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.

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.

Figure 1

Histology of axolotl hindlimbs. Longitudinal sections of axolotl hindlimbs regenerating from the mid-tibia/fibula, stained with Weigert's iron hematoxylin and light green SF: (a) Sections at 1 day post amputation (dpa). The amputation surface is covered with several layers of wound epidermal (WE) cells, including gland cells. The basal layer of the wound epidermis is in direct contact with underlying tissues. Some cell debris, red blood cells and lymphocytes are present under the wound epithelium. C = cartilage, M = muscle. (b) Sections at 4 dpa. The cartilage (C), muscle (M), and dermal tissue organization is breaking down, releasing cells that dedifferentiate (DC) and migrate toward the wound epithelium (WE). (c) Sections at 7 dpa. Blastema cells have accumulated under a thickened apical epidermal cap (AEC) to form an accumulation blastema (AB). C = cartilage. The arrows indicate the junction between the accumulation blastema and tissues still undergoing dedifferentiation. Magnification = 10 ×.

Figure 2

Functional and cellular categorization of proteins. Pie charts showing categories of 309 proteins according to (a) biological process, (b) molecular function, and (c) cellular location. Only the categories with at least five proteins have been included in the molecular function pie chart. Since a large number of categories were obtained from the Human Protein Reference Database (HPRD) for cellular localizations, they were classified into five major categories: cytoplasm (actin cytoskeleton, cytosol, and clathrin-coated vesicle), nucleus (centrosome, chromosome, and nucleolus), other intracellular organelles (ribosome, sarcoplasmic reticulum, sarcoplasm, mitochondrial matrix, mitochondrial membrane, mitochondrion, endoplasmic reticulum, golgi apparatus, lysosome and peroxisome), plasma membrane (integral to membrane) and extracellular (cell junction, extracellular matrix).

Figure 3

Global Expression intensity map. HeatMap showing upregulation (red) and downregulation (green) of priority 1 and 2 proteins identified as having significant fold changes relative to control. Numbers at bottom of each column indicate days post amputation (dpa). Left column: proteins upregulated on all dpa, or 1 dpa. Middle column: proteins downregulated on 1 and 4 dpa, and upregulated at 7 dpa; proteins upregulated at 1 and 4 dpa and downregulated at 7 dpa; and proteins downregulated at 1 dpa and upregulated at 4 and 7 dpa. Right column: proteins downregulated on all dpa or two of three dpa. Color intensity reflects fold change.

Figure 4

Immunostained sections of axolotl hindlimbs. Longitudinal sections of control (a, d, g) versus 1 day post amputation (dpa) (b, e, h) and 7 dpa (c, f, i) axolotl hindlimbs stained with primary antibodies to nitric oxide synthase 1 (NOS1) (a-c), fibronectin 1 (FN1) (d-f), α-actinin (ACTN) (g-i). Conjugated secondary antibodies were alexa-568 for fibronectin and NOS1, and alexa-488 for α-actinin. Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI). As expected from the proteomic data, fluorescence intensity of NOS1 showed a significant increase compared to control at 1 dpa, then decreased to a level slightly above control at 7 dpa. Fibronectin staining (red) at 1 and 7 dpa showed significant increases compared to controls, while α-actinin staining intensity (green) showed significant decreases.

Figure 5

Ecotropic viral integrative factor 5 (EVI5) network. Network of direct interactions of six proteins with EVI5. Green = positive regulation; orange = negative regulation. The 110-kDa form of EVI5 stabilizes Emi1 to prevent premature entry into mitosis. At the same time, EVI5 inhibits the vesicle trafficking function of Rab 11a and b. Polo-like kinase 1 (PLK1) is then activated by inner centromere protein (INCENP) to degrade both EVI5 and Emi1, allowing progression into mitosis (M). During M, 90-kDA and 20-kDa forms of EVI5 interact with the chromosomal passenger complex (CPC) proteins aurora B kinase, INCENP, and survivin, where EVI5 is necessary for cytokinesis.

Figure 6

Summary diagram of regeneration processes. (a) Amputation generates signals that result in histolysis and liberation of cells from their tissue matrix. At the same time, these cells are under hypoxic and endoplasmic reticulum (ER) stress, and use a variety of mechanisms to counter this stress and prevent apoptosis, including upregulation of antiapoptotic pathways that protect cell membranes and nuclei. Some proapoptotic pathways are upregulated but are co-opted to remodel or eliminate internal cell structure. Along with changes in transcription factors, chromatin modifying enzymes, microRNAs and polycomb proteins, these mechanisms lead to dedifferentiation. (b) Throughout histolysis, dedifferentiation and accumulation of blastema cells under the wound epidermis, ecotropic viral integrative factor 5 (EVI5) is highly upregulated, preventing blastema cells from undergoing mitosis until after the accumulation blastema has formed.