BACKGROUND
the regenerative capacity of the cns in adult mammals is limited, with minimal axonal re-growth, death of damaged neurons and long-term loss of function  <cit> .
by contrast, fish have the remarkable ability to repair most functional components of the cns  <cit>  making them a valuable model in which to identify key molecular mechanisms involved in neural regeneration .
anamniotes  and amniotes  have a high degree of conservation at the level of both nucleotide sequence and amino acid functional domains, resulting in similar molecular and cellular processes in neural development and function of both clades.
therefore, it is possible that genes required for successful cns regeneration are present in both fish and mammals, but that key regulatory differences in the expression of these genes underpin the differing levels of neuronal survival and axonal regeneration in different species.
micrornas  are an extensive subclass of regulatory non-coding rnas that repress gene expression at a post-transcriptional level by affecting mrna translation and stability  <cit> .
mirnas have been implicated in many aspects of development and homeostatic pathways, with their actions often becoming more pronounced under conditions of physiological or pathological stress  <cit> .
mirnas are highly abundant in the cns, which is perhaps unsurprising given the cellular and transcriptional complexity of this tissue  <cit> .
the high degree of conservation of mirnas across species, combined with their ability to target multiple genes, make them a likely regulator in fundamental processes, such as the ability to regenerate neural pathways in the cns  <cit> .
to gain further insight into the regulatory mechanisms activated following a cns lesion, the present study sought to identify mirna that were altered in zebrafish retina after an optic nerve  crush.
this model provides easy access to cns-derived retinal ganglion cells  and their axons that connect with the optic tectum, thereby allowing examination of both cell survival and axonal repair.
similar tissue structure across species has allowed comparison between on injury in a variety of vertebrate species  as a means to investigate the molecular basis of cns regeneration .
the most dynamic gene changes occur approximately 3– <dig> days post injury suggesting that this phase is a critical point that sets the scene for successful regeneration or failure  <cit> .
during this period in zebrafish, previous studies have suggested that the injured rgcs are geared towards maintaining and enhancing survival whilst simultaneously preparing for axonal outgrowth  <cit> .
in this study we performed a bioinformatic integration of mrna and mirna expression profiles of the zebrafish retina 3 days after injury, with the experimental outline represented in fig.
<dig>  we assessed changes in mirna expression within the same rna pool used for mrna profiling as means of increasing the likelihood of finding true biological mirna-gene relationships.
the integration of these lists revealed three mirnas that were significantly over-expressed after injury.
we focused on two of these, mir-29b and mir- <dig>  and validated seven of their target genes that were under-expressed in our dataset.
our results identified apoptotic signalling, cytoskeletal dynamics and extracellular matrix interactions as key processes activated in zebrafish following regeneration.
our data suggest that mirnas are potential molecular targets that may be used to regulate these multiple processes in an orchestrated fashion to promote cns regeneration.fig.
<dig> schematic of experimental outline.
schematic flow diagram of procedures used to identify inversely correlated putative target genes of the differentially expressed mirnas
RESULTS
injury-induced changes in gene expression and pathways
to assess the changes in gene expression after an optic nerve crush injury, we examined mrna from whole zebrafish retinae 3 days after injury.
using an absolute log2-fold change cut-off of  <dig>  and an adjusted p-value of ≤ <dig> ,  <dig> transcripts were identified as differentially expressed between uninjured  and injured  tissue and this equated to  <dig> genes .
of these,  <dig> genes were over-expressed due to nerve injury, whilst  <dig> genes were under-expressed.
the microarray data are available from the gene expression omnibus gse <dig>  http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gse <dig>  and full analyses provided in the additional files.
the top over- and under-expressed genes, ranked by a combination of p-value and absolute log fold change ≥ <dig> , are listed in tables  <dig> and  <dig> respectively.table  <dig> top upregulated genes after optic nerve crush
ranked by combined –log10
p-value and ≥ <dig>  absolute fold change
ago terms determined by zfin and uniprot databases
ranked by combined –log10
p-value and ≥ <dig>  absolute fold change
ago terms determined by zfin and uniprot databases
to determine the biological and functional implications of these expression changes by analysing over- versus under-expressed genes separately, we performed go analysis and functional enrichment on our differentially expressed genes using webgestalt and ipa, respectively.
figures  <dig> and  <dig> show the trimmed enriched go terms for over- and under-expressed genes.
additional file 2: table s <dig> and additional file 3: table s <dig> contain the untrimmed enriched go terms, including ids and p-values, with the full gene lists available in the supporting data.fig.
<dig> gene ontology of over-expressed genes.
go analysis of enriched  cellular components and  biological processes of the over-expressed gene set.
each part represents -log <dig> of the p-value associated with the go category from webgestalt.
webgestalt results were filtered through gotrim and presented only go terms with ≥ <dig> genes.
molecular function not shown as no terms passed this criteria.
fig.
<dig> gene ontology of under-expressed genes.
go analysis of enriched  cellular component,  biological processes, and  molecular functions of the under-expressed gene set.
each part represents -log <dig> of the p-value associated with the go category from webgestalt.
webgestalt results were filtered through gotrim and presented only go terms with ≥ <dig> genes.
enriched processes associated with over-expressed genes  included ribosomal complex biogenesis, microtubule-based processes, and cell cycle activities.
other relevant processes enriched in our dataset included response to wounding , and intracellular mediation of axon guidance signals .
our data also highlight for the first time that genes involved in non-coding rna processing were enriched following optic nerve crush, implying that non-coding rnas might orchestrate the regeneration process.
go terms over-represented in the under-expressed genes  revealed processes associated with eye development, dna binding activity, g-protein coupled receptor signaling, and ion transport.
in particular, transcription factors involved in eye development showed decreased expression after injury .
