BACKGROUND
mirnas are a class of small non-coding rnas that negatively regulate gene expression by binding to target sites in the 3' utr of mrnas.
this binding affects the stability and translation of the target transcript  <cit> .
to date more than six hundred highly-conserved mirnas have been identified in the mouse genome  proposing mirnome as a new layer of gene regulation.
mirnas are key mediators of basic biological processes.
in all plant and animal species examined, defects in mirna function have profound effects on development  <cit> .
in humans, deregulation of mirna expression caused by mutations in either the mirna or its target has been correlated with a number of clinically important diseases such as diabetes, neurodegenerative diseases and heart failure  <cit> , among others.
moreover, there is growing evidence supporting a role of mirnas in human cancers  <cit> .
in this regard, mirnas may constitute new targets of therapeutic interventions for a variety of diseases .
to gain insight into the functional role of mirnas, both in physiological and pathological processes, it is essential to have precise information on their temporal and spatial expression profile.
recent adaptations in tools for expression and functional analysis, developed to counteract the limitations posed by the small size of mirnas, have facilitated detection of the mirna content of several tissues  <cit> .
these studies revealed that about one third of mirnas are expressed in a tissue-restricted manner in vertebrates  <cit> .
the eye is a highly specialized organ whose development and function requires the precise coordination and timing of morphogenetic and cell differentiation events.
perturbation of mirna function in the eye of conditional dicer mouse mutants impairs the normal development of the retina, lens, cornea and optic chiasm  <cit> .
therefore, elucidation of the functional role of mirnas is expected to provide key information to decipher the complex regulatory circuits underlying these processes.
to define mirna function in the eye, it is essential to obtain a high-resolution profile of their spatial and temporal distribution.
this information, combined with in silico target prediction analysis, can also optimize the recognition of biologically significant mrna targets.
to date, only a limited number of reports are available on the expression profile of mirnas in the mammalian eye.
the majority of these publications analyze the mirna content of the retina  <cit>  while only a few address mirna expression in non-neural parts of the eye  <cit> .
furthermore, examples of the cellular distribution of mirnas in the eye are scarce, usually describing the spatial expression profiles of a limited number of mirnas at a few developmental stages  <cit> .
as a consequence, the information available on the complete set of mirnas expressed in ocular tissues is only partial and fragmented.
to obtain a global view of the differential mirna expression in the tissues that form the adult mouse eye, we performed microarray profiling of retina, retinal pigment epithelium , lens and cornea rna.
cluster analysis allowed us to identify subgroups of mirnas enriched in each of the above tissues.
these mirna clusters may reveal possible synergisms in the differentiation, cell-lineage commitment, development and maintenance of the analyzed eye structures.
moreover, to obtain a high-resolution atlas of the spatiotemporal distribution of these mirnas in the eye, we performed rna in situ hybridization  using locked nucleic acid -modified probes on sections of embryonic , postnatal  and adult  mouse eyes and implemented the data in a publicly available web database http://mirneye.tigem.it.
here, we describe the mirna expression profiles in the main tissues of the eye and show the spatiotemporal distribution of  <dig> mirnas.
finally, we propose a possible way on how cluster analysis of the mirna differential expression can be exploited to advance our understanding of the functional role of selected mirnas in a given tissue.
RESULTS
mirnas show differential expression in murine eye tissues
we decided to define the complete repertoire of mirnas that are expressed in any of four different eye tissues, i.e.
retina, lens, cornea and rpe.
as a first step, we collected retinas, lenses, corneas, rpe tissues and entire eyes from 8-week old c57bl/ <dig> mice.
we chose to use the c57bl/ <dig> strain that has a pigmented rpe in order to limit the carry-over contamination of the retina samples with rpe cells during tissue dissection.
following total rna extraction from these eye tissues, we carried out mirna profiling using the mircury lna™arrays provided by exiqon .
total rna from entire eyes was included as a reference to determine differential expression.
the mirnas that showed, in one or more tissues, a difference in log <dig> median ratios  equal or superior to  <dig>   compared to the 'entire eye' reference were considered for further analysis.
to demonstrate whether mirnas show tissue-enriched expression in a specific eye compartment, the  <dig> mirnas that passed the above threshold were used in a two-way hierarchical clustering analysis.
this analysis revealed distinct mirna clusters that are differentially expressed in one or more of the tissues analyzed .
the resulting dendrogram could be divided in five main clusters   enriched in mirnas predominantly expressed in retina , rpe , retina and lens , lens and cornea , and cornea and rpe .
within these five clusters, we defined  <dig> smaller groups  of mirnas with highly similar expression profiles .
the reliability of the clustering analysis of microarray data was confirmed by assessing the overall relationships among the analyzed tissues, which were in line with their developmental origin .
the significant number of mirnas that show differential expression profiles among the ocular tissues and their organization in distinct clusters suggest that the regulation of eye development and function by mirnas requires an intricate biological network.
our microarray results are overall concordant with similar global expression studies on single eye tissues previously reported in mouse or other vertebrates .
however, this is the first example of a simultaneous analysis of the main ocular structures, which therefore allows a direct semi-quantitative assessment of the differential mirna expression levels in the major components of the mouse eye.
to identify whether groups of mirnas with comparable expression profiles may be involved in the regulation of specific biological functions, we performed an in silico functional annotation analysis of their predicted target genes using the  <dig> above- described co-expression sub-clusters .
for each mirna present within these sub-clusters, we compiled a non-redundant list of predicted targets combining the output of miranda, targetscan and pictar  <cit> , three commonly used mirna target prediction software.
subsequently, we performed a gene ontology  and kegg pathway annotation analysis for the target lists of each single mirna, as described in methods.
