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Open Access Research article

Proteomic analysis of blastema formation in regenerating axolotl limbs

Nandini Rao1, Deepali Jhamb2, Derek J Milner3, Bingbing Li1, Fengyu Song4, Mu Wang5, S Randal Voss6, Mathew Palakal2, Michael W King5, Behnaz Saranjami1, Holly LD Nye3, Jo Ann Cameron3 and David L Stocum1*

Author affiliations

1 Department of Biology and Center for Regenerative Biology and Medicine, Indiana University-Purdue University Indianapolis, Indianapolis, IN, USA

2 School of Informatics and Center for Regenerative Biology and Medicine, Indiana University-Purdue University Indianapolis, Indianapolis, IN, USA

3 Department of Cell and Developmental Biology, and Regeneration Biology and Tissue Engineering Theme, Institute for Genomic Biology, University of Illinois-Urbana Champaign, Urbana, IL, USA

4 Department of Oral Biology, School of Dentistry and Center for Regenerative Biology and Medicine, Indiana University-Purdue University Indianapolis, Indianapolis, IN, USA

5 Department of Biochemistry, School of Medicine and Center for Regenerative Biology and Medicine, Indiana University-Purdue University Indianapolis, Indianapolis, IN, USA

6 Department of Biology and Spinal Cord and Brain Injury Center, University of Kentucky at Lexington, Lexington, KY, USA

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Citation and License

BMC Biology 2009, 7:83  doi:10.1186/1741-7007-7-83

Published: 30 November 2009

Abstract

Background

Following amputation, urodele salamander limbs reprogram somatic cells to form a blastema that self-organizes into the missing limb parts to restore the structure and function of the limb. To help understand the molecular basis of blastema formation, we used quantitative label-free liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS)-based methods to analyze changes in the proteome that occurred 1, 4 and 7 days post amputation (dpa) through the mid-tibia/fibula of axolotl hind limbs.

Results

We identified 309 unique proteins with significant fold change relative to controls (0 dpa), representing 10 biological process categories: (1) signaling, (2) Ca2+ binding and translocation, (3) transcription, (4) translation, (5) cytoskeleton, (6) extracellular matrix (ECM), (7) metabolism, (8) cell protection, (9) degradation, and (10) cell cycle. In all, 43 proteins exhibited exceptionally high fold changes. Of these, the ecotropic viral integrative factor 5 (EVI5), a cell cycle-related oncoprotein that prevents cells from entering the mitotic phase of the cell cycle prematurely, was of special interest because its fold change was exceptionally high throughout blastema formation.

Conclusion

Our data were consistent with previous studies indicating the importance of inositol triphosphate and Ca2+ signaling in initiating the ECM and cytoskeletal remodeling characteristic of histolysis and cell dedifferentiation. In addition, the data suggested that blastema formation requires several mechanisms to avoid apoptosis, including reduced metabolism, differential regulation of proapoptotic and antiapoptotic proteins, and initiation of an unfolded protein response (UPR). Since there is virtually no mitosis during blastema formation, we propose that high levels of EVI5 function to arrest dedifferentiated cells somewhere in the G1/S/G2 phases of the cell cycle until they have accumulated under the wound epidermis and enter mitosis in response to neural and epidermal factors. Our findings indicate the general value of quantitative proteomic analysis in understanding the regeneration of complex structures.