the genes involved in the g-protein signaling pathways appeared to be associated with neuropeptide signaling .
furthermore, there was a significant reduction in expression of ion transport genes, focusing on potassium and calcium channel activity.
pathway analysis of the  <dig> probe identifiers uploaded into ipa resulted in only a subset being used, as  <dig> probes were annotated but only  <dig> mapped to a biological function.
the over-expressed genes were associated with  <dig> significantly enriched canonical pathways , with the top ten pathways including cell cycle regulation, 14-3- <dig> and apoptosis signalling, consistent with go term results.
there were fewer canonical pathways enriched in the under-expressed gene set , but these pathways were associated with inflammatory and immune responses, as well as calcium signaling .
the majority of inflammation-associated genes were found in the acute phase response pathway .
genes specific to calcium signaling included chrna <dig>  chrna <dig>  ryr <dig>  camkk <dig> fig.
<dig> enriched ipa canonical pathways of genes that were differentially expressed after injury.
over-expressed and  under-expressed genes are shown
further functional analysis revealed the relationship between genes, by placing associated genes into networks related to specific biological processes.
over-expressed genes fell into networks associated with dermatological diseases and conditions, developmental and hereditary disorders additional file 4: figure s <dig>  in contrast, under-expressed genes were associated with networks involved in neurological disease, development and function of the nervous system, and organ morphology additional file 5: figure s <dig>
changes in mirna expression and integration with mrna profile identify candidate mirna-mrna target pairs involved in nerve regeneration
in order to examine the regulation of gene expression by mirnas after optic nerve injury, we assessed changes in mirna expression using the same rna pool used for mrna profiling.
of the  <dig> zebrafish specific mirnas on the platform, we found  <dig> to be significantly altered 3 days after nerve crush .
we focused our subsequent investigation on two of the mirnas over-expressed after nerve injury, mir-29b and mir- <dig>  as their increased expression  is pertinent when considering the role of mirnas is to negatively regulate their target genes  <cit> , and unlike mir- <dig>  <cit> , they have not been extensively studied in the brain.table  <dig> top mirnas differentially expressed after optic nerve
to identify genes that are regulated by these mirnas, we computationally identified target mrnas that contain a putative mirna binding site within their 3’utr using targetscan fish  <cit> , one of the few predictive databases to identify mirna targets for species other than humans and rodents.
to select gene candidates that were  most likely to be regulated by each mirna, we used stringent parameters whereby at least one mirna binding site had a context + score of ≤ <dig> .
this approach resulted in  <dig> and  <dig> unique putative ensembl target genes for mir-29b and mir- <dig>  respectively.
we then performed go analysis on our list of predicted target genes to determine if these mirnas where associated with any particular biological function.
these results suggest a propensity for mir-29b to target genes associated with dna modification and extracellular matrix  activities , in contrast to mir- <dig> whose putative targets appeared to fall into nucleoside catabolic processes  and gtpase regulators .
interestingly, glycoprotein metabolism processes  were also highlighted, with an emphasis on heparan sulfate proteoglycan  subtypes .
we then integrated the predicted gene list with our own differentially expressed gene set and this revealed  <dig> predicted target genes for mir-29b and  <dig> for mir- <dig>  we refined this list by focusing on target genes whose expression was inversely correlated with the mirna expression , and using a targetscan context + score of < − <dig>  for at least one binding site, we identified five predicted gene targets for mir-29b and four for mir- <dig> .fig.
<dig> gene ontology of mir-29b predicted targets from targetscan fish.
representation of go terms associated with  cellular component,  biological process, and  molecular function.
predicted gene targets contained at least one mirna binding site with a context score of ≤ − <dig> .
each part represents -log <dig> of the p-value of biological process and cellular component from the set of significant biological processes and cellular components.
the p-values were retrieved from gene ontology analysis in webgestalt.
a list of genes in each go category is in additional file 6: table s3
fig.
<dig> gene ontology of mir- <dig> predicted targets from targetscan fish.
representation of go terms associated with  biological process, and  molecular function.
predicted gene targets contained at least one mirna binding site with a context score of ≤ − <dig> .
each part represents -log <dig> of the p-value of biological process and cellular component from the set of significant biological processes and cellular components.
the p-values were retrieved from gene ontology analysis in webgestalt.
a list of genes in each go category is in additional file 7: table s4
eva1a
c
<dig>
layna
c
<dig>
nefmb
ina
c
<dig>
si:ch211-51a <dig>
smoc1
lrrn3
d
sb:cb252
afold change represents microarray expression.
all genes had an adjusted p-value ≤ <dig>
bsee methods for summary of targetscan fish context + score
cpredicted binding sites of mir- <dig> in genes with a context scores less stringent then our cut-off
dmicroarray results revealed sb:cb <dig> was the most upregulated gene on microarray, yet it was also one of the top predicted mir- <dig> target genes by targetscan fish; we chose to included it in subsequent experiments to determine if it was a valid mir- <dig> target
validation of putative target genes of mir- <dig> and mir-29b
to validate our computationally predicted mirna-gene targets, we first confirmed changes in their expression using rt-qpcr , which supported our microarray expression data for all but one mirna target, sb:cb <dig>  this gene was one of the top predicted targets for mir- <dig> identified in targetscan , but our array results showed strong over-expression .
however, rt-qpcr validation revealed a decrease in expression, supporting the idea that it is negatively regulated by mir- <dig> as predicted by targetscan fish; thus the gene was included in all subsequent analyses.
to functionally verify the putative mirna-mrna interaction, we cloned part of the 3’utr sequence that contained the mirna binding site of each gene into a pmirglo vector, which contains both luciferase and renilla reporter genes .
a mutated version of the mirna seed site was also created.
hek <dig> cells were transfected with either a wild type  or mutated construct , along with a mirna mimic  or negative control .
luciferase results revealed significant inhibition of luciferase activity for mir-29b with all of its predicted gene targets .
however, mir- <dig> showed statistically significant binding only to smoc <dig> and sb:cb <dig> .fig.
