BACKGROUND
salamanders are fascinating vertebrate organisms because they routinely regenerate complex tissues.
within only a few weeks of losing a piece of limb to a hungry predator or scalpel-wielding scientist, a salamander perfectly reforms the missing structure.
in the early history of salamander regeneration research, scientists innovated elegant experimental designs to probe the anatomical basis of regeneration  <cit> .
more recently and in parallel with the discovery of conserved regulatory genes and developmental pathways among metazoans, scientists have focused attention on candidate molecules and signaling pathways whose functions were deduced first from studies of model organisms.
in particular, much research has been devoted to understanding aspects of limb regeneration associated with wound healing that recapitulate limb development; this strategy has yielded many useful insights and molecular probes  <cit> .
although it is clear that key regulatory molecules play important roles in the development of all organisms, it is not clear that a framework for understanding regeneration can be constructed using a generic and limited molecular toolkit.
there is a need to go beyond candidate molecules and use unbiased approaches to characterize the molecular complexity underlying salamander regeneration.
recent research resource development for the mexican axolotl now allows classic regeneration experiments to be re-examined with powerful and unbiased genomic approaches.
one particularly elegant experiment performed almost  <dig> years ago showed that salamander limb regeneration requires the presence of peripheral nerves.
todd  <cit>  found in  <dig> that limb regeneration does not occur if the sciatic nerve of the hindlimb is severed shortly before or immediately after a more distal limb amputation.
subsequent research showed that the brachial nerves entering the forelimb are required to promote limb outgrowth and patterning of a new limb  <cit> .
within a few days of limb amputation, cells proximal to the amputation plane dedifferentiate and accumulate to form a blastema.
blastema cells proliferate and progressively differentiate during regeneration to give rise to all mesodermal structures of a typical vertebrate limb  <cit> .
upon amputation, nerve fibers in the vicinity of the amputation plane extend into the blastema and play a supportive role in cell proliferation  <cit> .
transection of the spinal nerves that enter the limb in the axolotl leads to a decrease in cycling cells and there is resorption of distal tissues of the amputated limb.
histological and cell proliferation analyses suggest that early cellular events are similar between denervated and innervated limbs, but denervated limbs are incapable of blastema formation because they do not support significant cell proliferation and outgrowth  <cit> .
although limb regeneration is a complex developmental process, nerve dependency and other aspects of regeneration have often been conceptualized as having a simple molecular basis, involving relatively few regulatory factors.
for example, ferretti and brockes  <cit>  hypothesized that in the absence of nerves, schwann cells produce an inhibitory factor that prevents blastema cell proliferation.
this mechanism is supported by experimental results although the hypothetical factor has not been identified  <cit> .
alternative mechanisms for nerve dependency have also been proposed for several factors with growth promoting effects  <cit> .
a recent study identified newt anterior gradient protein  as a blastema cell growth-promoting factor in vitro, whose over-expression was sufficient to rescue regeneration of denervated and amputated limbs in vivo  <cit> .
most recently, nerve-dependent expression of the transcription factor sp <dig> has been identified as an early marker of dedifferentiation of the wound epithelium and the initiation of limb regeneration  <cit> .
while considerable progress has been made in investigating the functions of candidate regulatory factors and signaling pathways, a broader systems-level perspective is needed to understand why multiple aspects of limb regeneration are dependent upon the presence of a nerve.
genomic tools are now available that allow global characterization of the regeneration process in salamanders.
expressed sequence tag  information has facilitated the development of an ambystoma salamander affymetrix microarray platform  <cit> .
this platform and a high-throughput  <dig> cdna sequencing approach were used in this study to compare transcript abundance among uninjured limbs, regenerating limbs, and limbs denervated at the time of amputation.
the results show that innervated  and denervated  limbs exhibited similar  gene expression patterns at five days post amputation  but then diverged as a blastema formed under the influence of nerves.
the results are discussed within the context of previous studies of nerve dependency, highlighting specific genes and biological processes that are associated with blastema formation and outgrowth, and more generally, the mexican axolotl's unparalleled ability to regenerate limbs.
RESULTS
morphology and histology of denervated and innervated limbs
histological staining verified our experimental procedures for creating innervated and denervated limbs on the same individual.
previous studies found few morphological or histological differences between innervated and denervated limbs during the first few days of regeneration  <cit> .
consistent with these observations, denervated and innervated limbs at  <dig> dpa  were histologically indistinguishable .
histological staining  showed hemorrhaging directly beneath the epithelium containing leydig cells, squamous cells, and basal keratinocytes .
all  <dig> dpa limbs resembled the wound-healing phase or early phase of dedifferentiation according to the staging of  <cit> .
a blastema was visible on innervated limbs  <dig> dpa  and histological staining showed an accumulation of blastemal cells under the wound epithelium.
these structures were not observed in denervated limbs at  <dig> dpa .
these results indicate that blastema formation and outgrowth only occurred in nr limbs.
an apical thickening of the epithelium characterized the distal end of nr <dig> limbs and this layer consisted of keratinocytes and leydig cells.
these histological traits indicate that  <dig> dpa limbs in this experiment corresponded to the early to mid bud stage of limb regeneration  <cit> .
immunological staining using rt- <dig> was performed to detect the presence or absence of nerve axons at  <dig> and  <dig> dpa.
at both time-points, neurofilament staining was positive in the nr limbs, but negative in dl limbs.
in pilot experiments, we determined that >  <dig> days is required for nerves to re-innervate limbs after denervation surgery .
thus, dl and nr limbs were created successfully and histology showed that  <dig> and  <dig> dpa time points correspond to wound-healing and early to mid bud phases of regeneration, respectively.
transcription during normal limb regeneration: deviations of nr limbs from baseline
at both  <dig> and  <dig> dpa, mrna levels for hundreds of genes were significantly different from baseline levels of genes expressed in whole limbs at day  <dig> .
many of the same genes  were identified as significant in nr <dig> and nr <dig> limbs; the deviation from baseline was in the same direction for all but one of these genes .
four matrix metalloproteinases  that are known to function during wound healing in many organisms were upregulated at both  <dig> and  <dig> dpa , while collagens  and muscle-specific genes  were downregulated .
upregulation of collagen catabolism genes coupled with downregulation of collagen structural genes suggests that transcriptional activation and repression are integrated to efficiently remodel the extracellular environment of damaged tissues.
downregulation of muscle genes at both time points suggests that the differentiated muscle gene expression phenotype changes by  <dig> dpa, and changes more dramatically by  <dig> dpa.
significant genes were identified by microarray analysis.
gene ontology terms are represented that were sampled more often than expected in nr <dig>  nr <dig>  dl <dig>  and dl <dig> limbs.
p-value is calculated by david  <cit> .
although many genes exhibited similar deviations from baseline at  <dig> and  <dig> dpa, unique gene expression changes were identified at each time point .
the unique nr <dig> genes were associated with gene ontology  terms that implicate extracellular protein changes and signal transduction pathways of the early wounding response.
these terms included response to stimulus, signal transduction, extracellular region, and ion transport.
the unique nr <dig> genes were associated with go terms that implicate cell division and dna metabolism including ccnd <dig>  ccnb <dig>  rrm <dig>  rrm <dig>  nasp, rrc <dig>  and cdc <dig>  the unique gene expression changes that were identified at  <dig> and  <dig> dpa support the idea of temporal progression from an early wound-healing phase to a blastema outgrowth phase during normal limb regeneration.
transcription within denervated limbs: deviations of dl limbs from baseline
as was observed in nr limbs, hundreds of genes were identified as significant when comparing dl <dig> and dl <dig> mrna levels with baseline levels measured at day  <dig> .
some of the same or similar go terms that were associated with nr limbs were identified as significantly enriched in dl limbs.
this was not unexpected because both dl and nr limbs undergo tissue histolysis at the limb stump and carry out an early wound-healing response .
