Mouse embryo dissection and culture

All mice used were intercrosses of strain CD1 and obtained through natural mating. The sex of embryos and the ages of mice using mating were not considered in this study. The mice were maintained in a state-of-the-art biofacility with daily health checks carried out by dedicated trained staff. The mice were maintained on a lighting regime of 12 h:12 h light:dark with food and water supplied ad libitum. This research has been regulated under the Animals (Scientific Procedures) Act 1986 Amendment Regulations 2012 following ethical review by the University of Cambridge Animal Welfare and Ethical Review Body (AWERB). Use of animals in this project was approved by the ethical review committee for the University of Cambridge, and relevant Home Office licences are in place. Embryonic staging assumed, that mating occurred at midnight. Hence, embryos were staged at E0.5 and noon of the following day. Pregnant females were killed on day 2.5, day 3.5, and day 4.5 post coitum (E2.5, E.3.5, and E4.5) by cervical dislocation. Oviduct and uterus were dissected and flushed with M2 medium (Merck) using Leica M165C stereo microscope. Post-implantation E5.5 and E6.5 embryos were carefully dissected from the implantation sites within the uterus and washed in M2 medium before fixation and subsequent processing.