<dig> validation of mir-29b and mir- <dig> putative gene targets from integration analysis.
microarray and corresponding rt-qpcr expression of genes predicted to be targeted by mir-29b  or mir- <dig> .
the latter is presented as expression fold change  relative to ppia
fig.
<dig> predicted mirna binding sites within 3’utr of predicted target genes.
sequence of mir-29b  and mir- <dig>  binding sites within 3’utr of predicted target genes .
seed region is bolded.
mutations predicted to disrupt mirna-mrna binding were made in the seed region, mutated nucleotides underlined
fig.
<dig> validation of mirna binding to 3’ utr of putative target genes.
luciferase reporter assay of hek <dig> cells cotransfected with pmirglo plasmid containing the wt or mt 3’utr mirna seed sequence from each gene, and either mir-29b, mir- <dig> or mir-nc .
samples were analysed 48 h after transfection and data normalised to the pmirglo only transfection.
columns represent the luciferase activity of either wt or mt constructs with mir-29b  or mir- <dig> , relative to transfection with the same construct and mir-nc.
data represents the mean ± sem, n =  <dig> independent experiments containing  <dig> replicates each.
student’s t test comparing wt or mt construct with mirna to mir-nc indicated as *p <  <dig> , **p <  <dig> , ***p <  <dig> .
student’s t test comparing wt with mirna to corresponding mt construct indicated as #p <  <dig> , ## p <  <dig> , ###p <  <dig>
discussion
here we used microarrays to examine and integrate expression of mirna and mrna in zebrafish retina after optic nerve crush to identify potential regulatory mechanisms involved in central nerve regeneration.
our integrated approach to studying gene regulation highlighted two mirnas that target genes in key biological processes associated with cell survival/apoptosis, ecm-cytoskeleton signaling, and hspg binding.
our study has provided unique information about the cellular context in which these genetic regulatory changes occur at a critical time point in the regeneration pathway.
zebrafish are a good model in which to delineate genes associated with regeneration and contrast with mammalian studies.
however, it appears there are a lot more genetic similarities between the species than first thought, with overlap between zebrafish and mammals observed in both up- and downregulated genes , and even in the expression of genes associated with inhibition of regeneration .
this further supports the idea that multiple pathway analysis is required, as differences are likely to be subtle and occur at multiple points across several processes  <cit> .
it is also likely that a successful response results from the synergistic activity of several cell types, given the interaction between rgcs and other retinal cell types that are reported to assist in a positive injury response.
our approach of profiling the expression of the whole retina takes into consideration the changes that occur to in the cells adjacent to the rgcs, such as muller glia and amacrine cells.
interestingly, the go processes altered in our dataset overlap with several mentioned in previous zebrafish studies that have utilised other ocular tissues .
however there were some processes that appeared unique to this study, in particular upregulation of non-coding rnas, which supports our original hypothesis that gene regulatory processes are key to promoting successful regeneration in zebrafish.
the mirnas: mir-29b and mir-223
the role of micrornas in lower vertebrates with a known regenerative ability is gaining a lot of attention, with several recent studies identifying mirnas associated with spinal cord repair  and appendage regeneration .
less is known about mirna-mediated regeneration within the eye, with most focus on the role of mirnas in muller glia cells  <cit> .
in this study, we show for the first time that mir-29b and mir- <dig>  are significantly over-expressed after optic nerve crush.
in zebrafish, mir-29b is part of the mir- <dig> family that comprises three intergenic members that map to chromosome  <dig> , and chromosome  <dig> .
mir-29b has been associated with ecm remodeling, and shown to have both pro- and anti-apoptotic properties depending on the cns injury/disease model .
the other mirna highlighted in this study, mir- <dig>  maps to chromosome  <dig> in zebrafish, and has been implicated in a wide range of pathophysiologies, including cancer, muscular dystrophy and atherosclerosis  <cit> .
within the cns, it has been implicated in inflammatory processes including autoimmune disorders and mammalian cns injury .
our gene ontology analysis confirmed a role for mir- <dig> in the zebrafish immune response following injury but additionally highlighted enhanced cell survival and altered cytoskeleton/ecm response.
the small number of significantly altered mirnas found in this study may result from looking at one specific time point.
on the other hand, the fact that a single mirna is able to regulate a large number of genes might negate the need for changes in many mirnas in order to bring about widespread biological change  <cit> .
further, it is likely that these two mirnas interact with each other in a synergistic manner, as evidenced by the overlapping targeting of the same genes when less stringent cut-offs were applied during our integration analysis.
mirna target genes: cell survival from altered mitochondrial function
after optic nerve injury, the viability of rgcs is paramount for axonal regeneration and restoration of function.
mammalian studies have shown a high degree of cell death in the retina 2– <dig> days after injury, in contrast to the limited death observed in zebrafish  <cit> .
this restrained cell death may be associated with several downregulated genes in our dataset that we functionally validated for the first time as targets genes of mir-29b and mir- <dig>  including eva1a, layna, si:ch211-51a <dig>  , smoc <dig> and es1-like homolog, sb:cb <dig>
eva1a is a strongly conserved transmembrane protein localized to the lysosome and endoplasmic reticulum membrane where it is thought to function as a regulator of programmed cell death processes as well as necrosis .
another target that may play a role in mitochondrial function  is the mir- <dig> target, sb:cb <dig>  this uncharacterized gene shares significant sequence homology with its homolog, zebrafish es <dig> and human c21orf <dig>  <cit> .
