Abstract As amniote vertebrates, lizards are the most closely related organisms to humans capable of appendage regeneration. Lizards can autotomize, or release their tails as a means of predator evasion, and subsequently regenerate a functional replacement. Green anoles (Anolis carolinensis) can regenerate their tails through a process that involves differential expression of hundreds of genes, which has previously been analyzed by transcriptomic and microRNA analysis. To investigate protein expression in regenerating tissue, we performed whole proteomic analysis of regenerating tail tip and base. This is the first proteomic data set available for any anole lizard. We identified a total of 2,646 proteins – 976 proteins only in the regenerating tail base, 796 only in the tail tip, and 874 in both tip and base. For over 90% of these proteins in these tissues, we were able to assign a clear orthology to gene models in either the Ensembl or NCBI databases. For 13 proteins in the tail base, 9 proteins in the tail tip, and 10 proteins in both regions, the gene model in Ensembl and NCBI matched an uncharacterized protein, confirming that these predictions are present in the proteome. Ontology and pathways analysis of proteins expressed in the regenerating tail base identified categories including actin filament-based process, ncRNA metabolism, regulation of phosphatase activity, small GTPase mediated signal transduction, and cellular component organization or biogenesis. Analysis of proteins expressed in the tail tip identified categories including regulation of organelle organization, regulation of protein localization, ubiquitin-dependent protein catabolism, small GTPase mediated signal transduction, morphogenesis of epithelium, and regulation of biological quality. These proteomic findings confirm pathways and gene families activated in tail regeneration in the green anole as well as identify uncharacterized proteins whose role in regrowth remains to be revealed. This study demonstrates the insights that are possible from the integration of proteomic and transcriptomic data in tail regrowth in the green anole, with potentially broader application to studies in other regenerative models. Keywords: Reptile, Squamate, Lizard, Anolis carolinensis, Anole, Regeneration, Tail, Proteome, Proteomic Introduction Lizards are the most closely related vertebrates to mammals with the ability to regenerate an entire appendage such as the tail as adults. Tail regeneration is extensively studied in lizards and despite important differences in anatomy, many species are able to regrow the spinal cord, peripheral nerves, cartilage, muscle, vasculature, skin as well as recover function as adults ([38]McLean & Vickaryous, 2011; [39]Fisher et al., 2012). In comparison, mammals are unable to regenerate functional appendages incorporating multiple tissue types, except during neonatal periods they can undergo scar-free healing to regrow digit tips after injury ([40]Han et al. 2003, [41]Choi et al. 2017). Since mammals and reptiles are both amniote vertebrates and have diverged relatively recently compared with other regeneration models (amphibians and teleosts), there are greater similarities for comparison of regulatory pathways. Transcriptomic studies of regeneration based on genome reference assemblies have been carried out in a number of model systems, ranging from teleosts such as the zebrafish, amphibians such as the axolotl and Xenopus frog, and in squamate reptiles such as the green anole lizard (reviewed in [42]Xu et al., 2020; [43]Hutchins and Kusumi, 2016), but proteomic analysis has been more limited ([44]Hong and Thornton 2015, [45]Patel et al. 2022). RNA-Seq based analysis has identified a number of common gene regulatory pathways involved in appendage regeneration, including the p38 MAPK and Wnt/Hippo signaling pathways ([46]Xu et al., 2020; [47]Hutchins et al., 2014). The green anole lizard, Anolis carolinensis, was the first squamate and non-avian reptile to have its genome sequenced ([48]Alföldi et al., 2011) and since then, there have been several transcriptomic studies analyzing the scar-free healing and regrowth phase of tail regeneration in this species ([49]Hutchins et al., 2014, [50]2016; [51]Palade et al., 2018; [52]Xu et al., 2020; [53]Londono et al., 2020). In addition to A. carolinensis, transcriptomic studies on regrowth have been conducted in at least four other lizard species (Liu et al. 2017a, Liu et al. 2017b, [54]Patel et al. 2022, Pawan et al. 2022). Together, these studies indicate that the genes related to limb regrowth are relatively conserved across lizards. Conserved processes include regulation of wnt and immune system pathways as well as the expression of various growth factors including VEGF and IGF proteins. Proteomic analyses of vertebrate appendage regeneration have been carried out in the zebrafish ([55]Saxena et al., 2012; [56]Rabinowitz et al., 2017), Xenopus ([57]King et al., 2009; Rao et al., 2014), axolotl ([58]Rao et al., 2009; [59]Demircan et al., 2017), and newts ([60]Looso et al., 2013; [61]Yu et al., 2019). Only twelve proteomic studies have been reported in squamate reptiles ([62]Table 1), including two analyses of tail regeneration in geckos ([63]Murawala et al., 2018; [64]Nagumantri et al., 2021). In addition, estimates of the full complement of proteins based on homology and ab initio gene model prediction can be comprehensive for a given species, protein products encoded from novel genes or annotated genes without clear orthology would be more likely to be missed. Table 