BACKGROUND
among amniotes, while mammals and birds display only limited capacity for regeneration in the adult, lizards retain the ability to regrow their tails, including the formation of multiple tissues such as spinal cord, skeletal muscle, vasculature, cartilage, and skin, throughout their lives .
transcriptomic analysis of the green anole lizard, a. carolinensis, regenerating tail revealed differential expression of genes involved in wound response, hormonal response, and musculoskeletal development as well as the wnt and mapk/fgf pathways  <cit> .
this study and others have demonstrated that the regenerating tail is not a recapitulation of development but a different structure with the same function  <cit> .
while many orthologous genes can be identified between the genomes of the green anole and mammals such as mouse and human  <cit> , a key question about the evolution of regeneration in vertebrates focuses on what genetic changes are responsible for lizards retaining their regenerative capacity and mammals and birds losing this ability.
changes in the coding or cis-regulatory sequences of multiple individual genes could account for the differential capacity for regeneration within vertebrates.
however, given the large number of genes regulating this process, regulators of multiple genes may be involved.
micrornas can modulate the expression levels of large numbers of genes, and divergent microrna regulation could contribute to differences in regeneration between reptilian and mammalian vertebrates.
micrornas are highly conserved across metazoa  <cit>  and play critical roles in regulating a variety of biological processes, including proliferation and differentiation of neurons as well as cardiac and skeletal muscle tissue during development  <cit> , hematopoietic and embryonic stem cell differentiation  <cit> , and t-cell development, maturation, differentiation, and activation  <cit> .
micrornas also play a key role in regulating muscle development and repair, which has been extensively studied in mouse and other model systems  <cit> .
the role of microrna regulation in adult regeneration is an active area of research in vertebrate models.
the expression of micrornas during development and regeneration has been investigated in amphibians  and in teleosts such as the zebrafish.
in the axolotl, micrornas regulate limb and tail regeneration .
in the newt, distinct sets of micrornas, specifically the let- <dig> family, are expressed during lens and inner ear hair cell regeneration  <cit> .
in zebrafish, micrornas play an important role in heart, spinal cord, and caudal tail fin regeneration .
micrornas from whole animal for the green anole lizard have been reported  <cit> , but no studies have been carried out to identify micrornas in tail regeneration of any lizard species.
to investigate the role of micrornas in lizard regeneration, we performed deep sequencing of rna smaller than 100 bp.
we targeted our analysis on micrornas from two distinct regenerating tail tissues, the growing tip and base, which yielded differentially expressed transcripts on total rna transcriptomic analysis  <cit> .
microrna profiles from adult brain and skeletal muscle were assayed to help in annotation of small rnas.
from this sequencing data and subsequent microrna annotation, we identified differentially expressed micrornas between the growing tip and base of the regenerating tail that may play important roles in regulating stem cell proliferation and differentiation during regeneration.
furthermore, we predicted the mrna targets of lizard micrornas and correlated their expression with mrna expression identified in a previous study  <cit> .
this study advances our understanding of which post-transcriptional regulators may regulate regenerative capacity in the lizard.
RESULTS
identification of micrornas in the regenerating lizard tail
during tail regeneration in the green anole lizard, there is rapid outgrowth at 25 days post autotomy .
we collected nine regenerating tails at this  <dig> dpa stage and dissected and pooled tissue from the tip and base to obtain sufficient rna for sequencing  .
these regenerating tail tissues and stages corresponded to our previous rna-seq gene expression analysis, permitting comparison of microrna and mrna levels  <cit> .
the  <dig> differentially expressed genes identified in our previous study clustered into two groups characterized by elevated gene expression in the regenerating tail tip or base.
therefore, we sought to identify micrornas in these tissues that could regulate the regenerative process.
to aid in annotating putative novel micrornas and confirm the presence of previously identified micrornas in the green anole, we sequenced micrornas in adult skeletal muscle and brain, which represent component tissues of the regenerating tail .fig.