we then compared the annotation term lists of all predicted target genes for each mirna belonging to each sub-cluster and counted the occurrence of shared individual go/kegg terms .
an arbitrary cut-off value of 70% was set and only terms enriched in the predicted target gene lists of at least 70% of the mirnas within a cluster were retained .
as a first result, we found a significant enrichment in several go/kegg terms related to neuronal development and function , which, considering the neural origin of the eye, is a good indication of the reliability of our analysis.
the above-mentioned enrichment in neuron-related terms was more prominent in sub-clusters that contained mirnas strongly expressed in retina .
another interesting example was sub-cluster  <dig>  strongly expressed in cornea and rpe, which showed enrichment for the go term 'epithelium development' .
overall, we found enrichment for specific processes, consistent with the physiology and function of the ocular tissues in which the analyzed mirnas were predominantly expressed.
this suggests that the above-described cluster analysis of mirna expression data, although requiring further experimental validation, can be used to gain insight into the specific functional processes regulated by mirna-target networks within the eye.
mirnas show distinct spatiotemporal distribution during eye development
to study the spatiotemporal distribution of the above-mentioned mirnas at the cellular level, we performed rna ish profiling on sections of murine eyes at different developmental stages.
to this purpose, we carried out high-throughput robotic rna ish on serial sections of whole heads and eyes  using lna-modified dna oligonucleotide probes .
the ish analysis was performed in the albino cd <dig> wild-type mouse strain in order to enable signal detection in the non-pigmented rpe.
the selection of mirnas to analyze mostly relied on the evaluation of microarray profiling data described above.
we prioritized for the analysis  <dig> mirnas that displayed the highest levels of differential expression in the adult eye by microarray.
moreover, we selected  <dig> mirnas that were not detected in a reliable manner by microarray.
given the different resolution and sensitivity of the two technologies, we decided to analyze also the latter ones to verify the ability of rna ish to detect mirnas expressed at very low or single-cell levels.
indeed, the ish protocol we used includes a signal amplification step which can reveal low abundance transcripts that would have been otherwise undetectable  <cit> .
as a final result, taking also into account technical failures and probe availability, we obtained reliable ish data for  <dig> mirnas .
for this study, we looked at time points that correspond to milestones of development and differentiation of the major eye structures.
in particular, we analyzed mirna expression at a prenatal stage , when the major structures of the eye are already formed, although not fully differentiated; at the date of birth, postnatal day   <dig>  when the newborn mouse eye is still developmentally immature; at p <dig>  when the majority of retinal cells have exited the cell-cycle and occupy their final positions within the retina and, finally, at the p <dig> stage that corresponds to full adulthood and maturation of the eye is complete.
the expression patterns obtained were annotated based on the overall signal distribution  and on the anatomical structures and/or cell-types that stained positive for mirna expression.
when a uniform signal was visible across the entire section, the pattern was defined 'ubiquitous'  .
whenever the signal was restricted to specific cells or structures the expression pattern was defined 'regional'  , while in the absence of a visible signal, the attribute 'not detected'  was assigned .
finally, an additional category, 'ubiquitous with pattern' , was defined for head sections at e <dig>  and p <dig> in cases where a signal was detected in most cells with some structures showing stronger staining .
overall, we identified a range of different spatially and/or temporally restricted expression profiles for  <dig> mirnas, while  <dig> did not yield any signal in the eye at any developmental stage.
we found a number of mirnas  that despite displaying significant differential expression by microarray, were not detected by rna ish.
this could be explained either by differences in sensitivity between the two approaches or by possible mirna expression level differences across mouse strains.
on the other hand, by rna ish we obtained a clear regional hybridization signal for  <dig> mirnas that were not detected in a reliable manner by microarray profiling .
these observations highlight the advantage of employing diverse approaches in determining mirna expression profiles.
a summary of the ish signal distributions observed at each developmental stage is shown in table  <dig>  a detailed annotation of the expression patterns at all time points is listed in additional file  <dig>  although broadly in agreement with previously published work, in some cases the ish images revealed differences in mirna distribution .
we believe that these discrepancies could be attributed to differences in the experimental conditions and, most importantly, to the improved sensitivity in the detection of low-abundance transcripts offered by the signal amplification step of the automated approach.
developmental
stage
with
detected
* na = non applicable
the ish data from the adult eye could be directly compared to the microarray profiling performed at the same stage .
a summary of mirna distribution in the different anatomical structures of the adult eye as well as in the retinal cell layers is shown in figure  <dig>  overall, the comparison between the rna ish annotation and the microarray data revealed a high degree of consistency and the combination of the two datasets largely contributes to the definition of the tissue distribution of mirnas in the eye.
1: mirnas that were annotated as 'ubiquitous' or 'not detected'
2: mirnas with Δlmr ≥  <dig>
3: mirnas labeled as na in at least one of the nine samples analyzed
4: total number of mirnas analyzed both by microarray and ish
a web atlas of mirna expression in eye structures
to make these data readily accessible we constructed mirneye, a web-searchable atlas of mirna expression patterns in the mouse eye that can be accessed at http://mirneye.tigem.it.
the atlas offers high-resolution images of the cellular distribution of mirnas in the mammalian eye and represents a useful tool in the study of mirna contribution in eye formation, maintenance and function.
combined with target prediction software, knowledge of the mirna cellular distribution, as well as of the mirna clusters of co-expression, can greatly aid in unraveling their role in the eye.
below we provide a more detailed description of the spatiotemporal mirna expression profiles we detected in each of the analyzed murine eye tissues.
mirnas show cell-type enriched expression in the retina
we found that the majority of mirnas showing, by rna ish, regional distribution in the adult eye  were detected in the retina, in one or more of its cell layers.
among them, we both found mirnas with previously reported retinal expression domains e.g.