the exact role of both sb:cb <dig> and es <dig> remains unknown, however they are postulated to be involved in mitochondrial function and es <dig> has been observed in zebrafish retina after a retinal lesion  <cit> .
downregulation of these targets may protect rgcs against cell death, consistent with the >90 % survival observed in zebrafish rgcs after crush  <cit> .
ecm-cell signaling and indirect cytoskeleton modification
interaction between the cell membrane and actin cytoskeleton is essential for many cellular processes, including cell shape, adhesion, migration and signal transduction  <cit> .
the role of ecm-cell signaling in regeneration is particularly pertinent as a means of restructuring the cellular architecture in response to injury.
the role of mirnas, including the mir- <dig> family, in modifying ecm-cell signaling has recently been highlighted  <cit> .
interestingly, three of the mirna targets we validated are associated with this process, layna, si:ch211-51a <dig>  and smoc <dig>
layilin  is a transmembrane receptor that mediates hyaluronan signaling by binding to the ecm cytoskeletal linker proteins, talin, radixin and merlin  <cit> , thus destabilising the cytoskeleton  <cit> .
downregulation of layna in zebrafish rgcs after crush may therefore reduce hyaluronan signaling and stabilise the cytoskeleton, promoting the early stages of axonal outgrowth.
interestingly, there were no significant changes in the expression of other hyaluronan receptors within our dataset .
another biological process highlighted in our study is heparan sulfate proteoglycan  signalling, which is known to influence cns regeneration  <cit> .
mir-29b and mir- <dig> target genes share common functions surrounding the hspg, agrin.
the mir-29b target, si:ch211-51a <dig> , is not characterised in zebrafish but is a homologue of the serine protease, neurotrypsin .
neurotrypsin cleaves agrin into two fragments which are involved in neural plasticity, axonal outgrowth and synaptogenesis , as well as cytoskeleton remodelling .
furthermore, the mir- <dig> target, secreted modular calcium binding  <dig> , is a member of the sparc  family and binds to hspgs  to modulate cell adhesion  <cit> .
although it is not clear how downregulation of their expression would impact on the regenerative process, key roles for these proteins in mediating the regenerative response in fish are supported by our finding that the agrin interactions canonical pathway is enriched with upregulated genes in our dataset.
interestingly, smoc <dig> has also been implicated in smad1-dependent bone morphogenetic protein  signaling which regulates axonal regrowth in injured dorsal root ganglion neurons .
thus the downregulation of smoc <dig> may also be part of bmp activation of pro-regenerative transcription program or manipulation of cytoskeletal dynamics at the growth cone  <cit> .
direct cytoskeleton modification
in addition to indirectly reorganizing the cytoskeleton by modulating membrane signaling mechanisms, our data highlight ways in which mir-29b may directly target members of the intermediate filaments of the cytoskeleton, by regulating expression of internexin  and neurofilament-medium homolog b   <cit> .
the latter is one of two homologs found in zebrafish , however nefmb is likely to represent the zebrafish version of nefh, as it shares a significant homology to human nfh.
although some neurofilament proteins are upregulated during successful regeneration in the mammalian peripheral nervous system, and in lower vertebrates , the overexpression of other intermediate filaments has been associated with several neurodegenerative and cns diseases , with specifically nefh and ina potentially being biomarkers for the likes of amyotrophic lateral sclerosis and neuropsychiatric systemic lupus erythematosus  <cit> .
both have been associated with the slowing of neurofilament axonal transport, resulting in neuronal aggregates which damage the endoplasmic reticulum and mitochondria in transit, ultimately leading to cell death .
thus their downregulation during the initial stages of axonal regeneration in our study may promote regeneration by facilitating transport of neurofilament components to required cellular areas.
it is interesting to note that other microtubule and actin components of the cytoskeleton, including b-actin, a- and b-tubulin, nefm and prph, were upregulated after injury in our dataset.
this suggests that the specific downregulation of ina and nefmb may play a significant role in zebrafish injury response.
zebrafish as a comparative model for mirna role in cns regeneration
examining the role of mirnas in regenerative permissive species, such as zebrafish, is an important step in delineating their role in higher order vertebrate injuries.
whilst the mirna sequence is strongly conserved between species, this does not necessarily guarantee conservation of their function  <cit> .
in many cases the target genes are not conserved, with the turnover of mirna binding sites within the 3’utr of genes thought to be a significant driver of evolutionary processes  <cit> .
however, the two mirs examined in this study are predicted to target human and rat orthologs of the genes we validated here , suggesting that the retention of mirna binding sites across species may provide an opportunity to manipulate gene regulation in mammals based on our findings.table  <dig> predicted mir-29b and mir- <dig> binding of human and rat orthologs to the validated zebrafish genes
zfish refseq
zfish ensembl
zebrafish gene
eva1a
layna
nefmb
ina
si:ch211-51a <dig>
smoc1
lrrn3
sb:cb252
acontext score calculated by targetscan for human and rat predictions
CONCLUSIONS
identifying the molecular and cellular factors associated with the successful regenerative response in zebrafish may aid in identifying therapeutic targets in the damaged mammalian cns  <cit> .
our results provide a basis from which to investigate the cellular processes required for central nervous system regeneration, and further studies will examine more extensively the complete repertoire of mrna targets of mir-29b and mir- <dig>   <cit> ).
the synergistic activities of multiple mirnas, and the redundancy in their regulation of specific target genes and signalling pathways, provides an opportunity to reengage 3’utr targets in order to instigate a fish-like regenerative response in the mammalian cns  <cit> .

