Gene expression profile of the regeneration epithelium during axolotl limb regeneration

Gene Expression Profile of the Regeneration Epithelium duringAxolotl Limb Regeneration

Leah J. Campbell1, Edna C. Suárez-Castillo1, Humberto Ortiz-Zuazaga2,3, Dunja Knapp4,Elly M. Tanaka4, and Craig M. Crews1,5,6,*

1Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT,USA2High Performance Computing facility, Río Piedras Campus, University of Puerto Rico, San Juan,PR, USA3Department of Computer Science, Río Piedras Campus, University of Puerto Rico, San Juan,PR, USA4Center for Regenerative Therapies, Dresden, Germany5Department of Chemistry, Yale University, New Haven, CT, USA6Department of Pharmacology, Yale University, New Haven, CT, USA

AbstractUrodele amphibians are unique amongst adult vertebrates in their ability to regenerate missinglimbs. The process of limb regeneration requires several key tissues including a regeneration-competent wound epidermis called the regeneration epithelium (RE). We used microarray analysisto profile gene expression of the RE in the axolotl, a Mexican salamander. A list of 125 genes andexpressed sequence tags (ESTs) showed a ≥1.5 fold expression in the RE than in a woundepidermis covering a lateral cuff wound. A subset of the RE ESTs and genes were furthercharacterized for expression level changes over the time-course of regeneration. This studyprovides the first large scale identification of specific gene expression in the RE.

Keywordslimb regeneration; urodele amphibian; gene expression microarray; regeneration epithelium

INTRODUCTIONLimb regeneration is a unique ability of urodele amphibians, which are the only vertebratesable to replace such a complex structure throughout their adult life. The process ofregenerating an amputated limb proceeds from the early phase of wound healing to digitdevelopment through formation of a mass of cells called the blastema. Until recently it wasbelieved that the blastema was comprised of dedifferentiated mesenchymal cells withpluripotent properties (Lo et al., 1993). On the contrary, the blastema appears to be aheterogeneous mass of cells that retain memory of their tissue origin and replenish theirrespective missing structures (Kragl et al., 2009) through a recapitulation of signalingmolecules and pathways used in development (Gardiner et al., 1999). Nevertheless, it isunclear how blastemal cells are recruited to the amputation plane and how urodele

*Correspondence to: [email protected].

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Published in final edited form as:Dev Dyn. 2011 July ; 240(7): 1826–1840. doi:10.1002/dvdy.22669.

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amphibians, such as newts and axolotls, are able to proceed past wound healing to replacemissing limbs.

The nerve has been extensively studied as the source of regeneration signaling moleculessince it was first described as required for regeneration (Singer, 1952). Recently, the newtAG protein (nAG) was identified as a secreted nerve factor that rescues regeneration indenervated newt limbs (Kumar et al., 2007). nAG is expressed in Schwann cells of the nervesheath as the severed axon regrows and it has been shown to promote proliferation ofblastemal cells in culture. Later in regeneration, nAG appears in the gland cells of theregeneration epithelium (RE). The RE is an epithelial structure covering the distal part of theregenerate and is also required for successful regeneration (Stocum, 2004). The RE is uniqueto the amputation wound and forms as a result of epidermal migration over the wound fromaround the circumference of the amputation plane (Repesh and Oberpriller, 1978). Inaddition to nAG, genes such as Sp9 (Satoh et al., 2008) and Dlx-3 (Mullen et al., 1996)display nerve-dependent expression patterns in the RE. Historically, the RE has beenreferred to as the wound epidermis (WE) and the apical epithelial cap (AEC), however it hasrecently been suggested that the structure be termed regeneration epithelium (RE) (Satoh etal., 2008) to distinguish it as a specialized structure that communicates with the nerve topromote regeneration. It has been demonstrated that the RE is required for successfulregeneration since removal of the structure delays the process (Thornton, 1957) andpreventing formation of the structure inhibits regeneration (Mescher, 1976). Markers for theRE are limited (Campbell and Crews, 2008), but include transcription factors such as Msx-2(Carlson et al., 1998), Dlx-3 (Mullen et al., 1996), and Sp9 (Satoh et al., 2008), FGFsignaling molecules (Han et al., 2001; Christensen et al., 2002), and matrixmetalloproteinases (Yang et al., 1999; Kato et al., 2003). Of these, Msx-2 is expressed theearliest, within hours after amputation, but is not RE-specific since it is also expressedduring healing of a lateral wound (Carlson et al., 1998). Sp9, another early marker, is RE-specific and expressed within 24 hours after amputation (Satoh et al., 2008). As moreinvestigations focus on exploring the molecular pathways involved in regeneration such aswith the Accessory Limb Model (Endo et al., 2004) or with in vitro work (Ferris et al.,2010), a larger set of RE-specific markers will be needed.

