Figure shows the histological appearance of regenerating limbs in longitudinal section at 1, 4 and 7 dpa. At 1 dpa, the wound epidermis, including gland cells, has migrated to cover the wound. Clotted plasma, muscle fragments, cellular debris and lymphocytes are present under the wound epidermis, which is 3 to 4 cells thick. At 4 dpa, histolysis is liberating cells from their tissue organization and blastema cells have begun to accumulate under the wound epidermis. Osteoclasts can be seen eroding the matrix of the periosteal bone shell that surrounds the cartilage. The basement membrane under the wound epidermis is absent and the wound epidermis is in direct contact with the underlying tissues. By 7 dpa, further histolysis and distal migration of dedifferentiated cells has produced an avascular accumulation blastema with a cell density distinctly higher than that of the more proximal tissue. Examination of serial sections revealed few mitotic figures from 1 to 7 dpa. Some pyknotic nuclei were observed at 1 dpa, but not at 4 and 7 dpa, suggesting a minimum of cell death during the latter time frame.
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 (more ...)
A total of 1,624 peptides were separated in the samples. In all, 138 from priority 1 and 285 peptides from priority 2 were statistically significant (Additional file 1
). Of these 423 statistically significant peptides, 114 peptides were not analyzed further for the reasons outlined in Methods. A total of 309 proteins (Additional file 2
) were analyzed for their role in biological processes. A comparison of non-redundant peptide sequences (N = 405) with the axolotl EST database identified 149 perfect-match peptides (36.8%) that were 100% identical to a translated EST contig from either Ambystoma mexicanum
or the closely related Ambystoma tigrinum
. These proteins are shown in bold in Additional file 1
Figure stratifies the proteins according to biological process, molecular function and cellular location. Figure is a global intensity map of fold changes at 1, 4 and 7 dpa. The peptides were grouped into 10 biological process categories (see Additional file 2
): (1) signaling, (2) Ca2+
binding and translocation, (3) transcription, (4) translation, (5) cytoskeleton, (6) ECM, (7) metabolism, (8) cell protection, (9) degradation, and (10) cell cycle. Table summarizes the ratios of the numbers of proteins upregulated (U) to downregulated (D) relative to controls (U/D ratios) at 1, 4, and 7 dpa for each category of biological process. Table lists those proteins with positive or negative fold changes greater than two with respect to controls, and which may thus have special biological significance. Below, we describe the results for each of the 10 biological categories in order, with those proteins upregulated or downregulated by a factor of 2.0 or more at any time point shown in bold type.
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 (more ...)
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 (more ...)
A key intracellular signaling pathway is the inositol triphosphate/diacylglycerol (IP3/DAG) pathway. IP3 and DAG are cleavage products of phosphatidylinositol-4, 5-bisphosphate (PIP2). A precursor to PIP2 is myoinositol (inositol). Inositol-3-phosphate synthase 1 (ISYNA1) is a key enzyme in the synthesis of inositol from glucose-6-phosphate, and it was upregulated on all dpa. Two regulators of Rho-type guanosine triphosphatases (GTPases) were detected. SYDE2, a GTPase activator, was upregulated at 4 dpa, but downregulated at 1 and 7 dpa, while NET1, a guanine nucleotide exchange factor, was upregulated on all dpa.
Several proteins involved in endocytotic trafficking were identified. CLTCL1, the major protein of the coat of coated pits and vesicles, was downregulated on all dpa. By contrast, ITSN2, which may regulate the formation of clathrin-coated vesicles, was upregulated on all dpa. Several Rab GTPases and associated factors exhibited differential regulation. The Rab family is involved in vesicular trafficking and signaling. RAB6B was upregulated on all dpa and ARL1 and XG28K were downregulated at 1 dpa and upregulated at 7 dpa. A Rab GTPase activator, TBCK, was upregulated at 4 and 7 dpa, while another, TBC1D17, was downregulated on all dpa. GDI2, which regulates the exchange reaction of most Rab proteins by inhibiting the dissociation of guanidine dihydrogen phosphate (GDP) from them, was downregulated at 1 dpa, then returned to control value at 4 and 7 dpa.
