Abstract
The role of programmed cell death during embryonic development has been described previously, but its specific contribution to peri- and post-implantation stages is still debatable. Here, we used transmission electron microscopy and immunostaining of E5.5–7.5 mouse embryos to investigate death processes during these stages of development. We report that in addition to canonical apoptosis observed in E5.5–E7.5 embryos, a novel type of cell elimination occurs in E7.5 embryos among the epiblasts at the apical side, in which cells shed membrane-enclosed fragments of cytosol and organelles into the lumen, leaving behind small, enucleated cell remnants at the apical surface. This process is caspase-independent as it occurred in Apaf1 knockout embryos. We suggest that this novel mechanism controls epiblast cell numbers. Altogether, this work documents the activation of two distinct programs driving irreversible terminal states of epiblast cells in the post-implantation mouse embryo.
Similar content being viewed by others
Log in or create a free account to read this content
Gain free access to this article, as well as selected content from this journal and more on nature.com
or
Data availability
Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study.
References
Zakeri Z, Penaloza CG, Smith K, Ye Y, Lockshin RA. What cell death does in development. Int J Devel Biol. 2015;59:11–22.
Hernández‐Martínez R, Covarrubias L. Interdigital cell death function and regulation: new insights on an old programmed cell death model. Dev Growth Differ. 2011;53:245–58.
Suzanne M, Steller H. Shaping organisms with apoptosis. Cell Death Diff. 2013;20:669–75.
Manjón C, Sánchez-Herrero E, Suzanne M. Sharp boundaries of dpp signalling trigger local cell death required for drosophila leg morphogenesis. Nat Cell Biol. 2007;9:57–63.
Kuan C-Y, Roth KA, Flavell RA, Rakic P. Mechanisms of programmed cell death in the developing brain. Trends Neurosci. 2000;23:291–7.
Brison DR, Schultz RM. Apoptosis during mouse blastocyst formation: evidence for a role for survival factors including transforming growth factor α. Biol Reprod. 1997;56:1088–96.
El-Shershaby A, Hinchliffe J. Cell redundancy in the zona-intact preimplantation mouse blastocyst: a light and electron microscope study of dead cells and their fate. Development. 1974;31:643–54.
Morris SA, Teo RT, Li H, Robson P, Glover DM, Zernicka-Goetz M. Origin and formation of the first two distinct cell types of the inner cell mass in the mouse embryo. Proc Natl Acad Sci USA. 2010;107:6364–9.
Byrne A, Southgate J, Brison D, Leese H. Analysis of apoptosis in the preimplantation bovine embryo using tunel. Reproduction. 1999;117:97–105.
Coucouvanis E, Martin GR. Signals for death and survival: a two-step mechanism for cavitation in the vertebrate embryo. Cell. 1995;83:279–87.
Coucouvanis E, Martin GR. Bmp signaling plays a role in visceral endoderm differentiation and cavitation in the early mouse embryo. Development. 1999;126:535–46.
Abud HE. Shaping developing tissues by apoptosis. Cell Death Differ. 2004;11:797.
Bedzhov I, Zernicka-Goetz M. Self-organizing properties of mouse pluripotent cells initiate morphogenesis upon implantation. Cell. 2014;156:1032–44.
Kim YS, Fan R, Kremer L, Kuempel-Rink N, Mildner K, Zeuschner D, et al. Deciphering epiblast lumenogenesis reveals proamniotic cavity control of embryo growth and patterning. Sci Adv. 2021;7:eabe1640.
Singla S, Iwamoto-Stohl LK, Zhu M, Zernicka-Goetz M. Autophagy-mediated apoptosis eliminates aneuploid cells in a mouse model of chromosome mosaicism. Nat Commun. 2020;11:1–15.
Molè MA, Weberling A, Fässler R, Campbell A, Fishel S, Zernicka-Goetz M. Integrin β1 coordinates survival and morphogenesis of the embryonic lineage upon implantation and pluripotency transition. Cell Rep. 2021;34:108834.
Snow MH. Cell death in embryonic development. Perspectives on mammalian cell death. Oxford University Press, Oxford; 1987. p. 202–28.
Gardner R, Cockroft D. Complete dissipation of coherent clonal growth occurs before gastrulation in mouse epiblast. Development. 1998;125:2397–402.
O’Farrell PH, Stumpff J, Su TT. Embryonic cleavage cycles: how is a mouse like a fly? Curr Biol. 2004;14:R35–R45.
Streffer C, Beuningen DV, Molls M, Zamboglou N, Schulz S. Kinetics of cell proliferation in the pre‐implanted mouse embryo in vivo and in vitro. Cell Prolif. 1980;13:135–43.
Poelmann R. Differential mitosis and degeneration patterns in relation to the alterations in the shape of the embryonic ectoderm of early post-implantation mouse embryos. Development. 1980;55:33–51.
Pereira PN, Dobreva MP, Graham L, Huylebroeck D, Lawson KA, Zwijsen A. Amnion formation in the mouse embryo: the single amniochorionic fold model. BMC Dev Biol. 2011;11:48.
Tam P, Beddington R. Establishment and organization of germ layers in the gastrulating mouse embryo. In: Derek J Chadwick JM, editor. Postimplantation development in the mouse: Ciba foundation symposium 165. Wiley: Chichester; 1992. p. 27–49.
