Integrin-alpha-6+ Stem Cells (ISCs) are responsible for whole body regeneration in an invertebrate chordate

Colonial ascidians are the only chordates able to undergo whole body regeneration (WBR), during which entire new bodies can be regenerated from small fragments of blood vessels. Here, we show that during the early stages of WBR in Botrylloides diegensis, proliferation occurs only in small, blood-borne cells that express integrin-alpha-6 (IA6), pou3 and vasa. Ablation of proliferating cells using Mitomycin C (MMC) blocks WBR in vascular fragments, but can be rescued by injection of cycling cells isolated from an untreated individual. Using prospective isolation and limit dilution analyes, we found that FACS-isolated IA6+ stem cells (ISCs) could rescue WBR in MMC treated vascular fragments, even when injecting only a single cell. Lineage tracing using EdU-labeling further revealed that donor-derived ISCs directly give rise to regenerating tissues. Inhibitors of either Notch or canonical Wnt signaling block WBR and reduce proliferation of ISCs, indicating that these two pathways regulate ISC activation.