<dig> experimental design of microrna analysis of lizard tail regeneration.
a: image of a green anole lizard with a fully regenerated tail .
b: a  <dig> dpa regenerating tail was divided into three equally sized segments, with the distal regenerating tip and proximal regenerating base collected for microrna sequencing .
for qrt-pcr analysis, five equally sized segments were collected .
c: venn diagram showing the distribution of micrornas expressed in the brain, skeletal muscle, and  <dig> dpa regenerating tail tip and base
annotation was carried out using mirdeep <dig>  <cit> , a tool designed to identify known and putative novel micrornas from small-rna sequencing together with the mirbase database of published micrornas  <cit> .
mirdeep <dig> takes into account predicted microrna secondary structure and uses the expression of 5p and 3p mature sequences in order to assign a score to each novel microrna precursor.
our mirdeep <dig> analysis identified a total of  <dig> precursor microrna families using a mirdeep <dig> score of  <dig>  for putative novel micrornas .
this compares to  <dig> microrna anole precursors already identified in mirbase  <cit> .
of the  <dig> precursor putative micrornas that we identified from regenerating tail, brain, and skeletal muscle,  <dig> of these precursors were also present in mirbase .
the remaining  <dig> putative microrna precursors were identified by mirdeep <dig> as potentially novel.
of these,  <dig> are most likely orthologs of micrornas found in other systems, displaying either 100 % seed identity or a reciprocal blast hit to vertebrate microrna precursors found in mirbase.
this left  <dig> putative microrna precursors with no currently known ortholog based on sequence alone   <cit> .
analysis of synteny conservation of these microrna precursors did not identify any clear orthologs in the mouse or human based on genomic location.
tissue-specific patterns of microrna gene expression
altogether,  <dig> micrornas are uniquely expressed in the regenerating tail base compared to only three anole micrornas identified in the regenerating tail tip .
most highly expressed micrornas in regenerating tissue are expressed in both the tip and the base of the regenerating tail .
while most micrornas are shared amongst tissues, the brain displayed the largest number of unique micrornas .
<dig> micrornas were expressed in brain,  <dig> are expressed in skeletal muscle, and  <dig> were expressed in regenerating tail tissue.
highly expressed micrornas in the brain include a number of regulators of neuronal development and differentiation such as mir-124a, mir-124b, mir- <dig>  and mir- <dig>  .
mir-124a, mir- <dig>  and mir-181a specifically are some of the most abundant micrornas expressed in the vertebrate central nervous system .
highly expressed micrornas in the skeletal muscle include the muscle specific micrornas, or myomirs, mir- <dig> and mir-133a  <cit> , along with mir- <dig>  mir-125b, and mir- <dig> all of which are involved in myogenesis and skeletal muscle repair  .
having identified the tissue specificity of the identified micrornas, we focused on differential expression within the regenerating tail.table  <dig> highly expressed micrornas in brain, skeletal muscle, and regenerating tail tip and base
differential expression analysis of regenerating tail micrornas and coordinated expression with mrnas
small-rna sequencing of the  <dig> dpa regenerating lizard tail tip and base identified the expression of  <dig> micrornas .
in general, most of the micrornas were highly correlated between these two tissues, with only  <dig> differentially expressed micrornas .
the impact of differential expression of  <dig> micrornas is of course amplified by a larger number of predicted target genes   <cit> .fig.
<dig> differential expression of micrornas in the  <dig> dpa regenerating lizard tail.
a: regression of normalized microrna expression in the regenerating tail tip and base .
each point on the graph represents a microrna.
dashed lines represent the cutoff for two-fold change.
differentially expressed micrornas, i.e., displayed significant expression differences as determined by deseq  are represented in red.
b: heatmap of differentially expressed micrornas.
expression in each replicate is shown.
micrornas were clustered by jensen-shannon divergence of deseq variance stabilization transformed expression data
the differentially expressed micrornas could be clustered into four groups, where many micrornas upregulated in the base share high levels of expression with skeletal muscle .
nine of these micrornas have elevated expression in the tail base, including mir- <dig>  mir-133a, mir-133b, and mir- <dig>  which have been shown to play key roles in regulating skeletal muscle differentiation and function .
in zebrafish, the mir- <dig> precursor family regulates regeneration in the tail fin  <cit> , the heart  <cit> , and spinal cord  <cit> .
in mice, mir- <dig> and mir- <dig> regulate satellite cell proliferation via repression of pax <dig> translation, thereby promoting myotube formation  <cit> .
mir- <dig>  which is differentially expressed in the regenerating tail tip, regulates proliferation and differentiation of neural stem cells  <cit> .
of the  <dig> differentially expressed micrornas, three were putatively novel.
in order to validate and study differential expression of mirnas in different portions of the regenerating tail we have followed expression levels in  <dig> mirnas identified by our sequencing efforts .
we sectioned four regenerated tails into five equal segments, extracted total rna from each segment, and followed mirna level changes between these segments using qrt-pcr .
each mirna was assayed in triplicate for each tail section, totaling  <dig> qrt-pcr reactions.
as shown in fig.