mir-124a, mir-29c, mir- <dig>  mir- <dig>  mir-181a  <cit> , among others, but we also identified at least  <dig> mirnas, which, to our knowledge, were not previously described to have a regional expression in the retina in any vertebrate species .
most of the mirna expression patterns in the retina had a developmental onset.
at e <dig> , retina-expressed mirnas stained mainly the inner neuroblastic layer  where newborn ganglion cell neurons are located .
likewise, in retinas of newborn mice , mirna expression was mainly detected in the vitreal cell layer of the retina  where differentiated ganglion and amacrine cells reside .
we did not encounter any case in which mirna expression was detected only in the mitotic progenitors of the outer neuroblastic layer  at either e <dig>  or at p <dig>  this was expected given that the mirnas we analyzed by rna ish were selected based on a microarray study carried out on fully-differentiated, adult ocular tissues and is also in line with the general view that mirnas principally promote cell differentiation  <cit> .
in a limited number of cases , staining at p <dig> was extended also to the onbl, where immature photoreceptors are found .
for these mirnas, photoreceptor-enriched expression was further confirmed at p <dig> and p <dig> .
interestingly, among the retina-expressed mirnas, we identified mir-542-3p, that showed an apparent low dorsal to high ventral gradient of expression in the developing  and newborn  retina .
the laminar organization of the mammalian retina - in particular at the adult stage when all differentiation events have been completed - can be exploited to deduce the possible identity of cells expressing a given mirna.
on that basis, we identified mirnas  that, in the adult retina, are clearly detected in the vitreal part of the inner nuclear layer  and are likely to be expressed in amacrine cells .
another class of mirnas  also stained, in the postnatal retina, the outer nuclear layer  where rod and cone photoreceptors reside .
many of the latter mirnas were also expressed at p <dig> in the olfactory epithelium, which, similarly to the retina, is equipped with sensory epithelia .
notably, the vast majority of the regional mirnas detected in the retina stained preferentially, though to a different extent, the ganglion cell layer  and the inner nuclear layer  and to a much lesser extent the outer nuclear layer .
this is consistent with the above-described preferential staining of the inbl at e <dig>  and p <dig> and suggests that the neuronal populations that are part of the innermost retinal cell layers need, for their proper function, the expression of a wider catalog of mirnas compared to photoreceptors cells.
finally, we observed subtle variations in the spatiotemporal expression patterns of polycistronically transcribed mirnas .
we believe that they could be explained either by the differential processing of the primary transcript or by the differential affinity of the probes to their respective mature mirna.
mirnas expressed in the retinal pigment epithelium
the rpe is a monolayer of pigmented epithelium that lies between the photoreceptors of the neural retina and the choroid.
similarly to the neural retina, the rpe derives from the anterior neural plate .
exogenous signals from the surrounding periocular mesenchyme induce rpe specification whereas inputs from the surface ectoderm drive differentiation of the prospective neural retina .
the initial regionalization between the presumptive rpe and neural retina is further consolidated by the expression of distinct sets of regulators, including well-established transcription factors and possibly tissue-specific mirnas  <cit> .
by ish we detected  <dig> mirnas in the adult rpe ,  <dig> of which also stained the neural retina.
instead, from the microarray analysis, we identified  <dig> mirnas that are enriched in the rpe.
by hierarchical clustering the majority of these mirnas grouped in cluster b  and some of them were found to share expression enrichment both in rpe and retina, similar to what we observed by ish.
this similarity in the mirna content of rpe and neural retinal cells could be attributed to the common origin of the two tissues.
instead, the mirnas that are differentially expressed between rpe and retina could be those that principally contribute in the establishment and maintenance of rpe identity.
finally, mirnas that were concurrently enriched in rpe, cornea and/or lens  may include mirna signatures associated with epithelial physiology.
mirnas expressed in the cornea
the cornea is an avascular, transparent structure within the anterior eye, of prime importance for light refraction and vision.
it consists of three distinct layers: the epithelium, the stroma and the endothelium.
the epithelium derives from the surface ectoderm whereas the corneal stroma and endothelium are formed by neural crest and mesoderm-derived mesenchymal cells  <cit> .
corneal function relies on the self-renewal capacity of the epithelium which in turn is ensured by a population of limbal epithelial stem cells  mostly present in the limbus, which is located at the corneo-scleral junction  <cit> .
to date, little is known about the mirna content of the cornea and on its contribution to corneal differentiation and homeostasis.
by rna ish, mirna expression in the cornea could be detected as early as e <dig>  and p <dig> in  <dig> and  <dig> cases, respectively .
at p <dig>  when the epithelium is actively proliferating and at p <dig>  when the cornea has reached adult thickness, we could detect  <dig> and  <dig> regional mirnas, respectively .
from the latter group,  <dig> stained clearly both the corneal epithelium  and endothelium  while the remaining ones  were detected only in the epithelium .
collectively, our results confirm the cornea expression of mirnas already reported in literature  <cit>  and reveal the corneal-enrichment of others that had not been previously described to be expressed in the eye .
most importantly, the ish analysis enhances the current scarce knowledge on the cellular distribution of these mirnas within the cornea.
in particular, we can differentiate between mirnas that are expressed exclusively in the ectoderm-derived epithelium and others that are expressed both in the epithelium and the endothelium.
interestingly, among the mirnas detected in the corneal epithelium, we could highlight different patterns of distribution in the limbus.
for instance,  <dig> mirnas clearly stained the entire corneal epithelium, including the limbus , while at least  <dig> were not detected in the limbal region despite being expressed in most of the corneal epithelium .
thus, it is tempting to speculate that such differences in mirna expression within the limbus may contribute to the establishment of the proper environment required for lescs maintenance.