 the regenerative capacity of the cns in adult mammals is limited, with minimal axonal re-growth, death of damaged neurons and long-term loss of function. by contrast, fish have the remarkable ability to repair most functional components of the cns making them a valuable model in which to identify key molecular mechanisms involved in neural regeneration. anamniotes and amniotes have a high degree of conservation at the level of both nucleotide sequence and amino acid functional domains, resulting in similar molecular and cellular processes in neural development and function of both clades. therefore, it is possible that genes required for successful cns regeneration are present in both fish and mammals, but that key regulatory differences in the expression of these genes underpin the differing levels of neuronal survival and axonal regeneration in different species. micrornas are an extensive subclass of regulatory non-coding rnas that repress gene expression at a post-transcriptional level by affecting mrna translation and stability.  mirnas have been implicated in many aspects of development and homeostatic pathways, with their actions often becoming more pronounced under conditions of physiological or pathological stress. mirnas are highly abundant in the cns, which is perhaps unsurprising given the cellular and transcriptional complexity of this tissue. the high degree of conservation of mirnas across species, combined with their ability to target multiple genes, make them a likely regulator in fundamental processes, such as the ability to regenerate neural pathways in the cns. to gain further insight into the regulatory mechanisms activated following a cns lesion, the present study sought to identify mirna that were altered in zebrafish retina after an optic nerve crush. this model provides easy access to cns-derived retinal ganglion cells and their axons that connect with the optic tectum, thereby allowing examination of both cell survival and axonal repair. similar tissue structure across species has allowed comparison between on injury in a variety of vertebrate species as a means to investigate the molecular basis of cns regeneration. the most dynamic gene changes occur approximately 3– days post injury suggesting that this phase is a critical point that sets the scene for successful regeneration or failure. during this period in zebrafish, previous studies have suggested that the injured rgcs are geared towards maintaining and enhancing survival whilst simultaneously preparing for axonal outgrowth. in this study we performed a bioinformatic integration of mrna and mirna expression profiles of the zebrafish retina 3 days after injury, with the experimental outline represented in fig.  we assessed changes in mirna expression within the same rna pool used for mrna profiling as means of increasing the likelihood of finding true biological mirna-gene relationships. the integration of these lists revealed three mirnas that were significantly over-expressed after injury. we focused on two of these, mir-29b and mir- and validated seven of their target genes that were under-expressed in our dataset. our results identified apoptotic signalling, cytoskeletal dynamics and extracellular matrix interactions as key processes activated in zebrafish following regeneration. our data suggest that mirnas are potential molecular targets that may be used to regulate these multiple processes in an orchestrated fashion to promote cns regeneration.fig. schematic of experimental outline. schematic flow diagram of procedures used to identify inversely correlated putative target genes of the differentially expressed mirnas  injury-induced changes in gene expression and pathways to assess the changes in gene expression after an optic nerve crush injury, we examined mrna from whole zebrafish retinae 3 days after injury. using an absolute log2-fold change cut-off of and an adjusted p-value of ≤, transcripts were identified as differentially expressed between uninjured and injured tissue and this equated to genes. of these, genes were over-expressed due to nerve injury, whilst genes were under-expressed. the microarray data are available from the gene expression omnibus gse http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gse and full analyses provided in the additional files. the top over- and under-expressed genes, ranked by a combination of p-value and absolute log fold change ≥, are listed in tables  and respectively.table top upregulated genes after optic nerve crush ranked by combined –log10 p-value and ≥ absolute fold change ago terms determined by zfin and uniprot databases ranked by combined –log10 p-value and ≥ absolute fold change ago terms determined by zfin and uniprot databases to determine the biological and functional implications of these expression changes by analysing over- versus under-expressed genes separately, we performed go analysis and functional enrichment on our differentially expressed genes using webgestalt and ipa, respectively. figures  and show the trimmed enriched go terms for over- and under-expressed genes. additional file 2: table s and additional file 3: table s contain the untrimmed enriched go terms, including ids and p-values, with the full gene lists available in the supporting data.fig. gene ontology of over-expressed genes. go analysis of enriched cellular components and biological processes of the over-expressed gene set. each part represents -log of the p-value associated with the go category from webgestalt. webgestalt results were filtered through gotrim and presented only go terms with ≥ genes. molecular function not shown as no terms passed this criteria. fig. gene ontology of under-expressed genes. go analysis of enriched cellular component, biological processes, and molecular functions of the under-expressed gene set. each part represents -log of the p-value associated with the go category from webgestalt. webgestalt results were filtered through gotrim and presented only go terms with ≥ genes. enriched processes associated with over-expressed genes included ribosomal complex biogenesis, microtubule-based processes, and cell cycle activities. other relevant processes enriched in our dataset included response to wounding, and intracellular mediation of axon guidance signals. our data also highlight for the first time that genes involved in non-coding rna processing were enriched following optic nerve crush, implying that non-coding rnas might orchestrate the regeneration process. go terms over-represented in the under-expressed genes revealed processes associated with eye development, dna binding activity, g-protein coupled receptor signaling, and ion transport. in particular, transcription factors involved in eye development showed decreased expression after injury. the genes involved in the g-protein signaling pathways appeared to be associated with neuropeptide signaling. furthermore, there was a significant reduction in expression of ion transport genes, focusing on potassium and calcium channel activity. pathway analysis of the probe identifiers uploaded into ipa resulted in only a subset being used, as probes were annotated but only mapped to a biological function. the over-expressed genes were associated with significantly enriched canonical pathways, with the top