Basal delamination during mouse gastrulation primes pluripotent cells for differentiation

Copyright and License

© 2024 MRC Laboratory of Molecular Biology. Published by Elsevier Under a Creative Commons license.

Acknowledgement

We are thankful to Emmanuel Derivery, Isabelle Migeotte, Katie McDole, and Madeline Lancaster for helpful discussions. We thank Claire Knox, Liam Bray, Lucy Tredgett, Chloe Watson, Jessica McCool, Michal Neumann, Victoria Syanda and Crystal Tong for their help with mouse husbandry, and Alicia Gonzalez Martinez for her help with data analysis. We also thank the light microscopy and flow cytometry facilities of the LMB for their experimental support. We thank Buzz Baum and Michele Petruzzelli for the critical reading of the manuscript. Work in the Shahbazi lab was supported by the Medical Research Council, as part of United Kingdom Research and Innovation (MC_UP_1201/24), the Engineering and Physical Sciences Research Council (Horizon Europe guarantee funding for projects selected by the European Research Council (ERC), EP/X023044/1), and an EMBO Advanced fellowship awarded to M.N.S. The Elosegui-Artola lab was supported by the ERC (851055) and the Francis Crick Institute, which receives its core funding from Cancer Research UK (CC2214), the Medical Research Council (CC2214), and the Wellcome Trust (CC2214). Work in the Zernicka-Goetz lab was supported by a Wellcome Trust grant (207415/Z/17/Z) and an ERC grant (669198). N.S. is supported by a JSPS Overseas Research fellowship, V.S.R. is supported by a Milstein fellowship, and A.M. is a recipient of an LMB Cambridge Scholarship (Cambridge Trust).

Contributions

V.S.R., N.S., and M.N.S. designed, performed, and analyzed most of the experiments. A.M. developed image analysis pipelines and analyzed immunofluorescent images. H.K. analyzed the WGBS, bulk, and scRNA-seq data. A.S.K. and S.G. designed the transcriptome and methylome experiments and collected and processed samples for sequencing. A.M. processed samples for WGBS. O.C. generated micromolds. S.W. and J.B. provided advice on image analysis. F.L., M.W., and D.K. performed embryo chimera and transfer experiments. A.E.-A. supervised and funded the generation of micromolds. A.M. supervised and funded the transcriptome and methylome experiments. M.Z.-G. supervised and funded the initial development of the 3D EpiSC culture. M.N.S. conceived the project, supervised the work, and wrote the manuscript with the help of V.S.R., N.S., A.M., and H.K.

Data Availability

Document S1. Figures S1–S7

Table S1. Morphological outcome of ESCs cultured in 3D Matrigel, related to Figure 1A and 1B. ESCs were cultured in 3D Matrigel using different combinations of basal medium and supplements (growth factors and inhibitors). The morphological outcome on day 6 is indicated

Table S2. Differentially expressed genes (DEGs) between ESCs, EpiLCs, 2D EpiSCs, and 3D EpiSCs, related to Figure 2B. The differentially expressed genes between all pair-wise comparisons of in vitro stem cell cultures are shown

Table S3. Marker genes for the clusters identified in the single-cell RNA sequencing data of 3D EpiSCs, related to Figures 2E and S3G. List of marker genes for clusters 0 (epiblast), 1 (primitive streak), and 2 (unknown)

Table S4. Expression of cell state and signaling markers in Brachyury− and Brachyury+ cells, related to Figures 2, S4N, and S4O

Table S5. List of oligonucleotides used in this study, related to STAR Methods

Conflict of Interest

The authors declare no competing interests.