mirnas expressed in the lens
the lens is an elastic, transparent structure located in the anterior segment of the eye with a role in light refraction and focal distance adjustment.
during embryogenesis, the lens derives from the surface ectoderm following inductions from the neuroectoderm and consists of the capsule, a layer of cuboidal epithelium and the lens fibers.
by rna ish we detected  <dig> regional mirnas in lens, especially at prenatal  and postnatal  stages when the lens structure remains intact after sectioning.
in all cases, staining was evident in the anterior lens epithelium, where cells actively proliferate during lifetime and in the equatorial zone, where cells differentiate to lens fiber cells .
interestingly, some of the lens-enriched mirnas are expressed at significant levels in the cornea  and by ish we frequently observe concurrent lens and cornea mirna expression , probably reflecting the common origin of these epithelial tissues.
mirnas detected in other ocular structure
rna ish analysis revealed a number  of regional mirnas that stained specifically the uveal tunics of the mouse eye in particular the iris and the ciliary body  .
among these, there were mirnas with a reported role in smooth muscle   <cit>  and stem cell differentiation   <cit>  .
this is in line with the fact that both the iris and ciliary body consist mainly of smooth muscle while their pigmented epithelia are niches of progenitor cells with stem-like properties of self-renewal and multi-potentiality  <cit> .
the remarkable plasticity of these cell populations allows them, under appropriate culture conditions, to be trans-differentiated into pigmented neurospheres that express neuronal or glial markers  <cit> .
in that respect, the mirnas we detected in the iris and ciliary body  could represent important mediators of stemness and plasticity of pluripotent cells with neurogenic potential.
this is particularly true for those that will turn out to be specifically expressed in the pigmented epithelia of these structures, though further experimental validation will be needed to confirm this hypothesis.
similarly, the strong and specific staining for mir- <dig> and mir- <dig> in the iris sphincter at all developmental stages analyzed  is consistent with recent reports that demonstrate the key role of these two mirnas in modulating smooth muscle cell proliferation and differentiation  <cit> .
by microarray profiling these two mirnas are highly enriched in the rpe .
that could in part explain the potential of rpe cells to lose epithelial characteristics and transdifferentiate into myofibroblasts during pathogenesis of proliferative vitreoretinopathy  <cit> .
CONCLUSIONS
as mirnas turn out to represent crucial regulatory elements, sharing common principles with transcription factors, it is fundamental to establish their distribution and decipher their functional role in the eye in order to obtain a complete picture of the gene networks operating therein in health and disease states.
over the past few years, several mirnas have been shown to be strongly and specifically expressed in the eye, suggesting that these small molecules play indispensable regulatory roles in eye biology.
however, most of the previously published reports focused on the analysis of the mirna component of the retina.
considerably less is known on the mirna content of the rpe and of the non-retinal parts of the eye such as the lens and cornea.
in addition, there is no report of a systematic analysis of mirna expression in the eye at a cell-specific resolution achieved by rna ish on tissue sections.
hence, the work described here provides a significant contribution to the expansion of our knowledge both in the field of eye and mirna biology.
this is the first atlas of the eye mirnome that offers the possibility to explore mirna expression profiles in the various ocular structures and cell-types during mouse development.
the amplification protocol of the ish procedure we used greatly enhanced the sensitivity of signal detection revealing novel, previously overlooked mirna patterns.
in particular, we analyzed the cellular distribution in the eye of  <dig> mirnas and demonstrated a regional localization for  <dig> of them.
furthermore, the parallel mirna array analysis of the retina, rpe, cornea and lens allows a direct and semi-quantitative comparison of the mirna content across the main ocular tissues and complements the qualitative data on mirna localization obtained by rna ish.
in particular, our approach highlights that eye development, maintenance and function is accompanied by the coordinated expression of a large set of mirnas, which display distinct and overlapping patterns among the various tissues of the eye.
as mirnas are proposed to act mainly as fine-tuners of the transcriptome , identifying groups of mirnas that are expressed in a given tissue offers a precious resource in evaluating targets and pathways that direct the differentiation and maintenance of the ocular structures.
by combining techniques with different sensitivity and data output we could identify distinct mirna clusters that show comparable expression patterns in the eye.
we coupled this information with target prediction and performed go term functional analysis in an attempt to shed light to the complex mirna regulatory networks that operate during eye development and function.
the analysis revealed that mirnas with highly correlated expression profiles may contribute, via their targets, to common pathways and cellular processes that are characteristic of the tissue in which these mirnas are predominantly expressed.
finally, deciphering the expression patterns of mirnas is not only an important step towards a better comprehension of the molecular mechanisms that underlie eye function but can possibly aid the elucidation of the molecular basis of eye disease.
although a direct involvement of mirnas in the pathogenesis of eye diseases has not yet been demonstrated, the latter hypothesis is extremely likely based on recent reports demonstrating that even alterations of a single mirna can be responsible for human genetic diseases  <cit> .
in that respect, precise knowledge on the sites and timing of mirna expression in the eye will allow to evaluate whether modulation of expression levels and/or cellular distribution of specific mirnas could constitute a means of therapeutic intervention in certain eye diseases.
overall, the described mirna atlas offers a much-needed, extensive and dynamic view on the mirna inventory of the eye during development.
this information will provide the foundation for a better understanding of the mirna-mediated regulation of specific developmental pathways in the eye with eventual implications in diagnostics and therapy.