for example, extracellular region and mmp genes were upregulated at  <dig> and  <dig> dpa, as was seen in nr limbs .
as in nr limbs, muscle contraction , cytoplasmic, and collagen genes  were downregulated in both dl <dig> and dl <dig> limbs .
moreover, 83% of the genes that were identified as significant in nr <dig> limbs were also significant  in dl <dig> limbs.
these results indicate that many transcriptional events are nerve-independent during early regeneration.
denervated limbs are known to cease growth following limb amputation.
several groups of genes may explain this observation including the downregulation of genes associated with the m phase of cell division, mitochondrial transcripts, and genes associated with glucose metabolism .
furthermore, dl <dig> limbs were morphologically similar to dl <dig> limbs because denervation prevented blastema formation .
consistent with this observation, many of the genes that were identified as upregulated in dl <dig> limbs were also identified as significant in dl <dig> limbs .
these genes are associated with the following go terms: lysosome, response to stress, hydrolyse activity, signal transducer activity, and ion transport.
overall, these terms suggest protraction and expansion of wound-healing responses in dl <dig> limbs, as well as changes in cellular metabolism and cell division.
transcript abundance differences between nr and dl limbs
in the preceding two sections, transcriptional patterns of nr and dl limbs were described relative to baseline levels at day  <dig>  here, we report genes found to be significant when comparing nr and dl limbs.
in general, few significant changes in gene expression were observed when nr <dig> and dl <dig> transcripts were compared directly.
transcripts for  <dig> genes were more abundant in nr <dig> limbs and  <dig> were more abundant in dl <dig> limbs, and these differences were small in terms of fold-level change .
eighteen of these genes exhibited significant sequence similarity to human or salamander presumptive gene sequences; the others are unknown.
the genes with significantly more transcripts in nr <dig> limbs are associated with intracellular  and extracellular functions, including membrane proteins  and collagen binding .
the genes with significantly more transcripts in dl <dig> limbs are associated with extracellular functions or the immune response .
all but one of the significant  <dig> dpa genes showed the same directional deviation at  <dig> dpa; myl1was  <dig> -fold higher in the nr limb at  <dig> dpa, but  <dig> -fold lower at  <dig> dpa.
thus, as was observed when comparing dl <dig> and nr <dig> mrna abundances with baseline levels, relatively few gene expression differences were identified between nr and dl limbs at  <dig> dpa, and the magnitude of these differences was small.
these results further support the idea that transcription during limb regeneration is predominantly nerve-independent at  <dig> dpa.
whereas only  <dig> transcriptional differences were observed between morphologically similar dl and nr limbs at  <dig> dpa,  <dig> differences were detected at  <dig> dpa .
k-means cluster analysis of these genes with significant human protein hits highlighted three clusters wherein genes exhibited similar patterns of expression .
genes in cluster  <dig> presented expression patterns with transcript abundances above baseline in nr <dig> limbs and abundances below baseline levels in dl <dig> limbs.
genes associated with cell cycling, a well-established characteristic of blastemal cells, were highly enriched in cluster  <dig> .
over 50% of the genes in cluster  <dig> are predicted to localize to the nucleus, including several transcriptional regulators .
thus, many of the genes in cluster  <dig> have functions that are consistent with blastema formation and outgrowth in nr limbs.
genes from clusters  <dig> and  <dig> were generally expressed in the same direction between nr <dig> and dl <dig> limbs; however, the magnitude of expression differed.
genes in cluster  <dig> presented transcript abundances that generally exceeded baseline levels, with higher levels observed in dl <dig> limbs.
this pattern suggests that most of these genes were activated in the same direction in the presence or absence of nerves, but denervation caused higher transcript abundances.
twenty-two percent of the genes in cluster  <dig> are associated with the go term cellular response to stimulus  and a significant proportion localized to the lysosome , including lgmn, ctsk, ctss, asah, atp6v0d <dig>  and neu <dig>  genes in cluster  <dig> presented transcript abundances that were generally lower than baseline levels, with much lower levels observed in nr <dig> limbs.
with relatively few genes in this cluster, no biological process was identified as significantly enriched.
however, inspection of the genes in cluster  <dig> again supports the idea that some genes may function in muscle contraction  and tissue repair .
in summary, mrna levels for genes from clusters  <dig> and  <dig> were quantitatively affected by the presence or absence of a nerve.
<dig> cdna sequence analysis of nerve dependency
to further explore nerve dependency and generate an unbiased collection of molecular probes for regenerating limbs, we sequenced cdnas derived from the same rna samples that were used in the microarray analysis.
over  <dig>  ×  <dig> reads were generated and this yielded approximately  <dig>  to  <dig>  high-quality sequence reads for each limb treatment, with an average of  <dig> base pairs in length .
more than half of the sequence reads correspond to mitochondrial transcripts and ribosomal rna.
this frequency of mtdna transcripts  approximates the number sampled in an earlier est screen of the ambystoma genome  <cit> .
the number of rrna transcripts was higher than expected.
assembly of all high-quality cdna reads yielded  <dig>  unique sequences.
these sequences were assembled with previous est contigs to produce  <dig>  contigs each containing at least two overlapping sequences.
the distribution of contig lengths is shown in figure  <dig>  all contigs and singletons were searched against ncbi databases to identify significant similarity matches that would suggest presumptive gene identities.
ambystoma contigs and singletons yielded  <dig>  significant hits to sequences in the human refseq database , including  <dig> unique human genes.
figure  <dig> shows the distribution of percent coverage to predicted human refseq proteins.
interestingly,  <dig> ambystoma queries that did not show significant amino acid sequence identity to a human reference sequence did show significant nucleotide identity to a xenopus sequence.
assembly of new  <dig> cdna sequences with existing est contigs from the ambystoma est database more than doubled  the number of non-redundant human-a.
mexicanum orthologous sequences.
this increase in sequence content was even among  <dig> go functional categories that are relevant to salamander wound healing and regeneration .
assuming that many of the anonymous  <dig> contigs and singletons  that were generated correspond to functional genes, significantly more than  <dig>  different transcripts are expressed during the first two weeks of axolotl limb regeneration.
the table shows the number of ambystoma estdb contigs with gene ontology annotations before and after  <dig> dna sequencing.
the total is less than the sum of each category because several genes may belong to multiple categories.
bp = biological process.
mf = molecular function.
mrna abundance estimates and gene discovery from  <dig> cdna sequence data
the number of times that a non-redundant transcript was sampled by  <dig> cdna sequencing was used to estimate mrna abundances.
the transcript counts for  <dig> ambystoma est contigs  differed significantly among limb cdna pools that were created for each of the limb types .
it was possible to assign a putative ortholog to  <dig> of these genes; the remaining genes were considered anonymous.
this final list of genes was compared with the significant gene lists from the microarray analysis.
it was determined that for  <dig> of the  <dig> significant genes from the  <dig> cdna sequencing analysis, a portion of the gene sequence was represented by a probe set on the ambystoma genechip.
of these,  <dig> genes were identified as significant by both methodologies and mrna abundances for these genes were highly positively correlated .
the  <dig> genes found to be significant by  <dig> cdna sequencing, but not by microarray analysis, were mostly characterized by low fold changes from baseline ; or conversely, registered low hybridization intensities in the microarray analysis .
gene functions that were identified as significantly enriched by microarray analysis were also identified as significant by  <dig> cdna sequencing.
for example, the muscle contraction go term was identified as highly enriched and the underlying genes were similarly downregulated relative to baseline .
also, genes sampled most often from nr <dig> limbs were associated with dna metabolism, a biological process associated with cell cycling , and transcripts for genes associated with cell proliferation and cell cycle progression  were either sampled more often among nr limbs or were only sampled from nr limbs .
thus,  <dig> cdna sequencing also identified genes in nr limbs that are consistent with blastema formation.
several functional terms that were not identified by microarray analysis were identified as enriched by  <dig> cdna sequencing.