The discovery and characterization of molecules and signaling pathways involved in limbregeneration has been improved by the development of genomic tools for salamanders.Significant effort has been put towards sequencing and organizing expressed sequence tags(ESTs) from Ambystoma mexicanum and Ambystoma tigrinum (Habermann et al., 2004;Putta et al., 2004). The Sal-Site at http://www.ambystoma.org (Smith et al., 2005) providesan Ambystoma gene collection and EST database that has allowed for approaches such asmicroarray analysis and high-throughput 454 cDNA sequencing to investigate aspects oflimb regeneration on a broader level (Monaghan et al., 2007; Monaghan et al., 2009). Thesestudies have introduced many new candidate genes that will enable future regenerationstudies.

In the present study we utilized the publicly available collections of Ambystoma genes andESTs to compare, by microarray analysis, the expression profiles of the RE and the woundepidermis covering a lateral cuff wound. From these results we focused on a list ofAmbystoma ESTs and genes that are significantly more highly expressed in the RE. A subsetof this list was further characterized using quantitative polymerase chain reaction (qPCR) toshow the temporal expression pattern during the time-course of regeneration. We discuss thecharacterized ESTs and genes in terms of best hit candidates discovered through BLASTsearches and with respect to previously published regeneration studies.

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RESULTS AND DISCUSSIONIdentification of RE-Specific Gene Expression

It is well documented that the RE is necessary for successful regeneration in urodeleamphibians (Thornton, 1957; Mescher, 1976), however, there are few markers for RE-specific gene expression. In order to identify RE-specific genes, we collected both theepidermis from a lateral cuff wound and from a RE after 7 days of healing, whichrepresented an early stage of regeneration between wound healing and early bud formation(Figure 1). Messenger RNA (mRNA) from the two populations were labeled and hybridizedto oligonucleotide arrays containing 42,553 elements that represented 16,257 sequencesfrom A. mexicanum and A. tigrinum and 396 sequences from other salamander species.

A total of 698 probe sets demonstrated significantly different (p≤0.0016) hybridizationintensities between the RE and the lateral cuff wound epidermis (LE) (266 in the RE; 432 inthe LE; Additional file 1). We focused on the significant probe sets that exhibited at least a1.5 fold intensity increase in the RE over the LE (n=195; Additional file 2). The Sal-Site(http://www.ambystoma.org, (Smith et al., 2005)) was used to BLAST the probe sets andidentify sequences targeted by the probes. Best hits were identified with NCBI BLAST(Table 1; Additional file 2).

Best hit identification revealed that blood-specific genes, such as globins, were over-represented in the RE list. Due to the extent of injury during amputation it is not surprisingto see this upregulation in the RE with respect to the LE. It is expected that there wascarryover of blood cells in the dissection and RNA isolation of the RE samples.Additionally, the best hit identification revealed that genes in the list are targeted by multipleprobe sets. In order to reduce the list of RE genes we averaged the multiple hits andachieved a list of 125 genes that are upregulated in the RE (Additional file 3).

Validation of RE Gene Overexpression by qPCRFor the purposes of validation and further investigation we focused on the top ten genes aswell as ten additional genes of interest (Table 1). Since we observed several blood-specificgenes in the RE list it was necessary to confirm that these selected genes are specific to REand not to blood. Total RNA was isolated from RE at 7 days post amputation, as well asfrom blood cells, and qPCR was used to examine expression levels of the genes of interest.The geometric mean of four endogenous genes, GAPDH, β-Actin, EF1α, and L27, was usedfor data normalization. A delta globin gene with Sal ID M001136 (Table 2) was used as aninternal control. The results confirmed a >5,000 fold level of expression for M001136 inblood over RE, while 16 of the 20 RE genes were significantly more highly expressed in REthan in blood (Figure 2A). Two genes, M006889 and Wnt-5a, tended towards higherexpression levels in RE than in blood but did not have significant p-values. QPCR analysiswith primers for M062282 and M062365 showed expression levels of 1.1 and 1.2 foldhigher, respectively, in blood over RE, suggesting that M062282 and M062365 may beexpressed by both blood and RE. Some of the fold difference in expression level ofM062282 and M062365 in RE over LE may be due to blood cell contamination.

QPCR was used as an independent verification method of RE gene expression levels ascompared to LE. The 20 genes of interest were tested in RE and LE at 7 days post woundingand also in normal unwounded epidermis. As with the tissue collection for the microarraydata set, the RE tissues were collected at a time-point corresponding to a phase betweenwound healing and early bud. RNA was isolated only from the epidermal tissue of theregenerate. Again, the analysis was performed using the geometric mean of four endogenouscontrols, GAPDH, βActin, EF1α, and L27, as reference. Two genes, M003674 andM032377, which were identified in the microarray as more highly expressed in LE, were



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used as internal controls (Table 2). The qPCR analysis confirmed overexpression of all 20genes of interest in the RE with respect to LE (Figure 2B). The two LE genes were validatedwith higher fold levels in the lateral cuff wound than the amputation wound. M011831,M064466, and M002949 demonstrated much higher fold level differences in RE than in LEby qPCR analysis than by microarray (Table 2), while the remaining genes displayedrelatively similar fold differences by both methods. These data suggest that the five geneswith the greatest fold difference between RE and LE are M011831, M064466, M002949,M003080, and M065526, respectively.