 mirnas are a class of small non-coding rnas that negatively regulate gene expression by binding to target sites in the 3' utr of mrnas. this binding affects the stability and translation of the target transcript. to date more than six hundred highly-conserved mirnas have been identified in the mouse genome proposing mirnome as a new layer of gene regulation. mirnas are key mediators of basic biological processes. in all plant and animal species examined, defects in mirna function have profound effects on development. in humans, deregulation of mirna expression caused by mutations in either the mirna or its target has been correlated with a number of clinically important diseases such as diabetes, neurodegenerative diseases and heart failure, among others. moreover, there is growing evidence supporting a role of mirnas in human cancers. in this regard, mirnas may constitute new targets of therapeutic interventions for a variety of diseases. to gain insight into the functional role of mirnas, both in physiological and pathological processes, it is essential to have precise information on their temporal and spatial expression profile. recent adaptations in tools for expression and functional analysis, developed to counteract the limitations posed by the small size of mirnas, have facilitated detection of the mirna content of several tissues. these studies revealed that about one third of mirnas are expressed in a tissue-restricted manner in vertebrates. the eye is a highly specialized organ whose development and function requires the precise coordination and timing of morphogenetic and cell differentiation events. perturbation of mirna function in the eye of conditional dicer mouse mutants impairs the normal development of the retina, lens, cornea and optic chiasm. therefore, elucidation of the functional role of mirnas is expected to provide key information to decipher the complex regulatory circuits underlying these processes. to define mirna function in the eye, it is essential to obtain a high-resolution profile of their spatial and temporal distribution. this information, combined with in silico target prediction analysis, can also optimize the recognition of biologically significant mrna targets. to date, only a limited number of reports are available on the expression profile of mirnas in the mammalian eye. the majority of these publications analyze the mirna content of the retina while only a few address mirna expression in non-neural parts of the eye. furthermore, examples of the cellular distribution of mirnas in the eye are scarce, usually describing the spatial expression profiles of a limited number of mirnas at a few developmental stages. as a consequence, the information available on the complete set of mirnas expressed in ocular tissues is only partial and fragmented. to obtain a global view of the differential mirna expression in the tissues that form the adult mouse eye, we performed microarray profiling of retina, retinal pigment epithelium, lens and cornea rna. cluster analysis allowed us to identify subgroups of mirnas enriched in each of the above tissues. these mirna clusters may reveal possible synergisms in the differentiation, cell-lineage commitment, development and maintenance of the analyzed eye structures. moreover, to obtain a high-resolution atlas of the spatiotemporal distribution of these mirnas in the eye, we performed rna in situ hybridization using locked nucleic acid -modified probes on sections of embryonic, postnatal and adult mouse eyes and implemented the data in a publicly available web database http://mirneye.tigem.it. here, we describe the mirna expression profiles in the main tissues of the eye and show the spatiotemporal distribution of mirnas. finally, we propose a possible way on how cluster analysis of the mirna differential expression can be exploited to advance our understanding of the functional role of selected mirnas in a given tissue.  mirnas show differential expression in murine eye tissues we decided to define the complete repertoire of mirnas that are expressed in any of four different eye tissues, i.e. retina, lens, cornea and rpe. as a first step, we collected retinas, lenses, corneas, rpe tissues and entire eyes from 8-week old c57bl/ mice. we chose to use the c57bl/ strain that has a pigmented rpe in order to limit the carry-over contamination of the retina samples with rpe cells during tissue dissection. following total rna extraction from these eye tissues, we carried out mirna profiling using the mircury lna™arrays provided by exiqon. total rna from entire eyes was included as a reference to determine differential expression. the mirnas that showed, in one or more tissues, a difference in log median ratios equal or superior to compared to the'entire eye' reference were considered for further analysis. to demonstrate whether mirnas show tissue-enriched expression in a specific eye compartment, the mirnas that passed the above threshold were used in a two-way hierarchical clustering analysis. this analysis revealed distinct mirna clusters that are differentially expressed in one or more of the tissues analyzed. the resulting dendrogram could be divided in five main clusters enriched in mirnas predominantly expressed in retina, rpe, retina and lens, lens and cornea, and cornea and rpe. within these five clusters, we defined smaller groups of mirnas with highly similar expression profiles. the reliability of the clustering analysis of microarray data was confirmed by assessing the overall relationships among the analyzed tissues, which were in line with their developmental origin. the significant number of mirnas that show differential expression profiles among the ocular tissues and their organization in distinct clusters suggest that the regulation of eye development and function by mirnas requires an intricate biological network. our microarray results are overall concordant with similar global expression studies on single eye tissues previously reported in mouse or other vertebrates. however, this is the first example of a simultaneous analysis of the main ocular structures, which therefore allows a direct semi-quantitative assessment of the differential mirna expression levels in the major components of the mouse eye. to identify whether groups of mirnas with comparable expression profiles may be involved in the regulation of specific biological functions, we performed an in silico functional annotation analysis of their predicted target genes using the above- described co-expression sub-clusters. for each mirna present within these sub-clusters, we compiled a non-redundant list of predicted targets combining the output of miranda, targetscan and pictar, three commonly used mirna target prediction software. subsequently, we performed a gene ontology and kegg pathway annotation analysis for the target lists of each single mirna, as described in methods. we then compared the annotation term lists of all predicted target genes for each mirna belonging to each sub-cluster and counted the occurrence of shared individual go/kegg terms. an arbitrary cut-off value of 70% was set and only terms enriched in the predicted target gene lists of at least 70% of the mirnas within a cluster were retained. as a first result, we found a significant enrichment in several go/kegg terms related to neuronal development and function, which, considering the neural origin of the eye, is a good indication of the reliability of our analysis. the above-mentioned enrichment in neuron-related terms was more prominent in sub-clusters that contained