transcripts for  <dig> genes associated with macromolecule metabolism were most abundant in nr <dig> limbs compared with other limbs.
transcripts for  <dig> genes associated with macromolecule catabolism were most abundant in dl <dig> limbs, including mmp <dig>  mmp3/10a, mmp3/10b, mmp <dig>  and mmp <dig>  this suggests that the presence or absence of nerves differentially affects transcriptional responses and regulation at  <dig> dpa.
also,  <dig> cdna sequencing identified additional psca-like genes that were not represented on the genechip, and these were also differentially expressed between nr <dig> and dl <dig> limbs.
these and other examples suggest that  <dig> cdna sequencing complemented the microarray analysis by providing deeper sampling of transcriptional programs and associated biological processes.
this revealed more candidate nerve-dependent gene expression changes at the earlier  <dig> dpa time point than was revealed by microarray analysis.
gene ontology analysis of  <dig> genes with presumptive human orthologs identified by  <dig> dna sequencing.
only the most significant gene ontology category was selected for the table.
in addition to providing estimates of mrna abundance,  <dig> cdna sequencing also discovered new gene sequences for the mexican axolotl.
these include genes that are known to affect developmental processes in other vertebrate models: notch , nrg <dig>  bmp <dig>  wnt <dig>  ctnna <dig>  btnna <dig>  dkk <dig>  axin  <dig>  nrg <dig>  fgf <dig>  sirt , stats , sema4f, tf, sfrp , rara, rarg, rxr, pdgf, acvri , bmpri, bmprii, smad , efna <dig>  ntn <dig>  slit <dig>  slit <dig>  robo <dig>  and robo <dig>  most of these genes were sampled less than  <dig> times and thus appear to be expressed at low levels.
other developmental genes that have not been previously associated with limb regeneration were sampled many more times than these candidates, and for the following examples, counts varied significantly among limb cdna libraries.
these include mdk, flii, tagln <dig>  ddx <dig>  umod, and cnot <dig> .
also, numerous retroelement-associated sequences were sampled differentially between dl and nr limbs .
overall, the  <dig> sequencing approach verified the primary results from the microarray analysis and identified many new candidate genes and functional pathways that are associated with limb regeneration.
genes identified as differentially expressed by  <dig> sequencing analysis that have high sequence similarity to retrovirus genes.
discussion
microarray analysis and  <dig> cdna sequencing were used to identify nerve-dependent and independent gene expression changes during limb regeneration in the mexican axolotl.
the results show that limb regeneration is associated with thousands of transcriptional changes.
considerable similarity was observed between the dl and nr transcriptional programs at  <dig> and  <dig> dpa.
for example, genes that are critical to wound healing were upregulated in both limb types  while genes that are associated with muscle structure and function were downregulated .
many of the transcriptional changes that were observed at  <dig> dpa were also observed at  <dig> dpa.
thus, many aspects of early limb regeneration are accomplished in the absence of nerves.
however, gene expression differences were identified between dl and nr limbs at  <dig> and  <dig> dpa.
many of the transcriptional differences correlated with blastema formation; cell numbers increased in nr limbs after  <dig> dpa and this yielded a distinct transcriptional signature of cell proliferation in nr <dig> limbs.
overall, this study identified genes that are associated with wound healing, early events of blastema formation, and subsequent blastema cell proliferation and outgrowth.
below, we discuss and expand upon these primary results and highlight genes whose functions appear to be important for understanding the basis of nerve dependency and limb regeneration.
fold-level change of upregulated genes that have functions associated with a wounding response .
early wound-healing response during limb regeneration
previous studies have documented anatomical similarities between innervated and denervated limbs at early stages of regeneration  <cit> .
this study shows that there are also many transcriptional similarities.
this suggests that many aspects of the early wound-healing phase of limb regeneration are not dependent upon post-amputation, nerve-derived factors .
instead, humoral immune and local tissue responses appear to be key.
many genes that are associated with wound-healing and tissue-repair activities including stress, inflammation, cell survival, immunity, and extracellular matrix remodeling were upregulated from baseline in  <dig> dpa limbs .
it is probably no coincidence that essentially all of the early stress-associated genes that were previously identified as significantly regulated  during early spinal cord regeneration  <cit> , and during the innate immune response of axolotls to a deadly viral pathogen  <cit> , were also identified as significant in this study.
many of these genes appear to be expressed similarly in all vertebrates in response to stress, including junb, irf <dig>  hmox <dig>  apoe, mmps, ptx <dig>  gal <dig>  gadd45g, and tgfb.
additionally, significantly more 'extracellular' genes were identified at  <dig> dpa than expected by chance, including genes that code for matrix remodeling proteins and secreted molecules whose functions are associated with growth factor binding, cell signaling, survival, death, adhesion, migration, and proliferation.
the early wound-healing response initiates local environmental changes of the injury site that are pivotal to subsequent phases of regeneration.
the  <dig> dpa time point was chosen in this study to identify critical nerve-dependent signaling events that are stimulated within the first few days of regeneration .
comparison of dl <dig> and nr <dig> data revealed few overall gene expression differences.
the  <dig> dpa time point captured many transcriptional responses that are induced by injury and/or amputation but relatively few that are associated with known or suspected neurotrophic signaling pathways.
more comprehensive sampling and deeper sequencing is needed to detail early nerve-dependent transcriptional responses because several genes that are known to be nerve-responsive during the wound-healing phase were not identified in this study .
downregulation of genes associated with differentiated muscle
this study documented dramatic decreases in the relative abundance of mrnas coding for skeletal muscle contractile proteins, including myosins, actins, actinins, titin, tropomyosins, and troponins.
many of these changes were also detected by  <dig> cdna sequence analysis .
this strong, muscle-specific transcriptional signature was observed because approximately half of uninjured forelimb nuclei in  <dig> to  <dig> cm axolotls, and likely more than half the cross-sectional area, derive from muscle  <cit> .
it is unlikely that the downregulation of muscle genes is due to retraction of the muscle towards the shoulder because muscle transcripts are much more downregulated at  <dig> dpa than  <dig> dpa in both denervated and innervated limbs.
considering that limb tissue samples in this study included ~ <dig> mm of undamaged tissue proximal to the amputation plane, the results suggest that injury to skeletal muscle induces tissue-wide loss of muscle contractile transcripts.
it is possible that the decrease in muscle transcripts is due to muscle wasting, caused by a lack of mechanical stress.
it is interesting to speculate that this response maybe associated with the degeneration or cellularization of multinucleated muscle fibers into mononucleated cells, which occurs in both denervated and innervated amputated limbs  <cit> .
it is interesting to note that muscle-specific genes, including actc <dig>  actn <dig>  atp2a <dig>  my1pf, tncc, tnni <dig>  tnni <dig>  are downregulated during early stages of mammalian skeletal muscle regeneration  <cit> , and this is accomplished without blastema formation.
skeletal muscle regeneration in mammals and other vertebrates involves resident stem  cells  <cit> .
it is possible that muscle-specific genes are downregulated as an integral step of a conserved, skeletal muscle regeneration program of vertebrates.
candidate transcriptional repressor genes were identified in this study including id <dig>  <cit> , tardbp  <cit> , cnot  <cit> , msx <dig>  <cit>  and msx <dig>  it will be important in future studies to determine if the dramatic decrease in muscle transcripts is due to activation of muscle stem  cells, muscle loss, muscle dysfunction/wasting, or whether these transcriptional patterns have an active role in regeneration.
genes associated with epigenetic reprogramming and genomic stability
blastema formation requires a large number of progenitor cells derived from quiescent stem cells or differentiated cell types.
it is generally known that reprogramming of differentiated cells is accompanied by epigenetic changes such as histone and dna modifications  <cit> .
few candidates have been identified previously as bringing about epigenetic changes necessary for cellular reprogramming during regeneration  <cit> .
this study identified several genes whose functions are associated with epigenetic phenomena, including chromatin remodeling, dna methylation, and transcriptional regulation.