Normal epidermis (NE) was also compared to RE using qPCR. The skin removed from thelimb to make the lateral cuff wound was soaked in dispase I, which promoted the separationof epidermis from the underlying dermis (Kitano and Okada, 1983). As before, analysis wasperformed using the geometric mean of four endogenous controls, GAPDH, βActin, EF1α,and L27. QPCR analysis confirmed that all but two genes were significantly overexpressedin RE over NE (Figure 2C). M002949 demonstrated the greatest fold difference at nearly200 fold (Table 2). The two genes that did not show significant difference were M062282and Dlx-3, with fold level differences of 1.08 and 1.06, respectively.

Given the technical difficulty of isolating epidermal tissue from the underlying stump andblastemal tissue, we cannot eliminate the possibility that gene expression differencesbetween RE and LE or RE and NE may be partially due to blastemal cell contamination.Whole mount in situ hybridization analysis shows that M002949, one of the genes with thegreatest difference between RE and LE, as well as RE and NE, is expressed over the distaltip of the regenerating limb at 7 days post amputation (Figure 3A). The sense controlshowed no stain (Figure 3B). Sectioning of the tissue showed that expression is limited tothe RE, with no expression in the underlying stump tissue (Figure 3C, D). The expression ofM002949 in the top layers of the RE (Figure 3E, F) confirms that the identification of thisgene by microarray analysis was due to tissue-specific expression in the RE as opposed tocontamination of blastemal or underlying stump tissues. It is important to note that this doesnot exclude the possibility that some microarray-identified RE genes may be expressed bythe mesenchymal tissue of the regenerate, however RE-specific expression of M002949demonstrates that mesenchymal cell contamination did not contribute to the identification ofone of the most highly expressed RE genes as compared to LE. The microarray results incombination with the qPCR data demonstrate that the RE is unique in terms of geneexpression as compared to the LE or NE. These results provide a set of regeneration markersfor the identification of the RE in regeneration studies.

RE-specific Genes Display Changes in Expression Levels throughout the RegenerationProcess

The temporal expression patterns of the RE genes of interest were characterized usingqPCR. Regenerating limbs representing six stages of regeneration were processed for RNAisolation and analyzed for each gene with two endogenous controls for normalization:GAPDH and EF1α. Regenerates were collected from slightly smaller animals than thoseused for the microarray and for the qPCR validation data in Table 2. Therefore, the 7 daypost amputation time-point used in the microarray study corresponds with the 2–4 day time-points used for the time-course expression profiling. Since the entire regenerate wascollected and assayed, expression level changes in the time-course experiments, unlike themicroarray and qPCR validation experiments, account for gene expression levels in the REas well as possible expression in the blastema tissue. Therefore it should be noted thatexpression level changes could account for gene expression in different or multiple tissuesof the regenerate during the various phases of limb regeneration. In the following sectionswe present the temporal expression patterns of all 20 genes of interest and discuss theiridentity as determined by BLAST search or previous characterization in limb regeneration.



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Temporal Expression Patterns of Genes Previously Described in LimbRegeneration—Five genes identified in the microarray have been previously described toplay a role during limb regeneration. Msx2 encodes a transcription factor that has beendescribed to be expressed in the RE and distal mesenchyme of the regenerating limb(Carlson et al., 1998; Koshiba et al., 1998).

The time course data show that the expression level gradually increases over time from thewound healing stage through late bud (Figure 4A). This result concurs with the previousreport that Msx2 is re-expressed after amputation and is highly expressed in the late budstage (Carlson et al., 1998).

Wnt-5a, a member of the Wnt family of secreted proteins, has been previously described toplay an important role in the early stages of limb regeneration during dedifferentiation(Ghosh et al., 2008). The temporal expression pattern described by the qPCR time courseagrees with this previous report that expression was detected from very early stages andthroughout regeneration (Figure 4B).

BMP2 has been described as an important factor in the onset of condensation during limbregeneration (Guimond et al., 2010). The qPCR time-course data show an increase inexpression from the stump through medium bud stages and a significant decrease frommedium bud to digit stage (Figure 4C). This concurs with the in situ hybridization patternsthat show strong staining in the medium to late bud and then a distinct localization to theinterdigital regions (Guimond et al., 2010).

Dlx-3, or distal-less 3, has been previously described to play a nerve-dependent role inregeneration with an expression pattern that is very low at early stages and peaks at the latebud stage of regeneration (Mullen et al., 1996). Contrary to the previous report, qPCR timecourse analysis indicates that Dlx-3 significantly increases early in expression level ascompared to stump tissue and tends to be expressed at a constant level throughoutregeneration with no significant increase or decrease over time (Figure 4D).

