Proteomic analysis of blastema formation in regenerating axolotl limbs

Abstract

BackgroundFollowing 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.ResultsWe 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.ConclusionOur 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.

Skeletal muscle regeneration in Xenopus tadpoles and zebrafish larvae

“…However, during muscle dedifferentiation, the expression of many muscle genes, mainly the structural ones, is expected to decrease [28,44-47]. Our Real Time PCR analysis showed that the expression of the late muscle genes car (alpha-cardiac actin) and myh4 (myosin heavy chain 4) was 5-fold lower in the 3 dpa distal tail stump compared to the 0 dpa distal stump (Figure 4a).…”

Fine-mapping quantitative trait loci affecting murine external ear tissue regeneration in the LG/J by SM/J advanced intercross line

“…Differentially expressed genes within our intervals and involved in metabolism include Cs, Rnf7 and Becn1. We focused on this group because of previous studies showing the importance of aerobic glycolysis and mitochondrial variations in MRL ear hole regeneration (Sachadyn et al, 2008;Naviaux et al, 2009) and the role of glycolytic enzymes during amphibian regeneration (Schmidt, 1958, Rao et al, 2009). We examined Cs or citrate synthase, a Kreb cycle enzyme and structural mitochondrial protein found in the mitochondrial matrix.…”

“…The first gene we examined in this group, Kif23, is a motor molecule known to be involved in cytokinesis (Nislow et al, 1992;Raich et al, 1998;Neef et al, 2006), a process important in axolotl (Rao et al, 2009, Heber-Katz et al, 2013 and zebrafish regeneration (Poss et al, 2002(Poss et al, , 2004. Kif23, also known as MKLP1, is part of a critical complex formed at the midzone or midbody and associates with CYK-4 in the central spindlin (Glotzer, 2005).…”

Amphibians as research models for regenerative medicine

“…43 Consistent with this scenario is the fact that the gene encoding AGP supports the growth of the blastema to fingerbud stages when electroporated into the tip of a denervated limb during histolysis and dedifferentiation. 42 Other important signaling molecules for blastema formation and growth are Fgf-2, 8, 10, BMPs and Wnt, 3,44 but how they are integrated with AGP is not known.…”

“…Proteomic analysis in our laboratory revealed high levels of the centrosomal protein EVI5 throughout blastema formation in the axolotl. 44 EVI5 functions in mammalian cells to stabilize the Emi1 protein, which prevents cells from entering the mitotic phase of the cell cycle while they replicate DNA. 62 Both EVI5 and Emi1 are destroyed at G 2 , allowing cells to enter mitosis.…”