Comparative Transcriptional Profiling of the Axolotl Limb Identifies a Tripartite Regeneration-Specific Gene Program Dunja Knapp 1,4 , Herbert Schulz 2 , Cynthia Alexander Rascon 4 , Michael Volkmer 1,3 , Juliane Scholz 4 , Eugen Nacu 1,4 , Mu Le 4 , Sergey Novozhilov 4 , Akira Tazaki 1,4 , Stephanie Protze 1 , Tina Jacob 1 , Norbert Hubner 2 , Bianca Habermann 1,3 , Elly M. Tanaka 1,4 * 1 Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany, 2 Max Delbrueck Center for Molecular Medicine, Berlin, Germany, 3 Max Planck Institute for Biology of Aging, Cologne, Germany, 4 Technical University Dresden, DFG Research Center for Regenerative Therapies Dresden, Dresden, Germany Abstract Understanding how the limb blastema is established after the initial wound healing response is an important aspect of regeneration research. Here we performed parallel expression profile time courses of healing lateral wounds versus amputated limbs in axolotl. This comparison between wound healing and regeneration allowed us to identify amputation- specific genes. By clustering the expression profiles of these samples, we could detect three distinguishable phases of gene expression – early wound healing followed by a transition-phase leading to establishment of the limb development program, which correspond to the three phases of limb regeneration that had been defined by morphological criteria. By focusing on the transition-phase, we identified 93 strictly amputation-associated genes many of which are implicated in oxidative-stress response, chromatin modification, epithelial development or limb development. We further classified the genes based on whether they were or were not significantly expressed in the developing limb bud. The specific localization of 53 selected candidates within the blastema was investigated by in situ hybridization. In summary, we identified a set of genes that are expressed specifically during regeneration and are therefore, likely candidates for the regulation of blastema formation. Citation: Knapp D, Schulz H, Rascon CA, Volkmer M, Scholz J, et al. (2013) Comparative Transcriptional Profiling of the Axolotl Limb Identifies a Tripartite Regeneration-Specific Gene Program. PLoS ONE 8(5): e61352. doi:10.1371/journal.pone.0061352 Editor: Mike O. Karl, Center for Regenerative Therapies Dresden, Germany Received April 10, 2012; Accepted March 12, 2013; Published May 1, 2013 Copyright: ß 2013 Knapp et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by an Alexander von Humboldt fellowship to DK, grant funding from the DFG, SPP1356 ‘‘Pluripotency and Reprogramming’’, and central funds from the Max Planck Society and the DFG Research Center for Regenerative Therapies. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: elly.tanaka@crt-dresden.de Introduction In salamander, limb amputation initiates a wound-healing response followed by the emergence of a proliferative zone of cells, called the blastema, that consists of mesenchymal progenitor cells covered by an epithelium [1]. Injuries trigger a wound-healing response as the first step in regeneration, but simple wounding is not sufficient to launch a full regeneration response. A number of axolotl limb studies have indicated that limb wounds in the absence of full amputation are repaired imperfectly, as in mammals (for review see [2]). Moreover, critical size bone defects are not repaired in the axolotl limb, similar to mammals [3–5]. Therefore, the specific conditions related to amputating the limb are critical to the accumulation of mesenchymal blastema cells that will regenerate the limb. An important question is what are the molecular factors that determine the establishment of a blastema only after amputation, in contrast to other injuries. In terms of a molecular perspective, a number of important studies have previously surveyed changes in gene or protein expression that occur during limb regeneration. Proteomic profiling at 1, 4 and 7 days after amputation and subtractive hybridization screen of the 4 day axolotl limb blastema compared to mature tissue have revealed a number of proteins and transcripts that are induced in a time course upon limb amputation [6,7]. In these studies, the identified transcripts could have been associated with wound healing, amputation or both. Three additional studies using microarrays applied comparative strategies to delineate progress of normal limb regeneration versus conditions where regeneration fails. One study compared normal and denervated limbs at 5 and 14 days after amputation [8,9]. Another study compared the regenerative versus laterally wound- ed epithelium at 7 days after injury, but the changes leading to the formation of mesenchymal blastema were not examined in this comparative approach [8,9]. The most recent study used microarrays to profile normal and denervated limbs at 1, 3 and 7 days and compared that to a skin injury at the body flank [10]. While the events associated with wound healing are doubtlessly a critical part of initiating regeneration, our aim was to identify an amputation-specific gene set that underlies the transition from the adult to the blastema state, distilled apart from the wound healing gene network. It is likely that many changes occur in the first hours or days after limb injury, and a detailed time course particularly at the early time points may help to define the relative kinetics of PLOS ONE | www.plosone.org 1 May 2013 | Volume 8 | Issue 5 | e61352