M002254 shows a significant increase in expression upon wound healing and decreases overtime from the wound healing stage to palette (Figure 4E). This gene shows similarity toXenopus tropicalis uromodulin-like. It has been linked to nerve-dependent blastemaoutgrowth in axolotl (Monaghan et al., 2009) and has been shown to be down-regulated inresponse to thyroid hormone-induced metamorphosis in Xenopus (Brown et al., 1996).

Temporal Expression Patterns of Three-Finger Protein Family Members—Three genes (M002949, M003964, and M061881) are structurally similar to Prod1 (Garza-Garcia et al., 2009). Prod1 is a three-finger protein (TFP) family member that is involved inpositional identity of the proximal-distal axis in the newt limb (da Silva et al., 2002) andbinds to nAG, the nerve factor that rescues regeneration in denervated newt limbs (Kumar etal., 2007). The structural similarity suggests that these factors may be secreted or anchoredto the cell membrane by glycosylphosphatidylinositol (GPI) linkage.

M002949 shows high expression level at the wound healing stage and takes a drop inexpression by the early bud stage (Figure 5A). The tendency for lower expression during theremaining stages of limb regeneration suggests that it may have a very early role inregeneration. M002949 shows similarity to sodefrin precursor-like factor from severalsalamander species, particularly at the N-terminal end and amongst the cysteine residues(Figure 6).

M003964 shows similarity to prostate stem cell antigen (PSCA), a GPI-anchored cellmembrane protein. The expression pattern of this PSCA-like gene takes a significant



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increase immediately upon wound healing and then a tendency towards low expressionlevels throughout regeneration (Figure 5B).

Similarly, the M061881 gene shows a significant increase upon wound healing and then asignificant drop in expression level at the early bud stage (Figure 5C). This gene showssimilarity to the LY6/PLAUR domain containing 2.

Temporal Expression Patterns of Genes with Roles in Cell Adhesion andOrganization—Four genes have similarities with cell adhesion factors. The M065526pattern shows higher expression in regenerating tissue as compared to stump tissue withfluctuating expression levels during regeneration (Figure 7A). This EST shows similarity todesmoglein 4 preprotein, a cadherin family member that is involved in the formation ofdesmosomes. More specifically it has been shown to localize to desmosomes in the humanhair follicle (Bazzi et al., 2006), a regenerating dermal appendage.

M008800 shows similarity to laminin, beta 1, which has been localized to epithelialbasement membranes (Virtanen et al., 2003). During limb regeneration its expressionincreases upon wound healing and significantly decreases by the palette stage (Figure 7B).

M062365 encodes a gene with strong similarity to the Krüppel-like zinc-finger transcriptionfactor 2. This gene is also described as lung KLF (LKLF). Knockout mouse studies haveshown that loss of LKLF results in a change in smooth muscle cell morphology and loss oforganization in the blood vessel wall (Kuo et al., 1997). The temporal expression patternshows a significant increase upon wound healing and a tendency towards relatively stableexpression level during regeneration (Figure 7C).

The M006889 gene shows similarity to a hyaluronan and proteoglycan link protein familymember. Alignment with Xenopus and human sequences shows high conservation in thesecond hyaluronan and proteoglycan binding link domain (Figure 8). This family of proteinsfunctions in cell adhesion and migration by binding hyaluronan, a glycosaminoglycan, withproteoglycans to modify the extracellular matrix or cell surfaces (Fraser et al., 1997). Thetemporal expression pattern demonstrates an increase in expression level at the medium budstage as compared to stump tissue and then a decrease in expression at the late bud stage(Figure 7D). The expression pattern suggests an interesting initial decrease in expressionlevel upon wound healing.

Temporal Expression Patterns of Genes with Lipid-Associated and NeuriteRegeneration Roles—M061758 shows similarity to dynein, a minus-end directed motorprotein (Figure 9A). Dynein has been described to play an important role in axonalregeneration by signaling to the cell body that an axon has been injured (Tuck and Cavalli,2010). The qPCR data for temporal limb regeneration expression shows that expression forthis gene peaks at the wound healing stage.

M003433 (Figure 9B) and M004510 (Figure 9C) are two lipid-associated proteins. Lipidrafts have been described as crucial for the signaling that drives neurite outgrowth andregeneration (Zhao et al., 2009). M003433 shows similarity to apolipoprotein C-I, which is alipid-binding protein believed to be involved in lipid transport. M004510 shows similarity tomal, T-cell differentiation protein-like, which is a protein that appears to localize in lipidrafts. Both of these genes show a significant increase in expression by the early bud stage.

