Abstract
The implanting mouse blastocyst invades the uterine stroma and undergoes a dramatic transformation into an egg cylinder. The morphogenetic and signaling events during this transition are largely unexplored, as the uterine tissues engulf the embryo. Here we describe a protocol supporting the development of the mouse embryo beyond the blastocyst stage in vitro. We established two types of medium to be applied sequentially, and we used a substrate permitting high-resolution imaging of the transition from blastocyst to egg cylinder. We developed two variants of this protocol: the first starts with intact early blastocysts that upon zona removal can attach to the substrate and develop into egg cylinders after 5 d, and the second starts with late blastocysts that upon dissection of the mural trophectoderm form egg cylinders in only 3 d. This method allows observation of a previously hidden period of development, and it provides a platform for novel research into peri-implantation embryogenesis and beyond.
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References
Ma, W.-G., Song, H., Das, S.K., Paria, B.C. & Dey, S.K. Estrogen is a critical determinant that specifies the duration of the window of uterine receptivity for implantation. Proc. Natl. Acad. Sci. 100, 2963–2968 (2003).
Song, H., Han, K. & Lim, H. Progesterone supplementation extends uterine receptivity for blastocyst implantation in mice. Reproduction 133, 487–493 (2007).
Parr, E.L., Tung, H.N. & Parr, M.B. Apoptosis as the mode of uterine epithelial cell death during embryo implantation in mice and rats. Biol. Reprod. 36, 211–225 (1987).
Ansell, J.D., Barlow, P.W. & McLaren, A. Binucleate and polyploid cells in the decidua of the mouse. J. Embryol. Exp. Morphol. 31, 223–227 (1974).
Bedzhov, I. & Zernicka-Goetz, M. Self-organizing properties of mouse pluripotent cells initiate morphogenesis upon implantation. Cell 156, 1032–1044 (2014).
Jenkinson, E.J. & Wilson, I.B. In vitro support system for the study of blastocyst differentiation in the mouse. Nature 228, 776–778 (1970).
Copp, A.J. The mechanism of mouse egg-cylinder morphogenesis in vitro. J. Embryol. Exp. Morphol. 61, 277–287 (1981).
Hsu, Y.C. Differentiation in vitro of mouse embryos beyond the implantation stage. Nature 239, 200–202 (1972).
Solter, D., Biczysko, W., Pienkowski, M. & Koprowski, H. Ultrastructure of mouse egg cylinders developed in vitro. Anat. Rec. 180, 263–279 (1974).
Wiley, L.M. & Pedersen, R.A. Morphology of mouse egg cylinder development in vitro: a light and electron microscopic study. J. Exp. Zool. 200, 389–402 (1977).
Hsu, Y.C. In vitro development of individually cultured whole mouse embryos from blastocyst to early somite stage. Dev. Biol. 68, 453–461 (1979).
Morris, S.A. et al. Dynamics of anterior-posterior axis formation in the developing mouse embryo. Nat. Commun. 3, 673 (2012).
Wu, T.C., Wan, Y.J. & Damjanov, I. Positioning of inner cell mass determines the development of mouse blastocysts in vitro. J. Embryol. Exp. Morphol. 65, 105–117 (1981).
Carson, D.D., Tang, J.P. & Gay, S. Collagens support embryo attachment and outgrowth in vitro: effects of the Arg-Gly-Asp sequence. Dev. Biol. 127, 368–375 (1988).
Hsu, Y.C. Differentiation in vitro of mouse embryos to the stage of early somite. Dev. Biol. 33, 403–411 (1973).
Salomon, D.S. & Sherman, M.I. Implantation and invasiveness of mouse blastocysts on uterine monolayers. Exp. Cell Res. 90, 261–268 (1975).
Kauma, S.W. & Matt, D.W. Coculture cells that express leukemia inhibitory factor (LIF) enhance mouse blastocyst development in vitro. J. Assist. Reprod. Gen. 12, 153–156 (1995).
Gonda, M.A. & Hsu, Y.-C. Correlative scanning electron, transmission electron, and light microscopic studies of the in vitro development of mouse embryos on a plastic substrate at the implantation stage. J. Embryol. Exp. Morphol. 56, 23–39 (1980).
Olovsson, M. & Nilsson, B.O. Structural and functional properties of trophoblast cells of mouse egg-cylinders in vitro. Anat. Rec. 236, 417–424 (1993).
Hsu, Y.C. In vitro development of whole mouse embryos beyond the implantation stage. in Methods in Mammalian Reproduction (ed. Daniel, J.C.) 229–245 (Academic Press, 1978).
Wilson, I.B. & Jenkinson, E.J. Blastocyst differentiation in vitro. J. Reprod. Fertil. 39, 243–249 (1974).
Tam, P.P. Postimplantation mouse development: whole embryo culture and micro-manipulation. Int. J. Dev. Biol. 42, 895–902 (1998).
Behringer, R., Gertsenstein, M., Nagy, K.V. & Nagy, A. Manipulating the Mouse Embryo: a Laboratory Manual 4th ed. (Cold Spring Harbor Laboratory Press, 2014).
Mesnard, D., Filipe, M., Belo, J.A. & Zernicka-Goetz, M. The anterior-posterior axis emerges respecting the morphology of the mouse embryo that changes and aligns with the uterus before gastrulation. Curr. Biol. 14, 184–196 (2004).
Acknowledgements
We are grateful to the Wellcome Trust for the Senior Research Fellowship to M.Z.-G. that funded this work. We also thank the European Molecular Biology Organization (EMBO) for supporting M.B.
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I.B., C.Y.L., M.B. and M.Z.-G. designed the protocol, carried out the work, analyzed the results and prepared the manuscript.
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Bedzhov, I., Leung, C., Bialecka, M. et al. In vitro culture of mouse blastocysts beyond the implantation stages. Nat Protoc 9, 2732–2739 (2014). https://doi.org/10.1038/nprot.2014.186
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DOI: https://doi.org/10.1038/nprot.2014.186
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