3mir-1a, mir-1b, mir-133a and mir- <dig> show increased expression in the proximal portion of the regenerating tail, while mir-184 and mir- <dig> display an opposite pattern.
importantly, we were able to also detect these positional changes in a small subset of putative novel mirnas  and putative novel anolis-specific mir-133b ortholog , detected by our sequencing results .
these three putative novel mirnas possess hairpin structures indicative of mirnas .
additionally, the sequencing reads preferentially stack onto one arm of the hairpin, and have 1– <dig> nucleotide overhangs, which is characteristic of pre-mirna processing by dicer  <cit>  .
of note, while we detected expression changes in the regenerating tail for 5_ <dig> by small rna-seq and qrt-pcr, this putative novel mirna maps to multiple regions of the anolis genome, making it difficult to determine the exact genomic origin of the transcript.
taken together, this data validates our mirna sequencing efforts, and importantly shows differential localization patterns of several mirnas in the regenerating tail, suggesting that mirnas may play a functional role in this process.fig.
<dig> mirna expression across regenerating tail sections by qrt-pcr.
total rna was extracted from regenerated tail sections from four biological replicates.
cdna was synthesized using primers specific to the mature mirna.
three qrt-pcr replicates were performed for each gene in each tail section .
expression is normalized to ef1a, and quantified using the ∆∆ct method.
a highly conserved mirnas with expression decreasing from the base to the tip.
b highly conserved mirnas with expression increasing from the base to the tip.
c expression of putative novel mirnas across regenerating tail sections
predicted targets of these putative novel micrornas are listed in table  <dig>  a number of genes predicted to be targeted by the three putative novel micrornas are involved in mitosis and cell cycle control, including antagonist of mitotic exit network  <dig> homolog , centrosomal protein 76 kda , jumonji, at rich interactive domain  <dig> , leucine rich repeat containing 4b , origin recognition complex, subunit  <dig> , protein phosphatase  <dig>  regulatory subunit 9b , proteasome macropain 26s subunit, atpase,  <dig> , proteasome macropain inhibitor subunit  <dig> , tousled-like kinase  <dig> , tumor protein translationally-controlled  <dig> , and the microtubule-associated gene tpx <dig>  in addition, a number of genes involved in neurogenesis or synapse formation were targets, including cholinergic receptor, nicotinic, alpha  <dig> , dihydropyrimidinase-like  <dig> , plexin a <dig> , sphingomyelin phosphodiesterase  <dig> neutral membrane , and ef-hand domain family member d <dig> .
finally, wnt pathway members fermitin family member  <dig> , naked cuticle homolog  <dig> , and spalt-like transcription factor  <dig>  were among the predicted targets.
given the cell proliferation and tissue formation taking place within the regenerating tail base, these putative novel micrornas may play a key role in regulating the regenerative process.
we have previously shown that there are at least  <dig> differentially expressed genes in the regenerating lizard tail, including genes in the wnt and fgf/mapk pathways as well as those involved in wound repair, hormonal regulation, and musculoskeletal development  <cit> .
we identified microrna/target mrna pairs that both have at least 2-fold change in expression between the regenerating tail tip and base  and performed david analysis of gene ontology biological processes on the targeted mrna transcripts to identify significant functional terms   <cit> .
of particular interest are coordinated profiles of expression where the microrna changes reinforce the mrna gene expression, i.e., micrornas levels are decreased where the expression of their mrna targets are increased, as these could represent post-transcriptional microrna repression .
additionally, the group of highly expressed mrna genes whose regulatory micrornas are also upregulated in the corresponding tissue are of interest as they could represent translational microrna repression .fig.
<dig> micrornas and their co-expressed mrna targets in the  <dig> dpa regenerating lizard tail.
a–b: a treemap overview of significant  gene ontology biological processes for downregulated micrornas and their upregulated mrna targets in the  <dig> dpa regenerating tail tip  and regenerating tail base .
c–d: a treemap overview of significant  gene ontology biological processes for upregulated micrornas and their upregulated mrna targets in the  <dig> dpa regenerating tail tip  and regenerating tail base .
the relative sizes of the treemap boxes are based on the |log10| of the respective go term.
related terms are visualized with the same color, with the representative category for each color group denoted in the legend
in the regenerating tail tip, genes involved in phosphorus metabolism, phosphorylation, development of tubular structures, cell motility, cell morphogenesis, lipid biosynthesis, and kinase activity are highly expressed.
this would be expected in the regenerating tail tip, where organization of structures with epithelial cell organization such as the vasculature, ependymal, and cartilage tube would require active signal transduction via phosphorylation .
micrornas that reinforce this pattern of expression are all putatively novel.