ten pathways including cell cycle regulation, 14-3- and apoptosis signalling, consistent with go term results. there were fewer canonical pathways enriched in the under-expressed gene set, but these pathways were associated with inflammatory and immune responses, as well as calcium signaling. the majority of inflammation-associated genes were found in the acute phase response pathway. genes specific to calcium signaling included chrna chrna ryr camkk fig. enriched ipa canonical pathways of genes that were differentially expressed after injury. over-expressed and under-expressed genes are shown further functional analysis revealed the relationship between genes, by placing associated genes into networks related to specific biological processes. over-expressed genes fell into networks associated with dermatological diseases and conditions, developmental and hereditary disorders additional file 4: figure s in contrast, under-expressed genes were associated with networks involved in neurological disease, development and function of the nervous system, and organ morphology additional file 5: figure s changes in mirna expression and integration with mrna profile identify candidate mirna-mrna target pairs involved in nerve regeneration in order to examine the regulation of gene expression by mirnas after optic nerve injury, we assessed changes in mirna expression using the same rna pool used for mrna profiling. of the zebrafish specific mirnas on the platform, we found to be significantly altered 3 days after nerve crush. we focused our subsequent investigation on two of the mirnas over-expressed after nerve injury, mir-29b and mir- as their increased expression is pertinent when considering the role of mirnas is to negatively regulate their target genes, and unlike mir-, they have not been extensively studied in the brain.table top mirnas differentially expressed after optic nerve to identify genes that are regulated by these mirnas, we computationally identified target mrnas that contain a putative mirna binding site within their 3’utr using targetscan fish, one of the few predictive databases to identify mirna targets for species other than humans and rodents. to select gene candidates that were  most likely to be regulated by each mirna, we used stringent parameters whereby at least one mirna binding site had a context + score of ≤. this approach resulted in and unique putative ensembl target genes for mir-29b and mir- respectively. we then performed go analysis on our list of predicted target genes to determine if these mirnas where associated with any particular biological function. these results suggest a propensity for mir-29b to target genes associated with dna modification and extracellular matrix activities, in contrast to mir- whose putative targets appeared to fall into nucleoside catabolic processes and gtpase regulators. interestingly, glycoprotein metabolism processes were also highlighted, with an emphasis on heparan sulfate proteoglycan subtypes. we then integrated the predicted gene list with our own differentially expressed gene set and this revealed predicted target genes for mir-29b and for mir- we refined this list by focusing on target genes whose expression was inversely correlated with the mirna expression, and using a targetscan context + score of < − for at least one binding site, we identified five predicted gene targets for mir-29b and four for mir-.fig. gene ontology of mir-29b predicted targets from targetscan fish. representation of go terms associated with  cellular component, biological process, and molecular function. predicted gene targets contained at least one mirna binding site with a context score of ≤ −. each part represents -log of the p-value of biological process and cellular component from the set of significant biological processes and cellular components. the p-values were retrieved from gene ontology analysis in webgestalt. a list of genes in each go category is in additional file 6: table s3 fig. gene ontology of mir- predicted targets from targetscan fish. representation of go terms associated with biological process, and molecular function. predicted gene targets contained at least one mirna binding site with a context score of ≤ −. each part represents -log of the p-value of biological process and cellular component from the set of significant biological processes and cellular components. the p-values were retrieved from gene ontology analysis in webgestalt. a list of genes in each go category is in additional file 7: table s4 eva1a c layna c nefmb ina c si:ch211-51a smoc1 lrrn3 d sb:cb252 afold change represents microarray expression. all genes had an adjusted p-value ≤ bsee methods for summary of targetscan fish context + score cpredicted binding sites of mir- in genes with a context scores less stringent then our cut-off dmicroarray results revealed sb:cb was the most upregulated gene on microarray, yet it was also one of the top predicted mir- target genes by targetscan fish; we chose to included it in subsequent experiments to determine if it was a valid mir- target validation of putative target genes of mir- and mir-29b to validate our computationally predicted mirna-gene targets, we first confirmed changes in their expression using rt-qpcr, which supported our microarray expression data for all but one mirna target, sb:cb this gene was one of the top predicted targets for mir- identified in targetscan, but our array results showed strong over-expression. however, rt-qpcr validation revealed a decrease in expression, supporting the idea that it is negatively regulated by mir- as predicted by targetscan fish; thus the gene was included in all subsequent analyses. to functionally verify the putative mirna-mrna interaction, we cloned part of the 3’utr sequence that contained the mirna binding site of each gene into a pmirglo vector, which contains both luciferase and renilla reporter genes. a mutated version of the mirna seed site was also created. hek cells were transfected with either a wild type or mutated construct, along with a mirna mimic or negative control. luciferase results revealed significant inhibition of luciferase activity for mir-29b with all of its predicted gene targets. however, mir- showed statistically significant binding only to smoc and sb:cb.fig. validation of mir-29b and mir- putative gene targets from integration analysis. microarray and corresponding rt-qpcr expression of genes predicted to be targeted by mir-29b or mir-. the latter is presented as expression fold change relative to ppia fig. predicted mirna binding sites within 3’utr of predicted target genes. sequence of mir-29b and mir- binding sites within 3’utr of predicted target genes. seed region is bolded. mutations predicted to disrupt mirna-mrna binding were made in the seed region, mutated nucleotides underlined fig. validation of mirna binding to 3’ utr of putative target genes. luciferase reporter assay of hek cells cotransfected with pmirglo plasmid containing the wt or mt 3’utr mirna seed sequence from each gene, and either mir-29b, mir- or mir-nc. samples were analysed 48 h after transfection and data normalised to the pmirglo only transfection. columns represent the luciferase activity of either wt or mt constructs with mir-29b or mir-, relative to transfection with