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Published March 5, 2024 | in press
Journal Article Open

Distinct pathways drive anterior hypoblast specification in the implanting human embryo


Development requires coordinated interactions between the epiblast, which generates the embryo proper; the trophectoderm, which generates the placenta; and the hypoblast, which forms both the anterior signalling centre and the yolk sac. These interactions remain poorly understood in human embryogenesis because mechanistic studies have only recently become possible. Here we examine signalling interactions post-implantation using human embryos and stem cell models of the epiblast and hypoblast. We find anterior hypoblast specification is NODAL dependent, as in the mouse. However, while BMP inhibits anterior signalling centre specification in the mouse, it is essential for its maintenance in human. We also find contrasting requirements for BMP in the naive pre-implantation epiblast of mouse and human embryos. Finally, we show that NOTCH signalling is important for human epiblast survival. Our findings of conserved and species-specific factors that drive these early stages of embryonic development highlight the strengths of comparative species studies.

Copyright and License

© The Author(s) 2024This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.


The authors thank patients at CARE, Herts & Essex, Bourn Hall Fertility and King’s Fertility Clinics for their generous donations, as well as the embryologists and members of each clinic for facilitating donations. We thank G. Serapio-García for advice on Bayesian statistical analysis and the bioinformaticians and data scientists who made their code, packages and vignettes available. This work is supported by Wellcome Trust (207415/Z/17/Z) and Open Atlas and NOMIS awards to M.Z.-G. B.A.T.W. was supported by the Gates Cambridge Trust. C.W.G. was supported by a Leverhulme Trust Early Career Fellowship. L.K.I.-S. was supported by the Rosetrees Trust.


These authors contributed equally: Bailey A. T. Weatherbee, Antonia Weberling, Carlos W. Gantner.

B.A.T.W., A.W., C.W.G. and L.K.I.-S. thawed and cultured human embryos for research. B.A.T.W. performed single-cell sequencing analyses. Z.B., A.B., A.C., P.C., C.D., P.E., S.F., S.G.V., M.L., R.O., C.P., N.C., L.R., A.M., L.W., L.C., K.E. and P.S. interfaced with patients, prepared informed consent documentation and prepared embryos for transfer from clinical to research setting. A.W. dissected and cultured mouse embryos. B.A.T.W. and C.W.G. cultured and differentiated human and mouse stem cells. B.A.T.W. and C.W.G. performed quantitative image analyses. B.A.T.W., C.W.G., A.W. and M.Z.-G. wrote the manuscript and conceived the study. B.A.T.W., A.W. and C.W.G. contributed equally.

Data Availability

All raw data used here are previously published and publicly available. For aligning sequencing data, GRCh38 (https://www.ncbi.nlm.nih.gov/assembly/GCF_000001405.26/), Genome assembly Macaca_fascicularis_5.0 (https://www.ncbi.nlm.nih.gov/datasets/genome/GCF_000364345.1/) and GRCm39 (https://www.ncbi.nlm.nih.gov/datasets/genome/GCF_000001635.27/) were used. For human data: Molè et al.10, ArrayExpress E-MTAB-8060; Xiang et al.26, Gene Expression Omnibus GSE136447; Zhou et al.27, Gene Expression Omnibus GSE109555; Petropoulos et al.45, ArrayExpress E-MTAB-3929; Blakely et al.36, Gene Expression Omnibus GSE66507. For cynomolgus monkey data: Yang et al.35, Gene Expression Omnibus GSE148683; Ma et al.28, Gene Expression Omnibus GSE130114; Nakamura et al.21, Gene Expression Omnibus GSE74767. For mouse data: Pijuan-Sala et al.71, ArrayExpress E-MTAB-6967; Mohammed et al.70, Gene Expression Omnibus GSE100597; Cheng et al.68, Gene Expression Omnibus GSE109071; Deng et al.69, Gene Expression Omnibus GSE45719. Scripts used for analysis are available at ref. 79. The integrated Seurat objects for each species are available on Zenodo100 (https://doi.org/10.5281/zenodo.7689580). All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

Conflict of Interest

The authors declare no competing interests.


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Additional details

March 6, 2024
March 6, 2024