Comparison of protein expression profile of limb regeneration between neotenic and metamorphic axolotl

Biochemical and Biophysical Research Communications xxx (xxxx) xxx

Contents lists available at ScienceDirect

Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc

Comparison of protein expression profile of limb regeneration between neotenic and metamorphic axolotl Mustafa Sibai a, 1, Ebru Altuntas¸ a, 1, Barıs¸ Ethem Süzek b, c, Betül S¸ahin d, a, f € , Ahmet Tarık Baykal d, g, Turan Demircan a, h, * Cüneyd Parlayan a, e, Gürkan Oztürk a

Regenerative and Restorative Medicine Research Center, REMER, Istanbul Medipol University, Istanbul, Turkey Department of Computer Engineering, Faculty of Engineering, Mugla Sitki Kocman University, Mugla, Turkey Department of Biochemistry and Molecular & Cellular Biology, Georgetown University Medical Center, Washington, DC, 20007, USA d Acibadem Labmed Research and Development Center, Istanbul, Turkey e Department of Biomedical Engineering, Faculty of Engineering, Istanbul Medipol University, Istanbul, Turkey f Department of Physiology, International School of Medicine, Istanbul Medipol University, Istanbul, Turkey g Department of Biochemistry, School of Medicine, Istanbul Acibadem Mehmet Ali Aydinlar University, Istanbul, Turkey h Department of Medical Biology, School of Medicine, Mugla Sitki Kocman University, Mugla, Turkey b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 7 November 2019 Accepted 18 November 2019 Available online xxx

The axolotl (Ambystoma mexicanum) salamander, a urodele amphibian, has an exceptional regenerative capacity to fully restore an amputated limb throughout the life-long lasting neoteny. By contrast, when axolotls are experimentally induced to metamorphosis, attenuation of the limb’s regenerative competence is noticeable. Here, we sought to discern the proteomic profiles of the early stages of blastema formation of neotenic and metamorphic axolotls after limb amputation by employing LC-MS/MS technology. We quantified a total of 714 proteins and qRT-PCR for selected genes was performed to validate the proteomics results and provide evidence for the putative link between immune system activity and regenerative potential. This study provides new insights for examination of common and distinct molecular mechanisms in regeneration-permissive neotenic and regeneration-deficient metamorphic stages at the proteome level. © 2019 Elsevier Inc. All rights reserved.

Keywords: Proteomics Axolotl Limb regeneration Metamorphosis Gene expression profile

1. Introduction Axolotl, a member of the urodele group of amphibians, possess spectacular abilities to restore the missing part of the body following an injury. Renewal of complex structures such as entire limb by axolotl [1,2], positions it as an attractive model for regeneration research. Axolotl limb regeneration occurs in three main steps: wound healing, neural-epithelial interactions to form the regeneration specific blastema tissue, and reprogramming of positional information [3e5]. Although axolotls are paedomorphic, metamorphosis can be induced by administration of thyroid hormones [6]. For a better understanding of morphological changes in axolotl, a comprehensive histological atlas of the morphological alterations taking place

* Corresponding author. Mugla Sitki Kocman University, School of Medicine, Mentese, Mugla, Turkey. E-mail address: [email protected] (T. Demircan). 1 These authors contributed equally.

within tissues and organs throughout metamorphosis has been described [7]. Interestingly, metamorphosis of axolotl brings about a sharp reduction in regeneration rate and leads to carpal and digit malformations [8,9]. Therefore, facultative metamorphosis in axolotl offers an ideal system to compare studies between regenerative and nonregenerative stages of axolotl to identify the molecular mechanism of the regeneration process. In order to utilize axolotl as a fruitful model, generation of reference resources for this animal is required. Recent efforts to elucidate the colossal axolotl genome [10], transcriptomics (e.g. Refs. [11,12]), proteomics [13,14] and the microbial diversity of axolotl during metamorphosis [9] and limb regeneration [15] have expanded the reference datasets. To the best of our current knowledge, there have not been omics-based studies aiming at unraveling the limb regenerative cues that distinguish regeneration-permissive from regenerationdeficient axolotl limbs. In this study, we, therefore, took the initiative of performing proteomic analysis of the neotenic and metamorphic axolotl limbs at different timepoints; 0,1,4, and 7 days

https://doi.org/10.1016/j.bbrc.2019.11.118 0006-291X/© 2019 Elsevier Inc. All rights reserved.