1. Summary of published proteomic analysis in squamate vertebrates, excluding studies of snake venom. No comprehensive proteomic reports have been published for anole lizards (family Dactyloidae). Several proteomic studies have been carried out analyzing snake venom, which are reviewed in [65]Tasoulis & Isbister, 2017. Proteomic analysis of regenerating tail has been carried out in two species of house geckos, which are in the infraorder Gekkota ([66]Murawala et al., 2018 and [67]Nagumantri et al., 2021) Family Species Common name Tissue Citation Gekkonidae Hemidactylus flaviviridis northern house gecko regenerating tail [68]Murawala et al., 2018 Gekkonidae Hemidactylus frenatus common house gecko regenerating tail [69]Nagumantri et al., 2021 Scincidae Euritropis carinata golden skink epididymal luminal fluid Medini et al., 2018 Lacertidae Takydromus sexlineatus, T. septentrionalis, T. wolteri grass lizards liver [70]Sun et al., 2022 Agamidae Uromastyx hardwickii Indian spiny-tailed lizard eye lens [71]Atta et al., 2016 Iguanidae Amblyrhynchus cristatus Marine iguana brain, erythrocytes, femoral gland secretions, heart, lung, muscle, serum, sperm, and skin tissue [72]Tellkamp et al., 2020 Shinisauridae Shinisaurus crocodilurus Chinese crocodile lizard mandibular gland [73]Calvete et al., 2023 Colubridae Natrix natrix grass snake skeletal muscle [74]Dubinska-Magiera et al., 2022 Colubridae Viperidae Elaphe carinata, E. taeniura, Lycodon rufozonatus, Ptyas dhumnades, Trimerodytes annularis Gloydius brevicaudus king ratsnake, beauty ratsnake, red-banded snake, Cantor’s rat snake, ringed water snake short-tailed pit viper, liver [75]Tan et al., 2017 Colubridae Pythonidae Nerodia rhombifer Python bivittatus Diamondback watersnake Burmese python intestine [76]Perry et al., 2019 [77]Open in a new tab Here, we constructed an inferred proteome from the high-quality green anole genome annotation coupled with differential detergent fractionation of tissues, in combination with HPLC and mass spectrometry (LC-MS/MS), to generate a comprehensive proteome of the regenerating tail at 25 days post autotomy (DPA). We identified a total of 2,646 proteins with roles in axonogenesis, myogenesis, chondrogenesis, extracellular matrix remodeling, cell proliferation, and immune signaling. A comparison of these proteins with other regenerative model datasets will enable the identification of conserved factors, unraveling the mechanisms that govern vertebrate structural regeneration. As the green anole regenerated tail is a complex structure capable of regrowing many different tissue types, these data also provide a valuable resource for future studies of regeneration as well as other developmental processes. Materials & Methods Animal care and tissue collection Adult green anole lizards (Anolis carolinensis) were purchased from vendors, Charles D. Sullivan Co., Inc. (Nashville, TN) or Marcus Cantos Reptiles (Fort Meyers, FL), and maintained according to the Institutional Animal Care and Use Committee guidelines at Arizona State University (Protocol #12–1247R). Animals were housed and cared for as previously described ([78]Fisher et al., 2012). Autotomy was induced 5 cm from the tail base by applying slight pressure until the tail was released but otherwise, animals were permitted to move freely. To collect regenerated tails, a second autotomy was induced at 25 days post autotomy. Regenerated tails were cut into three segmentsthat represented the tail base, middle, and tip. Six regenerating tail base and tip sections were respectively pooled for each base and tip tissue sample (n=4 biological replicates, 6 individuals pooled per replicate) for protein isolation. Protein sample preparation and mass spectrometry For the proteomic analysis, 100 mg of each tissue was subject to differential detergent fractionation and 20 μg of protein from each fraction was trypsin digested as previously described ([79]McCarthy et al., 2005, [80]2006). Following digestion, peptides were desalted using a peptide macrotrap (Michrom BioResources) according to the manufacturer’s instructions. After desalting, each fraction was further cleaned using a strong cation exchange macrotrap (Michrom BioResources) to remove any residual detergent, which could interfere with the mass spectrometry. Fractions were dried and resuspended in 10 μl of 2% acetonitrile, 0.1% formic acid and transferred to low retention vials in preparation for separation using reverse phase liquid chromatography. A Dionex Ultimate 3000 HPLC system coupled with a Thermo Scientific LTQ Velos Pro mass spectrometer was used for peptide analysis. The HPLC was operated at a flow rate of 333 nl per minute and equipped with a 75 μm × 10 cm fused silica column packed with Halo C18 reverse phase material (Mac-Mod Analytical). Each peptide sample was separated using a 4 h gradient from 2% to 50% acetonitrile with 0.1% formic acid as a proton source. The column was placed directly before a silica nanospray emitter with high voltage supplied via a liquid junction. Scan parameters for the LTQ Velos Pro were one MS scan followed by 20 MS/MS scans of the 20 most intense peaks, all collected in normal scan mode. High energy collisional dissociation (HCD) was chosen as the fragmentation method. Dynamic exclusion was enabled with a mass exclusion time of 3 min and a repeat count of 1 within 30 sec of initial m/z measurement. Peptide and protein identification A translated (frame 1) coding sequence protein database was generated from the longest isoform of each gene in the Anolis carolinensis ASU_Acar_v2.2.1 genome