these include uhrf <dig>  <cit> , lmnb <dig>  parp <dig>  <cit> , thymopoietin  <cit> , and a gene with high sequence identity to sam-dependent methyltransferases .
after cellular reprogramming and during limb outgrowth, blastemal cells undergo tremendous cell proliferation.
during blastema cell proliferation, telomere lengths and overall genome stability must be maintained to prevent cell death.
this study identified several candidate genes from nr <dig> limbs that are known to function in genome stability, telomere homeostasis, and dna repair.
these include parp <dig>  <cit> , hmgb <dig>  <cit> , fen <dig>  <cit> , aurka  <cit> , aurkb  <cit> , and pif <dig>  a dna helicase that maintains genome stability and binds to telomerase from yeast to humans  <cit> .
it is also important to note that many transcripts were identified from nr and dl limbs that code for retroelement components .
retrotransposons are normally transcriptionally silenced in differentiated somatic cells by epigenetic mechanisms, but become active upon changes in epigenetic status; these may also regulate nearby gene expression  <cit> .
it is unclear if upregulation of retroelement transcripts affects genome stability and/or is necessary for regeneration.
genes associated with nerve-dependent blastema outgrowth
in this study, blastemas were not observed in nr <dig> limbs or dl <dig> limbs, and only formed on nr <dig> limbs.
nerve-dependent limb outgrowth occurs as a result of blastemal cell proliferation.
two early cell proliferation biomarkers, umod and psca, were identified as significantly different between nr <dig> and dl <dig> limbs.
umod probably locates to the wound epithelium as it is upregulated in apical skin cells during thyroid hormone-induced metamorphosis of axolotl epidermis .
it is possible that psca is a membrane receptor of blastema cells because it shows structural similarity to prod <dig>  a surface protein that is implicated in proximal-distal positional identity of blastemal cells during newt limb regeneration  <cit> .
most of the cell proliferation biomarkers were identified at  <dig> dpa, when a blastema was present in nr limbs but absent in dl limbs.
thus, between  <dig> and  <dig> dpa, blastemal cells underwent considerable cell proliferation in the presence of nerves.
because the limb blastema continues to expand after  <dig> dpa, the blastema-specific genes that were identified in this study are probably transcribed at much higher levels at later time points.
a clear signature of cell proliferation, including genes that function in the cell cycle, mitosis, and nucleotide synthesis, was observed in nr <dig> limbs.
in contrast, these genes were slightly downregulated in denervated limbs at this time .
thus, transcripts associated with cell proliferation are maintained at steady state  in nr and dl limbs for at least five days.
this early, nerve-independent portion of the limb regeneration program may allow time for re-innervation of the injury site and production of nerve-derived molecules in sufficient quantity to initiate and sustain blastemal cell proliferation.
multiple gene products have been hypothesized to be neurotrophic factors provided by nerves to sustain blastema cell proliferation.
these include growth-promoting factors such as fibroblast growth factors  <cit> , substance p  <cit> , neuregulin  <cit> , and transferrin  <cit> .
transferrin and neuregulin were sampled by  <dig> sequencing, but were not identified as differentially expressed by our analysis.
fgf <dig> and fgf <dig> were screened out of the microarray analysis due to low expression, but post-hoc analysis suggested that both are upregulated in nr <dig> limbs compared to control and dl <dig> limbs .
expression of these molecules is known to be nerve-dependent in blastemas of xenopus  <cit>  and axolotls  <cit> .
other molecules that have previously been associated with the blastema during limb regeneration were not included in our statistical analyses due to low hybridization intensity, but were later found to be differentially expressed in nr <dig> limbs, including hoxd <dig>  hoxa <dig>  hoxa <dig>  and msx <dig> .
recently, kumar et al  <cit>  identified a growth-promoting extracellular ligand  that rescues aspects of nerve dependency of limb regeneration in newts.
probesets for nag are represented on the ambystoma genechip and transcripts for this gene were sampled by  <dig> cdna sequencing.
nag mrna was transcribed at a high level in all tissues, but did not differ significantly between nr, dl, or control limbs.
it is possible that the effects of nag and other neurotrophic candidates are associated with quantitative variation of mrna transcript abundances over fine temporal and spatial scales.
such variation would have been missed in this analysis of three time points and sampling of mrnas from heterogeneous tissues.
furthermore, it is possible that the increase in nag immunoreactivity observed by kumar et al  <cit>  is regulated at the level of translation and would not be observed using microarray or sequencing approaches.
overall, we found that several, but not all, genes previously shown to be directly downstream of neurotrophic factors are expressed at higher mrna levels in the blastema versus control and denervated samples.
singer  <cit>  emphasized that the neurotrophic factor underlying nerve dependency was growth promoting and quantitative in effect.
he showed that the trophic effect of nerves could be titred by surgically manipulating the number of nerves innervating the limb  <cit> .
other lines of research showed that nerve-derived factors were not necessary for traversing cell cycle checkpoints, as an equivalent number of presumptive blastema cells are initially observed to enter s phase in denervated and innervated limbs  <cit> .
instead, the neurotrophic factor appears to be necessary to complete the cell cycle and this may be non-trivial considering the cost of replicating a large salamander genome.
these studies suggest that nerves either directly or indirectly provide limiting macromolecules that are needed to accomplish cell division.
according to this reasoning and assuming a correlation between transcript and protein abundance for specific mrna species, transcripts associated with nerve dependency would be expected to exceed baseline levels in nr limbs during regeneration, and decrease in abundance in dl limbs.
this study identified many human-axolotl presumptive orthologs and anonymous axolotl transcripts that showed this pattern.
for example, psat codes for a protein that regulates the second enzymatic step of the phosphorylated pathway in mammals, which produces l-serine  <cit> .
psat expression  is high among cell types with high rates of proliferation, including cancer cell lines  <cit> .
strikingly, psat registered the largest mrna abundance difference between nr <dig> and dl <dig> limbs among probesets on the ambystoma genechip , and this result was verified by both  <dig> cdna sequencing  and real-time pcr .
we also found that the third enzyme in the phosphorylated pathway, psph, was  <dig> -fold higher in nr <dig> versus dl <dig> by real-time pcr .
psat and psph may have neurotrophic potential but more likely function to provide proliferating cells with a limiting and conditionally important substance  that is required for synthesis of macromolecules, amino acids, and purines that are needed to accomplish mitosis  <cit> .
the fact that macromolecular synthesis and cell proliferation are both depressed in denervated limbs  <cit>  supports the idea that psat activity is associated with nerve-derived factors that contribute to blastema outgrowth.
comparison of microarray and cdna sequencing approaches
microarray analysis and  <dig> cdna sequencing offer different advantages for dissecting complex biological processes.
the strength of microarray analysis is the precision that this approach provides for estimating transcript abundances for a specific panel of genes.
such gene panels provide standardized markers for identifying shared and unique patterns of transcription among experiments.
in turn, identification of such patterns provides systems-level insight.
for example and as was discussed above, cross-experiment comparisons of axolotl transcription helped distinguish general stress responses from local regenerative responses.
a relatively conservative fold level threshold  was used in this study to identify significant genes in the microarray analysis and lowly expressed genes  were removed from consideration.
these conservative approaches potentially exclude important genes but likely discover 'real' transcriptional differences between samples.
a lower threshold could be applied to extract more information from the dataset and future studies would likely benefit by increasing replication of genechips to detect significant differences for lowly expressed genes.
with respect to  <dig> cdna sequencing, two goals were accomplished: gene discovery and estimation of transcript abundance.
however, accomplishment of both goals required a trade-off in the allocation of resources toward deep sequencing versus replication.
sequencing resources were used to deeply sequence day  <dig>  nr, and dl cdna libraries instead of shallowly sequencing replicate libraries for these time points.
this strategy identified thousands of new gene sequences that will greatly enrich future regeneration studies.
future studies will benefit from experimental designs that replicate deep sequencing of cdna libraries and this will be possible as sequencing costs decrease.
still, it was encouraging to find in this study that 38% of genes that were identified as differentially expressed by  <dig> cdna sequencing and found on the microarray genechip were identified as significant by both of these technologies, and transcript abundances for these genes were highly positively correlated.
