Whole Body Regeneration in a Colonial Ascidian, Botrylloides violaceus FEDERICO D. BROWN 1–3ç , ELENA L. KEELING 1,2,4§ , ANNA D. LE 1,2 , AND BILLIE J. SWALLA 1,2 1 Biology Department, Center for Developmental Biology, and Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, Washington 2 Friday Harbor Laboratories, University of Washington, Friday Harbor, Washington 3 Evolutionary Biology—Division 4, Max Planck Institute for Developmental Biology, Tu¨bingen, Germany 4 Biological Sciences Department, California Polytechnic State University, San Luis Obispo, California ABSTRACT Colonial ascidians are the only chordates to undergo whole body regeneration (WBR), the ability to form an entirely new individual from the peripheral vasculature. Here we describe WBR in Botrylloides violaceus, a colonial ascidian that reliably regenerates after ablation of all zooids and buds of young colonies. During early regeneration several buds develop within the tunic vasculature, but only one continues development into a complete zooid. We describe some of the first events of vascular budding leading to the vesicle stage with phase contrast microscopy, time-lapse video recording and detailed histological studies of regenerating colonies. The first conspicuous stage of vascular budding is when a single-layered sphere of cells becomes enclosed by vascular epithelium. We report the appearance of Piwi-positive cells in hemocytes surrounding the regenerates. We observed an increase of proliferating cell nuclear antigen (PCNA)-positive cells in circulatory hemocytes in late regenerates, and found double-labeled nuclear expression with Piwi in a subset of large circulatory cells. We rarely found Piwi or PCNA in differentiating tissues during vascular budding, suggesting that cells that form the epithelial tissues during budding and WBR originate mostly from circulatory hemocyte precursors. We propose that multiple stem cell types are circulating within B. violaceus and that they undergo proliferation in the peripheral vasculature before differentiating into epithelial tissues for all three germ layers during WBR. J. Exp. Zool. (Mol. Dev. Evol.) 312B:885-900, 2009. r 2009 Wiley-Liss, Inc. How to cite this article: Brown FD, Keeling EL, Le AD, Swalla BJ. 2009. Whole body regeneration in a colonial ascidian, Botrylloides violaceus. J. Exp. Zool. (Mol. Dev. Evol.) 312B:885–900. Many organisms have the capacity to regenerate organs or tissues through the proliferation and differentiation of stem cells (Weissman, 2000). Stem cells are self-renewing and unspecialized, but their daughter cells can develop into specia- lized cells (Hyslop et al., 2005; Wong et al., 2005). Studies documenting regeneration by stem cells in the metazoans have mostly been reported for planarians, hydra, and salamanders, but regen- eration is widespread in metazoans (Morgan, ’01; Sanchez Alvarado, 2000). Some classically described vertebrate stem cell lineages are the hematopoietic blood cell lineage and primordial germ cells (PGCs). A small pool of hematopoietic stem cell precursors become determined during Published online 8 July 2009 in Wiley InterScience (www. interscience.wiley.com). DOI: 10.1002/jez.b.21303 Received 16 January 2009; Revised 22 April 2009; Accepted 21 May 2009 § Brown and Keeling contributed equally to the work. Grant sponsors: Dudley Research Fellowship; The UW Royalty Research Fund; The Lerner Gray Fund for Marine Research; The American Heart Association. Additional Supporting Information may be found in the online version of this article. à Correspondence to: Federico D. Brown , Max Planck Institute for Developmental Biology—Evolutionary Biology, Spemmanstrasse 37, Tu ¨bingen D-72076, Germany. E-mail: federico.brown@tuebingen.mpg.de r 2009 WILEY-LISS, INC. JOURNAL OF EXPERIMENTAL ZOOLOGY (MOL DEV EVOL) 312B:885–900 (2009)