Please cite this article as: M. Sibai et al., Comparison of protein expression profile of limb regeneration between neotenic and metamorphic axolotl, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.118

2

M. Sibai et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx

post-amputation (dpa). Our study provides novel insights on regulation of limb regeneration, and the generated, processed proteomics data would contribute to a better understanding of decline in regenerative capacity after metamorphosis. 2. Materials and methods 2.1. Experimental design, induced metamorphosis and sample collection Axolotls were obtained from the Ambystoma Genetic Stock Center and bred at the Istanbul Medipol University Medical Research Center as described elsewhere [16]. The experimental design followed in this study is shown in Fig. 1a. A total of 72 adult wild-type axolotls (12e15 cm in length, 1 year old) were randomly chosen among the siblings. Out of 72 axolotls, 36 were kept in neoteny and the other 36 were induced to metamorphosis using Lthyroxine (Sigma-Aldrich, St Louis, MO, USA, Cat. No. T2376) as described in Ref. [9]. The 36 neotenic animals were randomly split into four groups representing timepoint amputations (0, 1, 4, and 7dpa). In each group, 9 animals were sub-grouped into three biological replicates (R1, R2, and R3) to obtain repeatable accuracy. The same grouping was applied for metamorphic axolotls. All animals were anesthetized prior to amputations and sample collection using 0.1% Ethyl 3-aminobenzoate methanesulfonate (MS-222, Sigma-Aldrich, St Louis, MO, USA). 2.2. Protein extraction and sample preparation Filter aided sample preparation (FASP) method was followed for LC-MS/MS according to previously published protocols [14,17]. Briefly, 50 mg of protein lysate was incubated with dithiothreitol (DTT) and iodoacetamide (IAA) respectively for reduction and alkylation steps. Protein concentration was determined by Bradford Protein Assay prior to trypsinization step. Trypsin (Promega) was then added at 1:50 (w/w) and digestion was carried out for 18 h. Peptide concentrations were measured by Quantitative Fluorometric Peptide Assay (Pierce) prior to LC-MS/MS analysis. 2.3. Label-free quantitative nano-LC-MS/MS proteomics analysis LC-MS/MS analysis was performed as in our previous study [17]. In brief, 200 ng tryptic peptide mixture was analyzed by nano-LC-

MS/MS system (Acquity UPLC M-Class and SYNAPT G2-si HDMS; Waters. Milford, MA, USA). The peptide samples were separated from the trap column (Symmetry C18 5 mm, 180 mm i. d.
20 mm) onto the analytic column (CSH C18, 1.7 mm, 75 mm i. d.
250 mm) by a linear 2-h gradient (4%e40% Acetonitrile 0.1% (v/v) FA, 0,300 ml/min flow rate). 100 fmol/ul Glu-q-fibrinopeptide-B was used as lock mass reference at 0,500 ml/min flow rate with 60 s intervals. Positive ionization mode was used at 50e2000 m/z in the full scan mode. Acquisition parameters were used from the previously published methods [14,17].

2.4. LC-MS/MS data processing Progenesis QI for proteomics (v.4.0, Waters) was used for the quantitative analysis of peptide features and protein identification. Processing parameters were 150 counts for the low energy threshold and 30 counts for the elevated energy threshold. The principle of the search algorithm and the followed parameters for data analysis has been described in detail previously [17,18] and an in-house generated axolotl proteome database was used as a reference. Expressional changes and p values were calculated with the statistical package included in Progenesis QI for proteomics and protein normalization was performed according to the relative quantitation using non-conflicting peptides. The resulting data set was filtered by ANOVA p-value  0.01 and only proteins with a differential expression level between the two conditions greater than or equal to 2.0-fold change were considered.

2.5. qRT-PCR analyses Total RNA isolation from frozen tissues that were collected at 0, 1, 4, and 7 dpa was performed using TRIzol Reagent (Invitrogen™, Cat. No. 15596018) according to the manufacturer’s protocol. Complementary DNA (cDNA) was synthesized from total RNA of all time points with M-MuLV Reverse Transcriptase (NEB, M0253S) by following the manual of the manufacturer. qRT-PCR was carried out with specific primer sets (listed in Table S1) using CFX Connect Real-Time PCR Detection System (BIO-RAD) and SensiFAST™ SYBR® No-ROX Kit (BIOLINE, BIO-98005) as described in its protocol. cDNA concentrations were normalized with elongation factor 1-alpha (Ef1-a) gene.