Manova K, Tomihara‐Newberger C, Wang S, Godelman A, Kalantry S, Witty‐Blease K, et al. Apoptosis in mouse embryos: elevated levels in pregastrulae and in the distal anterior region of gastrulae of normal and mutant mice. Dev Dyn. 1998;213:293–308.
Sanders E, Torkkeli P, French A. Patterns of cell death during gastrulation in chick and mouse embryos. Anat Embryol. 1997;195:147–54.
Los M, Wesselborg S, Schulze-Osthoff K. The role of caspases in development, immunity, and apoptotic signal transduction: lessons from knockout mice. Immunity. 1999;10:629–39.
Lee SC, Chan J, Clement MV, Pervaiz S. Functional proteomics of resveratrol‐induced colon cancer cell apoptosis: caspase‐6‐mediated cleavage of lamin a is a major signaling loop. Proteomics. 2006;6:2386–94.
Menon V, Ghaffari S. Erythroid enucleation: a gateway into a “bloody” world. Exp Hematol. 2021;95:13–22.
Yoshida H, Kong Y-Y, Yoshida R, Elia AJ, Hakem A, Hakem R, et al. Apaf1 is required for mitochondrial pathways of apoptosis and brain development. Cell. 1998;94:739–50.
Nagasaka A, Kawane K, Yoshida H, Nagata S. Apaf-1-independent programmed cell death in mouse development. Cell Death Diff. 2010;17:931–41.
Yuan J, Kroemer G. Alternative cell death mechanisms in development and beyond. Genes Dev. 2010;24:2592–602.
Cocucci E, Meldolesi J. Ectosomes and exosomes: shedding the confusion between extracellular vesicles. Trends Cell Biol. 2015;25:364–72.
Williams J, Duckworth C, Burkitt M, Watson A, Campbell B, Pritchard D. Epithelial cell shedding and barrier function: a matter of life and death at the small intestinal villus tip. Vet Pathol. 2015;52:445–55.
Bullen TF, Forrest S, Campbell F, Dodson AR, Hershman MJ, Pritchard DM, et al. Characterization of epithelial cell shedding from human small intestine. Lab Invest. 2006;86:1052.
Gudipaty SA, Rosenblatt J. Epithelial cell extrusion: pathways and pathologies. Semin Cell Dev Biol. 2017;67:132–40.
Farkaš R. The complex secretions of the salivary glands of drosophila melanogaster, a model system. In: Cohen E. MB, (ed). Extracellular composite matrices in arthropods. Cham: Springer; 2016. p. 557–600.
Farkaš R, Ďatková Z, Mentelova L, Löw P, Beňová-Liszeková D, Beňo M, et al. Apocrine secretion in drosophila salivary glands: Subcellular origin, dynamics, and identification of secretory proteins. PloS ONE. 2014;9:e94383.
Basilicata MF, Frank M, Solter D, Brabletz T, Stemmler MP. Inappropriate cadherin switching in the mouse epiblast compromises proper signaling between the epiblast and the extraembryonic ectoderm during gastrulation. Sci Rep. 2016;6:1–15.
Mathiah N, Despin‐Guitard E, Stower M, Nahaboo W, Eski ES, Singh SP, et al. Asymmetry in the frequency and position of mitosis in the mouse embryo epiblast at gastrulation. EMBO Rep. 2020;21:e50944.
Ichikawa T, Nakazato K, Keller PJ, Kajiura-Kobayashi H, Stelzer EH, Mochizuki A, et al. Live imaging of whole mouse embryos during gastrulation: migration analyses of epiblast and mesodermal cells. PLoS ONE. 2013;8:e64506.
Acknowledgements
We thank the Stem Cells and Advanced Cell Technologies Unit at the Weizmann Institute for LMD equipment and guidance. We thank Katya Rehav (Department of Chemical Research Support, Weizmann Institute) for her expertise in performing the FIB-SEM volume imaging. We thank Dr. Yonatan Stelzer (Department of Molecular and Cellular Biology, Weizmann Institute) for reading the paper and consultations. We thank Prof. Tak Wah Mak (University of Toronto) for providing the Apaf-1 mice. We thank Prof. Yaqub (Jacob) Hanna and Alejandro Aguilera Castrejon for consulting and assistance. This work was supported by grants from the Israel Science Foundation, Grant No. 679/17, and from the European Research Council under the European Union’s Seventh Framework Program (FP7/2007-2013/ERC grant agreement 322709) to A.K.
Author information
Authors and Affiliations
Contributions
R.H. and A.K. conceived and designed the overall project; R.H. performed the experiments; S.L.-Z. performed and analyzed the EM studies; R.H. and A.K. interpreted the results; R.H., V.L.-S., S.B., and A.K. wrote and edited the paper; R.H. and S.B. generated figures and graphs; A.K. supervised the work and acquired funding for the project. All authors critically reviewed and revised the paper and approved the final paper.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Ethics statement
Animal studies were conducted in compliance with the Institutional Animal Care and Use Committee (IACUC) regulations (Permission# 12410419-2).
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Edited by M. Piacentini
Rights and permissions
About this article
Cite this article
Halimi, R., Levin-Zaidman, S., Levin-Salomon, V. et al. Epiblast fragmentation by shedding—a novel mechanism to eliminate cells in post-implantation mouse embryos. Cell Death Differ 29, 1255–1266 (2022). https://doi.org/10.1038/s41418-021-00918-5
Received:
Revised:
Accepted:
Published:
Version of record:
Issue date:
DOI: https://doi.org/10.1038/s41418-021-00918-5