It has also been suggested that an epidermal growth factor-like molecule and lipid rafts areinvolved in cutaneous wound healing (Mathay et al., 2007). The M062282 sequence showssimilarity to an epidermal growth factor repeat superfamily member, EGFL6. Genes of this



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type have been shown to be expressed during early development (Buchner et al., 2000b) andin the anterior part of the dermal placode, which induces hair follicle formation (Buchner etal., 2000a). The qPCR temporal expression pattern for EGFL6 shows a very low expressionlevel after amputation and then a significant increase at each time point thereafter, with thehighest expression at the digit stage (Figure 9D). Taken in concert with the comparison datathat suggested nearly equal expression levels between RE and NE (Figure 2C), M062282may play a role in the maturation of the epidermis covering the regenerate.

Temporal Expression Patterns of Sequences with Unknown or HypotheticalIdentity—The M011831 sequence does not show strong similarity to any known genes butdemonstrates a high fold difference of expression level in RE over LE (Table 2, Figure 2B).The temporal expression pattern shows a significant increase in expression at the woundhealing stage and then a tendency for higher expression through medium bud and lowerexpression from late bud through digit stage (Figure 10A).

The expression pattern of M064466 is highest at the wound healing stage and thensignificantly decreases by the late bud and digit stages (Figure 10B), suggesting that thegene product plays a role in wound healing and early blastema formation.

M065735, another sequence with no identifiable similarities, shows a significant increaseupon wound healing followed by a decrease at early bud and then an increase at mediumbud stage, suggesting a dynamic expression pattern (Figure 10C).

M003080, which presented in the microarray with the highest fold difference in RE over LE,demonstrated an increase in expression upon wound healing followed by a tendency towardshigher expression during medium and late bud stages and then a decrease to the digit stage(Figure 10D). The gene shows similarity to a putative S-adenosylmethionine-dependentmethyltransferase (AdoMet-MTase), class I (Figure 11). The class I family of AdoMet-MTases is the largest and most diverse including members with substrate specificity to smallmolecules, lipids, proteins, and nucleic acids (Martin and McMillan, 2002; Schubert et al.,2003). Further investigation is needed to identify the substrate of this potential axolotlAdoMet-MTase and its role in limb regeneration.

CONCLUSIONSThis study provides the first expression profiling of the RE in urodele amphibian limbregeneration. The analysis identified 125 genes that demonstrate higher expression in theregenerative epithelium than in wound epidermis covering a lateral cuff wound, suggestingthat the expression is specific to the regeneration response of an amputation wound asopposed to general wound healing. QPCR data for a subset of the genes support themicroarray findings and show that they are significantly more highly expressed in RE thanin NE. Additional qPCR data show interesting expression changes for the genes during thetime-course of regeneration. These markers will provide an important tool for studying theearly events in limb regeneration. Further study into the function of the individual genes willilluminate the role of the RE for successful limb regeneration.

EXPERIMENTAL PROCEDURESAnimal procedures

Axolotls (Ambystoma mexicanum) were spawned at Yale University or obtained from theAmbystoma Genetic Stock Center at the University of Kentucky. Amputations and tissuecollections were performed on animals measuring 8–15 cm from snout to tip of tail. Allanimals were anesthetized in 0.1% MS222 solution (Ethyl 3-aminobenzoate



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methanesulfonate salt, Sigma-Aldrich, St. Louis, MO, USA). Radial lateral wounds werecreated by cutting through full thickness skin around the circumference of the limb withspring scissors and peeling away the full thickness skin from the underlying stump tissue.Animal care and use protocols were approved by the Yale University Institutional AnimalCare and Use Committee.

Tissue collectionAmputations and lateral wounds were made in the zeugopod region of the limbs (betweenthe wrist/ankle joint and the elbow/knee joint). RE and LE were collected at 7 days postamputation/wounding. NE was collected by soaking full thickness skin from the radiallateral wounding in a 1% solution of dispase I (Sigma-Aldrich, St. Louis, MO, USA) in 0.8xPBS for 5 hours at room temperature, washing with 0.8x PBS, and then gently peeling theepidermis from the dermis layer. Dispase treatment was only performed on the NE tissuesample. Blood was collected from the radial lateral and amputation wounds at the time ofRE and LE collection. Limb regenerates for qPCR were collected at 0, 2, 4, 7, 10, 14, and 21days post amputation. Tissues were soaked in RNAlater® (Ambion, Foster City, CA, USA)prior to RNA isolation. For microarray analysis, 3 pools of 7 animals each were used. ForqPCR validation studies, 3 pools of 4 animals each were used. For regeneration time-courseqPCR studies, 3 pools of 3 animals each were used.