Fig. 1. Experimental design and workflow. a) Protein datasets in this study were generated by collecting post-amputated limb tissue samples derived from neotenic and metamorphic axolotls. b) The workflow of proteomic data analysis from sample collection to data visualization is shown.

Please cite this article as: M. Sibai et al., Comparison of protein expression profile of limb regeneration between neotenic and metamorphic axolotl, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.118

M. Sibai et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx

2.6. Generation of reference protein database A non-redundant set of 119,347 axolotl proteins is computed from 181,431 RNA transcripts [10] in multiple steps. First, the pairwise identities of transcripts are computed through all-againstall BLAST searches using the transcripts database. Next, the longest transcript for the transcripts with 100% pairwise sequence identity is selected as the representative. Finally, for each representative, the longest open reading frame among all six reading frames is translated into a protein sequence to generate the protein set. In order to support functional annotation of these axolotl proteins, the most similar mouse protein orthologs for axolotl proteins were identified by running BLAST searches against the mouse proteins from NCBI nr database. 2.7. GO and KEGG enrichment analyses DE proteins were selected based on an adjusted p-value < 0.01 and |FC| > 2 and were subjected to GO and KEGG enrichment analyses using “clusterProfiler” package in R (version: 3.5.1) [19,20]. The p-value and q-value cutoffs of the enrichment test were set to 0.05. The “org.Mm.eg.db” (mouse) database was used as the background gene list. Benjamini & Hochberg (BH) as the adjusted pvalue method. 2.8. Visualization of GO and KEGG terms Heatmap-like plots and dot plots were generated using “clusterProfiler” R package [19,20] Pathway-based data integration and visualization graphs were plotted using “pathview” R package [19,20]. Bar plots were generated using “ggplot2” R package [19]. 3. Results and discussion Previous studies [8,9] have shown that, unlike neotenic axolotl, metamorphic axolotl exhibits delayed limb blastema formation associated with restricted regeneration capacity. The difference in timing of limb blastema formation between regenerative and nonregenerative stages of axolotls prompted us to study early blastema protein expression profile. In total 714 differentially expressed (fold change greater than or equal to 2.0 between at least two conditions) proteins were identified with an adjusted p-value of <0.01 (Fig. 1b, Table S2). qRT-PCR results for Drebrin (DRBN1), LaminB1 (LMNB1), Max gene-associated protein (MGA), and Perforin (PRF1) validated the proteome results (Fig. S1). 3.1. GO enrichment analyses at 7dpa/0dpa Since molecular content of blastema is decisive for a successful regeneration process, we decided to focus on DE proteins of 7dpa samples for further analyses. GO enrichment analysis was separately carried out on the lists of up and downregulated proteins of neotenic and metamorphic samples at 7dpa/0dpa. The complete lists of enriched biological processes (BP), cellular components (CC), and molecular function (MF) terms are provided in Tables S3e6 and the top 10 of each of the ontology types are visualized in Fig. S2. A similar trend of muscle activity (related BPs are highlighted in Tables S3e6) in neotenic and metamorphic 7dpa/0dpa samples was observed based on GO results. In neotenic samples, the vast majority of the top 10 BPs along with other significant BPs enriched by upregulated proteins center around muscle tissues. Notably, though, many of them were enriched by very few proteins (a max of 5 proteins), most of which were found in almost every process. ‘Myosin filament assembly’, ‘myosin filament organization’,