annotation ([81]Hutchins et al., 2014) using the Transeq application in EMBOSS ([82]Rice et al., 2000). Mass spectra were searched against this protein fasta database using spectrum matching programs X!Tandem ([83]Craig & Beavis, 2003) and OMSSA ([84]Geer et al., 2004) at the University of Arizona High Throughput Computing Center (Tucson, AZ). Raw spectra were converted to Mascot generic format (mgf) for analysis. X!tandem was run with 12 threads, precursor and fragment tolerance of 0.5 Da, and up to two missed tryptic cleavages. OMSSA was run with 12 threads, precursor and fragment tolerance of 0.5 Da, up to two missed tryptic cleavages, and set to XML output format. A custom Perl script was used to parse XML search results from both X!tandem and OMSSA and organize them by tail segment. Peptides, as well as proteins, with E-values ≤ 0.05 were accepted and single spectrum identifications were rejected unless they were identified by both search engines. To evaluate data set quality, a randomized version of the target database was concatenated to the original. False discovery rates were less than 5% for all searches. Functional enrichment and pathway analysis All identified proteins were analyzed using PANTHER to obtain GO Slim categories, which provide higher-level terms that are considered the most informative and evolutionarily conserved for biological process (BP), cellular component (CC), and molecular function (MF) categories. Proteins that were shared or unique to the tail base or tip were further assessed using g:Profiler ([85]Raudvere et al., 2019) to obtain all possible BP, CC, MF categories as well as Kyoto Encyclopedia of Gene and Genomes (KEGG) and Reactome pathways. Enrichment p-values were corrected for multiple testing using the Benjamini-Hochberg method and adjusted p-values < 0.05 were considered significant. Adjusted p-values were used to summarize and visualize biological process GO terms based on the mouse database using REVIGO ([86]Supek et al., 2011) ([87]http://revigo.irb.hr), which uses semantic similarity to assign parent-child GO term relationships and remove redundant gene ontology assignments that meets the simRel score specified by the user (C = 0.7). For both PANTHER and g:Profiler, enrichment analysis was generated using mouse databases. All treemaps and enrichment plots were generated with R. Treemaps were created using the treemap v2.4–2 (Tennekes et al., 2017) and all plots were customized using the ggplot2 package ([88]Villaneuva and Chen, 2019). Data sharing and data availability All proteomic data can be accessed from the Mass Spectrometry Interactive Virtual Environment (MassIVE) at [89]ftp://massive.ucsd.edu/ under accession number/directory MSV000091830. Results and Discussion Defining the green anole regenerating tail proteome To date, there is no published proteome for any anole lizard. In order to identify proteins present in the green anole regenerating tail, we generated a frame 1 coding protein database that is based on the high-quality ASU_Acar_v.2.2.1 genome annotation in which 91% of transcripts have notated start and stop codons ([90]Eckalbar et al., 2013; [91]Hutchins et al., 2014). Using this database, a total of 2,646 proteins (e-value < 0.05) were identified in the regenerating tail of the green anole lizard at 25 days post autotomy (DPA) and of these, 581 (22%) proteins initially did not have an orthologous gene assignment to other vertebrate species. Additional othologous gene assignments were made based on matches with Ensembl and NCBI gene models. There were 976 (37%) and 796 (30%) proteins unique to the regenerating tail base and tip, respectively ([92]Figure 1; [93]Supplementary Table S1, [94]S2). Shared between the regenerating tail base and tip, we identified 874 proteins (33%) in common ([95]Supplementary Table S1, [96]S2). After matching for gene orthology, we identified 13 proteins in the tail base (1.3%, [97]Table 2), 9 proteins in the tail tip (1.1%, [98]Table 3), and 10 proteins in both regions (1.1%, [99]Table 4), that matched an uncharacterized protein gene model in Ensembl and NCBI, confirming that these predicted proteins are present in the proteome. Figure 1. Identified proteins in the green anole regenerating tail at 25 DPA. Figure 1. [100]Open in a new tab A Venn diagram of the number of proteins unique to the tail base (red), tail tip (blue), and number of proteins shared between the two regenerating segments (purple). Table 2. Uncharacterized proteins matching with Ensembl or NCBI gene models specifically expressed in the regenerating tail base. Genome location Strand ASU Gene ID Gene ID Coverage (%) e-value Peptides Unique Peptides 1:121302119–121328380 − ASU_Acar_G.2389 LOC103279406 1.22 8.82E-07 2 2 1:180522379–180525001 − ASU_Acar_G.580 ENSACAG00000036959 10.34 2.57E-19 4 4 2:195304522–195324548 − ASU_Acar_G.4685 ENSACAG00000010478 4.96 2.83E-15 1 1 2:195458396–195487184 − ASU_Acar_G.3608 ENSACAG00000027601^[101]1 2.79 6.32E-07 2 1 2:76471083–76501104 − ASU_Acar_G.4292 ENSACAG00000015559 4.73 8.35E-14 3 3 3:59412829–59512354 + ASU_Acar_G.6498 ENSACAG00000021034^[102]2 2.74 2.62E-06 1 1 4:40620966–40628881 − ASU_Acar_G.7092 ENSACAG00000039577 16.67 1.45E-19 1 1 6:14768492–14779877 + ASU_Acar_G.9710 LOC103279099 1.42 1.56E-09 2 2 [103]GL343244.1:943493–981087 − ASU_Acar_G.13894 ENSACAG00000020939 1.69 6.95E-09 2 2 [104]GL343434.1:651786–667158 + ASU_Acar_G.17621 ENSACAG00000029436 7.39 1.71E-06 2 2 [105]GL343502.1:597553–602388 + ASU_Acar_G.18507 ENSACAG00000022610 2.48 2.89E-09 1 1 [106]GL343557.1:44773–51960 − ASU_Acar_G.19068 