Vandetanib Embryonic Development

At E13, the eggs in the incubator were candled to remove unfertilized eggs and dead embryos. Embryonic eggs from the layer lineage were randomly selected for intra-ovo injection. Briefly, a small hole was drilled at the wide end of the egg after cleaning the eggshell with 75% alcohol. Vandetanib (Beyotime Biotechnology, Shanghai, China) diluted in PBS (75 μg/mL) was injected into the air chamber through the hole without passing through the chamber, with a dose equivalent to 15 μg per egg. PBS-injected eggs were used as control. After the inoculations were completed, the hole was sealed with paraffin solution and the eggs were placed back into the same incubator to allow them to continue developing.