3

‘skeletal muscle thin filament assembly’, ‘skeletal myofibril assembly’, ‘cardiac muscle fiber development’, and ‘muscle fiber development’ were enriched by the same three proteins (TTN, MYBPC1, MYBPC3). Other muscle-related processes in the GO list were enriched by the same former three proteins and CHGA, TGFB3 and MYH1. On the other hand, only three muscle-related processes were enriched by downregulated proteins in neotenic 7dpa/0dpa samples. ‘Myofibril assembly’, ‘striated muscle cell development’, and ‘actomyosin structure organization’ share seven downregulated proteins (NEB, ACTN2, MYOM1, ACTG1, ACTC1, KRT19, CASQ2). The latter two processes had an extra Pdlim5 and SERPINF2 downregulated proteins, respectively. For neotenic samples, at 7dpa, there appears to be a somewhat moderate muscle activity through upregulation of same set of proteins. Since dedifferentiation of specialized cells to the progenitor and stem cells takes place at blastema stage, an observed tendency for initiation of further muscle degradation through a highly connected set of downregulated muscle-related proteins is consistent with a previously published study [13]. ‘Muscle cell migration’ proteins (FGA, ADIPOQ, VTN, IQGAP1, ANXA1) and ‘regulation of smooth muscle cell migration’ proteins (FGA, ADIPOQ, VTN, IQGAP1) were upregulated in metamorphic samples at 7dpa/0dpa and were enriched in only these two musclespecific BPs. On the other hand, a plethora of muscle-related BPs (10 in total) were enriched by high number of downregulated proteins (31 non-redundant in total), with the top six of muscle processes (such as ‘muscle cell development’ and ‘muscle contraction’) sharing a set of five proteins (MYBPC3, ACTC1, MYBPC2, MYOM1, ACTN2). Metamorphic samples at 7dpa hence clearly undergo a dramatic decrease in muscle tissues. Interestingly, metamorphic samples exhibited an overall higher number of up and downregulated muscle-related proteins than did the neotenic samples. This might be due to adaptation to terrestrial conditions whereby axolotls undergo dramatic tissue remodeling including the limb muscles during metamorphosis. 3.2. KEGG pathway analyses at 7dpa/0dpa KEGG enrichment analysis was carried out on the lists of DE proteins (upregulated and downregulated ones combined) of neotenic and metamorphic samples at 7dpa/0dpa which is shown in Fig. S3. Among the identified pathways, the ‘coagulation and complement cascades’ were further investigated due to their relevance to immunity processes. The coagulation cascade proteins included coagulation factor XI (F11), coagulation factor V (F5), prothrombin (F2), kininogen (KNG), plasminogen activator inhibitor (PAI), a-1antityrpsin (A1AT), and a2-antiplasmin (a2AP) were found downregulated in neotenic samples at 7dpa/0dpa (Fig. 2a), however only PAI protein was found downregulated in metamorphic samples (Fig. 2b). Fibrinogen was found upregulated in the coagulation cascade in both neotenic and metamorphic samples at 7dpa/0dpa (Fig. 2). Regarding complement cascade, an upregulated plasma protease C1 inhibitor (C1INH) and a downregulated CR4 were found in both neotenic and metamorphic samples at 7dpa/0dpa (Fig. 2). Furthermore, FI, C4, C6, C7, C8, C9, vitronectin proteins were upregulated and FH protein was downregulated in metamorphic samples (Fig. 2b). In order to further explore the differences from a different angle in regenerative capacity between neotenic and metamorphic axolotls, KEGG enrichment analysis was implemented on the 259 proteins DE genes in metamorphic samples relatively to neotenic samples at 7dpa (Table S7). Out of 259 proteins, 163 were upregulated (FC > 2) and 96 were downregulated (FC > 2) in metamorphic samples compared to neotenic samples. KEGG pathways such as ‘estrogen signaling’, ‘complement and

Please cite this article as: M. Sibai et al., Comparison of protein expression profile of limb regeneration between neotenic and metamorphic axolotl, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.118

4

M. Sibai et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx

Fig. 2. Differential enrichment of complement and coagulation cascades between neotenic and metamorphic stages. DE proteins were rendered and visualized as part of the whole two cascades on a KEGG graph for a) neotenic and b) metamorphic samples. The colored bar of the KEGG graphs represents a status of up-regulation (red) and downregulation (green) of DE proteins. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

coagulation cascades’, ‘dilated cardiomyopathy’ and ‘hypertrophic cardiomyopathy’ were enriched mostly by upregulated proteins in metamorphic samples with respect to neotenic samples (Fig. 3a). By contrast, KEGG pathways such as ‘carbon metabolism’, ‘glycolysis/gluconeogenesis’, and ‘biosynthesis of amino acids’ were mostly enriched by downregulated proteins. The complement and coagulation cascades were also further investigated. The coagulation cascade proteins F11, F2, A1AT, a2AP, A2M, and fibrinogen were found upregulated at 7dpa in metamorphic samples compared to neotenic samples (Fig. S4). The complement cascade, however, was enriched by the downregulated protein FH and the upregulated proteins C1INH, C5, C6, C7, C8, C9, and vitronectin at 7dpa in metamorphic samples compared to neotenic ones. The complement and coagulation cascades appear to be not fully functional in neotenic samples at 7dpa/0dpa. Proteins which are downregulated at 7dpa is crucial to negatively regulate the fibrinolytic system and initiate the inflammatory reaction. Since their decreased expression level is accompanied by upregulated fibrinogen level, degradation of fibrin products ensues, which is required for proper tissue remodeling. Concomitantly, the