ENSACAG00000042309^[107]3 5.26 9.10E-08 2 2 [108]GL343891.1:42522–51296 + ASU_Acar_G.21103 ENSACAG00000010449 7.69 4.25E-15 3 3 [109]Open in a new tab These gene models displayed sequence similarity to ^1VWA7, ^2GABRP, ^3ZNF394. Table 3. Uncharacterized proteins matching with Ensembl or NCBI gene models specifically expressed in the regenerating tail tip. Genome location Strand ASU Gene ID Gene ID Coverage (%) e-value Peptides Unique Peptides 1:45101221–45137861 − ASU_Acar_G.368 LOC103277971 2.19 1.75E-10 2 1 3:99848231–99863751 − ASU_Acar_G.5680 LOC107982582 1.12 6.01E-14 1 1 4:62041734–62042582 + ASU_Acar_G.8088 ENSACAG00000040668 7.69 4.50E-17 1 1 4:90784309–90971839 + ASU_Acar_G.7759 LOC103278649 6.2 1.79E-05 2 2 [110]GL343237.1:1224837–1235463 + ASU_Acar_G.13713 ENSACAG00000027156 2.7 2.63E-07 1 1 [111]GL343713.1:32861–44724 − ASU_Acar_G.20201 ENSACAG00000002585 7.71 2.52E-09 2 2 [112]GL343257.1:982508–1062983 − ASU_Acar_G.14298 ENSACAG00000002945 1.29 7.99E-23 4 4 [113]GL343393.1:232639–235666 + ASU_Acar_G.17063 ENSACAG00000014891 4.16 3.91E-38 3 0 [114]GL343717.1:232803–253078 + ASU_Acar_G.20221 ENSACAG00000017499 3.39 1.29E-08 1 0 [115]Open in a new tab Table 4. Uncharacterized proteins matching with Ensembl or NCBI gene models expressed in both the regenerating tail base and tip. Genome location Strand ASU Gene ID Gene ID Coverage (%) e-value Peptides Unique Peptides 1:6422669–6431518 − ASU_Acar_G.1861 ENSACAG00000022474 17.29 2.41E-156 5 5 1:65383303–65406330 + ASU_Acar_G.835 LOC103278635 10.27 2.60E-09 1 1 2:193675702–193682076 − ASU_Acar_G.4541 ENSACAG00000007391^[116]1 1.68 4.70E-12 1 1 3:42788173–43105682 + ASU_Acar_G.5450 ENSACAG00000037409 1.35 4.31E-11 2 2 4:41559354–41650847 − ASU_Acar_G.7831 ENSACAG00000036653 0.86 3.17E-07 2 1 5:27642826–27643234 − ASU_Acar_G.9688 ENSACAG00000010716^[117]2 28.68 3.67E-39 4 0 6:70702068–70711291 − ASU_Acar_G.22992 ENSACAG00000029628^[118]3 46.04 0 39 38 [119]GL343194.1:9749996–9786939 + ASU_Acar_G.12149 ENSACAG00000040254 18.1 0 35 25 [120]GL343252.1:1828391–1828769 + ASU_Acar_G.14129 ENSACAG00000015318 55.91 0 10 0 [121]GL343713.1:15166–19968 − ASU_Acar_G.20207 ENSACAG00000029476 2.49 1.48E-19 1 1 [122]Open in a new tab These gene models displayed sequence similarity to ^1gastrula zinc finger protein XlCGF26.1, ^2histone H3, ^3keratin 10. Of the 2,646 proteins, 111 proteins were encoded by genes that were differentially expressed between proximal to distal regenerating tail segments by transcriptomic analysis of the 25 DPA regenerating tail ([123]Hutchins et al., 2014). These include corneous beta proteins (LI-AC-X, LI-AC-14, LI-AC-17), which are expressed in lizard scale epidermis, cartilage-specific collagens (COL2A1, COL9A1, COL9A2, COL9A3), and many muscle structural proteins (ACTA1, ACTN3, LMOD3, MYL1, MYL2, MYL3, MYL4, MYL6, MYL10, MYH6, MYOM1, MYOM2, MYOZ2, TNNT3). Additionally, neural-associated genes include transcription factor SALL1, which is required for neural tube closure ([124]Böhm et al., 2008) and may be involved in neuroependyma outgrowth, and GAS7, which plays a putative role in neuronal development and differentiation. Uncharacterized proteins without orthology to known vertebrate genes While appendage regrowth is a conserved trait among vertebrates, there are examples of novel protein families identified in the regrowth process in the newt ([125]Looso et al., 2013). To examine whether there are proteins present in the regenerating lizard tail without orthology to known vertebrate genes, we screened for proteins which corresponded to gene models in either Ensembl or NCBI databases but that represented uncharacterized entries. The green anole genome is comprised of 6 macrochromosomes (1–6), 6 minichromosomes (7, 8, 9, g, h, and X), and at least 28 microchromosomes. It was notable that of these uncharacterized proteins, 9 out of 14 in the tail base, 4 out of 9 in the tail tip, and 7 out of 10 shared between the base and tip were located on macrochromosomes. The remainder proteins were located on scaffolds that had not yet been assembled into existing chromosomes, suggesting that many of the uncharacterized proteins may derive from genes on less characterized smaller chromosomal elements. Comparison of proteins present in the regenerating tail tip versus base Proteins unique to the regenerating tail base and tail tip were categorized into biological process (BP), cellular component (CC), and molecular function (MF) Gene Ontology categories and characterized for KEGG and Reactome pathway enrichment categories using the g:Profiler tool. Regenerating tail base Proteins in the tail base were significantly enriched (adj. p-value < 0.05) for 553 BPs, 162 CCs, and 101 MFs ([126]Figure 2, [127]Supplementary Table S3). Summarization of GO biological processes revealed 18 major categories including actin filament-based process, ncRNA metabolism, regulation of phosphatase activity, small GTPase mediated signal transduction, and cellular component organization or biogenesis ([128]Figure 3). All GO term parent-child relationships, i.e., relationships referring to terms encompassing multiple categories closer to the root of the graphs versus more specific terms closer to the leaf nodes, are presented in [129]Supplementary Table S4. Actin filament-based process is expected as muscle and cartilage differentiation are evident at 25 DPA ([130]Hutchins et al., 2014). Unique tail base proteins were also enriched for cellular components such as sarcomere, A band, I band, and Z disc in the tail base but not tail tip which supports a pattern of proximal