure, CNRS, INSERM, Université PSL, Paris, France
Kamal Bouhali & Xavier Morin
Contributions
S.N. and S.G. conceived the project. S.N., S.G., X.M., A.v.O. and R.R. designed experiments. S.G., S.N., R.R. and C.M. performed most wet lab experiments. K.B., R.R., S.N. and X.M. performed time-lapse imaging. J.F. performed clearing and 3D imaging. A.v.O. and A.L. performed scRNA-seq and tomo-seq experiments. A.v.O. and B.v.S. performed scRNA-seq and tomo-seq analysis. Project administration: S.N., X.M. and A.v.O. Supervision: S.N., A.v.O. and X.M. Writing—original draft: S.G. and S.N. Writing—review and editing: all authors.
Corresponding author
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Competing interests
The authors declare no competing interests.
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Extended data
Extended Data Fig. 1 Morphological evolution of hiPSC-derived aggregates over time.
a, Representative bright field images of day 7 structures derived from WTS2 or WTC11 cell lines and from independent experiments, n = 13 for WTS2 and n = 7 for WTC11. b, Aspect ratio (Length (L)/Width (W)) of WTS2-derived structures over time. At day 7, the mean aspect ratio is 1.7 ± 0.76. c, Distribution of (Lmax-Wmax) of WTS2-derived structures over time: bin center=50 µm. d, Box-plot of WTC11-derived structures width and length over time. At day 7, the mean length is 516 ± 285 µm. e, WTC11-derived structures aspect ratio over time. At day 7, the mean aspect ratio is 1.7 ± 0.75. f, Distribution of (Lmax-Wmax) of WTC11-derived structures over time: bin center=50 µm. For box plots, the central line indicates the median; box limits the 1st and 3rd quartiles and whiskers the range. Each dot corresponds to a single structure and different colors indicate independent experiments. For WTS2, n = 11 for day 3; n = 12 for day 4; n = 13 for day 7. For WTC11, n = 2 for day 3; n = 3 for day 4; n = 3 for day 7. Scale bar, 200 µm (a).
Extended Data Fig. 2 Cell fate specification and topographical organization.
a, Schematic of the caudal region of the mammalian embryo. Axial progenitors generate both spinal neural and somitic lineages. n.t: neural tube. pPSM, aPSM: posterior and anterior PSM. b-b’, Maximum-intensity projection of whole-mount immunostaining on day 4 for markers of axial (SOX2/TBXT/CDX2), neural (SOX2) and mesodermal (TBX6) progenitors. WTS2 (b) and WTC11 (b’) derived structures. c, Percentage of structures with regionalized SOX2/TBXT/CDX2 progenitors in the entire population (day 4, n = 7, 159 structures) or among the elongated structures (at day 6, n = 3, 74 structures; day 7, n = 4, 48 structures and day 8, n = 3, 35 structures). Error bars represents the standard deviation. d-g, Immunostaining of sections at day 7 for mitotic cells (P-H3), F-Actin (Phalloidin) and neural progenitors (SOX1/3) markers (d, n = 2), or for the ventral neural progenitor marker (NKX6.1) (e, n = 4), or for the somitic markers FOXC2, SOX9, PAX3 (f, n = 2) and MEOX1 (g, n = 5). h, Low magnification view of sections of structures at day 7, immunostained for SOX9, PAX3 and SOX1/3, n = 4 i,j, 3D maximum-intensity projections of whole mount immunostaining at day 7 for the neuronal (SOX1/3, PAX3 in (i) or SOX1/3, OLIG2 in (j)) and somitic (FOXC2, SOX9, PAX3) markers. Optical plane in j. Scale bars, 50 µm (b, d-f), 100 µm (g, i, j), 500 µm (h).