the same construct and mir-nc. data represents the mean ± sem, n =  independent experiments containing replicates each. student’s t test comparing wt or mt construct with mirna to mir-nc indicated as *p < , **p < , ***p < . student’s t test comparing wt with mirna to corresponding mt construct indicated as #p < , ## p < , ###p <  discussion here we used microarrays to examine and integrate expression of mirna and mrna in zebrafish retina after optic nerve crush to identify potential regulatory mechanisms involved in central nerve regeneration. our integrated approach to studying gene regulation highlighted two mirnas that target genes in key biological processes associated with cell survival/apoptosis, ecm-cytoskeleton signaling, and hspg binding. our study has provided unique information about the cellular context in which these genetic regulatory changes occur at a critical time point in the regeneration pathway. zebrafish are a good model in which to delineate genes associated with regeneration and contrast with mammalian studies. however, it appears there are a lot more genetic similarities between the species than first thought, with overlap between zebrafish and mammals observed in both up- and downregulated genes, and even in the expression of genes associated with inhibition of regeneration. this further supports the idea that multiple pathway analysis is required, as differences are likely to be subtle and occur at multiple points across several processes. it is also likely that a successful response results from the synergistic activity of several cell types, given the interaction between rgcs and other retinal cell types that are reported to assist in a positive injury response. our approach of profiling the expression of the whole retina takes into consideration the changes that occur to in the cells adjacent to the rgcs, such as muller glia and amacrine cells. interestingly, the go processes altered in our dataset overlap with several mentioned in previous zebrafish studies that have utilised other ocular tissues. however there were some processes that appeared unique to this study, in particular upregulation of non-coding rnas, which supports our original hypothesis that gene regulatory processes are key to promoting successful regeneration in zebrafish. the mirnas: mir-29b and mir-223 the role of micrornas in lower vertebrates with a known regenerative ability is gaining a lot of attention, with several recent studies identifying mirnas associated with spinal cord repair and appendage regeneration. less is known about mirna-mediated regeneration within the eye, with most focus on the role of mirnas in muller glia cells. in this study, we show for the first time that mir-29b and mir- are significantly over-expressed after optic nerve crush. in zebrafish, mir-29b is part of the mir- family that comprises three intergenic members that map to chromosome, and chromosome. mir-29b has been associated with ecm remodeling, and shown to have both pro- and anti-apoptotic properties depending on the cns injury/disease model. the other mirna highlighted in this study, mir- maps to chromosome in zebrafish, and has been implicated in a wide range of pathophysiologies, including cancer, muscular dystrophy and atherosclerosis. within the cns, it has been implicated in inflammatory processes including autoimmune disorders and mammalian cns injury. our gene ontology analysis confirmed a role for mir- in the zebrafish immune response following injury but additionally highlighted enhanced cell survival and altered cytoskeleton/ecm response. the small number of significantly altered mirnas found in this study may result from looking at one specific time point. on the other hand, the fact that a single mirna is able to regulate a large number of genes might negate the need for changes in many mirnas in order to bring about widespread biological change. further, it is likely that these two mirnas interact with each other in a synergistic manner, as evidenced by the overlapping targeting of the same genes when less stringent cut-offs were applied during our integration analysis. mirna target genes: cell survival from altered mitochondrial function after optic nerve injury, the viability of rgcs is paramount for axonal regeneration and restoration of function. mammalian studies have shown a high degree of cell death in the retina 2– days after injury, in contrast to the limited death observed in zebrafish. this restrained cell death may be associated with several downregulated genes in our dataset that we functionally validated for the first time as targets genes of mir-29b and mir- including eva1a, layna, si:ch211-51a, smoc and es1-like homolog, sb:cb eva1a is a strongly conserved transmembrane protein localized to the lysosome and endoplasmic reticulum membrane where it is thought to function as a regulator of programmed cell death processes as well as necrosis. another target that may play a role in mitochondrial function is the mir- target, sb:cb this uncharacterized gene shares significant sequence homology with its homolog, zebrafish es and human c21orf. the exact role of both sb:cb and es remains unknown, however they are postulated to be involved in mitochondrial function and es has been observed in zebrafish retina after a retinal lesion. downregulation of these targets may protect rgcs against cell death, consistent with the >90 % survival observed in zebrafish rgcs after crush. ecm-cell signaling and indirect cytoskeleton modification interaction between the cell membrane and actin cytoskeleton is essential for many cellular processes, including cell shape, adhesion, migration and signal transduction. the role of ecm-cell signaling in regeneration is particularly pertinent as a means of restructuring the cellular architecture in response to injury. the role of mirnas, including the mir- family, in modifying ecm-cell signaling has recently been highlighted. interestingly, three of the mirna targets we validated are associated with this process, layna, si:ch211-51a and smoc layilin is a transmembrane receptor that mediates hyaluronan signaling by binding to the ecm cytoskeletal linker proteins, talin, radixin and merlin, thus destabilising the cytoskeleton. downregulation of layna in zebrafish rgcs after crush may therefore reduce hyaluronan signaling and stabilise the cytoskeleton, promoting the early stages of axonal outgrowth. interestingly, there were no significant changes in the expression of other hyaluronan receptors within our dataset. another biological process highlighted in our study is heparan sulfate proteoglycan signalling, which is known to influence cns regeneration. mir-29b and mir- target genes share common functions surrounding the hspg, agrin. the mir-29b target, si:ch211-51a, is not characterised in zebrafish but is a homologue of the serine protease, neurotrypsin. neurotrypsin cleaves agrin into two fragments which are involved in neural plasticity, axonal outgrowth and synaptogenesis, as well as cytoskeleton remodelling. furthermore, the mir- target, secreted modular calcium binding, is a member of the sparc family and binds to hspgs to modulate cell adhesion. although it is not clear how downregulation of their expression would impact on the regenerative process, key roles for these proteins