CONCLUSIONS
this study addressed the nerve dependency of limb regeneration by characterizing downstream cellular events that are affected when an intact nerve supply is removed from an amputated salamander limb.
microarray analysis showed that the early wound response is largely nerve-independent, but transcriptional profiles diverge between denervated and innervated limbs when the innervated limb starts to regenerate.
pyrosequencing supported these microarray results while substantially increasing sequence information from the salamander transcriptome.
this study shows the utility of next-generation sequencing platforms for gaining transcriptome information  <cit> .
this new dna sequence information will greatly enrich future regeneration studies using the axolotl.

 salamanders are fascinating vertebrate organisms because they routinely regenerate complex tissues. within only a few weeks of losing a piece of limb to a hungry predator or scalpel-wielding scientist, a salamander perfectly reforms the missing structure. in the early history of salamander regeneration research, scientists innovated elegant experimental designs to probe the anatomical basis of regeneration. more recently and in parallel with the discovery of conserved regulatory genes and developmental pathways among metazoans, scientists have focused attention on candidate molecules and signaling pathways whose functions were deduced first from studies of model organisms. in particular, much research has been devoted to understanding aspects of limb regeneration associated with wound healing that recapitulate limb development; this strategy has yielded many useful insights and molecular probes. although it is clear that key regulatory molecules play important roles in the development of all organisms, it is not clear that a framework for understanding regeneration can be constructed using a generic and limited molecular toolkit. there is a need to go beyond candidate molecules and use unbiased approaches to characterize the molecular complexity underlying salamander regeneration. recent research resource development for the mexican axolotl now allows classic regeneration experiments to be re-examined with powerful and unbiased genomic approaches. one particularly elegant experiment performed almost years ago showed that salamander limb regeneration requires the presence of peripheral nerves. todd found in that limb regeneration does not occur if the sciatic nerve of the hindlimb is severed shortly before or immediately after a more distal limb amputation. subsequent research showed that the brachial nerves entering the forelimb are required to promote limb outgrowth and patterning of a new limb. within a few days of limb amputation, cells proximal to the amputation plane dedifferentiate and accumulate to form a blastema. blastema cells proliferate and progressively differentiate during regeneration to give rise to all mesodermal structures of a typical vertebrate limb. upon amputation, nerve fibers in the vicinity of the amputation plane extend into the blastema and play a supportive role in cell proliferation. transection of the spinal nerves that enter the limb in the axolotl leads to a decrease in cycling cells and there is resorption of distal tissues of the amputated limb. histological and cell proliferation analyses suggest that early cellular events are similar between denervated and innervated limbs, but denervated limbs are incapable of blastema formation because they do not support significant cell proliferation and outgrowth. although limb regeneration is a complex developmental process, nerve dependency and other aspects of regeneration have often been conceptualized as having a simple molecular basis, involving relatively few regulatory factors. for example, ferretti and brockes hypothesized that in the absence of nerves, schwann cells produce an inhibitory factor that prevents blastema cell proliferation. this mechanism is supported by experimental results although the hypothetical factor has not been identified. alternative mechanisms for nerve dependency have also been proposed for several factors with growth promoting effects. a recent study identified newt anterior gradient protein as a blastema cell growth-promoting factor in vitro, whose over-expression was sufficient to rescue regeneration of denervated and amputated limbs in vivo. most recently, nerve-dependent expression of the transcription factor sp has been identified as an early marker of dedifferentiation of the wound epithelium and the initiation of limb regeneration. while considerable progress has been made in investigating the functions of candidate regulatory factors and signaling pathways, a broader systems-level perspective is needed to understand why multiple aspects of limb regeneration are dependent upon the presence of a nerve. genomic tools are now available that allow global characterization of the regeneration process in salamanders. expressed sequence tag information has facilitated the development of an ambystoma salamander affymetrix microarray platform. this platform and a high-throughput cdna sequencing approach were used in this study to compare transcript abundance among uninjured limbs, regenerating limbs, and limbs denervated at the time of amputation. the results show that innervated and denervated limbs exhibited similar gene expression patterns at five days post amputation but then diverged as a blastema formed under the influence of nerves. the results are discussed within the context of previous studies of nerve dependency, highlighting specific genes and biological processes that are associated with blastema formation and outgrowth, and more generally, the mexican axolotl's unparalleled ability to regenerate limbs.  morphology and histology of denervated and innervated limbs histological staining verified our experimental procedures for creating innervated and denervated limbs on the same individual. previous studies found few morphological or histological differences between innervated and denervated limbs during the first few days of regeneration. consistent with these observations, denervated and innervated limbs at dpa were histologically indistinguishable. histological staining showed hemorrhaging directly beneath the epithelium containing leydig cells, squamous cells, and basal keratinocytes. all dpa limbs resembled the wound-healing phase or early phase of dedifferentiation according to the staging of. a blastema was visible on innervated limbs dpa and histological staining showed an accumulation of blastemal cells under the wound epithelium. these structures were not observed in denervated limbs at dpa. these results indicate that blastema formation and outgrowth only occurred in nr limbs. an apical thickening of the epithelium characterized the distal end of nr limbs and this layer consisted of keratinocytes and leydig cells. these histological traits indicate that dpa limbs in this experiment corresponded to the early to mid bud stage of limb regeneration. immunological staining using rt- was performed to detect the presence or absence of nerve axons at and dpa. at both time-points, neurofilament staining was positive in the nr limbs, but negative in dl limbs. in pilot experiments, we determined that > days is required for nerves to re-innervate limbs after denervation surgery. thus, dl and nr limbs were created successfully and histology showed that and dpa time points correspond to wound-healing and early to mid bud phases of regeneration, respectively. transcription during normal limb regeneration: deviations of nr limbs from baseline at both and dpa, mrna levels for hundreds of genes were significantly different from baseline levels of genes expressed in whole limbs at day. many of the same genes were identified as significant in nr and nr limbs; the deviation from baseline was in the same direction for all but one of these genes. four matrix metalloproteinases that are known to function during wound healing in many organisms were upregulated at both and dpa, while collagens and muscle-specific genes were downregulated. upregulation of collagen catabolism genes coupled with downregulation of collagen structural genes suggests that transcriptional activation and repression are integrated to efficiently remodel the extracellular environment of damaged tissues. downregulation of muscle genes at both time points suggests that the differentiated muscle gene expression phenotype changes by dpa, and changes more dramatically by dpa. significant genes were identified by microarray analysis. gene ontology terms are represented that were sampled more often than expected in nr nr dl and dl limbs. p-value is calculated by david. although many genes exhibited similar deviations from baseline at and dpa, unique gene expression changes were identified at each time point. the unique nr genes were associated with gene ontology terms that implicate extracellular protein changes and signal transduction pathways of the early wounding response. these terms included response to stimulus, signal transduction, extracellular region, and ion transport. the unique nr genes were associated with go terms that implicate cell division and dna metabolism including ccnd ccnb rrm rrm nasp, rrc and cdc the unique gene expression changes that were identified at and dpa support the idea of temporal progression from an early wound-healing phase to a blastema outgrowth phase during normal limb regeneration. transcription within denervated limbs: deviations of dl limbs from baseline as was observed in nr limbs, hundreds of genes were identified as significant when comparing dl and dl mrna levels with baseline levels measured at day. some of the same or similar go terms that were associated with nr limbs were identified as significantly enriched in dl limbs. this was not unexpected because both dl and nr limbs undergo tissue histolysis at the limb stump and carry out an early wound-healing response. for example, extracellular region and mmp genes were upregulated at and dpa, as was seen in nr limbs. as in nr limbs, muscle contraction, cytoplasmic, and collagen genes were downregulated in both dl and dl limbs. moreover, 83% of the genes that were identified as significant in nr