complement system seems to be barely activated as represented by only upregulated C1INH and downregulated CR4 which may lead to decreased level of phagocytosis. On the other hand, metamorphic samples at 7dpa/0dpa have a much less activated fibrinolytic system manifested in upregulated fibrinogen and downregulated PAI levels. This may lead to inadequate clearance of fibrin clots, and consequently, tissue remodeling might be interfered. Activation of complement system by especially upregulating C5, C6, C7, C8, and C9 proteins would result in formation of membrane attack complex, leading to cell lysis. It may be conceivable, therefore, that at 7dpa metamorphic axolotls start to exhibit regeneration deficient symptoms at the molecular level due to the incomplete activation of the fibrinolytic system and the relatively-strong activation of the complement system. This notion can be further asserted when comparing metamorphic samples to neotenic samples at 7dpa. Indeed, the relative upregulation of F2, a2AP, and A2M inhibit plasmin from fully activating the fibrinolytic degradation system, and the relative upregulation of C5, C6, C7, C8, C9 leads to further cell lysis through the formation of membrane attack complexes.

Fig. 3. KEGG analysis and correlation testing of DE protein. a) All KEGG enriched pathways by DE proteins in metamorphic samples compared to neotenic ones at 7dpa were visualized as clusters of up and down-regulated proteins using a dotplot. (adj P < 0.05) b) Scatter plot showing the direction of fold changes of DE proteins at 7dpa/0dpa shared in metamorphic (x-axis) and neotenic (y-axis) samples.

Please cite this article as: M. Sibai et al., Comparison of protein expression profile of limb regeneration between neotenic and metamorphic axolotl, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.118

M. Sibai et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx

3.3. Oppositely regulated common DE proteins between animal stages suggests important roles in regeneration The 204 DE proteins in neotenic samples at 7dpa/0dpa were merged with the 296 DE proteins in metamorphic samples at 7dpa/ 0dpa. A total of 130 genes were found common between the two sample types at this time point comparison. A spearman correlation test was conducted on the fold changes of those common genes (rs ¼ 0.39), wherein 47 had an opposite direction of protein expression (Fig. 3b). Among the latter, a couple of proteins were considered as good candidates to suggest possible explanations for regenerative competence which might be contributed by the observed negative correlation of common DE proteins. FAT atypical cadherin 1 (FAT1) is relatively upregulated in metamorphic compared to neotenic samples at 7dpa. FAT1 takes role in assembly and activation of Hippo pathway [21], which regulates cell growth, proliferation, differentiation and organ size. This finding aligns well with reported cell division rate differences in neotenic and metamorphic axolotl limb regeneration [8]. On the other hand, FAT4 is found relatively upregulated at 7dpa in neotenic samples compared to metamorphic ones. One of the essential functions of FAT4 is the maintenance of planar cell polarity (PCP) [22], a process which

5

blastema cells require for exhibiting their positional information. FAT4 might act on PCP pathway to provide apical-basal and anterior-posterior positioning of blastema cells for neotenic axolotl to support successful regeneration. At 7dpa/0dpa, neotenic samples upregulate cathepsin S (CTSS), a lysosomal enzyme that cleaves some extracellular matrix proteins [23], downregulate coiled-coil domain-containing protein 80 (CCDC80) and desmoglein 1 beta precursor (DSG1B), both of which promote cell adhesion [24,25], and also downregulate Tensin3 (TNS3), which inhibits cell migration and matrix invasion [26]. Furthermore, downregulation of CTSS and upregulation of CCDC80, TNS3, and DSG1B were observed in metamorphic samples at 7dpa/ 0dpa. Since alteration of extracellular matrix (ECM) and cell-cell adhesion is essential for successful regeneration, based on these results, we can speculate that ECM remodeling is achieved effectively in neotenic axolotl rather than in metamorphic animals. 3.4. qRT-PCR validates variations of some immunity markers between both stages The striking distinction in the complement and coagulation cascades prompted us to analyze the expression level of some proand anti-inflammatory markers in neotenic and metamorphic

Fig. 4. Expression levels of immune-modulating genes. qRT-PCR of selected immune-modulating genes was performed for a) neotenic and b) metamorphic samples at 1dpa and 7 dpa compared to 0dpa. TCELLB3: T cell receptor, TGFB1: transforming growth factor beta 1, IL-6: Interleukin 6, IL-8: Interleukin 8, IL-10: Interleukin 10, CSF1R: colony stimulating factor 1 receptor, CD11b: cluster of differentiation molecule 11B, NeuElas: Neutrophil elastase, VEGFR: Vascular Endothelial Growth Factor Receptor.

