Journal article
Ex utero mouse embryogenesis from pre-gastrulation to late organogenesis
Nature (London)
17/Mar/2021
PMID: 33731940
Abstract
Establishment of the mammalian body plan occurs shortly after the embryo implants into the maternal uterus, and our understanding of post-implantation developmental processes remains limited. While methods for in vitro culture of pre- and peri-implantation mouse embryos are routinely utilized, approaches for robust culture of post-implantation embryos from egg cylinder stages until advanced organogenesis remain to be established. Here we develop highly conducive ex utero post-implantation mouse embryo culture platforms, that enable appropriate development of embryos before gastrulation (E5.5) until the hind limb formation stage (E11). Late gastrulating embryos (E7.5) are grown in 3D rotating bottles settings, while extended culture from pre-gastrulation stages (E5.5 or E6.5) requires a combination of novel static and rotating bottle culture platforms. Histological, molecular, and single-cell RNA-seq analyses validate that the ex utero cultured embryos recapitulate in utero development precisely. This culture system is amenable to introducing a variety of embryonic perturbations and micro-manipulations that can be followed ex utero for up to six days. Establishment of a system to robustly grow normal mouse embryos ex utero from pre-gastrulation to advanced organogenesis represents a valuable tool to investigate embryogenesis, eliminating the uterine barrier to mechanistically interrogate post-implantation morphogenesis and tissue specification in mammals.
Details
- Title
- Ex utero mouse embryogenesis from pre-gastrulation to late organogenesis
- Creators
- Alejandro Aguilera-Castrejon (Corresponding Author) - 972WIS_INST___111Bernardo Oldak - Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, IsraelTom Shani - 972WIS_INST___111Nadir Ghanem - Rambam Health Care CampusChen Itzkovich - Rambam Health Care CampusSharon Slomovich - Technion – Israel Institute of TechnologyShadi Tarazi - 972WIS_INST___111Jonathan Bayerl - 972WIS_INST___111Valeriya Chugaeva - 972WIS_INST___111Muneef Ayyash - 972WIS_INST___111Shahd Ashouokhi - 972WIS_INST___111Daoud Sheban - 972WIS_INST___121Nir Livnat - 972WIS_INST___111Lior Lasman - 972WIS_INST___111Sergey Viukov - 972WIS_INST___111Mirie Zerbib - 972WIS_INST___130Yoseph Addadi - 972WIS_INST___113Yoach Rais - 972WIS_INST___122Saifeng Cheng - 972WIS_INST___122Hadas Keren-Shaul - 972WIS_INST___971Yonatan Stelzer - 972WIS_INST___122Raanan Shlomo - 972WIS_INST___ARAD_TECHNOLOGIES_(ISRAEL,_ASHDOD)Rada Massarwa - 972WIS_INST___111Noa Novershtern - 972WIS_INST___111Itay Maza (Corresponding Author) - Gastroenterology Unit, Rambam Health Care Campus, Bruce Rappaport faculty of Medicine, Israel Institute of Technology - Technion, Haifa, Israel. i_maza@rambam.health.gov.ilJacob H Hanna (Corresponding Author) - 972WIS_INST___111
- Resource Type
- Journal article
- Publication Details
- Nature (London); 17/Mar/2021
- Publisher
- England
- Language
- English
- DOI
- https://doi.org/10.1038/s41586-021-03416-3
- PMID
- 33731940
- Grant note
- This work was funded by Pascal and Ilana Mantoux; the European Research Council (ERC-CoG-2016 726497-Cellnaivety); the Flight Attendant Medical Research Council (FAMRI); an Israel Cancer Research Fund (ICRF) professorship; BSF; the Helen and Martin Kimmel Institute for Stem Cell Research; the Helen and Martin Kimmel Award for Innovative Investigation; the Israel Science Foundation (ISF); Minerva; the Sherman Institute for Medicinal Chemistry; the Nella and Leon Benoziyo Center for Neurological Diseases; the David and Fela Shapell Family Center for Genetic Disorders Research; the Weizmann–U. Michigan program; the Kekst Family Institute for Medical Genetics; the Dr. Beth Rom-Rymer Stem Cell Research Fund; the Edmond de Rothschild Foundations; the Zantker Charitable Foundation; and the Estate of Zvia Zeroni. We thank O. Reiner and T. Sapir for help with mouse embryo electroporations; the Crown Genomics institute of the Nancy and Stephen Grand Israel National Center for Personalized Medicine at the Weizmann Institute for support with scRNA-seq; and the Weizmann Institute management and board for providing critical financial and infrastructural support. We dedicate this paper to the memories of R. Massarwa and H. Garty. Author contributions A.A.-C. designed and conducted most of the wet lab, embryology, sequencing and imaging experiments, established the ex utero culture protocol and co-wrote the manuscript. B.O. conducted embryo injections, performed human microglia cultures and generated human–mouse chimeras, assisted in culture condition testing and processed cryosections. T.S. conducted bioinformatics analysis with N.N. supervising. R.M. helped to reproduce previously published protocols for ex utero culture and taught immunohistochemical protocols to our team. I.M. submitted Helsinki approval, collected cord blood and calibrated human cord serum production. N.G. recruited donors and performed cord blood extraction during caesarean sections. C.I. and S.S. assisted with human cord serum production. S.T. generated lentiviruses and assisted in embryo immunostaining and lentiviral infection of embryos. J.B., D.S. and S.V. performed tissue culture and bulk RNA sequencing of mouse pluripotent stem cells. V.C., S.A. and L.L. assisted with embryo immunostaining. N.L. performed characterization of cultured cells by qPCR. M.A. and H.K.-S. assisted with library preparation and single-cell RNA sequencing. Y.A. assisted with light sheet microscopy and live imaging. Y.R., S.C. and Y.S. generated tdTomato reporter embryos and assisted with allele imprinting experiments. M.Z. assisted with embryo injections. R.S. assembled and maintained the gas-pressure regulator module. J.H.H. conceived the idea for this project, conceptually designed the gas regulator module, established the ex utero culture protocol, supervised execution of experiments and adequate analysis of data, and wrote the manuscript. These authors contributed equally: Alejandro Aguilera-Castrejon, Bernardo Oldak. These authors jointly supervised this work: Alejandro Aguilera-Castrejon, Rada M
- Record Identifier
- 993261020003596
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