The University of Sheffield is a licensed establishment under the Animals (Scientific Procedures) Act 1986. All animals were culled by approved methods cited under Schedule 1 to the Act. Fertilized Bovan brown chicken eggs (Henry Stewart & Co., Norfolk, UK) were incubated at 37.5°C before overnight fixation in Carnoy’s solution between E6 and E9. S. canicula embryos (North Wales Biologicals, Bangor, UK) were raised in oxygenated artificial saltwater (Instant Ocean) at 16°C. Shark embryos were culled with MS-222 (tricaine) at 300 mg/liter and fixed overnight in 4% paraformaldehyde in phosphate-buffered saline (PBS). After fixation, chicken and shark embryos were dehydrated through a graded series of PBS to ethanol (EtOH) and stored at −20°C.

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