RNA isolation and microarray analysisRNA was isolated using Trizol Reagent (Invitrogen, Carlsbad, CA, USA). Followingisolation, RNA was purified and DNase treated using RNeasy minicolumns (Qiagen,Valencia, CA, USA). RNA quality was assessed by spectrophotometry using a NanoDropND-1000 (NanoDrop, Wilmington, DE, USA). RNA samples were also analyzed on aBioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). Microarray analysis wasperformed on custom eArrays (Agilent Technologies, Santa Clara, CA, USA) using 60-merprobes designed against genes and ESTs from Ambystoma mexicanum, Ambystoma tigrinumand other salamander species. Hybridization and data collection were performed by theWhitehead Institute Genome Technology Core (Cambridge, MA, USA). RE samples werelabeled with Cy5; LE samples were labeled with Cy3. Each of the three pools of tissuescollected for RE and LE were hybridized to three arrays as biological replicates, while afourth array was used as a technical replicate of one of the biological samples. Analysis ofthe arrays was done using the limma (Smyth, 2005) package in Bioconductor (Gentleman etal., 2004). Multiple probes for the same clone were averaged. Arrays were normalized withthe loess routine (Yang et al., 2001; Yang et al., 2002; Smyth and Speed, 2003) withoutbackground correction (Zahurak et al., 2007). A linear model was fit to the expressionvalues, and an empirical Bayes routine was used to moderate the t-statistic (Smyth, 2004).Genes were selected as differentially expressed after adjusting p-values for repeated tests bycontrolling the false discovery rate (Benjamini and Hochberg, 1995).

Quantitative PCRReverse transcription was performed with iScript™ Reverse Transcription Supermix (Bio-Rad, Hercules, CA, USA). Quantitative PCR assays were run using Power SYBR® GreenPCR Master Mix (Applied Biosystems, Carlsbad, CA, USA) on a C1000 Thermal Cycler(Bio-Rad, Hercules, CA, USA) and analyzed with the CFX96 Real-Time System. Primersequences and annealing temperatures for the genes assayed are listed in Additional File 4.

Whole mount in situ hybridization and HistologyWhole mount in situ hybridization was performed as previously described (Gardiner et al.,1995) with modifications. Tissues were fixed overnight at 4°C with gentle rocking in



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freshly-made MEMFA (0.1 M MOPS, pH 7.4, 2 mM EGTA, 1 mM MgSO4, 3.7%formaldehyde) and then dehydrated and stored at −20°C in 100% methanol. Proteinase K(New England BioLabs, Ipswich, MA, USA) treatment was performed at 10 μg/mL for 30minutes at 4°C followed by 15 minutes at 37°C to permeabilize tissue. Prehybridization wasperformed at 60°C overnight. Probes were prepared from pBluescript SK- vector containingthe 1062 bp M002949 fragment. The vector was linearized with PstI and anti-sense probewas transcribed with T7 RNA polymerase (New England BioLabs, Ipswich, MA, USA) anddigoxigenin RNA labeling mix (Roche Applied Science, Indianapolis, IN, USA). Senseprobe was transcribed with T3 RNA polymerase (Roche Applied Science, Indianapolis, IN,USA) and digoxigenin RNA labeling mix after linearization with XhoI. Hybridization wasperformed at 60°C for 72 hours. Alkaline-phosphatase (AP) conjugated anti-digoxigeninantibody was obtained from Roche Applied Science (Indianapolis, IN, USA). Thecolorimetric alkaline-phosphatase reaction was developed with BM Purple (Roche AppliedScience, Indianapolis, IN, USA). Tissues from whole mount in situ hybridization werecryoembedded and sectioned at 14 μm. Hematoxylin and eosin stains (Sigma-Aldrich, St.Louis, MO, USA) were used to stain nuclei blue and cytoplasm and extracellular proteinsshades of pink.

Supplementary MaterialRefer to Web version on PubMed Central for supplementary material.

AcknowledgmentsWe thank the Crews laboratory for helpful discussion. We thank Randall Voss for providing the vector containingM002949. This work was supported by the NIH (GM094944) and the ARO (Army US #W911NF-07-1-0252).HOZ is supported, in part, by P20RR016470. We acknowledge the services of the Ambystoma Genetic StockCenter, which is supported by NSF-DBI-0443496, and the resources available at the Sal-Site, which are supportedby NIH-NCRR-R24RR16344.

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Fig. 1.Schematic depicting tissue dissection and RNA isolation for microarray hybridization.Axolotl limbs were amputated or wounded with a lateral cuff wound. After 7 days ofhealing, the RE and the LE were dissected and RNA was isolated. mRNA was labeled withCy3 and Cy5 dyes and hybridized to custom microarray slides designed from ESTassemblies for A. mexicanum, A. tigrinum, and other salamander species.



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Fig. 2.qPCR validation of fold change gene expression of RE upregulated hits. A: Fold changeexpressions in blood over RE. B: Fold change expressions in RE over LE. C: Fold changeexpressions in RE over NE. Genes are represented by Sal ID or gene name as in Table 1.Data represents the mean of three biological replicates and standard error of the mean.*p≤0.05; **p≤0.01.