limbs were also significant in dl limbs. these results indicate that many transcriptional events are nerve-independent during early regeneration. denervated limbs are known to cease growth following limb amputation. several groups of genes may explain this observation including the downregulation of genes associated with the m phase of cell division, mitochondrial transcripts, and genes associated with glucose metabolism. furthermore, dl limbs were morphologically similar to dl limbs because denervation prevented blastema formation. consistent with this observation, many of the genes that were identified as upregulated in dl limbs were also identified as significant in dl limbs. these genes are associated with the following go terms: lysosome, response to stress, hydrolyse activity, signal transducer activity, and ion transport. overall, these terms suggest protraction and expansion of wound-healing responses in dl limbs, as well as changes in cellular metabolism and cell division. transcript abundance differences between nr and dl limbs in the preceding two sections, transcriptional patterns of nr and dl limbs were described relative to baseline levels at day here, we report genes found to be significant when comparing nr and dl limbs. in general, few significant changes in gene expression were observed when nr and dl transcripts were compared directly. transcripts for genes were more abundant in nr limbs and were more abundant in dl limbs, and these differences were small in terms of fold-level change. eighteen of these genes exhibited significant sequence similarity to human or salamander presumptive gene sequences; the others are unknown. the genes with significantly more transcripts in nr limbs are associated with intracellular and extracellular functions, including membrane proteins and collagen binding. the genes with significantly more transcripts in dl limbs are associated with extracellular functions or the immune response. all but one of the significant dpa genes showed the same directional deviation at dpa; myl1was -fold higher in the nr limb at dpa, but -fold lower at dpa. thus, as was observed when comparing dl and nr mrna abundances with baseline levels, relatively few gene expression differences were identified between nr and dl limbs at dpa, and the magnitude of these differences was small. these results further support the idea that transcription during limb regeneration is predominantly nerve-independent at dpa. whereas only transcriptional differences were observed between morphologically similar dl and nr limbs at dpa, differences were detected at dpa. k-means cluster analysis of these genes with significant human protein hits highlighted three clusters wherein genes exhibited similar patterns of expression. genes in cluster presented expression patterns with transcript abundances above baseline in nr limbs and abundances below baseline levels in dl limbs. genes associated with cell cycling, a well-established characteristic of blastemal cells, were highly enriched in cluster. over 50% of the genes in cluster are predicted to localize to the nucleus, including several transcriptional regulators. thus, many of the genes in cluster have functions that are consistent with blastema formation and outgrowth in nr limbs. genes from clusters and were generally expressed in the same direction between nr and dl limbs; however, the magnitude of expression differed. genes in cluster presented transcript abundances that generally exceeded baseline levels, with higher levels observed in dl limbs. this pattern suggests that most of these genes were activated in the same direction in the presence or absence of nerves, but denervation caused higher transcript abundances. twenty-two percent of the genes in cluster are associated with the go term cellular response to stimulus and a significant proportion localized to the lysosome, including lgmn, ctsk, ctss, asah, atp6v0d and neu genes in cluster presented transcript abundances that were generally lower than baseline levels, with much lower levels observed in nr limbs. with relatively few genes in this cluster, no biological process was identified as significantly enriched. however, inspection of the genes in cluster again supports the idea that some genes may function in muscle contraction and tissue repair. in summary, mrna levels for genes from clusters and were quantitatively affected by the presence or absence of a nerve. cdna sequence analysis of nerve dependency to further explore nerve dependency and generate an unbiased collection of molecular probes for regenerating limbs, we sequenced cdnas derived from the same rna samples that were used in the microarray analysis. over × reads were generated and this yielded approximately to high-quality sequence reads for each limb treatment, with an average of base pairs in length. more than half of the sequence reads correspond to mitochondrial transcripts and ribosomal rna. this frequency of mtdna transcripts approximates the number sampled in an earlier est screen of the ambystoma genome. the number of rrna transcripts was higher than expected. assembly of all high-quality cdna reads yielded unique sequences. these sequences were assembled with previous est contigs to produce contigs each containing at least two overlapping sequences. the distribution of contig lengths is shown in figure all contigs and singletons were searched against ncbi databases to identify significant similarity matches that would suggest presumptive gene identities. ambystoma contigs and singletons yielded significant hits to sequences in the human refseq database, including unique human genes. figure shows the distribution of percent coverage to predicted human refseq proteins. interestingly, ambystoma queries that did not show significant amino acid sequence identity to a human reference sequence did show significant nucleotide identity to a xenopus sequence. assembly of new cdna sequences with existing est contigs from the ambystoma est database more than doubled the number of non-redundant human-a. mexicanum orthologous sequences. this increase in sequence content was even among go functional categories that are relevant to salamander wound healing and regeneration. assuming that many of the anonymous contigs and singletons that were generated correspond to functional genes, significantly more than different transcripts are expressed during the first two weeks of axolotl limb regeneration. the table shows the number of ambystoma estdb contigs with gene ontology annotations before and after dna sequencing. the total is less than the sum of each category because several genes may belong to multiple categories. bp = biological process. mf = molecular function. mrna abundance estimates and gene discovery from cdna sequence data the number of times that a non-redundant transcript was sampled by cdna sequencing was used to estimate mrna abundances. the transcript counts for ambystoma est contigs differed significantly among limb cdna pools that were created for each of the limb types. it was possible to assign a putative ortholog to of these genes; the remaining genes were considered anonymous. this final list of genes was compared with the significant gene lists from the microarray analysis. it was determined that for of the significant genes from the cdna sequencing analysis, a portion of the gene sequence was represented by a probe set on the ambystoma genechip. of these, genes were identified as significant by both methodologies and mrna abundances for these genes were highly positively correlated. the genes found to be significant by cdna sequencing, but not by microarray analysis, were mostly characterized by low fold changes from baseline; or conversely, registered low hybridization intensities in the microarray analysis. gene functions that were identified as significantly enriched by microarray analysis were also identified as significant by cdna sequencing. for example, the muscle contraction go term was identified as highly enriched and the underlying genes were similarly downregulated relative to baseline. also, genes sampled most often from nr limbs were associated with dna metabolism, a biological process associated with cell cycling, and transcripts for genes associated with cell proliferation and cell cycle progression were either sampled more often among nr limbs or were only sampled from nr limbs. thus, cdna sequencing also identified genes in nr limbs that are consistent with blastema formation. several functional terms that were not identified by microarray analysis were identified as enriched by cdna sequencing. transcripts for genes associated with macromolecule metabolism were most abundant in nr limbs compared with other limbs. transcripts for genes associated with macromolecule catabolism were most abundant in dl limbs, including mmp mmp3/10a, mmp3/10b, mmp and mmp this suggests that the presence or absence of nerves differentially affects transcriptional responses and regulation at dpa. also, cdna sequencing identified additional psca-like genes that were not represented on the genechip, and these were also differentially expressed between nr and dl limbs. these and other examples suggest that cdna sequencing complemented the microarray analysis by providing deeper sampling of transcriptional programs and associated biological processes. this revealed more candidate nerve-dependent gene expression changes at the earlier dpa time point than was revealed by microarray analysis. gene ontology analysis of genes with presumptive human orthologs identified by dna sequencing. only the most significant gene ontology category was selected for the table. in addition to providing estimates of mrna abundance, cdna sequencing also discovered new gene sequences for the mexican axolotl. these include genes that are known to affect developmental processes in other vertebrate models: notch, nrg bmp wnt ctnna btnna dkk axin nrg fgf sirt, stats, sema4f, tf, sfrp, rara, rarg, rxr, pdgf, acvri, bmpri, bmprii, smad, efna ntn slit slit robo and robo most of these genes were sampled less than times and thus appear to be expressed at low levels. other developmental genes that have not been previously associated with limb regeneration were sampled many