in the regenerating tail base, genes involved in musculoskeletal development, enzyme catalysis, response to organic substances, muscle contraction, and extracellular matrix organization display increased expression, as might be expected in differentiating skeletal muscle and cartilage present in that tissue .
micrornas that reinforce this pattern of expression included many putative novel micrornas, including two of the differentially expressed putative novel micrornas , as well as let-7b, which is regulates neural stem cell proliferation and is additionally expressed during lens regeneration in the newt  <cit> .
discussion
this study describes the first microrna transcriptome analysis of regeneration in the green anole.
we identified  <dig> microrna precursors from regenerating tail and adult brain and skeletal muscle, with  <dig> microrna precursors orthologous to families in other vertebrate species.
in addition, we have validated the presence of a subset of differentially expressed mirnas detected by our sequencing efforts, including three putative novel mirnas.
given previous analysis finding a distributed pattern of cell proliferation throughout the regenerating green anole tail  <cit> , we did not expect that the tail tip would be enriched for stem cell proliferation or reveal a gradient of differentiation.
the cellular organization at the tail tip differs from the base in being enriched for forming vasculature, growing ependyma, and coalescing cartilage tube.
in addition, we have validated the presence of a subset of mirnas detected by our sequencing efforts, including three putative novel mirnas.
differentially expressed mrnas and micrornas both reflect this enrichment for those tissues in the tail tip.
conversely, the regenerating tail base is enriched for differentiating skeletal muscle groups, and this pattern was observed in mrna and microrna expression.
the finding that three putative novel micrornas are differentially expressed in the regenerating tail is intriguing.
however, these putative novel micrornas likely have homologues in other vertebrates, but the lack of reptilian genomes and micrornas sequenced to date limits our ability to clearly identify orthology.
though microrna target prediction is a useful tool, prediction algorithms often have varying target lists, and their false positive and false negative rates are difficult to assess  <cit> .
while comparing microrna expression with the expression of its mrna target helps resolve and identify microrna/mrna target pairs for further analysis, it would be beneficial to further verify these genes for downstream analyses.
since micrornas are an example of post-transcriptional regulation, the addition of proteomic data would provide a unique insight into verification of microrna targets.
specifically, proteomic data would help assess whether certain micrornas act at a post-transcriptional or translational level; aiding in resolution of identifying micrornas that inhibit translation versus false positives in cases where both a microrna and its targeted mrna transcript are upregulated.
given that most microrna precursors expressed in lizard tail regeneration have orthologs in other vertebrates, comparison with micrornas identified in other regenerative models could be instructive.
for example, the small rna mir- <dig> is downregulated during heart regeneration and in the tip of the regenerating tail in zebrafish  <cit> .
in the anole, we identified high levels of mir-133a in the regenerating tail base compared to the tail tip.
the small rna mir- <dig>  which is differentially expressed in the tip of anole regenerating tail, has also been identified in zebrafish tail fin regeneration  <cit> .
in addition to regulating neural stem cell proliferation and differentiation, mir- <dig> targets the rna-induced silencing complex  member argonaute <dig>  <cit> .
during newt lens regeneration, mir- <dig> and mir- <dig> regulate cell proliferation  <cit> .
orthologs of these two micrornas are both differentially expressed in the regenerating anole tail base.
while previous studies did not identify novel microrna precursors specific to regeneration, we identified  <dig> previously unknown differentially expressed micrornas in the regenerating tail base.
this may reflect the ability of rna-seq to identify novel sequences, while microarray analysis is limited by probe sets included in the arrays.
comparative analysis of the role of micrornas in vertebrate regeneration would be advanced by further deep sequencing of small rna populations in other model systems.
CONCLUSIONS
given that micrornas are able to regulate a large number of genes, it is possible that microrna regulation during the regenerative process can contribute to differences in regenerative capacity among vertebrates.
divergence in vertebrate microrna regulation could arise by a number of possible models including, i.)
the deletion or loss of micrornas regulating regeneration within the mammalian lineage, ii.)
the change in downstream transcripts targeted by micrornas in the mammalian lineage, and iii.)
the emergence of novel reptile-specific micrornas that promote regeneration.
the latter model appears less parsimonious given the conservation of regeneration across vertebrates, including teleosts, amphibians, and amniotes .
in addition to microrna-based regulation, genomic changes may of course affect coding genes and non-coding regulatory sites such as enhancers, silencers, and insulators.
further analysis in the lizard and comparison with other regenerative models will allow us to further distinguish between these possibilities.