Extended Data Fig. 3 Analysis of structures derived from other hiPSC lines and factors impacting neuro/mesodermal differentiation.
a, Representative bright field images of day 7 structures generated from SOX2-GFP, WTC11 and KUTE4 cell lines (n = 3, n = 3, n = 2). b-d, Immunostaining of sections of day 7 structures for neural progenitors (SOX1/3) and somitic (PAX3) markers, n = 3 for SOX2-GFP and WTC11, n = 2 for KUTE4. e-h, Representative bright field images (e, f; n = 3 and n = 4) and sections immunostained for neural progenitors (SOX2) and somitic (FOXC2) markers (g, h; n = 3 and n = 4) of day 7 structures generated by aggregating different cell numbers at day 0 (120, 160 and 300 cells). i, Representative bright field images (left panel) and sections immunostained for SOX2 and FOXC2 markers (right panel) of day 7 structures generated by aggregating different cell numbers at day 0 (300 or 600 cells) in presence of CHIR or with the addition of LDN and SB (n = 3). j, Representative bright field images (left panel) and sections immunostained for SOX2 and FOXC2 markers (right panel) of day 7 structures in presence of CHIR or with the addition of bFGF (20 ng/ml) for the first 2 days of differentiation (n = 3). k, Immunostaining of plated day 3 aggregates for neural progenitor (SOX2) and PSM (TBX6) markers. Aggregates obtained with different initial cell numbers were cultured using different B27 supplement (percentage of aggregates with TBX6 staining at day 3: B27 control, 50 ± 13% (120 cells) versus 83 ± 8% (300 cells), n = 2; B27 batch #2450484, 14 ± 9% (120 cells) versus 32 ± 21% (300 cells), n = 2; B27 batch #2450486, 2% (120 cells) versus 8% (300 cells), n = 1). l, Representative bright field images of day 7 structures obtained with the different initial cell numbers and different B27 supplements (n as in k). Scale bars, 200 µm (a, e, f, g, h, i, j, l), 100 µm (b, c, d), 50 µm (k).
Extended Data Fig. 4 Morphometric analysis of day 7 matrigel embedded structures.
a, Percentage of elongation for structures derived from 3 different cells lines (WTS2, n = 9, 892 structures total; WTC11, n = 3, 161 structures total; and SOX2-GFP, n = 2, 76 structures total). b, Box-plot of aspect ratio (length/width) of elongated structures at day 7 (WTS2, n = 8, 100 structures total; WTC11, n = 4, 57 structures total; SOX2-GFP, n = 3, 34 structures total). In the box plots, the central line indicates the median; box limits the 1st and 3rd quartiles and whiskers the range. Each dot corresponds to a single aggregate and different colors indicate independent differentiations. c-e, Gallery of bright field images of day 7 structures derived from 3 different hiPSC lines. Numbers indicate series from independent experiments. Scale bars, 200 µm (c, d, e).
Extended Data Fig. 5 Live imaging of matrigel embedded structures.
a, Time-lapse imaging showing dynamic elongation of matrigel-embedded SOX2-GFP derived structures between day 4 and 5.5. Red arrows pinpoint SOX2 negative cells. b, Time-lapse of matrigel-embedded WTS2 derived structures between day 4 and 7. Post-imaging whole mount immunostaining at day 7 for axial progenitors (SOX2, CDX2) and somitic (FOXC2) markers for the corresponding structure. #3 and #4, examples of non-elongating aggregates. Scale bars, 100 µm (a, b).
Extended Data Fig. 6 WNT, FGF and RA pathways control neural tube formation.
a, Experimental design. b, Representative images of SOX2-GFP whole mount structures in the different culture conditions at day 7, n = 3. c-d, Quantification of the aspect ratio (length/width) in the different conditions for day 7 organoids derived from SOX2-GFP (c) (n = 3, 2 to 6 wells/n/condition) and WTS2 (d) (n = 1, 2 wells/n/condition) hiPSC lines. ERKi and FGFRi=FGF pathways inhibitors. IWP2=Wnt pathway inhibitor. In the box plots, the central line indicates the median; box limits the 1st and 3rd quartiles and whiskers the range. Each dot represents a single organoid. Statistical test in c: Kruskal-Wallis followed by Dunn’s multiple comparison test. *** P
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