mirnas strongly expressed in retina. another interesting example was sub-cluster strongly expressed in cornea and rpe, which showed enrichment for the go term'epithelium development'. overall, we found enrichment for specific processes, consistent with the physiology and function of the ocular tissues in which the analyzed mirnas were predominantly expressed. this suggests that the above-described cluster analysis of mirna expression data, although requiring further experimental validation, can be used to gain insight into the specific functional processes regulated by mirna-target networks within the eye. mirnas show distinct spatiotemporal distribution during eye development to study the spatiotemporal distribution of the above-mentioned mirnas at the cellular level, we performed rna ish profiling on sections of murine eyes at different developmental stages. to this purpose, we carried out high-throughput robotic rna ish on serial sections of whole heads and eyes using lna-modified dna oligonucleotide probes. the ish analysis was performed in the albino cd wild-type mouse strain in order to enable signal detection in the non-pigmented rpe. the selection of mirnas to analyze mostly relied on the evaluation of microarray profiling data described above. we prioritized for the analysis mirnas that displayed the highest levels of differential expression in the adult eye by microarray. moreover, we selected mirnas that were not detected in a reliable manner by microarray. given the different resolution and sensitivity of the two technologies, we decided to analyze also the latter ones to verify the ability of rna ish to detect mirnas expressed at very low or single-cell levels. indeed, the ish protocol we used includes a signal amplification step which can reveal low abundance transcripts that would have been otherwise undetectable. as a final result, taking also into account technical failures and probe availability, we obtained reliable ish data for mirnas. for this study, we looked at time points that correspond to milestones of development and differentiation of the major eye structures. in particular, we analyzed mirna expression at a prenatal stage, when the major structures of the eye are already formed, although not fully differentiated; at the date of birth, postnatal day when the newborn mouse eye is still developmentally immature; at p when the majority of retinal cells have exited the cell-cycle and occupy their final positions within the retina and, finally, at the p stage that corresponds to full adulthood and maturation of the eye is complete. the expression patterns obtained were annotated based on the overall signal distribution and on the anatomical structures and/or cell-types that stained positive for mirna expression. when a uniform signal was visible across the entire section, the pattern was defined'ubiquitous'. whenever the signal was restricted to specific cells or structures the expression pattern was defined'regional', while in the absence of a visible signal, the attribute'not detected' was assigned. finally, an additional category,'ubiquitous with pattern', was defined for head sections at e and p in cases where a signal was detected in most cells with some structures showing stronger staining. overall, we identified a range of different spatially and/or temporally restricted expression profiles for mirnas, while did not yield any signal in the eye at any developmental stage. we found a number of mirnas that despite displaying significant differential expression by microarray, were not detected by rna ish. this could be explained either by differences in sensitivity between the two approaches or by possible mirna expression level differences across mouse strains. on the other hand, by rna ish we obtained a clear regional hybridization signal for mirnas that were not detected in a reliable manner by microarray profiling. these observations highlight the advantage of employing diverse approaches in determining mirna expression profiles. a summary of the ish signal distributions observed at each developmental stage is shown in table a detailed annotation of the expression patterns at all time points is listed in additional file although broadly in agreement with previously published work, in some cases the ish images revealed differences in mirna distribution. we believe that these discrepancies could be attributed to differences in the experimental conditions and, most importantly, to the improved sensitivity in the detection of low-abundance transcripts offered by the signal amplification step of the automated approach. developmental stage with detected * na = non applicable the ish data from the adult eye could be directly compared to the microarray profiling performed at the same stage. a summary of mirna distribution in the different anatomical structures of the adult eye as well as in the retinal cell layers is shown in figure overall, the comparison between the rna ish annotation and the microarray data revealed a high degree of consistency and the combination of the two datasets largely contributes to the definition of the tissue distribution of mirnas in the eye. 1: mirnas that were annotated as'ubiquitous' or'not detected' 2: mirnas with Δlmr ≥ 3: mirnas labeled as na in at least one of the nine samples analyzed 4: total number of mirnas analyzed both by microarray and ish a web atlas of mirna expression in eye structures to make these data readily accessible we constructed mirneye, a web-searchable atlas of mirna expression patterns in the mouse eye that can be accessed at http://mirneye.tigem.it. the atlas offers high-resolution images of the cellular distribution of mirnas in the mammalian eye and represents a useful tool in the study of mirna contribution in eye formation, maintenance and function. combined with target prediction software, knowledge of the mirna cellular distribution, as well as of the mirna clusters of co-expression, can greatly aid in unraveling their role in the eye. below we provide a more detailed description of the spatiotemporal mirna expression profiles we detected in each of the analyzed murine eye tissues. mirnas show cell-type enriched expression in the retina we found that the majority of mirnas showing, by rna ish, regional distribution in the adult eye were detected in the retina, in one or more of its cell layers. among them, we both found mirnas with previously reported retinal expression domains e.g. mir-124a, mir-29c, mir- mir- mir-181a, among others, but we also identified at least mirnas, which, to our knowledge, were not previously described to have a regional expression in the retina in any vertebrate species. most of the mirna expression patterns in the retina had a developmental onset. at e, retina-expressed mirnas stained mainly the inner neuroblastic layer where newborn ganglion cell neurons are located. likewise, in retinas of newborn mice, mirna expression was mainly detected in the vitreal cell layer of the retina where differentiated ganglion and amacrine cells reside. we did not encounter any case in which mirna expression was detected only in the mitotic progenitors of the outer neuroblastic layer at either e or at p this was expected given that the mirnas we analyzed by rna ish were selected based on a microarray study carried out on fully-differentiated, adult ocular tissues and is also in line with the general view that mirnas principally promote cell differentiation. in a limited number of cases, staining at p was extended also to the onbl, where immature photoreceptors are found. for these