 among amniotes, while mammals and birds display only limited capacity for regeneration in the adult, lizards retain the ability to regrow their tails, including the formation of multiple tissues such as spinal cord, skeletal muscle, vasculature, cartilage, and skin, throughout their lives. transcriptomic analysis of the green anole lizard, a. carolinensis, regenerating tail revealed differential expression of genes involved in wound response, hormonal response, and musculoskeletal development as well as the wnt and mapk/fgf pathways. this study and others have demonstrated that the regenerating tail is not a recapitulation of development but a different structure with the same function. while many orthologous genes can be identified between the genomes of the green anole and mammals such as mouse and human, a key question about the evolution of regeneration in vertebrates focuses on what genetic changes are responsible for lizards retaining their regenerative capacity and mammals and birds losing this ability. changes in the coding or cis-regulatory sequences of multiple individual genes could account for the differential capacity for regeneration within vertebrates. however, given the large number of genes regulating this process, regulators of multiple genes may be involved. micrornas can modulate the expression levels of large numbers of genes, and divergent microrna regulation could contribute to differences in regeneration between reptilian and mammalian vertebrates. micrornas are highly conserved across metazoa and play critical roles in regulating a variety of biological processes, including proliferation and differentiation of neurons as well as cardiac and skeletal muscle tissue during development, hematopoietic and embryonic stem cell differentiation, and t-cell development, maturation, differentiation, and activation. micrornas also play a key role in regulating muscle development and repair, which has been extensively studied in mouse and other model systems. the role of microrna regulation in adult regeneration is an active area of research in vertebrate models. the expression of micrornas during development and regeneration has been investigated in amphibians and in teleosts such as the zebrafish. in the axolotl, micrornas regulate limb and tail regeneration. in the newt, distinct sets of micrornas, specifically the let- family, are expressed during lens and inner ear hair cell regeneration. in zebrafish, micrornas play an important role in heart, spinal cord, and caudal tail fin regeneration. micrornas from whole animal for the green anole lizard have been reported, but no studies have been carried out to identify micrornas in tail regeneration of any lizard species. to investigate the role of micrornas in lizard regeneration, we performed deep sequencing of rna smaller than 100 bp. we targeted our analysis on micrornas from two distinct regenerating tail tissues, the growing tip and base, which yielded differentially expressed transcripts on total rna transcriptomic analysis. microrna profiles from adult brain and skeletal muscle were assayed to help in annotation of small rnas. from this sequencing data and subsequent microrna annotation, we identified differentially expressed micrornas between the growing tip and base of the regenerating tail that may play important roles in regulating stem cell proliferation and differentiation during regeneration. furthermore, we predicted the mrna targets of lizard micrornas and correlated their expression with mrna expression identified in a previous study. this study advances our understanding of which post-transcriptional regulators may regulate regenerative capacity in the lizard.  identification of micrornas in the regenerating lizard tail during tail regeneration in the green anole lizard, there is rapid outgrowth at 25 days post autotomy. we collected nine regenerating tails at this dpa stage and dissected and pooled tissue from the tip and base to obtain sufficient rna for sequencing. these regenerating tail tissues and stages corresponded to our previous rna-seq gene expression analysis, permitting comparison of microrna and mrna levels. the differentially expressed genes identified in our previous study clustered into two groups characterized by elevated gene expression in the regenerating tail tip or base. therefore, we sought to identify micrornas in these tissues that could regulate the regenerative process. to aid in annotating putative novel micrornas and confirm the presence of previously identified micrornas in the green anole, we sequenced micrornas in adult skeletal muscle and brain, which represent component tissues of the regenerating tail.fig. experimental design of microrna analysis of lizard tail regeneration. a: image of a green anole lizard with a fully regenerated tail. b: a dpa regenerating tail was divided into three equally sized segments, with the distal regenerating tip and proximal regenerating base collected for microrna sequencing. for qrt-pcr analysis, five equally sized segments were collected. c: venn diagram showing the distribution of micrornas expressed in the brain, skeletal muscle, and dpa regenerating tail tip and base annotation was carried out using mirdeep, a tool designed to identify known and putative novel micrornas from small-rna sequencing together with the mirbase database of published micrornas. mirdeep takes into account predicted microrna secondary structure and uses the expression of 5p and 3p mature sequences in order to assign a score to each novel microrna precursor. our mirdeep analysis identified a total of precursor microrna families using a mirdeep score of for putative novel micrornas. this compares to microrna anole precursors already identified in mirbase. of the precursor putative micrornas that we identified from regenerating tail, brain, and skeletal muscle, of these precursors were also