Please cite this article as: M. Sibai et al., Comparison of protein expression profile of limb regeneration between neotenic and metamorphic axolotl, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.118

6

M. Sibai et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx

samples at 0,1, and 7dpa to unravel the immune system activity during blastema formation. (Fig. 4). The T-cell receptor (TCELLB3), IL-6, IL-8, colony stimulating factor 1 receptor (CSF1R), CD11B, and VEGFR serve as reliable readout markers for pro-inflammatory actions. Those markers are upregulated at 1dpa/0dpa, which aligns well with wound healing phase of neotenic axolotl regeneration (Fig. 4a). Upregulation of anti-inflammatory markers, such as IL-10, at 7dpa/1dpa in neotenic axolotls considerably contributes to the immune modulation by suppressing the inflammation during the blastema formation. For metamorphic axolotls, on the other hand, downregulation or moderate upregulation of pro-inflammatory markers are observed at 1dpa/0dpa (Fig. 4b), signifying a limited activation of the immune system at the wound healing phase in metamorphic animals. Significant upregulation of proinflammatory markers, such as IL-6, at 7dpa compared to 0dpa and 1dpa indicates a clear shift in immune response timing which might interfere with blastema formation in metamorphic axolotls. TGFB1 and VEGFR had similar expression profiles in neotenic samples, in which they were upregulated at 1dpa and 7dpa compared to 0dpa (Fig. 4a). Although these genes were similarly upregulated at 7dpa with respect to 1dpa and 0dpa in metamorphic samples, TGFB1 was upregulated and VEGFR was downregulated at 1dpa compared to 0dpa (Fig. 4b). The observed mRNA expression levels of pro- and anti-inflammatory markers indicate a negative correlation between the persistent immune system activity and the regeneration capacity in metamorphic axolotls as opposed to neotenic ones. 4. Conclusion Presented data here is the first comparison of axolotl limb tissues for regeneration-permissive and regeneration-deficient stages at the proteome level. Our analyses provide new clues to clarify the biological processes which facilitate or hinder axolotl limb regeneration capacity before and after metamorphosis. ECM degradation, muscle reorganization and degradation, cell division, differentiation and migration pathways are regulated differentially during axolotl limb regeneration at neotenic and metamorphic stages. Beside these specified pathways, strikingly, immune system associated genes are expressed at different levels for neotenic and metamorphic animals throughout the regeneration period. To clarify the putative roles of immune system in axolotl limb regeneration, gain and loss of function studies should be performed prior to and post metamorphosis. Abbreviations BP, biological processes; CC, cellular components; DE, differentially expressed; ECM, extracellular matrix; GO, gene ontology; KEGG, kyoto encyclopedia of genes and genomes; LC-MS/MS, liquid chromatography-tandem mass spectrometry; MF, molecular functions; qRT-PCR, quantitative reverse transcription polymerase chain reaction. Ethics approval and consent to participate All animal experiments were approved by the local ethics committee of Istanbul Medipol University with authorization number 38828770-E15936. National guidelines for the care and use of laboratory animals were followed in animal protocols and experimental procedures described in this article. Availability of data and materials The datasets generated and/or analyzed during the current