Other signaling-related proteins that were upregulated on all three or two of three dpa were: (1) EZR, a peripheral membrane protein that may act to organize transmembrane receptors and binds to signal transduction molecules such as phosphoinositol 3 (PI3) kinase, (2) the receptor for nicotinic acid GPR109B, (3) IRS4, which interfaces between many growth factors and intracellular signaling molecules, (4) TYK2, which phosphorylates receptors of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway to transduce cytokine signals, (5) guanine nucleotide binding protein β polypeptide 2-like 1 (GNB2L1), which anchors protein kinase C to the cytoskeleton, (6) EPHA7, the receptor for the A1 to 5 members of the ephrin A family of ligands, and (7) neuronal nitric oxide synthase (NOS1), the enzyme that synthesizes nitric oxide (NO), a gas with a wide variety of signaling functions. Of all the proteins detected, NOS1 exhibited the highest upregulation at 1 dpa (4.93), after which the level of upregulation declined below 2.0 at 4 and 7 dpa. PPP2CB, the catalytic subunit for phosphatase 2A, a major serine/threonine phosphatase implicated in the negative control of cell growth and division, was downregulated at 1 and 4 dpa, and upregulated at 7 dpa. YWHAZ, an adaptor protein that mediates signal transduction by binding to phosphoserine-containing proteins, was also downregulated at 1 and 4 dpa, with no change at 7 dpa. Another adaptor protein, YWHAE, as well as IRF6.2 and tyrosine-protein kinase 6 (PTK6), were downregulated at all dpa. PTK6 is a cytoplasmic protein kinase that may function as an intracellular signal transducer in epithelia.
Five proteins associated with Wnt signaling were detected. Wnt8a, a ligand for the canonical Wnt pathway, was upregulated on all three dpa. Adenomatous polyposis coli (APC), part of the complex that destabilizes β-catenin in the canonical pathway was upregulated at 4 and 7 dpa. CCDC88C, a Disheveled-binding protein that negatively regulates the canonical pathway, was upregulated on all dpa, while DIXDC1, a positive effector of the canonical pathway, was downregulated on all dpa. Inversin (INVS), which acts to switch Wnt signaling from the canonical to the non-canonical pathway by targeting the Disheveled protein for degradation by the ubiquitin proteasome, was upregulated at 4 and 7 dpa.
Two olfactory receptors were detected, with opposite fold change. OR2AT4 was upregulated on all dpa, whereas OR4D10 was downregulated on all dpa. Follicle stimulating hormone receptor (FSHB) was also upregulated on all days. The potential functions of these proteins in limb regeneration are unknown. The latent transforming growth factor (TGF)β-binding protein was upregulated at 7 dpa, and ectodermin (ECTO), a SMAD4 ubiquitin ligase that attenuates the TGFβ response was downregulated on all dpa.
Ca2+ binding and translocation proteins
The cell maintains cytosolic calcium homeostasis by channels that translocate Ca2+ between the cytosol and the endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR), and between the inside and outside of the cell. Overall, the patterns of fold change for Ca2+-binding proteins suggest a significant increase in cytosolic Ca2+ during blastema formation. Channel proteins in the plasma membrane that mediate extracellular Ca2+ influx into the cytosol were upregulated on all dpa (CACNA1A, ATP11A) or at 7 days (CACNA2D3), while proteins that translocate Ca2+ from the cytosol to the ER/SR (ATP2A3, SRL, ASPH), or buffer cytosolic Ca2+ during muscle contraction (PVALB) were downregulated on all dpa. CAMK2D, a kinase that regulates transport of Ca2+ into and out of cells, was downregulated at 4 and 7 dpa. Another kinase that is covalently linked to ion channels and regulates Ca2+ influx is heart α-protein kinase (HAK), which was downregulated at 1 and 4 dpa, but strongly upregulated at 7 dpa. MYLC2PL, a mitochondrial Ca2+ binding myosin light chain, was downregulated on all dpa. By contrast, CASQ1, which complexes to Ca2+ for storage in the ER/SR and mitochondria, was upregulated on all dpa. Another protein upregulated at 4 and 7 dpa was the Ca2+ binding mitochondrial solute carrier (SLC25A24), which shuttles metabolites, nucleotides and cofactors through the mitochondrial inner membrane.
An interesting group of calcium/phospholipid-binding proteins was the annexins. ANXA1, which is thought to reduce inflammation and promote fibrinolysis, was downregulated at 1 and 4 dpa. ANXA2 was upregulated at 1 and 4 dpa and ANXA4 and 6 were upregulated at 7 dpa. ANXA2 is an autocrine factor that promotes osteoclast formation and bone resorption, and ANXA4 and 6 promote exocytosis in epithelial cells.
