Microinjection and 4D fluorescence imaging in the eggs and embryos of the ascidian Phallusia mammillata.

Time-lapse 4D imaging of fluorescently tagged proteins to follow the dynamics of cellular structures (chromosomes, microtubules, actin, centrosomes, cortical structures like the CAB in ascidians, etc.) combined with targeted gene knockdown during embryonic development is a powerful technique to understand the mechanisms of embryonic development. The eggs and embryos of the primitive marine chordate Phallusia mammillata are an excellent model system for combining live cell imaging with gene knockdown experiments. Here we describe simple methods for microinjecting Phallusia eggs with mRNA encoding fluorescent fusion proteins combined with 4D time-lapse imaging techniques we use to follow all of embryonic development from the egg to late tailbud stage.