mirnas, photoreceptor-enriched expression was further confirmed at p and p. interestingly, among the retina-expressed mirnas, we identified mir-542-3p, that showed an apparent low dorsal to high ventral gradient of expression in the developing and newborn retina. the laminar organization of the mammalian retina - in particular at the adult stage when all differentiation events have been completed - can be exploited to deduce the possible identity of cells expressing a given mirna. on that basis, we identified mirnas that, in the adult retina, are clearly detected in the vitreal part of the inner nuclear layer and are likely to be expressed in amacrine cells. another class of mirnas also stained, in the postnatal retina, the outer nuclear layer where rod and cone photoreceptors reside. many of the latter mirnas were also expressed at p in the olfactory epithelium, which, similarly to the retina, is equipped with sensory epithelia. notably, the vast majority of the regional mirnas detected in the retina stained preferentially, though to a different extent, the ganglion cell layer and the inner nuclear layer and to a much lesser extent the outer nuclear layer. this is consistent with the above-described preferential staining of the inbl at e and p and suggests that the neuronal populations that are part of the innermost retinal cell layers need, for their proper function, the expression of a wider catalog of mirnas compared to photoreceptors cells. finally, we observed subtle variations in the spatiotemporal expression patterns of polycistronically transcribed mirnas. we believe that they could be explained either by the differential processing of the primary transcript or by the differential affinity of the probes to their respective mature mirna. mirnas expressed in the retinal pigment epithelium the rpe is a monolayer of pigmented epithelium that lies between the photoreceptors of the neural retina and the choroid. similarly to the neural retina, the rpe derives from the anterior neural plate. exogenous signals from the surrounding periocular mesenchyme induce rpe specification whereas inputs from the surface ectoderm drive differentiation of the prospective neural retina. the initial regionalization between the presumptive rpe and neural retina is further consolidated by the expression of distinct sets of regulators, including well-established transcription factors and possibly tissue-specific mirnas. by ish we detected mirnas in the adult rpe, of which also stained the neural retina. instead, from the microarray analysis, we identified mirnas that are enriched in the rpe. by hierarchical clustering the majority of these mirnas grouped in cluster b and some of them were found to share expression enrichment both in rpe and retina, similar to what we observed by ish. this similarity in the mirna content of rpe and neural retinal cells could be attributed to the common origin of the two tissues. instead, the mirnas that are differentially expressed between rpe and retina could be those that principally contribute in the establishment and maintenance of rpe identity. finally, mirnas that were concurrently enriched in rpe, cornea and/or lens may include mirna signatures associated with epithelial physiology. mirnas expressed in the cornea the cornea is an avascular, transparent structure within the anterior eye, of prime importance for light refraction and vision. it consists of three distinct layers: the epithelium, the stroma and the endothelium. the epithelium derives from the surface ectoderm whereas the corneal stroma and endothelium are formed by neural crest and mesoderm-derived mesenchymal cells. corneal function relies on the self-renewal capacity of the epithelium which in turn is ensured by a population of limbal epithelial stem cells mostly present in the limbus, which is located at the corneo-scleral junction. to date, little is known about the mirna content of the cornea and on its contribution to corneal differentiation and homeostasis. by rna ish, mirna expression in the cornea could be detected as early as e and p in and cases, respectively. at p when the epithelium is actively proliferating and at p when the cornea has reached adult thickness, we could detect and regional mirnas, respectively. from the latter group, stained clearly both the corneal epithelium and endothelium while the remaining ones were detected only in the epithelium. collectively, our results confirm the cornea expression of mirnas already reported in literature and reveal the corneal-enrichment of others that had not been previously described to be expressed in the eye. most importantly, the ish analysis enhances the current scarce knowledge on the cellular distribution of these mirnas within the cornea. in particular, we can differentiate between mirnas that are expressed exclusively in the ectoderm-derived epithelium and others that are expressed both in the epithelium and the endothelium. interestingly, among the mirnas detected in the corneal epithelium, we could highlight different patterns of distribution in the limbus. for instance, mirnas clearly stained the entire corneal epithelium, including the limbus, while at least were not detected in the limbal region despite being expressed in most of the corneal epithelium. thus, it is tempting to speculate that such differences in mirna expression within the limbus may contribute to the establishment of the proper environment required for lescs maintenance. mirnas expressed in the lens the lens is an elastic, transparent structure located in the anterior segment of the eye with a role in light refraction and focal distance adjustment. during embryogenesis, the lens derives from the surface ectoderm following inductions from the neuroectoderm and consists of the capsule, a layer of cuboidal epithelium and the lens fibers. by rna ish we detected regional mirnas in lens, especially at prenatal and postnatal stages when the lens structure remains intact after sectioning. in all cases, staining was evident in the anterior lens epithelium, where cells actively proliferate during lifetime and in the equatorial zone, where cells differentiate to lens fiber cells. interestingly, some of the lens-enriched mirnas are expressed at significant levels in the cornea and by ish we frequently observe concurrent lens and cornea mirna expression, probably reflecting the common origin of these epithelial tissues. mirnas detected in other ocular structure rna ish analysis revealed a number of regional mirnas that stained specifically the uveal tunics of the mouse eye in particular the iris and the ciliary body. among these, there were mirnas with a reported role in smooth muscle and stem cell differentiation. this is in line with the fact that both the iris and ciliary body consist mainly of smooth muscle while their pigmented epithelia are niches of progenitor cells with stem-like properties of self-renewal and multi-potentiality. the remarkable plasticity of these cell populations allows them, under appropriate culture conditions, to be trans-differentiated into pigmented neurospheres that express neuronal or glial markers. in that respect, the mirnas we detected in the iris and ciliary body could represent important mediators of stemness and plasticity of pluripotent cells with neurogenic potential. this is particularly true for those that will turn out to be specifically expressed in the pigmented epithelia of these structures, though further experimental validation will be needed to confirm this hypothesis. similarly, the strong and specific staining for mir- and mir- in the iris sphincter at all developmental stages analyzed is consistent with recent reports that demonstrate the key role of these two mirnas in modulating smooth muscle cell proliferation and differentiation. by microarray profiling these two mirnas are highly enriched in the rpe. that could in part explain the potential of rpe cells to lose epithelial characteristics and transdifferentiate into myofibroblasts during pathogenesis of proliferative vitreoretinopathy.  