study are available in the PRIDE Archive database with project DOI: 10.6019/PXD014806 and Figshare repository: https://figshare.com/ s/3370004a3bed362863a0. Authors’ contributions T.D. conceived the study. T.D. and E.A. performed animal and qRT-PCR experiments. B.S¸., C.P. and A.T.B. carried out LC-MS/MS analysis. M.S., B.E.S. and T. D performed bioinformatic analyses € analyzed the reand data visualization. T.D., B.E.S., A.T.B. and G.O. sults. T.D., M.S. and E.A. wrote the manuscript, with contributions from the remaining co-authors. All authors read and approved the final manuscript. Declaration of competing interest The authors declare that they have no competing interests. Acknowledgements Authors would like to thank Emel Akgün and Deniz Duralı for their helpful comments on manuscript. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.11.118. References [1] K. Echeverri, E.M. Tanaka, Ectoderm to mesoderm lineage switching during axolotl tail regeneration, Science 298 (2002) 1993e1996, https://doi.org/ 10.1126/science.1077804. [2] M. Kragl, D. Knapp, E. Nacu, S. Khattak, M. Maden, H.H. Epperlein, E.M. Tanaka, Cells keep a memory of their tissue origin during axolotl limb regeneration, Nature 460 (2009) 60e65, https://doi.org/10.1038/nature08152. [3] V. French, P.J. Bryant, S.V. Bryant, Pattern regulation in epimorphic fields, Science 193 (1976) 969e981, https://doi.org/10.1126/science.948762. [4] D.M. Gardiner, K. Muneoka, S.V. Bryant, The migration of dermal cells during blastema formation in axolotls, Dev. Biol. 118 (1986) 488e493, https:// doi.org/10.1016/0012-1606(86)90020-5. [5] M. Singer, The influence of the nerve in regeneration of the Amphibian extremity, Q. Rev. Biol. 27 (2004) 169e200, https://doi.org/10.1086/398873. [6] C. McCusker, D.M. Gardiner, The axolotl model for regeneration and aging research: a mini-review, Gerontology 57 (2011) 565e571, https://doi.org/ 10.1159/000323761. € _ _ Keskin, [7] T. Demircan, A.E. Ilhan, N. Aytürk, B. Yıldırım, G. Oztürk, I. A histological atlas of the tissues and organs of neotenic and metamorphosed axolotl, Acta Histochem. 118 (2016) 746e759, https://doi.org/10.1016/ j.acthis.2016.07.006. [8] J.R. Monaghan, A.C. Stier, F. Michonneau, M.D. Smith, B. Pasch, M. Maden, A.W. Seifert, Experimentally induced metamorphosis in axolotls reduces regenerative rate and fidelity, Regeneration 1 (2014) 2e14, https://doi.org/ 10.1002/reg2.8. _ Keskin, A.E. Ilhan, _ lu, [9] T. Demircan, G. Ovezmyradov, B. Yıldırım, I. E.C. Fesçiog € G. Oztürk, S. Yıldırım, Experimentally induced metamorphosis in highly regenerative axolotl (ambystoma mexicanum) under constant diet restructures microbiota, Sci. Rep. 8 (2018) 10974, https://doi.org/10.1038/ s41598-018-29373-y. [10] S. Nowoshilow, S. Schloissnig, J.-F. Fei, A. Dahl, A.W.C. Pang, M. Pippel, S. Winkler, A.R. Hastie, G. Young, J.G. Roscito, F. Falcon, D. Knapp, S. Powell, A. Cruz, H. Cao, B. Habermann, M. Hiller, E.M. Tanaka, E.W. Myers, The axolotl genome and the evolution of key tissue formation regulators, Nature 554 (2018) 50e55, https://doi.org/10.1038/nature25458. [11] D.M. Bryant, K. Johnson, T. DiTommaso, T. Tickle, M.B. Couger, D. PayzinDogru, T.J. Lee, N.D. Leigh, T.H. Kuo, F.G. Davis, J. Bateman, S. Bryant, A.R. Guzikowski, S.L. Tsai, S. Coyne, W.W. Ye, R.M. Freeman, L. Peshkin, C.J. Tabin, A. Regev, B.J. Haas, J.L. Whited, A tissue-mapped axolotl de novo transcriptome enables identification of limb regeneration factors, Cell Rep. 18 (2017) 762e776, https://doi.org/10.1016/j.celrep.2016.12.063. [12] N.D. Leigh, G.S. Dunlap, K. Johnson, R. Mariano, R. Oshiro, A.Y. Wong, D.M. Bryant, B.M. Miller, A. Ratner, A. Chen, W.W. Ye, B.J. Haas, J.L. Whited, Transcriptomic landscape of the blastema niche in regenerating adult axolotl limbs at single-cell resolution, Nat. Commun. 9 (2018) 5153, https://doi.org/ 10.1038/s41467-018-07604-0.

Please cite this article as: M. Sibai et al., Comparison of protein expression profile of limb regeneration between neotenic and metamorphic axolotl, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.118