A total of 58 proteins were associated with transcription. Changes in 14 chromatin-associated proteins were detected. In all, 10 of these were H1 and H2 histones, with four being upregulated on all dpa and two downregulated at all dpa. Another was upregulated at 1 dpa, returning to control level at 7 dpa. Of the remaining three histones, one was upregulated at 1 and 4 dpa before returning to control level at 7 dpa, another was downregulated at 1 and 4 dpa, but was upregulated at 7 dpa and the other showed no change at 1 dpa, then was downregulated at 4 and 7 dpa. Two proteins that regulate gene expression by covalent modification of histone proteins (MTA1) and nucleosome assembly (NAP1L1-A) were upregulated on all dpa and at 4 and 7 dpa, respectively. JMJD1B (Jumonji domain), a lysine-specific histone demethylase, was downregulated at all dpa (over sixfold at 7 dpa). Hairless (HR), a Jumonji domain-containing transcription factor that recruits histone acetylases to repress transcription, was upregulated at 1 and 4 dpa, and downregulated at 7 dpa.
Of the transcription-associated proteins, 21 were transcription factors. The majority of these were upregulated at all three or two of three dpa, particularly at 4 and 7 dpa. Of six factors expected to act in a general fashion, CBTF122, a subunit of the Xenopus laevis CCAAT box transcription factor, was the only one upregulated on all dpa. SND1 and TRIM29 were downregulated at 1 dpa but upregulated at 7 dpa while E4F1 and TAF4 were downregulated at 1 and 4 dpa and upregulated at 7 dpa. ATF1 was downregulated on all dpa. FUBP1, an ATP-dependent DNA helicase that stimulates c-myc expression in undifferentiated cells was upregulated at 7 dpa. MNT, an E-box (CANNTG) binding transcriptional repressor of c-myc was upregulated at 1 dpa, but downregulated at 4 and 7 dpa.
Six zinc finger transcription factors designated by number were noted, four of which were upregulated at all, or two of three dpa. Of the other two, ZNF777 was downregulated on all dpa, and ZNF559 was downregulated at 1 and 4 dpa, and then upregulated at 7 dpa. The Kruppel-like factor 6 is a ubiquitously expressed zinc finger tumor suppressor that was upregulated at 1 and 4 dpa, and then downregulated at 7 dpa. Several factors (AHCTF1, nuclear receptor subfamily 2, group C member 2 (NR2C2), nuclear factor of activated T-cells cytoplasmic 4 (NFATC4), sex determining region Y box 6 (SOX6), and LIN28 that were upregulated on all, or two of three dpa, induce transcription of specific sets of genes. For example, NR2C2 is a nuclear receptor for mineralocorticoids and glucocorticoids, NFATC4 plays a role in inducing cytokine gene expression in T cells, and SOX6 is required for neurogenic and skeletal differentiation. LIN28 is a transcription factor active in embryonic stem cells [48
]. NEUROD2, a neuronal differentiation factor, was downregulated on all dpa.
In all, 23 of the transcriptional proteins were associated with mRNA processing. At 1 and 4 dpa, downregulation predominated over upregulation. By 7 dpa, however, the U/D ratio was 1.5. The majority of the processing proteins were heterogeneous nuclear ribonucleoproteins, small nuclear riboproteins, and splicing factors. One of these proteins, CWC15, was downregulated over threefold at 7 dpa. Two DEAD box helicases, which unwind RNA structure for accessibility by splicing enzymes, were detected. DEAD box polypeptide 10 (DDX10) was upregulated on all dpa, while DDX46 was upregulated at 1 dpa and downregulated at 4 and 7 dpa. MATR3 anchors mRNA to the nuclear matrix, and was upregulated on all dpa. RBM, a RNA-binding protein of unknown function, was upregulated at 4 and 7 dpa.
Many of the 20 proteins involved in translation, particularly ribosome structural proteins, were upregulated. We detected 13 ribosome structural proteins, about evenly divided between the 60S and 40S subunits. Two of these, RPL7L1 and RPS20, were upregulated at all the time points. Factors for initiation (PABPC1), binding of mRNA to the ribosome (E1F4B), and translocation of nascent protein from the A site to the B site of the ribosome (EEF2) were downregulated or unchanged at 1 dpa, but were upregulated at 4 and 7 dpa. Another initiation factor, E1F4A1, was downregulated at 1 dpa, returned to control level at 4 dpa, and was upregulated at 7 dpa. The elongation factor EEF1A2 was upregulated on all dpa. TARSL2, which is involved in tRNA aminoacylation, was upregulated at 1 dpa, and downregulated at 4 and 7 dpa. Lastly, a translation termination factor, ETF1, was upregulated at 4 and 7 dpa.
