Lebel, M.; Sancerini, T.; Rabiteau, S.; Marchal, S.; Sharma, P.; Scully, T. D.; Balissat, E.; Hiebert, L. S.; De Tomaso, A. W.; Klein, A. M.; Alie, A.; Tiozzo, S.

Colonial tunicates are the only chordates capable of forming fully functional bodies from somatic tissues through non-embryonic development, known as budding. In Botryllus schlosseri, this agametic process, termed peribranchial budding, generates new zooids in a stereotyped, cyclical manner from a cluster of cells of the peribranchial epithelium. Despite detailed morphological characterization, the molecular and cellular underpinnings of budding initiation remain poorly understood. Here, we present the first single-cell transcriptomic atlas of B. schlosseri peribranchial budding, encompassing multiple stages from pre-budding to near-mature zooid. This high-resolution atlas captures the diversity of cell types involved in budding and their dynamics enabling precise cluster annotation and lineage trajectory inference. While circulating mesenchymal cells exhibit transcriptional hallmarks of stem-like states, we found no evidence of broad contribution to bud onset and morphogenesis beyond hematopoietic and gonadal lineages. Instead, we identified a distinct founder cell population arising from peribranchial epithelium, marked by a unique transcriptional profile and progressive acquisition of developmental potency. This supports a model in which budding is initiated by dedifferentiation of committed epithelial cells rather than activation of multi- or pluripotent stem cells. Furthermore, we highlight signaling pathways, including GNRHR-like receptors, that may couple metabolic state with developmental progression. Altogether, our data suggest that peribranchial budding in B. schlosseri is driven by potential reprogramming within epithelial tissues. This work provides a foundational resource for studying non-embryonic development and the evolution of regenerative strategies in chordates.