present in mirbase. the remaining putative microrna precursors were identified by mirdeep as potentially novel. of these, are most likely orthologs of micrornas found in other systems, displaying either 100 % seed identity or a reciprocal blast hit to vertebrate microrna precursors found in mirbase. this left putative microrna precursors with no currently known ortholog based on sequence alone. analysis of synteny conservation of these microrna precursors did not identify any clear orthologs in the mouse or human based on genomic location. tissue-specific patterns of microrna gene expression altogether, micrornas are uniquely expressed in the regenerating tail base compared to only three anole micrornas identified in the regenerating tail tip. most highly expressed micrornas in regenerating tissue are expressed in both the tip and the base of the regenerating tail. while most micrornas are shared amongst tissues, the brain displayed the largest number of unique micrornas. micrornas were expressed in brain, are expressed in skeletal muscle, and were expressed in regenerating tail tissue. highly expressed micrornas in the brain include a number of regulators of neuronal development and differentiation such as mir-124a, mir-124b, mir- and mir-. mir-124a, mir- and mir-181a specifically are some of the most abundant micrornas expressed in the vertebrate central nervous system. highly expressed micrornas in the skeletal muscle include the muscle specific micrornas, or myomirs, mir- and mir-133a, along with mir- mir-125b, and mir- all of which are involved in myogenesis and skeletal muscle repair. having identified the tissue specificity of the identified micrornas, we focused on differential expression within the regenerating tail.table highly expressed micrornas in brain, skeletal muscle, and regenerating tail tip and base differential expression analysis of regenerating tail micrornas and coordinated expression with mrnas small-rna sequencing of the dpa regenerating lizard tail tip and base identified the expression of micrornas. in general, most of the micrornas were highly correlated between these two tissues, with only differentially expressed micrornas. the impact of differential expression of micrornas is of course amplified by a larger number of predicted target genes.fig. differential expression of micrornas in the dpa regenerating lizard tail. a: regression of normalized microrna expression in the regenerating tail tip and base. each point on the graph represents a microrna. dashed lines represent the cutoff for two-fold change. differentially expressed micrornas, i.e., displayed significant expression differences as determined by deseq are represented in red. b: heatmap of differentially expressed micrornas. expression in each replicate is shown. micrornas were clustered by jensen-shannon divergence of deseq variance stabilization transformed expression data the differentially expressed micrornas could be clustered into four groups, where many micrornas upregulated in the base share high levels of expression with skeletal muscle. nine of these micrornas have elevated expression in the tail base, including mir- mir-133a, mir-133b, and mir- which have been shown to play key roles in regulating skeletal muscle differentiation and function. in zebrafish, the mir- precursor family regulates regeneration in the tail fin, the heart, and spinal cord. in mice, mir- and mir- regulate satellite cell proliferation via repression of pax translation, thereby promoting myotube formation. mir- which is differentially expressed in the regenerating tail tip, regulates proliferation and differentiation of neural stem cells. of the differentially expressed micrornas, three were putatively novel. in order to validate and study differential expression of mirnas in different portions of the regenerating tail we have followed expression levels in mirnas identified by our sequencing efforts. we sectioned four regenerated tails into five equal segments, extracted total rna from each segment, and followed mirna level changes between these segments using qrt-pcr. each mirna was assayed in triplicate for each tail section, totaling qrt-pcr reactions. as shown in fig. 3mir-1a, mir-1b, mir-133a and mir- show increased expression in the proximal portion of the regenerating tail, while mir-184 and mir- display an opposite pattern. importantly, we were able to also detect these positional changes in a small subset of putative novel mirnas and putative novel anolis-specific mir-133b ortholog, detected by our sequencing results. these three putative novel mirnas possess hairpin structures indicative of mirnas. additionally, the sequencing reads preferentially stack onto one arm of the hairpin, and have 1– nucleotide overhangs, which is characteristic of pre-mirna processing by dicer. of note, while we detected expression changes in the regenerating tail for 5_ by small rna-seq and qrt-pcr, this putative novel mirna maps to multiple regions of the anolis genome, making it difficult to determine the exact genomic origin of the transcript. taken together, this data validates our mirna sequencing efforts, and importantly shows differential localization patterns of several mirnas in the regenerating tail, suggesting that mirnas may play a functional role in this process.fig. mirna expression across regenerating tail sections by qrt-pcr. total rna was extracted from regenerated tail sections from four biological replicates. cdna was synthesized using primers specific to the mature mirna. three qrt-pcr replicates were performed for each gene in each tail section. expression is normalized to ef1a, and quantified using the ∆∆ct method. a highly conserved mirnas with expression decreasing from the base to the tip. b highly conserved mirnas with expression increasing from the base to the tip. c expression of putative novel mirnas across regenerating tail sections