About one-third of the cytoskeletal proteins were sarcomeric proteins of skeletal muscle, and these were heavily downregulated. Many, such as TNNT3A, TM7, myosin light chain 3 (MYL3) and MYL5, were downregulated at all the time points.
Of the 40 non-sarcomeric proteins, 25 had functions related to cell motility and maintenance of cell shape and structural integrity. The U/D ratio of these proteins strongly favored downregulation at 1 dpa, but the ratio shifted in favor of upregulation at 4 and 7 dpa. Proteins that were downregulated on all dpa were ACTN1 and 4, GOLGA1, PLS3, XAK-B, and cytokeratin type II. Proteins downregulated at 1 and 4 dpa were desmoplakin isoform II, KRT 12 and KRT5.5. NAV1 was upregulated at all dpa. Seven proteins, FLNB, SYNE2, TUBA, TUBA4B, KRT 19, ACTR2-A and TUBB2C, were downregulated or showed no change at 1 dpa, then were upregulated at 4 and 7 dpa. The remaining proteins MYO9A, MYH9, ACTG1, TUBB4, desmoplakin (DSP), XAK-C and EPPK1, showed a mixture of fold change patterns.
In all, 10 proteins are involved in intracellular movement. MYO1C and MYO5A were downregulated at 1 and 4 dpa, but upregulated at 7 dpa. DYNC1LI2 was upregulated at 1 and 4 dpa, but downregulated at 7 dpa, DNAH3 was downregulated at 4 and 7 dpa, and DYNLL1 was downregulated on all dpa. MYH1 was upregulated at 1, then downregulated at 4 and 7 dpa. MYO1E was upregulated at 1 dpa, downregulated at 4 dpa, and returned to control level at 7 dpa. Two proteins that move or anchor kinases to the cytoskeleton (PDLIM1, PALM2) were downregulated at 4 and 7 dpa. The major vault protein (MVP), which may act as a scaffold for kinases involved in signal transduction and may also play a role in nucleocytoplasmic transport, was downregulated at 1 and 4 dpa, returning to control level at 7 dpa.
There were five adhesion proteins. CDH5 (vascular endothelial cadherin), SCARF2, and ST3GAL5, a type II membrane protein that also maintains fibroblast morphology, were upregulated at all dpa, while CNTNAP4 and FHDC1were downregulated at all dpa.
Of the remaining five non-sarcomeric proteins, KPNA2, which is involved in the import of nuclear proteins, and MYOF, a Ca2+/phospholipid-binding protein that promotes rapid resealing of damaged endothelial cell membranes, were downregulated on 4 and 7 dpa. Sorbin (SORBS1), which plays a role in insulin-stimulated glucose transport, was downregulated on all dpa. By contrast, piccolo (PCLO), which organizes the cytoskeleton in synaptic zones, and PMFBP1, a general cytoskeletal organizing protein, were upregulated at all dpa.
Components of collagen 1 and collagen 13 were upregulated at all or two of three dpa. Collagen 5 was upregulated at 1 and 4 dpa, and then downregulated at 7 dpa. Components of cartilage matrix (collagen 2) and basement membrane (collagen 4) were downregulated at all dpa, as was decorin, which interacts with collagen1 fibrils and may affect the rate of their formation. However, matrilin (MATN) 4, a major component of cartilage matrix, was upregulated at 1 and 4 dpa, then downregulated at 7 dpa. FBN1, a large glycoprotein that associates with elastin to provide force-bearing support in the ECM, was upregulated at 1 and 7 dpa, with no change at 4 dpa. MATN 2, a von Willebrand family member involved in matrix assembly, was upregulated at 1 and 4 dpa, then returned to control level at 7 dpa. FGB, FGG, and fibronectin 1 (FN1) form part of the provisional wound matrix (clot) and were upregulated at all dpa, whereas another provisional matrix protein, tenascin, was downregulated at 1 dpa, showed no change at 4 dpa, and was upregulated at 7 dpa. Periostin, an osteoblast specific factor, was downregulated at 1 and 4 dpa, but upregulated at 7 dpa. EHD4, an endosomal transport protein that promotes assembly and stabilization of collagen 6 filaments, showed no change at 1 dpa and was downregulated at 4 and 7 dpa. Tubulointerstitial nephritis antigen (TINAG), a basement membrane glycoprotein that mediates adhesion of proximal tubule epithelial cells via cell surface integrins, was downregulated on all dpa.