to distal tissue differentiation. As genes are being actively transcribed and translated during regeneration, enrichment for ncRNA metabolism is consistent with the processing of rRNAs, tRNAs, and regulatory miRNAs. Indeed, previous transcriptomic studies identified several highly expressed miRNAs in the regenerating tail base with roles in muscle differentiation and function ([131]Hutchins et al., 2016). Moreover, post-translational modifications, such as phosphatase activity, is critical for regulating many cellular processes including signal transduction, protein synthesis, and cell division. Proteins unique to the regenerating tail base were also enriched for 14 signaling pathways in KEGG and one from Reactome ([132]Supplementary Table S3). Extracellular matrix related networks were identified as the most significant pathway in both KEGG and Reactome analyses. Proteins associated with these pathways include laminins, type IX collagens, and glycoproteins such as TNC, TNXB, SPARC, and VTN. These proteins are all expressed by cartilage-forming cells, which coalesce around the neuroependyma at 25 DPA. Figure 2. Gene Ontology enrichment for proteins present in the regenerating tail base. Figure 2. [133]Open in a new tab The top 10 most highly represented biological process, cellular compartment, and molecular function GO categories in the green anole regenerating tail base. Enrichment scores, calculated as −log10(adjusted p-value), are shown for each GO term. Full data set available in [134]Supplementary Table S3. Figure 3. Biological processes enriched in the regenerating tail base are summarized into major functional categories as a treemap. Figure 3. [135]Open in a new tab Each rectangle shows a single, representative GO term where size is determined by the absolute log10 p-values of related, enriched biological processes within each group. Legend for biological processes terms summarized by letters: A, developmental process; B, metabolism; C, cellular process; D, cell-cell adhesion; E, localization; F, biological adhesion; G, multicellular organismal process; H, locomotion; I, methylation; J, establishment or maintenance of cell polarity; K, establishment of cell polarity. All enriched GO terms can be viewed in [136]Supplementary Tables S3 and [137]S4. Regenerating tail tip Analysis of proteins unique to the tail tip revealed 464 BPs, 94 CCs, 112 MFs, but no significant pathways ([138]Figure 4, [139]Supplementary Table S5). While the top represented GO categories in the tail tip and tail base are very similar despite having unique proteins, these terms are highly generalized and represented by a large number of genes. However, significant enrichment for nervous system development is a major category of interest. Higher level grouping of tail tip biological processes identified a total of 22 clusters including those involved in regulating organelles, regulation of protein localization, ubiquitin-dependent protein catabolism, small GTPase mediated signal transduction, morphogenesis of epithelium, and regulation of biological quality ([140]Figure 5, [141]Supplementary Table S6). Small GTPase mediated signal transduction indicates that there is active intracellular signaling, which was also a major functional category in the tail base. Other categories are likely associated with cellular processes related to changes in tissue size and mass as the regenerating tail elongates, including the outgrowth of specialized nerve and epithelial structures like the neuroependyma and vasculature. This is further supported by representative categories such as developmental process, growth, regulation of growth, stem cell division, and regulation of neural precursor cell proliferation. These include transcription factors, MYB and GLI1, which are involved in regulating cell proliferation. Additionally, NOTCH1, and Notch inhibitor, NUMB are involved in proliferation and cell fate specification in neural precursor and muscle satellite cells ([142]Conboy and Rando, 2002; [143]Zhong et al., 1997). We also identified GJC2 and ULK4, which are respectively associated with axonal myelination and neuronal branching. This likely reflects the robust axonal outgrowth and concurrent myelination observed during green anole tail regrowth ([144]Tokuyama et al., 2018). Figure 4. Gene Ontology enrichment for proteins unique to the regenerating tail tip. Figure 4. [145]Open in a new tab The top 10 most highly represented biological process, cellular compartment, and molecular function GO categories in the green anole regenerating tail tip. GO terms are displayed on the x-axis and enrichment scores, calculated as −log10(adjusted p-value), are shown on the y-axis. Full data set available in [146]Supplementary Table S5. Figure 5. Biological processes enriched to the regenerating tail tip are summarized into major functional categories as a treemap. Figure 5. [147]Open in a new tab Each rectangle shows a single, representative GO term where size is determined by the absolute log10 p-values of related, enriched biological processes within each group. Legend for biological processes terms summarized by letters: A, regulation of autophagy; B, autophagy; C, developmental process; D, biological regulation; E, growth; F, regulation of growth; G, regulation of neural precursor cell proliferation. All enriched GO terms can be viewed in [148]Supplementary Tables S5 and [149]S6. Enrichment and pathway analysis of proteins expressed throughout the regenerating tail Enrichment analysis of proteins shared between the regenerating tail base and tip segments