predicted targets of these putative novel micrornas are listed in table  a number of genes predicted to be targeted by the three putative novel micrornas are involved in mitosis and cell cycle control, including antagonist of mitotic exit network homolog, centrosomal protein 76 kda, jumonji, at rich interactive domain, leucine rich repeat containing 4b, origin recognition complex, subunit, protein phosphatase regulatory subunit 9b, proteasome macropain 26s subunit, atpase,, proteasome macropain inhibitor subunit, tousled-like kinase, tumor protein translationally-controlled, and the microtubule-associated gene tpx in addition, a number of genes involved in neurogenesis or synapse formation were targets, including cholinergic receptor, nicotinic, alpha, dihydropyrimidinase-like, plexin a, sphingomyelin phosphodiesterase neutral membrane, and ef-hand domain family member d. finally, wnt pathway members fermitin family member, naked cuticle homolog, and spalt-like transcription factor were among the predicted targets. given the cell proliferation and tissue formation taking place within the regenerating tail base, these putative novel micrornas may play a key role in regulating the regenerative process. we have previously shown that there are at least differentially expressed genes in the regenerating lizard tail, including genes in the wnt and fgf/mapk pathways as well as those involved in wound repair, hormonal regulation, and musculoskeletal development. we identified microrna/target mrna pairs that both have at least 2-fold change in expression between the regenerating tail tip and base and performed david analysis of gene ontology biological processes on the targeted mrna transcripts to identify significant functional terms. of particular interest are coordinated profiles of expression where the microrna changes reinforce the mrna gene expression, i.e., micrornas levels are decreased where the expression of their mrna targets are increased, as these could represent post-transcriptional microrna repression. additionally, the group of highly expressed mrna genes whose regulatory micrornas are also upregulated in the corresponding tissue are of interest as they could represent translational microrna repression.fig. micrornas and their co-expressed mrna targets in the dpa regenerating lizard tail. a–b: a treemap overview of significant gene ontology biological processes for downregulated micrornas and their upregulated mrna targets in the dpa regenerating tail tip and regenerating tail base. c–d: a treemap overview of significant gene ontology biological processes for upregulated micrornas and their upregulated mrna targets in the dpa regenerating tail tip and regenerating tail base. the relative sizes of the treemap boxes are based on the |log10| of the respective go term. related terms are visualized with the same color, with the representative category for each color group denoted in the legend in the regenerating tail tip, genes involved in phosphorus metabolism, phosphorylation, development of tubular structures, cell motility, cell morphogenesis, lipid biosynthesis, and kinase activity are highly expressed. this would be expected in the regenerating tail tip, where organization of structures with epithelial cell organization such as the vasculature, ependymal, and cartilage tube would require active signal transduction via phosphorylation. micrornas that reinforce this pattern of expression are all putatively novel. in the regenerating tail base, genes involved in musculoskeletal development, enzyme catalysis, response to organic substances, muscle contraction, and extracellular matrix organization display increased expression, as might be expected in differentiating skeletal muscle and cartilage present in that tissue. micrornas that reinforce this pattern of expression included many putative novel micrornas, including two of the differentially expressed putative novel micrornas, as well as let-7b, which is regulates neural stem cell proliferation and is additionally expressed during lens regeneration in the newt. discussion this study describes the first microrna transcriptome analysis of regeneration in the green anole. we identified microrna precursors from regenerating tail and adult brain and skeletal muscle, with microrna precursors orthologous to families in other vertebrate species. in addition, we have validated the presence of a subset of differentially expressed mirnas detected by our sequencing efforts, including three putative novel mirnas. given previous analysis finding a distributed pattern of cell proliferation throughout the regenerating green anole tail, we did not expect that the tail tip would be enriched for stem cell proliferation or reveal a gradient of differentiation. the cellular organization at the tail tip differs from the base in being enriched for forming vasculature, growing ependyma, and coalescing cartilage tube. in addition, we have validated the presence of a subset of mirnas detected by our sequencing efforts, including three putative novel mirnas. differentially expressed mrnas and micrornas both reflect this enrichment for those tissues in the tail tip. conversely, the regenerating tail base is enriched for differentiating skeletal muscle groups, and this pattern was observed in mrna and microrna expression. the finding that three putative novel micrornas are differentially expressed in the regenerating tail is intriguing. however, these putative novel micrornas likely have homologues in other vertebrates, but the lack of reptilian genomes and micrornas sequenced to date limits our ability to clearly identify orthology. though microrna target prediction is a useful tool, prediction algorithms often have varying target lists, and their false positive and false negative rates are difficult to assess. while comparing microrna expression with the expression of its mrna target