INTRODUCTIONAxis and germ-layer formations are central issues in vertebrate embryology. An intriguing question is how the mechanisms that existed in an ancestral vertebrate have been modified during vertebrate evolution. A major stream of vertebrates (Osteichthyes) evolved in two monophyletic lineages: the Sarcopterygii and Actinopterygii. There are many differences in the molecular and cellular mechanisms of embryogenesis between teleosts (actinopterygians) and amphibians (sarcopterygians), including the differences in bauplan (body plan). Polypterus diverged from all other actinopterygians ~400 million years ago (Mya) during the Devonian period, soon after the divarication of an ancestral bony fish into Actinopterygii and Sarcopterygii. Polypterus is thus uniquely well suited for studies assessing the ancestral state or bauplan of Osteichthyes and Actinopterygii, as well as the divergence of embryogenetic processes in teleosts and amphibians. This protocol describes the collection of Polypterus embryos and the method for microinjection.