more times than these candidates, and for the following examples, counts varied significantly among limb cdna libraries. these include mdk, flii, tagln ddx umod, and cnot. also, numerous retroelement-associated sequences were sampled differentially between dl and nr limbs. overall, the sequencing approach verified the primary results from the microarray analysis and identified many new candidate genes and functional pathways that are associated with limb regeneration. genes identified as differentially expressed by sequencing analysis that have high sequence similarity to retrovirus genes. discussion microarray analysis and cdna sequencing were used to identify nerve-dependent and independent gene expression changes during limb regeneration in the mexican axolotl. the results show that limb regeneration is associated with thousands of transcriptional changes. considerable similarity was observed between the dl and nr transcriptional programs at and dpa. for example, genes that are critical to wound healing were upregulated in both limb types while genes that are associated with muscle structure and function were downregulated. many of the transcriptional changes that were observed at dpa were also observed at dpa. thus, many aspects of early limb regeneration are accomplished in the absence of nerves. however, gene expression differences were identified between dl and nr limbs at and dpa. many of the transcriptional differences correlated with blastema formation; cell numbers increased in nr limbs after dpa and this yielded a distinct transcriptional signature of cell proliferation in nr limbs. overall, this study identified genes that are associated with wound healing, early events of blastema formation, and subsequent blastema cell proliferation and outgrowth. below, we discuss and expand upon these primary results and highlight genes whose functions appear to be important for understanding the basis of nerve dependency and limb regeneration. fold-level change of upregulated genes that have functions associated with a wounding response. early wound-healing response during limb regeneration previous studies have documented anatomical similarities between innervated and denervated limbs at early stages of regeneration. this study shows that there are also many transcriptional similarities. this suggests that many aspects of the early wound-healing phase of limb regeneration are not dependent upon post-amputation, nerve-derived factors. instead, humoral immune and local tissue responses appear to be key. many genes that are associated with wound-healing and tissue-repair activities including stress, inflammation, cell survival, immunity, and extracellular matrix remodeling were upregulated from baseline in dpa limbs. it is probably no coincidence that essentially all of the early stress-associated genes that were previously identified as significantly regulated during early spinal cord regeneration, and during the innate immune response of axolotls to a deadly viral pathogen, were also identified as significant in this study. many of these genes appear to be expressed similarly in all vertebrates in response to stress, including junb, irf hmox apoe, mmps, ptx gal gadd45g, and tgfb. additionally, significantly more'extracellular' genes were identified at dpa than expected by chance, including genes that code for matrix remodeling proteins and secreted molecules whose functions are associated with growth factor binding, cell signaling, survival, death, adhesion, migration, and proliferation. the early wound-healing response initiates local environmental changes of the injury site that are pivotal to subsequent phases of regeneration. the dpa time point was chosen in this study to identify critical nerve-dependent signaling events that are stimulated within the first few days of regeneration. comparison of dl and nr data revealed few overall gene expression differences. the dpa time point captured many transcriptional responses that are induced by injury and/or amputation but relatively few that are associated with known or suspected neurotrophic signaling pathways. more comprehensive sampling and deeper sequencing is needed to detail early nerve-dependent transcriptional responses because several genes that are known to be nerve-responsive during the wound-healing phase were not identified in this study. downregulation of genes associated with differentiated muscle this study documented dramatic decreases in the relative abundance of mrnas coding for skeletal muscle contractile proteins, including myosins, actins, actinins, titin, tropomyosins, and troponins. many of these changes were also detected by cdna sequence analysis. this strong, muscle-specific transcriptional signature was observed because approximately half of uninjured forelimb nuclei in to cm axolotls, and likely more than half the cross-sectional area, derive from muscle. it is unlikely that the downregulation of muscle genes is due to retraction of the muscle towards the shoulder because muscle transcripts are much more downregulated at dpa than dpa in both denervated and innervated limbs. considering that limb tissue samples in this study included ~ mm of undamaged tissue proximal to the amputation plane, the results suggest that injury to skeletal muscle induces tissue-wide loss of muscle contractile transcripts. it is possible that the decrease in muscle transcripts is due to muscle wasting, caused by a lack of mechanical stress. it is interesting to speculate that this response maybe associated with the degeneration or cellularization of multinucleated muscle fibers into mononucleated cells, which occurs in both denervated and innervated amputated limbs. it is interesting to note that muscle-specific genes, including actc actn atp2a my1pf, tncc, tnni tnni are downregulated during early stages of mammalian skeletal muscle regeneration, and this is accomplished without blastema formation. skeletal muscle regeneration in mammals and other vertebrates involves resident stem cells. it is possible that muscle-specific genes are downregulated as an integral step of a conserved, skeletal muscle regeneration program of vertebrates. candidate transcriptional repressor genes were identified in this study including id, tardbp, cnot, msx and msx it will be important in future studies to determine if the dramatic decrease in muscle transcripts is due to activation of muscle stem cells, muscle loss, muscle dysfunction/wasting, or whether these transcriptional patterns have an active role in regeneration. genes associated with epigenetic reprogramming and genomic stability blastema formation requires a large number of progenitor cells derived from quiescent stem cells or differentiated cell types. it is generally known that reprogramming of differentiated cells is accompanied by epigenetic changes such as histone and dna modifications. few candidates have been identified previously as bringing about epigenetic changes necessary for cellular reprogramming during regeneration. this study identified several genes whose functions are associated with epigenetic phenomena, including chromatin remodeling, dna methylation, and transcriptional regulation. these include uhrf, lmnb parp, thymopoietin, and a gene with high sequence identity to sam-dependent methyltransferases. after cellular reprogramming and during limb outgrowth, blastemal cells undergo tremendous cell proliferation. during blastema cell proliferation, telomere lengths and overall genome stability must be maintained to prevent cell death. this study identified several candidate genes from nr limbs that are known to function in genome stability, telomere homeostasis, and dna repair. these include parp, hmgb, fen, aurka, aurkb, and pif a dna helicase that maintains genome stability and binds to telomerase from yeast to humans. it is also important to note that many transcripts were identified from nr and dl limbs that code for retroelement components. retrotransposons are normally transcriptionally silenced in differentiated somatic cells by epigenetic mechanisms, but become active upon changes in epigenetic status; these may also regulate nearby gene expression. it is unclear if upregulation of retroelement transcripts affects genome stability and/or is necessary for regeneration. genes associated with nerve-dependent blastema outgrowth in this study, blastemas were not observed in nr limbs or dl limbs, and only formed on nr limbs. nerve-dependent limb outgrowth occurs as a result of blastemal cell proliferation. two early cell proliferation biomarkers, umod and psca, were identified as significantly different between nr and dl limbs. umod probably locates to the wound epithelium as it is upregulated in apical skin cells during thyroid hormone-induced metamorphosis of axolotl epidermis. it is possible that psca is a membrane receptor of blastema cells because it shows structural similarity to prod a surface protein that is implicated in proximal-distal positional identity of blastemal cells during newt limb regeneration. most of the cell proliferation biomarkers were identified at dpa, when a blastema was present in nr limbs but absent in dl limbs. thus, between and dpa, blastemal cells underwent considerable cell proliferation in the presence of nerves. because the limb blastema continues to expand after dpa, the blastema-specific genes that were identified in this study are probably transcribed at much higher levels at later time points. a clear signature of cell proliferation, including genes that function in the cell cycle, mitosis, and nucleotide synthesis, was observed in nr limbs. in contrast, these genes were slightly downregulated in denervated limbs at this time. thus, transcripts associated with cell proliferation are maintained at steady state in nr and dl limbs for at least five days. this early, nerve-independent portion of the limb regeneration program may allow time for re-innervation of the injury site and production of nerve-derived molecules in sufficient quantity to initiate and sustain blastemal cell proliferation. multiple gene products have been hypothesized to be neurotrophic factors provided by nerves to sustain blastema cell proliferation. these include growth-promoting factors such as