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Fig. 3.Whole mount in situ hybridization analysis of M002949 (sodefrin precursor factor)expression in 7 day post amputation regenerating axolotl limbs. All panels are forelimbswith distal at the top. A: M002949 expression covering distal tip of regenerating limbindicated in blue as determined with anti-sense probe. B: Negative control using M002949sense probe. C, D: Sister sections of tissue from panel A. Dashed line represents level ofamputation. Tissue sectioned at 14 μm. C: Expression in the regeneration epitheliumindicated in blue. D: Hematoxylin and eosin. E, F: Higher magnification of the distal tip ofthe regenerating limb shows that the M002949 expression is localized to the top layers of theregeneration epidermis.



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Fig. 4.Expression level time-courses of genes previously reported to play a role in limbregeneration. The y-axis represents normalized RNA level and the x-axis represents dayspost amputation. Genes are represented by Sal ID and/or gene name as in Table 1. Each darkcircle represents the mean of three samples ± standard error of the mean. *p≤0.05;**p≤0.01.



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Fig. 5.Expression level time-courses of three finger protein family members. The y-axis representsnormalized RNA level and the x-axis represents days post amputation. Genes arerepresented by Sal ID and gene name as in Table 1. Each dark circle represents the mean ofthree samples ± standard error of the mean.



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Fig. 6.Alignment of axolotl (A. mexicanum) M002949 amino acid sequence with sodefrinprecursor-like factor from four species of salamander: seal salamander (D. monticola;accession: AAZ06333), clouded salamander (A. ferreus; accession: AAZ06336), Blue Ridgetwo-lined salamander (E. wilderae; accession: AAZ06337), and Siskiyou Mountainsalamander (P. stormi, accession: AAZ06325). Conserved residues are indicated by blue andnon-conserved residues are indicated by red. Gaps are indicated by a dash (−).



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Fig. 7.Expression level time-courses of genes involved in cell adhesion and organization. The y-axis represents normalized RNA level and the x-axis represents days post amputation. Genesare represented by Sal ID and gene name as in Table 1. Each dark circle represents the meanof three samples ± standard error of the mean. *p≤0.05; **p≤0.01.



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Fig. 8.Alignment of axolotl (A. mexicanum) M006889 amino acid sequence with hyaluronan andproteoglycan link protein 3 from X. laevis (accession: NP_001079631) and human (H.sapiens; accession: NP_839946). Conserved residues are indicated in blue and non-conserved residues are indicated by red. Gaps are indicated by a dash (−). The shaded boxesidentify the hyaluronan and proteoglycan link domains.



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Fig. 9.Expression level time-courses of genes with lipid-associated and neurite regeneration roles.The y-axis represents normalized RNA level and the x-axis represents days post amputation.Genes are represented by Sal ID and gene name as in Table 1. Each dark circle representsthe mean of three samples ± standard error of the mean. *p≤0.05; **p≤0.01.



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Fig. 10.Expression level time-courses of genes with unknown or hypothetical identity. The y-axisrepresents normalized RNA level and the x-axis represents days post amputation. Genes arerepresented by Sal ID and gene name as in Table 1. Each dark circle represents the mean ofthree samples ± standard error of the mean. *p≤0.05; **p≤0.01.



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Fig. 11.Alignment of axolotl (A. mexicanum) M003080 amino acid sequence with putativemethyltransferase sequences from chicken (G. gallus; accession: XP_001232694) and X.laevis (accession: NP_001136263). Conserved residues are indicated by blue and residuesthat are not conserved are indicated by red. Gaps are indicated by a dash (−). The shadedboxes identify the S-adenosylmethionine-dependent methyltransferase, class I regions inchicken and Xenopus.



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TAB

LE 1

List

of I

nter

estin

g H

its U

p-re

gula

ted

in R

egen

erat

ion

Epith

eliu

m a

s Com

pare

d to

Rad

ial L

ater

al W

ound

Epi

derm

is

Fold

cha

nge

(RE

/LE

)Sa

l ID

/acc

essi

on n

o.G

ene

nam

eB

LA

STX

(E)

Bes

t hit

acce

ssio

n no

.

7.79

M00

3080

Sim

ilar t

o m

ethy

ltran

sfer

ase

24 (G

allu

s gal

lus)

3E-6

6X

M_4

2187

1

6.47

D82

576

Msx

2 (A

mby

stom

a m

exic

anum

)

5.73

M01

1831

N/A

N/A

No

hit

5.30

M06

4466

N/A

N/A

No

hit

5.18

M00

2949

Sode

frin

pre

curs

or-li

ke fa

ctor

(Des

mog

nath

us m

ontic

ola)

3E-2

6D

Q09

7063

5.11

M00

6889

hyal

uron

an a

nd p

rote

ogly

can

link

prot

ein

3 (X

enop

us la

evis

)8E

-42

NM

_001

0861

62

5.02

M06

5735

N/A

N/A

No

hit

4.53

M06

1758

Dyn

ein,

cyt

opla

smic

1, i

nter

med

iate

cha

in 1

(Hom

o sa

pien

s)2E

-39

NM

_004

411

4.08

Z140

47W

nt-5

a (A

mby

stom

a m

exic

anum

)