yielded 992 BPs, 331 CCs, 193 MFs, and a total of 183 (29 KEGG, 154 Reactome) signaling pathways ([150]Figure 6, [151]Supplementary Table 7). PANTHER pathway enrichment identified a total of 117 signaling pathways in this group ([152]Supplementary Table S8), with the top 35 pathways related to integrin, Wnt, and inflammation signaling ([153]Table 5). During vertebrate regeneration, these pathways are involved in maintaining tissue integrity, cell adhesion, cell proliferation, and wound healing (Kawakimi et al. 2006, [154]Abu-Daya et al. 2011, [155]Hutchins et al., 2014, [156]Bosak et al. 2018). Moreover, shared proteins were enriched for molecular function categories such as mRNA 3’-UTR binding and mRNA 5’-UTR binding, which can post-transcriptionally regulate gene expression to ensure that genes are expressed in the appropriate cell and at the correct time. A large proportion of detected proteins were either cytoskeletal or extracellular matrix related. As the most abundant class of proteins, structural proteins could bias proteomic analyses and mask regeneration candidate factors detected by gene expression studies. However, post-transcriptional and post-translational changes could also explain the absence of certain proteins and this possibility cannot be ruled out. While the ECM is recognized for its architectural role, its components affect the regenerative process by i) regulating the timing and release of growth factors and signaling ligands, which modulates gene expression, ii) maintain tissue homeostasis through mechanotransduction, and iii) modulates cell behavior ([157]Prestwich and Healy, 2015). Taken together, the extracellular matrix is a major regulatory and dynamic component of tissue regeneration and warrants further study, as indicated by our study and previous literature ([158]Tassava et al. 1996, [159]Calve et al. 2010, [160]Godwin et al. 2014). Figure 6. Gene Ontology enrichment for proteins shared between the regenerating tail base and tip. Figure 6. [161]Open in a new tab The top 10 most highly represented biological process, cellular compartment, and molecular function GO categories for shared proteins in the green anole regenerating tail base and tip. GO terms are displayed on the x-axis and enrichment scores, calculated as −log10(adjusted p-value), are shown on the y-axis. Full data set available in [162]Supplementary Table S7. Table 5. Top 20 signaling pathways in the 25 DPA regenerating tail tip and base. PANTHER pathways enrichment analysis of 2,646 proteins identified in regenerating tail tip and base samples. Pathway Accession Pathway Name Percent [163]P00034 Integrin signalling pathway 6.4 [164]P00057 Wnt signaling pathway 4.5 [165]P00031 Inflammation mediated by chemokine and cytokine signaling pathway 4.4 [166]P06664 Gonadotropin-releasing hormone receptor pathway 3.3 [167]P00029 Huntington disease 3.0 [168]P00016 Cytoskeletal regulation by Rho GTPase 2.6 [169]P00044 Nicotinic acetylcholine receptor signaling pathway 2.4 [170]P00026 Heterotrimeric G-protein signaling pathway-Gi alpha and Gs alpha mediated pathway 2.4 [171]P00004 Alzheimer disease-presenilin pathway 2.3 [172]P00018 EGF receptor signaling pathway 2.1 [173]P00012 Cadherin signaling pathway 2.1 [174]P00049 Parkinson disease 1.9 [175]P00005 Angiogenesis 1.8 [176]P06959 CCKR signaling map 1.8 [177]P00021 FGF signaling pathway 1.6 [178]P00053 T cell activation 1.5 [179]P00027 Heterotrimeric G-protein signaling pathway-Gq alpha & Go alpha mediated pathway 1.5 [180]P00060 Ubiquitin proteasome pathway 1.4 [181]P00043 Muscarinic acetylcholine receptor 2 and 4 signaling pathway 1.4 [182]P00039 Metabotropic glutamate receptor group III pathway 1.4 [183]Open in a new tab As expected, there were many proteins enriched for biological GO terms related to the growth and differentiation of central and peripheral nerves, muscle, cartilage, epithelium, and vasculature ([184]Table 5), which are all tissues regenerated in the tail. Proliferation is also distributed along the proximo-distal axis and GO terms including mitotic cell cycle, mitotic cell process, cell population proliferation, epithelial cell as well as mesenchymal cell proliferation were significantly represented. We identified proliferation marker, Ki67, and keratin proteins, K8 and K18, which are involved in mesenchymal progenitor cell proliferation and differentiation during newt limb regeneration ([185]Corcoran & Ferretti, 1997) as well as expressed during zebrafish caudal fin regeneration ([186]Saxena et al., 2012). Revigo analysis of biological processes produced 24 semantic clusters of proteins, which include supramolecular fiber organization, muscle contraction, response to wounding, peptide metabolism, regulation of catabolism, developmental process, and growth, and are all canonical processes of regeneration ([187]Figure 7, [188]Supplementary Table S9). Supramolecular fiber organization is further supported by CC, MF, and KEGG/Reactome pathway analysis, which reveal that structural proteins are most likely to be shared between the regenerating tail base and tip. While ECM components provide support for differentiating cell populations, its composition plays a key role in modulating cell behaviors such as proliferation, migration, and adhesion as well. As regeneration requires a large investment of energy and resources, representative categories for peptide metabolism and regulation of catabolism are also expected. Figure 7. Biological processes enriched in and shared in the regenerating tail base and tip are summarized into major functional categories as a