fibroblast growth factors, substance p, neuregulin, and transferrin. transferrin and neuregulin were sampled by sequencing, but were not identified as differentially expressed by our analysis. fgf and fgf were screened out of the microarray analysis due to low expression, but post-hoc analysis suggested that both are upregulated in nr limbs compared to control and dl limbs. expression of these molecules is known to be nerve-dependent in blastemas of xenopus and axolotls. other molecules that have previously been associated with the blastema during limb regeneration were not included in our statistical analyses due to low hybridization intensity, but were later found to be differentially expressed in nr limbs, including hoxd hoxa hoxa and msx. recently, kumar et al identified a growth-promoting extracellular ligand that rescues aspects of nerve dependency of limb regeneration in newts. probesets for nag are represented on the ambystoma genechip and transcripts for this gene were sampled by cdna sequencing. nag mrna was transcribed at a high level in all tissues, but did not differ significantly between nr, dl, or control limbs. it is possible that the effects of nag and other neurotrophic candidates are associated with quantitative variation of mrna transcript abundances over fine temporal and spatial scales. such variation would have been missed in this analysis of three time points and sampling of mrnas from heterogeneous tissues. furthermore, it is possible that the increase in nag immunoreactivity observed by kumar et al is regulated at the level of translation and would not be observed using microarray or sequencing approaches. overall, we found that several, but not all, genes previously shown to be directly downstream of neurotrophic factors are expressed at higher mrna levels in the blastema versus control and denervated samples. singer emphasized that the neurotrophic factor underlying nerve dependency was growth promoting and quantitative in effect. he showed that the trophic effect of nerves could be titred by surgically manipulating the number of nerves innervating the limb. other lines of research showed that nerve-derived factors were not necessary for traversing cell cycle checkpoints, as an equivalent number of presumptive blastema cells are initially observed to enter s phase in denervated and innervated limbs. instead, the neurotrophic factor appears to be necessary to complete the cell cycle and this may be non-trivial considering the cost of replicating a large salamander genome. these studies suggest that nerves either directly or indirectly provide limiting macromolecules that are needed to accomplish cell division. according to this reasoning and assuming a correlation between transcript and protein abundance for specific mrna species, transcripts associated with nerve dependency would be expected to exceed baseline levels in nr limbs during regeneration, and decrease in abundance in dl limbs. this study identified many human-axolotl presumptive orthologs and anonymous axolotl transcripts that showed this pattern. for example, psat codes for a protein that regulates the second enzymatic step of the phosphorylated pathway in mammals, which produces l-serine. psat expression is high among cell types with high rates of proliferation, including cancer cell lines. strikingly, psat registered the largest mrna abundance difference between nr and dl limbs among probesets on the ambystoma genechip, and this result was verified by both cdna sequencing and real-time pcr. we also found that the third enzyme in the phosphorylated pathway, psph, was -fold higher in nr versus dl by real-time pcr. psat and psph may have neurotrophic potential but more likely function to provide proliferating cells with a limiting and conditionally important substance that is required for synthesis of macromolecules, amino acids, and purines that are needed to accomplish mitosis. the fact that macromolecular synthesis and cell proliferation are both depressed in denervated limbs supports the idea that psat activity is associated with nerve-derived factors that contribute to blastema outgrowth. comparison of microarray and cdna sequencing approaches microarray analysis and cdna sequencing offer different advantages for dissecting complex biological processes. the strength of microarray analysis is the precision that this approach provides for estimating transcript abundances for a specific panel of genes. such gene panels provide standardized markers for identifying shared and unique patterns of transcription among experiments. in turn, identification of such patterns provides systems-level insight. for example and as was discussed above, cross-experiment comparisons of axolotl transcription helped distinguish general stress responses from local regenerative responses. a relatively conservative fold level threshold was used in this study to identify significant genes in the microarray analysis and lowly expressed genes were removed from consideration. these conservative approaches potentially exclude important genes but likely discover'real' transcriptional differences between samples. a lower threshold could be applied to extract more information from the dataset and future studies would likely benefit by increasing replication of genechips to detect significant differences for lowly expressed genes. with respect to cdna sequencing, two goals were accomplished: gene discovery and estimation of transcript abundance. however, accomplishment of both goals required a trade-off in the allocation of resources toward deep sequencing versus replication. sequencing resources were used to deeply sequence day nr, and dl cdna libraries instead of shallowly sequencing replicate libraries for these time points. this strategy identified thousands of new gene sequences that will greatly enrich future regeneration studies. future studies will benefit from experimental designs that replicate deep sequencing of cdna libraries and this will be possible as sequencing costs decrease. still, it was encouraging to find in this study that 38% of genes that were identified as differentially expressed by cdna sequencing and found on the microarray genechip were identified as significant by both of these technologies, and transcript abundances for these genes were highly positively correlated.  this study addressed the nerve dependency of limb regeneration by characterizing downstream cellular events that are affected when an intact nerve supply is removed from an amputated salamander limb. microarray analysis showed that the early wound response is largely nerve-independent, but transcriptional profiles diverge between denervated and innervated limbs when the innervated limb starts to regenerate. pyrosequencing supported these microarray results while substantially increasing sequence information from the salamander transcriptome. this study shows the utility of next-generation sequencing platforms for gaining transcriptome information. this new dna sequence information will greatly enrich future regeneration studies using the axolotl. 
considerable similarity was observed between nr and dl transcriptional programs at and days post amputation. cdna sequencing complemented the microarray analysis by providing deeper sampling of transcriptional programs and associated biological processes. the majority of the transcriptional differences between nr and dl limbs were correlated with blastema formation; cell numbers increased in nr limbs after dpa and this yielded distinct transcriptional signatures of cell proliferation in nr limbs at dpa. these transcriptional signatures were not observed in dl limbs. instead, gene expression changes within dl limbs suggest more diverse and protracted wound-healing responses. microarray analysis and cdna sequencing were used to investigate a centuries-old problem in regenerative biology: the basis of nerve-dependent limb regeneration in salamanders. thus, many processes that are regulated during early limb regeneration do not depend upon nerve-derived factors. many new candidate gene sequences were discovered for the first time and these will greatly enable future studies of wound healing, epigenetics, genome stability, and nerve-dependent blastema formation and outgrowth using the axolotl model. assembly of new cdna sequences with existing expressed sequence tag contigs from the ambystoma est database more than doubled the number of non-redundant human-a. genes with extracellular functions that are critical to wound healing were upregulated while muscle-specific genes were downregulated.
 microarray analysis and cdna sequencing were used to investigate a centuries-old problem in regenerative biology: the basis of nerve-dependent limb regeneration in salamanders. innervated and denervated forelimbs of mexican axolotls were amputated and transcripts were sampled after and days of regeneration.  considerable similarity was observed between nr and dl transcriptional programs at and days post amputation. genes with extracellular functions that are critical to wound healing were upregulated while muscle-specific genes were downregulated. thus, many processes that are regulated during early limb regeneration do not depend upon nerve-derived factors. the majority of the transcriptional differences between nr and dl limbs were correlated with blastema formation; cell numbers increased in nr limbs after dpa and this yielded distinct transcriptional signatures of cell proliferation in nr limbs at dpa. these transcriptional signatures were not observed in dl limbs. instead, gene expression changes within dl limbs suggest more diverse and protracted wound-healing responses. cdna sequencing complemented the microarray analysis by providing deeper sampling of transcriptional programs and associated biological processes. assembly of new cdna sequences with existing expressed sequence tag contigs from the ambystoma est database more than doubled the number of non-redundant human-a. mexicanum orthologous sequences.  many new candidate gene sequences were discovered for the first time and these will greatly enable future studies of wound healing, epigenetics, genome stability, and nerve-dependent blastema formation and outgrowth using the axolotl model. 