as mirnas turn out to represent crucial regulatory elements, sharing common principles with transcription factors, it is fundamental to establish their distribution and decipher their functional role in the eye in order to obtain a complete picture of the gene networks operating therein in health and disease states. over the past few years, several mirnas have been shown to be strongly and specifically expressed in the eye, suggesting that these small molecules play indispensable regulatory roles in eye biology. however, most of the previously published reports focused on the analysis of the mirna component of the retina. considerably less is known on the mirna content of the rpe and of the non-retinal parts of the eye such as the lens and cornea. in addition, there is no report of a systematic analysis of mirna expression in the eye at a cell-specific resolution achieved by rna ish on tissue sections. hence, the work described here provides a significant contribution to the expansion of our knowledge both in the field of eye and mirna biology. this is the first atlas of the eye mirnome that offers the possibility to explore mirna expression profiles in the various ocular structures and cell-types during mouse development. the amplification protocol of the ish procedure we used greatly enhanced the sensitivity of signal detection revealing novel, previously overlooked mirna patterns. in particular, we analyzed the cellular distribution in the eye of mirnas and demonstrated a regional localization for of them. furthermore, the parallel mirna array analysis of the retina, rpe, cornea and lens allows a direct and semi-quantitative comparison of the mirna content across the main ocular tissues and complements the qualitative data on mirna localization obtained by rna ish. in particular, our approach highlights that eye development, maintenance and function is accompanied by the coordinated expression of a large set of mirnas, which display distinct and overlapping patterns among the various tissues of the eye. as mirnas are proposed to act mainly as fine-tuners of the transcriptome, identifying groups of mirnas that are expressed in a given tissue offers a precious resource in evaluating targets and pathways that direct the differentiation and maintenance of the ocular structures. by combining techniques with different sensitivity and data output we could identify distinct mirna clusters that show comparable expression patterns in the eye. we coupled this information with target prediction and performed go term functional analysis in an attempt to shed light to the complex mirna regulatory networks that operate during eye development and function. the analysis revealed that mirnas with highly correlated expression profiles may contribute, via their targets, to common pathways and cellular processes that are characteristic of the tissue in which these mirnas are predominantly expressed. finally, deciphering the expression patterns of mirnas is not only an important step towards a better comprehension of the molecular mechanisms that underlie eye function but can possibly aid the elucidation of the molecular basis of eye disease. although a direct involvement of mirnas in the pathogenesis of eye diseases has not yet been demonstrated, the latter hypothesis is extremely likely based on recent reports demonstrating that even alterations of a single mirna can be responsible for human genetic diseases. in that respect, precise knowledge on the sites and timing of mirna expression in the eye will allow to evaluate whether modulation of expression levels and/or cellular distribution of specific mirnas could constitute a means of therapeutic intervention in certain eye diseases. overall, the described mirna atlas offers a much-needed, extensive and dynamic view on the mirna inventory of the eye during development. this information will provide the foundation for a better understanding of the mirna-mediated regulation of specific developmental pathways in the eye with eventual implications in diagnostics and therapy. 
we initially determined the expression profiles of mirnas in the retina, lens, cornea and retinal pigment epithelium of the adult mouse eye by microarray. in this report, we present the first comprehensive survey of mirna expression in ocular tissues, using both microarray and rna in situ hybridization procedures. each tissue exhibited notably distinct mirna enrichment patterns and cluster analysis identified groups of mirnas that showed predominant expression in specific ocular tissues or combinations of them. this analysis revealed mirnas with differential expression in ocular tissues and provided a detailed atlas of their tissue-specific distribution during development of the murine eye. we found that mirnas displayed restricted expression domains in the eye at different developmental stages, with the majority of them expressed in one or more cell layers of the neural retina. to define mirna function in the eye, it is essential to determine a high-resolution profile of their spatial and temporal distribution. the combination of the two approaches offers a valuable resource to decipher the contributions of specific mirnas and mirna clusters to the development of distinct ocular structures. next, we performed rna ish for over mirnas, including those showing the highest expression levels by microarray, and generated a high-resolution expression atlas of mirnas in the developing and adult wild-type mouse eye, which is accessible in the form of a publicly available web database.
 micrornas are key regulators of biological processes. to define mirna function in the eye, it is essential to determine a high-resolution profile of their spatial and temporal distribution.  in this report, we present the first comprehensive survey of mirna expression in ocular tissues, using both microarray and rna in situ hybridization procedures. we initially determined the expression profiles of mirnas in the retina, lens, cornea and retinal pigment epithelium of the adult mouse eye by microarray. each tissue exhibited notably distinct mirna enrichment patterns and cluster analysis identified groups of mirnas that showed predominant expression in specific ocular tissues or combinations of them. next, we performed rna ish for over mirnas, including those showing the highest expression levels by microarray, and generated a high-resolution expression atlas of mirnas in the developing and adult wild-type mouse eye, which is accessible in the form of a publicly available web database. we found that mirnas displayed restricted expression domains in the eye at different developmental stages, with the majority of them expressed in one or more cell layers of the neural retina.  this analysis revealed mirnas with differential expression in ocular tissues and provided a detailed atlas of their tissue-specific distribution during development of the murine eye. the combination of the two approaches offers a valuable resource to decipher the contributions of specific mirnas and mirna clusters to the development of distinct ocular structures. 