helps resolve and identify microrna/mrna target pairs for further analysis, it would be beneficial to further verify these genes for downstream analyses. since micrornas are an example of post-transcriptional regulation, the addition of proteomic data would provide a unique insight into verification of microrna targets. specifically, proteomic data would help assess whether certain micrornas act at a post-transcriptional or translational level; aiding in resolution of identifying micrornas that inhibit translation versus false positives in cases where both a microrna and its targeted mrna transcript are upregulated. given that most microrna precursors expressed in lizard tail regeneration have orthologs in other vertebrates, comparison with micrornas identified in other regenerative models could be instructive. for example, the small rna mir- is downregulated during heart regeneration and in the tip of the regenerating tail in zebrafish. in the anole, we identified high levels of mir-133a in the regenerating tail base compared to the tail tip. the small rna mir- which is differentially expressed in the tip of anole regenerating tail, has also been identified in zebrafish tail fin regeneration. in addition to regulating neural stem cell proliferation and differentiation, mir- targets the rna-induced silencing complex member argonaute. during newt lens regeneration, mir- and mir- regulate cell proliferation. orthologs of these two micrornas are both differentially expressed in the regenerating anole tail base. while previous studies did not identify novel microrna precursors specific to regeneration, we identified previously unknown differentially expressed micrornas in the regenerating tail base. this may reflect the ability of rna-seq to identify novel sequences, while microarray analysis is limited by probe sets included in the arrays. comparative analysis of the role of micrornas in vertebrate regeneration would be advanced by further deep sequencing of small rna populations in other model systems.  given that micrornas are able to regulate a large number of genes, it is possible that microrna regulation during the regenerative process can contribute to differences in regenerative capacity among vertebrates. divergence in vertebrate microrna regulation could arise by a number of possible models including, i.) the deletion or loss of micrornas regulating regeneration within the mammalian lineage, ii.) the change in downstream transcripts targeted by micrornas in the mammalian lineage, and iii.) the emergence of novel reptile-specific micrornas that promote regeneration. the latter model appears less parsimonious given the conservation of regeneration across vertebrates, including teleosts, amphibians, and amniotes. in addition to microrna-based regulation, genomic changes may of course affect coding genes and non-coding regulatory sites such as enhancers, silencers, and insulators. further analysis in the lizard and comparison with other regenerative models will allow us to further distinguish between these possibilities. 
regeneration in lizards involves differential expression of hundreds of genes that regulate wound healing, musculoskeletal development, hormonal response, and embryonic morphogenesis. microrna sequencing of green anole lizard regenerating tail and associated tissues revealed putative novel and known microrna precursors. eleven micrornas were differentially expressed between the regenerating tail tip and base during maximum outgrowth, including mir-133a, mir-133b, and mir- which have been reported to regulate regeneration and stem cell proliferation in other model systems. three putative novel differentially expressed micrornas were identified in the regenerating tail tip. while micrornas are able to regulate large groups of genes, their role in lizard regeneration has not been investigated. differentially expressed micrornas were identified in the regenerating lizard tail, including known regulators of stem cell proliferation.
 lizards are evolutionarily the most closely related vertebrates to humans that can lose and regrow an entire appendage. regeneration in lizards involves differential expression of hundreds of genes that regulate wound healing, musculoskeletal development, hormonal response, and embryonic morphogenesis. while micrornas are able to regulate large groups of genes, their role in lizard regeneration has not been investigated.  microrna sequencing of green anole lizard regenerating tail and associated tissues revealed putative novel and known microrna precursors. eleven micrornas were differentially expressed between the regenerating tail tip and base during maximum outgrowth, including mir-133a, mir-133b, and mir- which have been reported to regulate regeneration and stem cell proliferation in other model systems. three putative novel differentially expressed micrornas were identified in the regenerating tail tip.  differentially expressed micrornas were identified in the regenerating lizard tail, including known regulators of stem cell proliferation. the identification of putative novel micrornas suggests that regulatory networks, either conserved in vertebrates and previously uncharacterized or specific to lizards, are involved in regeneration. these findings suggest that differential regulation of micrornas may play a role in coordinating the timing and expression of hundreds of genes involved in regeneration. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords micrornaregenerationtranscriptomelizardreptilegene expressiontailmusclebrainhttp://dx.doi.org/ /100000069national institute of arthritis and musculoskeletal and skin diseasesr ar064935kusumi kenro http://dx.doi.org/ /100000097national center for research resourcesr rr031305kusumi kenro http://dx.doi.org/ /100008335arizona biomedical research commission1113kusumi kenro issue-copyright-statement© the author 2016 