Eight proteins directly or indirectly involved in oxidative phosphorylation were detected. ATP5B, COX-Va, ECHS1, GLUD1 and CS function in the citric acid cycle; most were downregulated at all or two of three dpa. The only mitochondrial metabolic protein that was upregulated at all dpa was SLC25A4, an adenine nucleotide translocator that catalyzes the exchange of adenosine di- and triphosphate (ADP and ATP) across the inner mitochondrial membrane, but a second translocator, SLC25A13, was downregulated at all dpa. Eight proteins involved in the glycolytic pathway were detected, most of which were downregulated at all or two of three dpa. Two proteins, PGM1 and PYGM, are involved in glycogen metabolism; both were downregulated at all dpa.
In all, 15 other metabolic proteins were detected. Most were downregulated at 1 and 4 dpa, with the U/D ratio rising to 1.00 at 7 dpa. Three exceptions were DAGLB, which catalyzes DAG to the endocannabinoid 2-arachidonoyl glycerol (2-AG), DHRS4, which is involved in retinoid metabolism, and PAPPA2 a matrix metalloproteinase that cleaves IGFBP-5. All were upregulated on all dpa.
Seven proteins associated with the post amputation inflammatory response were antioxidants or antipathogens, proinflammatory enzymes, or detoxicants. The antioxidants PXDN and PRDX1 were upregulated on all dpa, while antioxidant TLR6 was upregulated at 1 and 4 dpa. OAS2 and GSTP1, which activate responses to pathogens, were upregulated at 4 and 7 dpa. The proinflammatory enzyme AOX1, by contrast, was downregulated at 1 and 4 dpa. CYP2F1, which plays a role in detoxification, was downregulated on all dpa.
A total of 13 apoptotic pathway-related proteins were detected. Six of these are involved in proapoptotic pathways, and all but one was downregulated on all or two of three dpa. The four downregulated proapoptotic proteins were MICB, a stress induced self-antigen that leads to cell lysis by T cells, VDAC1, a mitochondrial ion channel that promotes apoptosis when open, FASTKD5, which initiates caspase activity, and AK2, which is located in the mitochondrial intermembrane space. Exceptions were microtubule associated serine/threonine kinase 3 (MAST3), which was upregulated at 1 and 4 dpa, and ABTB1, which was upregulated at 4 and 7 dpa. ABTB1 mediates the phosphatase and tensin homolog (PTEN) growth-suppressive signaling pathway. Both negatively regulate the Akt cell survival pathway. Of the seven antiapoptotic proteins, three were downregulated at all or two of three dpa (AKT1S1, BIRC6, and PDCD6IP). Antiapoptotic proteins upregulated at two of three dpa were NEK11 (genotoxic stress reponse), tumor necrosis factor receptor-associated factor 1 (TRAF1; mediates antiapoptotic signals from TNF receptors), and PAIRBP1 (mediates the antiapoptotic action of progesterone in mammalian cells). Interleukin 7 receptor (IL7R), which blocks apoptosis during the differentiation and activation of T lymphocytes, was downregulated at 1 dpa and upregulated at 7 dpa.
A total of 15 proteins that promote or stabilize protein folding in the ER were detected. Four were isomerases. FKBP10 and P4HB were downregulated at all dpa and protein disulfide isomerase A3 (PDIA3) at 1 and 4 dpa. PPIA was upregulated at all dpa and PDIA6 was upregulated at 4 and 7 dpa. A total of 10 proteins were members of chaperone families that accelerate protein folding in the ER. Two of these were upregulated at all dpa (heat shock protein (HSP)B3, TOR1A), three were upregulated at 4 and 7 dpa (HSP90B1, HSP90AB2P, CCT2), one was upregulated at 1 dpa and downregulated at 4 and 7 dpa (HSP27), and two were downregulated at 1 and 4 dpa, but upregulated at 7 dpa (PCMT1, HSP90AA1). Two other chaperones (SSR1 and HSP90AA1) were downregulated at 1 dpa and upregulated at 7 dpa.