3.53

M06

5526

Sim

ilar t

o de

smog

lein

4 p

repr

opro

tein

(Gal

lus g

allu

s)2E

-05

XM

_426

082

3.38

M00

3433

Apo

lipop

rote

in C

-I (H

emib

arbu

s myl

odon

)6E

-06

FJ17

0109

3.26

M00

2254

Uro

mod

ulin

-like

(Xen

opus

trop

ical

is)

1E-1

6X

M_0

0293

4636

2.86

M00

8800

lam

inin

, bet

a 1

(Hom

o sa

pien

s)9E

-67

NM

_002

291

2.66

M00

3964

Pros

tate

stem

cel

l ant

igen

(Hom

o sa

pien

s)8E

-16

NM

_005

672

2.25

M00

4510

Mal

, T-c

ell d

iffer

entia

tion

prot

ein-

like

(Hom

o sa

pien

s)2E

-27

NM

_005

434

2.13

M06

2282

EGF-

like-

dom

ain,

mul

tiple

6 (H

omo

sapi

ens)

7E-2

6N

M_0

1550

7

2.06

AY

3262

72B

MP2

/4-li

ke (A

mby

stom

a m

exic

anum

)

1.88

U59

480

Dis

tal-l

ess (

Dlx

-3) (

Amby

stom

a m

exic

anum

)

1.68

M06

1881

LY6/

PLA

UR

dom

ain

cont

aini

ng 2

(Hom

o sa

pien

s)6E

-17

NM

_205

545

1.58

M06

2365

Krü

ppel

-like

fact

or 2

(Hom

o sa

pien

s)3E

-19

NM

_016

270


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TAB

LE 2

Sum

mar

y of

Fol

d Le

vel C

hang

es in

Exp

ress

ion

Bet

wee

n Ti

ssue

sa

Sal I

D/g

ene

nam

e (b

est h

it ge

ne n

ame)

RE

/LE

(mic

roar

ray)

RE

/LE

(qPC

R)

Blo

od/R

E (q

PCR

)R

E/N

E (q

PCR

)

M00

3080

(met

hyltr

ansf

eras

e)7.

7910

.64

0.00

377.

44

Msx

26.

473.

110.

3995

6.17

M01

1831

5.73

34.5

00.

0118

60.3

3

M06

4466

5.30

32.3

70.

0468

54.1

5

M00

2949

(sod

efrin

pre

curs

or-li

ke fa

ctor

)5.

1819

.91

0.00

8319

9.06

M00

6889

(hya

luro

nan

and

prot

eogl

ycan

link

pro

tein

3)

5.11

2.43

0.74

4114

.23

M06

5735

5.02

2.58

0.04

4120

.97

M06

1758

(dyn

ein)

4.53

3.07

0.00

1528

.15

Wnt

-5a

4.08

2.28

0.28

9922

.65

M06

5526

(sim

ilar t

o de

smog

lein

4 p

repr

otei

n)3.

533.

290.

0119

23.0

7

M00

3433

(apo

lipop

rote

in C

-I)

3.38

1.68

0.04

7428

.49

M00

2254

(uro

mod

ulin

-like

)3.

261.

890.

0278

33.2

3

M00

8800

(lam

inin

, bet

a 1)

2.86

2.68

0.00

1240

.71

M00

3964

(pro

stat

e st

em c

ell a

ntig

en)

2.66

1.84

0.01

904.

32

M00

4510

(mal

, T-c

ell d

iffer

entia

tion

prot

ein-

like)

2.25

1.23

0.00

892.

66

M06

2282

(EG

F-lik

e do

mai

n, m

ultip

le 6

)2.

131.

381.

1405

1.08

BM

P2/4

-like

2.06

1.83

0.38

723.

03

Dlx

-31.

881.

550.

0974

1.06

M06

1881

(LY

6/PL

AU

R d

omai

n co

ntai

ning

2)

1.68

2.88

0.07

5618

.05

M06

2365

(Kru

ppel

-like

fact

or 2

)1.

582.

311.

2014

4.05

M00

1136

(hem

oglo

bin,

del

ta)

2.09

N/A

5225

N/A

M00

3674

(myo

sin,

hea

vy c

hain

3)

0.05

0.09

N/A

N/A

M03

2377

0.04

0.15

N/A

N/A

a RE,

rege

nera

tion

epith

eliu

m; L

E, la

tera

l cuf

f wou

nd e

pide

rmis

; qPC

R, q

uant

itativ

e po

lym

eras

e ch

ain

reac

tion

(qPC

R).


Documents

Gene expression profile of the regeneration epithelium during axolotl limb regeneration