title: Proteomic analysis of blastema formation in regenerating axolotl limbs.
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.
  
  
  CONCLUSIONS
  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.
authors: Department of Biology and Center for Regenerative Biology and Medicine, Indiana University-Purdue University Indianapolis, Indianapolis, IN, USA. narao@iupui.edu
authors: Nandini
authors: N
authors: Rao
authors: Deepali
authors: D
authors: Jhamb
authors: Derek J
authors: DJ
authors: Milner
authors: Bingbing
authors: B
authors: Li
authors: Fengyu
authors: F
authors: Song
authors: Mu
authors: M
authors: Wang
authors: S Randal
authors: SR
authors: Voss
authors: Mathew
authors: M
authors: Palakal
authors: Michael W
authors: MW
authors: King
authors: Behnaz
authors: B
authors: Saranjami
authors: Holly L D
authors: HL
authors: Nye
authors: Jo Ann
authors: JA
authors: Cameron
authors: David L
authors: DL
authors: Stocum
mesh_terms: D000557:Ambystoma / Q000502:physiology*; D000671:Amputation, Surgical; D000818:Animals; D020013:Calcium Signaling / Q000235:genetics; D002851:Chromatography, High Pressure Liquid; D005109:Extracellular Matrix / Q000378:metabolism; D005121:Extremities / Q000502:physiology* / Q000601:surgery; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D010449:Peptide Mapping; D040901:Proteomics; D012038:Regeneration / Q000502:physiology*; D053719:Tandem Mass Spectrometry; D014945:Wound Healing
chemical_list: D015544:Inositol 1,4,5-Trisphosphate