Systemic Bud Induction and Retinoic Acid Signaling Underlie Whole Body Regeneration in the Urochordate Botrylloides leachi

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Abstract
Regeneration in adult chordates is confined to a few model cases and terminates in restoration of restricted tissues and organs. Here, we study the unique phenomenon of whole body regeneration (WBR) in the colonial urochordate Botrylloides leachi in which an entire adult zooid is restored from a miniscule blood vessel fragment. In contrast to all other documented cases, regeneration is induced systemically in blood vessels. Multiple buds appear simultaneously in newly established regeneration niches within vasculature fragments, stemming from composites of pluripotent blood cells and terminating in one functional zooid. We found that retinoic acid (RA) regulates diverse developmental aspects in WBR. The homologue of the RA receptor and a retinaldehyde dehydrogenase-related gene were expressed specifically in blood cells within regeneration niches and throughout bud development. The addition of RA inhibitors as well as RNA interference knockdown experiments resulted in WBR arrest and bud malformations. The administration of all-trans RA to blood vessel fragments resulted in doubly accelerated regeneration and multibud formation, leading to restored colonies with multiple zooids. The Botrylloides system differs from known regeneration model systems by several fundamental criteria, including epimorphosis without the formation of blastema and the induction of a “multifocal regeneration niche” system. This is also to our knowledge the first documented case of WBR from circulating blood cells that restores not only the soma, but also the germ line. This unique Botrylloides WBR process could serve as a new in vivo model system for regeneration, suggesting that RA signaling may have had ancestral roles in body restoration events. Whole body regeneration (WBR) in Animalia is rare, confined to morphologically less complex taxa such as sponges, cnidarians, and flatworms. In the chordate phylum, only colonial ascidians (invertebrate chordates also known as sea squirts) have the documented ability to wholly regenerate. Once separated from the colony, any minute fragment of peripheral blood vessel (about 1 mm in length, containing 100–300 blood cells) of the colonial ascidian Botrylloides leachi regenerates an entire functional adult within one to three weeks. By following cellular and molecular events in Botrylloides WBR, we revealed that this system proceeds differently from regeneration events in other model organisms by several fundamental criteria. This is, for example, to our knowledge the first documented case of WBR initiating from circulating blood cells that restore not only the body tissue, but also the germ line. We found that retinoic acid (RA) signaling, previously reported in the regeneration of specific vertebrate tissues and organs, plays a major role in WBR via RA receptor expression throughout the entire regenerating animal. This suggests that RA signaling may have had ancestral roles in body restoration events. Elucidating the processes involved in this WBR system will improve our understanding of the nature of regeneration and the reduced regeneration capabilities represented in so many vertebrates.