treemap. Figure 7. [189]Open in a new tab Each rectangle shows a single, representative GO term where size is determined by the absolute log10 p-values of related, enriched biological processes within each group. Legend for biological processes terms summarized by letters: A, cellular component organization or biogenesis; B, regulation of locomotion; C, biological adhesion; D, cellular process; E, localization; F, locomotion; G, metabolism; H, multicellular organismal process; I, intermediate filament-based process; J, protein folding; K, sperm-egg recognition; L, establishment or maintenance of cell polarity; M, response to stimulus. All enriched GO terms can be viewed in [190]Supplementary Tables S7 and [191]S9. Regeneration is only possible by modulation of the adult immune response ([192]Julier et al. 2017, Vitulo et al. 2017). This, along with successive studies, indicates that the lizard blastema is transiently immuno-supressed ([193]Alibardi 2018). In the green anole lizard, tail autotomy induces changes to both innate and adaptive immune systems, which facilitates scar-free healing of the wound stump and initiates the regrowth process ([194]Xu et al., 2020). Previous transcriptomic analysis of the 25 DPA tail revealed that the wound and immune response extends through the regrowth phase ([195]Hutchins et al., 2014). Indeed, proteomic analysis of the regenerated tail identified many immune relevant proteins shared between proximal and distal tail segments ([196]Table 3). The Revigo category, response to wounding, was composed of several biological processes and includes wound healing, response to endogenous stimulus, response to stress, and response to interleukin-7 among others ([197]Supplementary Table S9). Additionally, shared proteins were significantly associated with KEGG phagosome and leukocyte transendothelial migration pathways, and Reactome neutrophil degranulation, MHC class II antigen presentation, innate immune system, and adaptive immune system pathways too. These include proteins related to wound healing (CD9, CD44, PKM), coagulation (F5, VWF), the complement system (C1QBP, C3, C9, CFH), non-canonical Wnt pathway (PTK7, NFATC2), and immunomodulation (ANXA1, ANXA2, ANXA5, ANXA6). Annexin proteins are also expressed during zebrafish fin and larval Xenopus limb regeneration ([198]King et al., 2009; [199]Saxena et al., 2012). In addition, several annexin proteins were detected in both green anole regenerating tail base and tip segments (ANXA1, ANXA2, ANXA5, and ANXA6). In Xenopus and axolotl regenerating limbs, ANXA1 and ANXA2 are considered to be key players in resolving inflammation and fibrinolysis ([200]King et al., 2009; [201]Rao et al., 2009). In comparison with the regenerating zebrafish caudal fin, ANXA1 also displayed differential patterns of phosphorylation state and expression shortly after injury ([202]Saxena et al., 2012). Moreover, ANXA5 is used to identify apoptotic cells but can also bind to extracellular phosphatidylserine, preventing macrophage recognition and phagocytosis ([203]Kenis et al., 2006; [204]van Genderen et al., 2008). This is thought to inhibit the auto-immune response against self-antigens of the dying cell ([205]Munoz et al., 2007). We also identified several members of the innate complement system. During urodele limb regeneration, C3 is expressed in the blastema ([206]Del Rio-Tsonis et al., 1998; [207]Kimura et al., 2003) and in later stages, in differentiating muscle ([208]Del Rio-Tsonis et al., 1998). In mice, C3 signaling initiates the recruitment of monocytes or macrophages to the site of injury and is critical to stimulating muscle regeneration ([209]Zhang et al., 2017). Lastly, CFH prevents complement activation against resident host cells by blocking the alternative pathway and assembly of the membrane attack complex, which includes C9, and C1QBP inhibits C1 activation ([210]Józsi et al., 2019; [211]Wang et al., 2022). These findings, coupled with previous research, suggests that a persistent and likely polarized immune response toward M2 healing inflammation promotes regrowth and may protect new tissues by maintaining self-recognition or survival ([212]Alibardi 2020, [213]Alibardi 2022, He et al. 2022). Comparison of green anole and gecko regenerative proteomes [214]Nagumantri et al., 2021, identified 128 proteins expressed during tail regeneration at 0, 2 and 5 days post autotomy in the common house gecko (Hemidactylus frenatus), which after consolidating multiple protein isoforms, resulting in a list of 110 proteins. In contrast, our analysis of 25 DPA tails, which is a much later stage, identified 2,646 proteins in the green anole regenerating tail. We cross-referenced proteins identified in the green anole regenerative data set at 25 days post- autotomy with this gecko list ([215]Supplementary Table S10). As [216]Nagumantri et al. (2021) did not subdivide the early regenerating tail into tip and base regions, we cross referenced the green anole tail tip and tail base separate against this gecko data set. We identified 61 proteins in both the green anole and gecko data sets, with 41 proteins expressed in both tail base and tip regions and 5 proteins only the tip and 13 only in the base ([217]Supplementary Table S10). Proteins shared between the gecko tail and green anole tail base included muscle-related proteins such as ACTN1 (alpha-actinin 1), ATP2A1 (sarcoplasmic/endoplasmic reticulum calcium ATPase 1), CAPNS1 (calpain small subunit 1), CKM (creatine kinase M-type), MYLPF3 (myosin regulatory light chain 2, skeletal muscle), MYOM1 (myomesin-2), and TNNT3 (troponin T, fast skeletal muscle). Proteins shared between the gecko tail and green anole tail tip included metabolic enzymes GAPDHS (glyceraldehyde-3-phosphate dehydrogenase 2), LDHA (L-lactate dehydrogenase A chain), and PGK1 (phosphoglycerate mutase 1). These enzymes are all glycolytic, which is a conserved process typical of wound healing ([218]Alibardi 2014). Proteins shared between the gecko tail and identified in both the green anole tail tip and base included collagens (COL1A1, COL1A2, COL6A1, COL6A2, COL6A3), muscle proteins (ACTN1, DES, MYH7B, MYL1, NEB, TTN, TUBA1A), heat shock protein HSPA8, and tissue regeneration and wound healing protein periostin (POSTN). Similar collagen proteins were found in other lizard blastemas, suggesting that collagen expression may be a conserved characteristic of lizard regeneration ([219]Liu et al. 2015, [220]Vitulo et al. 2017a). While the 110 proteins identified in the regenerating gecko tail at 0, 2, and 5 days post autotomy is only a fraction of the 2,646 proteins found in the regenerating green anole tail at 25 DPA, our comparison of the two data sets identified 61/110 proteins in common despite the different stages examined.While [221]Nagumantri et al., 2021 examined earlier stages of regeneration, specifically 2 and 5 days post autotomy in the common house gecko, we have previously carried out RNA-Seq analysis of the regrowing green anole tail from 0.5 to 5 days post autotomy ([222]Xu et al., 2020). In comparing the proteomic dataset from gecko with the RNA-Seq dataset from the green anole, we found that 1/3^rd of our identified proteins (553 out of 1,673) were also found by Xu et al. 2021 ([223]Supplementary Table S11). Biological processes associated with these shared genes included actin filament organization, cell-matrix adhesion, and cell differentiation ([224]Supplementary Table S12). Comparison of the green anole regenerative proteome with analyses in other squamate species Unlike some urodele amphibians, squamate reptiles can regrow tail appendages but not the limb after amputation, with molecular profiles of proteins such as FGFs and noncoding RNAs differing between in early stages post injury (reviewed in [225]Alibardi, 2018). Conclusion While protein mapping and profiling of appendage regeneration has been carried out for many anamniote vertebrates, these data provide insight into the regenerative proteome in a non-avian reptile and amniote vertebrate. In this study, we utilized high-throughput proteomics to identify and describe proteins expressed during tail regeneration in the green anole lizard, capturing the end products of gene activity. This proteomic analyses has identified 2,646 proteins with roles in axonogenesis, myogenesis, chondrogenesis, extracellular matrix remodeling, cell proliferation, and immune signaling in tail regeneration in the green anole lizard. These results are largely consistent with findings from previous studies in multiple lizard species, indicating that the pathways underlying regeneration in lizards are relatively conserved. Specifically, there is an inflammatory immune response immediately after wounding that subsides with time. Genes involved in apoptosis, leukocyte trafficking, phagocytosis, T-cell stimulation, and tumor necrosis factor production, in particular TNF-α, are upregulated, while genes involved in cell cycle regulation, DNA replication and repair, and immune system downregulation are commonly found to expressed later during the regenerative process. In addition, we have identified 32 uncharacterized proteins that also match Ensembl or NCBI gene models, pointing to the need for further analysis to determine if these gene products play a role in regeneration. These findings provide a framework for future regenerative studies which include but are not limited to post-translational modification enrichment proteomics or differential protein expression analysis. Integration of proteomic data with previous mRNA and miRNA transcriptomes of the anole regenerated tail could also provide valuable insight into the genetic regulatory networks that enable regeneration in an amniote model with the potential for translational applications in mammals. Lastly, although reptiles are the sister taxon to mammals and represent a highly diverse group of animals, -omic resources for reptiles remain largely under-represented among all vertebrate classes. However, the proteomic data presented here is a useful, multi-tissue resource that contributes to a growing list of sequencing efforts and collectively, can be utilized to understand the phylogenetic diversity and evolutionary history of different reptile groups, filling a major gap of amniote vertebrate history. Supplementary Material Table S1 [226]NIHMS1925360-supplement-Table_S1.xlsx^ (248.9KB, xlsx) Table S3 [227]NIHMS1925360-supplement-Table_S3.xlsx^ (144.9KB, xlsx) Table S2 [228]NIHMS1925360-supplement-Table_S2.xlsx^ (223.6KB, xlsx) Table S4 [229]NIHMS1925360-supplement-Table_S4.xlsx^ (24KB, xlsx) Table S5 [230]NIHMS1925360-supplement-Table_S5.xlsx^ (105.2KB, xlsx) Table S6 [231]NIHMS1925360-supplement-Table_S6.xlsx^ (24KB, xlsx) Table S8 [232]NIHMS1925360-supplement-Table_S8.xlsx^ (14KB, xlsx) Table S7 [233]NIHMS1925360-supplement-Table_S7.xlsx^ (234KB, xlsx) Table S9 [234]NIHMS1925360-supplement-Table_S9.xlsx^ (24.1KB, xlsx) Table S10 [235]NIHMS1925360-supplement-Table_S10.xlsx^ (13.2KB, xlsx) Table S12 [236]NIHMS1925360-supplement-Table_S12.xlsx^ (12.7KB, xlsx) Table S11 [237]NIHMS1925360-supplement-Table_S11.xlsx^ (21.1KB, xlsx) Acknowledgements