Misfolded or damaged proteins that cannot be salvaged are polyubiquinated in the ER, transferred to the Golgi, and then to a cytosolic complex of proteins called the 26S proteasome, where they are degraded [49
]. In our samples, we detected seven proteins of the proteasome pathway. HACE1 (a ubiquitin protein ligase) was upregulated at all dpa, and ubiquitin specific protease 3 (USP3), was upregulated at 1 and 7 dpa. Ubiquitin-like modifier activating enzyme 1 (UBA1) was upregulated at 1 dpa and downregulated at 4 and 7 dpa. We detected four proteins that are part of the proteasome itself. Three of the four were upregulated only at 7 dpa (PSMB8, PSMD 2,7), whereas PSMC4 was downregulated at 1 and 4 dpa before returning to control level at 7 dpa.
Cell debris produced by histolysis, necrosis or apoptosis, is degraded by cytosolic proteases and lysosomal enzymes, and removed by exocytotic pathways. EXOC7, a component of the exocyst, a protein complex essential for docking exocytotic vesicles to the plasma membrane, was upregulated at all three dpa, suggesting the removal of degraded material by this pathway. Other degradative enzymes were TMPRSS9 (a serine protease) and membrane metalloendopeptidase (MME), both of which degrade small peptides. The former was downregulated at all dpa (by nearly sevenfold at 7 days), while the latter was upregulated at 1 and 4 dpa.
NME1, a kinase involved in the synthesis of nucleoside triphosphates other than ATP was upregulated at all dpa. MMCM3 (required for DNA replication) was downregulated at all dpa and FUS (a heterogeneous nuclear protein that promotes annealing of complementary DNA strands) was downregulated at 1 and 4 dpa, but upregulated at 7 dpa. Five cell cycle progression proteins were identified. WDR36 and MARK4 were downregulated on all dpa, whereas ULA1 was upregulated on all dpa. LOH11CR2A, a von Willebrand family member, acts as a tumor suppressor and a negative regulator of the cell cycle. It was downregulated at 1 and 4 dpa, returning to control level at 7 dpa. PPP1C, a protein phosphatase required for chromatin condensation and maintenance of histone H3 phosphorylation during mouse oocyte meiosis [50
], showed no change at 1 dpa, and then was downregulated at 4 and 7 dpa.
Several proteins implicated in mitotic spindle formation were detected. CROCC, which contributes to centrosome cohesion before mitosis and NDEL1, which anchors microtubules to the centrosome during interphase and localizes to mitotic spindles during mitosis were upregulated on all dpa. However, XMAP215 and Ras-related nuclear protein (RAN), which regulate microtubule assembly during the cell cycle, were downregulated on all dpa and on 1 and 4 dpa, respectively. RAN has other functions as well, including translocation of RNA and proteins through the nuclear pore complex, DNA synthesis, and cell cycle progression. Titin (TTN), which in skeletal muscle serves as an adhesion template for the assembly of contractile machinery, and may play a role in chromosome condensation and segregation in non-muscle cells, was upregulated at 1 and 4 dpa. EVI5, a centrosomal oncoprotein implicated in the prevention of premature entry of cells into mitosis, and in the completion of cytokinesis, was upregulated at 1 dpa nearly to the level attained by NOS1, but unlike NOS1 its level remained exceptionally high at 4 and 7 dpa as well.
Validation of proteomic methods
Antibodies to axolotl proteins are not available. Most commercially available antibodies are directed against human and mouse antigens. We therefore tested antibodies to a variety of mammalian proteins that were upregulated in our study on control and regenerating limb tissue. Antibodies to three of these, NOS1, FN, and α-actinin, reacted strongly enough on longitudinal sections of axolotl limb tissue for direct validation by immunohistochemistry. We therefore tested the expression of these proteins at 1 and 7 dpa relative to control tissue (Figure ). NOS1 and fibronectin were upregulated at 1 and 7 dpa, whereas α-actinin was downregulated. Table shows the densitometric quantification of these proteins in immunostained sections. The fold changes determined by liquid chromatography/mass spectrometry/mass spectrometry (LC-MS/MS) were congruent with the densitometric measurements, indicating that quantitative LC-MS/MS data accurately reflect the levels of specific proteins. Indirect validation from the literature provides further support for this conclusion. For example, the upregulation of retinoids and chaperones observed in regenerating urodele limbs [51
] matches a similar upregulation of DHRS4 and multiple chaperones in our study, as does the downregulation of citric acid cycle enzymes observed by Schmidt [54
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 (more ...)
Liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) versus densitometry measurements