in mediating the regenerative response in fish are supported by our finding that the agrin interactions canonical pathway is enriched with upregulated genes in our dataset. interestingly, smoc has also been implicated in smad1-dependent bone morphogenetic protein signaling which regulates axonal regrowth in injured dorsal root ganglion neurons. thus the downregulation of smoc may also be part of bmp activation of pro-regenerative transcription program or manipulation of cytoskeletal dynamics at the growth cone. direct cytoskeleton modification in addition to indirectly reorganizing the cytoskeleton by modulating membrane signaling mechanisms, our data highlight ways in which mir-29b may directly target members of the intermediate filaments of the cytoskeleton, by regulating expression of internexin and neurofilament-medium homolog b. the latter is one of two homologs found in zebrafish, however nefmb is likely to represent the zebrafish version of nefh, as it shares a significant homology to human nfh. although some neurofilament proteins are upregulated during successful regeneration in the mammalian peripheral nervous system, and in lower vertebrates, the overexpression of other intermediate filaments has been associated with several neurodegenerative and cns diseases, with specifically nefh and ina potentially being biomarkers for the likes of amyotrophic lateral sclerosis and neuropsychiatric systemic lupus erythematosus. both have been associated with the slowing of neurofilament axonal transport, resulting in neuronal aggregates which damage the endoplasmic reticulum and mitochondria in transit, ultimately leading to cell death. thus their downregulation during the initial stages of axonal regeneration in our study may promote regeneration by facilitating transport of neurofilament components to required cellular areas. it is interesting to note that other microtubule and actin components of the cytoskeleton, including b-actin, a- and b-tubulin, nefm and prph, were upregulated after injury in our dataset. this suggests that the specific downregulation of ina and nefmb may play a significant role in zebrafish injury response. zebrafish as a comparative model for mirna role in cns regeneration examining the role of mirnas in regenerative permissive species, such as zebrafish, is an important step in delineating their role in higher order vertebrate injuries. whilst the mirna sequence is strongly conserved between species, this does not necessarily guarantee conservation of their function. in many cases the target genes are not conserved, with the turnover of mirna binding sites within the 3’utr of genes thought to be a significant driver of evolutionary processes. however, the two mirs examined in this study are predicted to target human and rat orthologs of the genes we validated here, suggesting that the retention of mirna binding sites across species may provide an opportunity to manipulate gene regulation in mammals based on our findings.table predicted mir-29b and mir- binding of human and rat orthologs to the validated zebrafish genes zfish refseq zfish ensembl zebrafish gene eva1a layna nefmb ina si:ch211-51a smoc1 lrrn3 sb:cb252 acontext score calculated by targetscan for human and rat predictions  identifying the molecular and cellular factors associated with the successful regenerative response in zebrafish may aid in identifying therapeutic targets in the damaged mammalian cns. our results provide a basis from which to investigate the cellular processes required for central nervous system regeneration, and further studies will examine more extensively the complete repertoire of mrna targets of mir-29b and mir- ). the synergistic activities of multiple mirnas, and the redundancy in their regulation of specific target genes and signalling pathways, provides an opportunity to reengage 3’utr targets in order to instigate a fish-like regenerative response in the mammalian cns. 
gene ontology analysis placed the mirna-regulated genes in key biological processes that included cell survival/apoptosis, ecm-cytoskeleton signaling, and heparan sulfate proteoglycan binding, we focused on two over-expressed mirnas, and functionally validated seven of their predicted gene targets using rt-qpcr and luciferase assays to confirm mirna-mrna binding. we used microarrays to analyse and integrate the mrna and microrna expression profiles of zebrafish retina after optic nerve crush to identify potential regulatory mechanisms that underpin central nerve regeneration. although many studies have described differences in gene expression in zebrafish during cns regeneration, the regulatory mechanisms underpinning the differential expression of these genes have not been examined. our results suggest a key role for mir-29b and mir- in zebrafish regeneration. we then combined inverse correlations between our mirna expression and mrna expression, and integrated these findings with target predictions from targetscan fish to identify putative mirna-gene target pairs. the identification of mirna regulation in a zebrafish injury model provides a framework for future studies in which to investigate not only the cellular processes required for cns regeneration, but also how these mechanisms might be regulated to promote successful repair and return of function in the injured mammalian brain.
 unlike mammals, zebrafish have the ability to regenerate damaged parts of their central nervous system and regain functionality of the affected area. a better understanding of the molecular mechanisms involved in zebrafish regeneration may therefore provide insight into how cns repair might be induced in mammals. although many studies have described differences in gene expression in zebrafish during cns regeneration, the regulatory mechanisms underpinning the differential expression of these genes have not been examined.  we used microarrays to analyse and integrate the mrna and microrna expression profiles of zebrafish retina after optic nerve crush to identify potential regulatory mechanisms that underpin central nerve regeneration. bioinformatic analysis identified mirnas and mrnas that were differentially expressed after injury. we then combined inverse correlations between our mirna expression and mrna expression, and integrated these findings with target predictions from targetscan fish to identify putative mirna-gene target pairs. we focused on two over-expressed mirnas, and functionally validated seven of their predicted gene targets using rt-qpcr and luciferase assays to confirm mirna-mrna binding. gene ontology analysis placed the mirna-regulated genes in key biological processes that included cell survival/apoptosis, ecm-cytoskeleton signaling, and heparan sulfate proteoglycan binding,  our results suggest a key role for mir-29b and mir- in zebrafish regeneration. the identification of mirna regulation in a zebrafish injury model provides a framework for future studies in which to investigate not only the cellular processes required for cns regeneration, but also how these mechanisms might be regulated to promote successful repair and return of function in the injured mammalian brain. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords zebrafishmicrornamrnaoptic nerveregenerationcnsissue-copyright-statement© the author 2015 