Temporal BMP4 effects on mouse embryonic and extraembryonic development

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Acknowledgement

We thank the Tanay and Stelzer group members for discussions and advice; and G. J. Shin, S. J. Schulte and N. A. Pierce for support using multiplex HCR–RNA-FISH. Y.S. is the incumbent of the Louis and Ida Rich Career Development Chair and a member of the European Molecular Biology Organization Young Investigator Program. Research in the Stelzer laboratory is supported by a research grant from the Estate of Betty Weneser, European Research Council (ERC_StG 852865), Helen and Martin Kimmel Stem Cell Institute, ISF (1610/18), Weizmann–Caltech Schwartz/Reisman Collaborative Science Program and Abisch Frenkel Foundation. This research was also supported by B. and J. Lang, the Hadar Impact Fund, the Lord Sieff of Brimpton Memorial Fund, J. and S. Anixter, J. Silva, Maurice and the Vivienne Wohl Biology Endowment, and the Lester and Edward Anixter Family. Work in the lab of M.B.E. was supported by US National Institutes of Health grant R01 HD075335A and by the Paul G. Allen Frontiers Group and Prime Awarding Agency under award no. UWSC10142. M.Z.-G. is a Bren Professor of Biology and Biological Engeneering and Nomis Foundation Distinguish Fellow and this research in her laboratory was supported by the US National Institutes of Health (R01 HD101489A and DP1 HD104575A). The J.H.H. laboratory is funded by Pascal and Ilana Mantoux, the Flight Attendant Medical Research Institute (FAMRI) and the European Union (ERC-COG-2022 no. 101089297–ExUteroEmbryogenesis). M.M. was a postdoctoral fellow of the Minerva Stiftung and is supported by the Walter Benjamin program of the German Research Foundation (DFG). A.T. is supported by the European Research Council (ERC CoG scAssembly), the EU BRAINTIME project, the Israel Science Foundation and the Chen-Zuckerberg Foundation. This research was further supported Israeli Council for Higher Education Data Science program and by a grant from Madame Olga Klein-Astracha.

Data Availability

All sequencing data supporting the conclusions of this study have been meticulously archived and are publicly accessible through the NCBI Gene Expression Omnibus (GEO). These data are catalogued under the GEO Series accession number GSE267870. This ensures comprehensive availability and transparency of the data supporting our research findings. Source data are provided with this paper.

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Code Availability

All custom scripts used in this study have been made openly accessible and can be found at GitHub (https://github.com/tanaylab/EmbExe). Moreover, these scripts have been deposited for permanent archiving and citation at Zenodo⁷¹ (https://doi.org/10.5281/zenodo.11240229). This ensures transparency and reproducibility of the computational methods employed in our research.

Conflict of Interest

M.B.E. is a co-founder, scientific advisory board member, or consultant at TeraCyte, Primordium Labs, Spatial Genomics, and Asymptote Genetic Medicines. J.H.H. is an inventor on patents and patent applications related to ex utero embryogenesis, and a co-founder and chief scientific advisor of Renewal Bio, which has licensed the latter technologies. The other authors declare no competing interests.

Supplemental Material

• Supplementary Table 1: EPC lineage marker genes. Absolute expression (log₂) of variable differentially expressed genes within the EPC lineage (see Fig. 2 and Extended Data Fig. 3) annotated with their corresponding cluster.
• Supplementary Table 2: Specialized TGCs marker genes. Absolute expression (log₂) of differentially expressed genes within specialized TGCs annotated with their corresponding cell type mark.
• Supplementary Table 3: Chorion lineage marker genes. Absolute expression (log₂) of variable differentially expressed genes within the Chorion lineage (see Fig. 3 and Extended Data Fig. 4) annotated with their corresponding cluster. 
• Supplementary Table 4: gRNA and Primers. Primers and guide RNAs used for this study.
• Supplementary Table 5: Differential expression in ExM of embryonic Bmp4-KO. List of genes that showed significant differential expression between WT ExM and embryonic Bmp4-KO ExM (Extended Data Fig. 12b,c).
• Supplementary Table 6: ExM/Allantois marker genes. List of genes showing enrichment within the ExM/Allantois (Fig. 5f,h,i and Extended Data Fig. 13a).
• Supplementary Table 7: PGC marker genes. List of genes showing enrichment within the PGCs (Fig. 5f,h,i and Extended Data Fig. 13a).
• Supplementary Table 8: ExE explants BMP4/NOG RT–qPCR results. RT–qPCR results for explants cultured for 24/36 h in the presence or absence of BMP4/NOG (Extended Data Fig. 9c).
• Supplementary Table 9: ExE Bmp4-KO E12.5 placental data. Measurements of placentas isolated from E12.5 ExE Bmp4-KO embryos (Extended Data Fig. 9d).