M. Sibai et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx [13] N. Rao, D. Jhamb, D.J. Milner, B. Li, F. Song, M. Wang, S.R. Voss, M. Palakal, M.W. King, B. Saranjami, H.L.D. Nye, J.A. Cameron, D.L. Stocum, Proteomic analysis of blastema formation in regenerating axolotl limbs, BMC Biol. 7 (2009) 83, https://doi.org/10.1186/1741-7007-7-83. lu, E. Akgün, [14] T. Demircan, I. Keskin, S.N. Dumlu, N. Aytürk, M.E. Avs¸arog € G. Oztürk, A.T. Baykal, Detailed tail proteomic analysis of axolotl (Ambystoma mexicanum) using an mRNA-seq reference database, Proteomics 17 (2017) 1600338, https://doi.org/10.1002/pmic.201600338. € _ [15] T. Demircan, A.E. Ilhan, G. Ovezmyradov, G. Oztürk, S. Yıldırım, Longitudinal 16S rRNA data derived from limb regenerative tissue samples of axolotl Ambystoma mexicanum, Sci. Data. 6 (2019) 70. [16] S. Khattak, P. Murawala, H. Andreas, V. Kappert, M. Schuez, T. Sandovaln, K. Crawford, E.M. Tanaka, Optimized axolotl (Ambystoma mexGuzma icanum) husbandry, breeding, metamorphosis, transgenesis and tamoxifenmediated recombination, Nat. Protoc. 9 (2014) 529e540, https://doi.org/ 10.1038/nprot.2014.040. [17] O. Cevik, A.T. Baykal, A. Sener, Platelets proteomic profiles of acute ischemic stroke patients, PLoS One 11 (2016) e0158287, https://doi.org/10.1371/ journal.pone.0158287. [18] U. Distler, J. Kuharev, P. Navarro, Y. Levin, H. Schild, S. Tenzer, Drift timespecific collision energies enable deep-coverage data-independent acquisition proteomics, Nat. Methods 11 (2014) 167e170, https://doi.org/10.1038/ nmeth.2767. [19] R.C. Team, R: A Language and Environment for Statistical Computing, 2018. Vienna, Austria. [20] W. Huber, V.J. Carey, R. Gentleman, S. Anders, M. Carlson, B.S. Carvalho, H.C. Bravo, S. Davis, L. Gatto, T. Girke, R. Gottardo, F. Hahne, K.D. Hansen,

[21]

[22]

[23]

[24]

[25]

[26]

7

R.A. Irizarry, M. Lawrence, M.I. Love, J. MaCdonald, V. Obenchain, A.K. Oles, s, A. Reyes, P. Shannon, G.K. Smyth, D. Tenenbaum, L. Waldron, H. Page M. Morgan, Orchestrating high-throughput genomic analysis with Bioconductor, Nat. Methods 12 (2015) 115e121, https://doi.org/10.1038/ nmeth.3252. D. Martin, M.S. Degese, L. Vitale-Cross, R. Iglesias-Bartolome, J.L.C. Valera, Z. Wang, X. Feng, H. Yeerna, V. Vadmal, T. Moroishi, R.F. Thorne, M. Zaida, B. Siegele, S.C. Cheong, A.A. Molinolo, Y. Samuels, P. Tamayo, K.L. Guan, S.M. Lippman, J.G. Lyons, J.S. Gutkind, Assembly and activation of the Hippo signalome by FAT1 tumor suppressor, Nat. Commun. 9 (2018) 2372, https:// doi.org/10.1038/s41467-018-04590-1. X. Zhang, J. Liu, X. Liang, J. Chen, J. Hong, L. Li, Q. He, X. Cai, History and progression of fat cadherins in health and disease, OncoTargets Ther. 9 (2016) 7337e7343, https://doi.org/10.2147/OTT.S111176. M. Fonovi c, B. Turk, Cysteine cathepsins and extracellular matrix degradation, Biochim. Biophys. Acta Gen. Subj. 1840 (2014) 2560e2570, https://doi.org/ 10.1016/j.bbagen.2014.03.017. L. Pulkkinen, Y.W. Choi, A. Kljuic, J. Uitto, M.G. Mahoney, Novel member of the mouse desmoglein gene family: dsg1-b, Exp. Dermatol. 12 (2003) 11e19, https://doi.org/10.1034/j.1600-0625.2003.120102.x. G. Pei, Y. Lan, W. Lu, L. Ji, Z.C. Hua, The function of FAK/CCDC80/E-cadherin pathway in the regulation of B16F10 cell migration, Oncol. Lett. 16 (2018) 4761e4767, https://doi.org/10.3892/ol.2018.9159. D. Martuszewska, B. Ljungberg, M. Johansson, G. Landberg, C. Oslakovic, B. Dahlb€ ack, S. Hafizi, Tensin3 is a negative regulator of cell migration and all four Tensin family members are downregulated in human kidney cancer, PLoS One 4 (2009) e4350, https://doi.org/10.1371/journal.pone.0004350.

Please cite this article as: M. Sibai et al., Comparison of protein expression profile of limb regeneration between neotenic and metamorphic axolotl, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.118