neural stem cells ( nscs ) are multipotent cells that can give rise to various cell types in the central nervous system ( cns ) , including neurons , astrocytes , and oligodendrocytes ( alvarez - buylla et al . , 2002 ) .
in adult mammals , neurogenesis is limited to two brain regions ; the subventricular zone ( svz ) by the lateral ventricles and the subgranular zone ( sgz ) of the dentate gyrus in the hippocampus ( reviewed in zhao et al . , 2008 ) .
nscs in the svz , so called type - b cells , are of astrocytic lineage and generate transient amplifying type - c cells .
neuroblasts then migrate along a defined pathway , the rostral migratory stream , to mature and form interneurons in the olfactory bulb ( doetsch et al . , 1999 ) .
they generate type 2 intermediate proliferating progenitors that subsequently give rise to type 3 neuroblasts migrating into the subgranular layer , to become mature functional glutamatergic granule neurons ( kempermann et al . , 2004 ) .
although the functional role of adult neurogenesis still is debated , it is undoubtedly a complex and highly regulated process .
recently , micrornas ( mirnas ) have been suggested as players of neurogenesis , controlling expression of key regulatory genes ( gao , 2010 ; shi et al . , 2010 ; luikart et al . , 2011 ) .
mirnas are one of the central determinants of mrna abundance , functioning as standby mediators of mrna regulation .
they are small ( 2123 nucleotides long ) , non - coding endogenously expressed rna that bind to complementary mrna targets , resulting in a decrease in target mrna activity ( bartel , 2009 ) . since the discovery of the first mirna in 1993 , they have been identified in animals , plants , and viruses and more than 1000 mirna sequences have so far been found in humans ( www.mirbase.org ) . many are conserved across species .
some have a general expression pattern , others are specifically expressed in certain tissues or cell types and expression can be spatially and temporally restricted .
several labs have utilized conditional deletion of dicer , a key enzyme in mirna biogenesis , as a tool to study the role of mirnas in neurogenesis .
disruption of the function of dicer during brain development results in gross anatomical changes and in some cases it is embryonic lethal ( giraldez et al . , 2005 ; davis et al . , 2008 ; de pietri tonelli et al .
, nscs can be cultured in the absence of dicer but are unable to generate neurons or astrocytes upon differentiation ( andersson et al . , 2010 ) .
although , dicer knockout ( ko ) studies indicate that mirnas are important regulators of neurogenesis and nsc self - renewal , they are difficult to interpret . not only because the simultaneous deficiency of all mirnas , but the long half - life of mature mirna makes conditional dicer ko experiments difficult to control from a temporal aspect , moreover there are concerns that non - mirna related functions of dicer contribute to the observed phenotypes ( konopka et al .
. for example , when dicer was conditionally deleted in the retinal pigmented epithelium , cell death occurred via a mechanism that is independent on mirna , rather depending on the accumulation of retrotransposon transcripts ( kaneko et al . , 2011 ) .
with this in mind , studies of the functional role of individual mirnas are a necessity . in 2007 there were four simultaneous reports of mirna ko mice ( rodriguez et al . , 2007 ; thai et al . , 2007 ; van rooij et al .
, 2007 ; zhao et al . , 2007 ) , which enabled loss - of - function studies of specific mirnas .
however , for several mirna , including many of those involved in neurogenesis , a classic ko strategy is complicated to apply due to single mirna species being located in multiple copies in separate regions of the genome , in clusters or located within other genes .
one example of such a mirna family is mir-124 , which is transcribed as three different primary transcripts from three independent loci .
mir-124 - 1 , mir-124 - 2 , and mir-124 - 3 all produce the same mature mirna ( griffiths - jones , 2006 ) .
another example is mir-9 which is expressed from three independent loci , mir-9 - 1 , mir-9 - 2 , and mir-9 - 3 .
the mature sequence is identical , and conserved in vertebrates and mammals . with let-7 the situation is even more complex with 12 human let-7 genes encoding for nine distinct , but closely related , mature forms of the mirna ( griffiths - jones , 2006 ) .
recently , a ko mouse for mir-124 - 1 was described showing that reduction of mir-124 has severe consequences for neuronal survival and axonal outgrowth ( sanuki et al . , 2011 ) .
while this study demonstrates the importance of mir-124 , it also highlights the problems of using a classic ko strategy .
( 2011 ) that compensation of mir-124 - 2 and mir-124 - 3 influence the phenotype of the mir-124 - 1 ko mouse . likewise , a ko mice that have two of the three copies of mir-9 deleted has been reported ( shibata et al . ,
these two reports suggest that conditional deletion of all three copies of either mir-9 or mir-124 is necessary in order to fully understand the role of these mirnas in adult neurogenesis .
although challenging and time consuming , such a strategy may be feasible for mir-9 and mir-124 .
however , for let-7 such a strategy appears unlikely to be successful given the large size of the let-7-family . adding to this , in situ analysis of the expression profile of individual mirna is difficult due to the small size of the mature mirna , which leads to poor resolution obtained in the brain with current histological techniques . as such , the study of the functional role of individual mirnas in vivo is complicated and makes interpretation and comparison between different studies challenging .
we describe the current status of the field , existing attempts to study loss of mirna function , and point out technical limitations that need to be circumvented in order to move the field forward .
it is fairly straightforward to profile mirna - expression patterns from bulk rna samples , either at single species resolution using for example northern blot or pcr - techniques , or at a global level using mirna arrays , pcr - array , deep sequencing of small rnas or other more specialized platforms .
these methods all have their innate differences and parallel analysis of the same samples using different techniques may give significantly different results ( see , e.g. , hebert and nelson , 2011 for a discussion on this matter ) .
since there is currently no  gold standard  for transcriptional profiling of mirna , the use of independent techniques to verify results is therefore necessary .
nevertheless , these approaches have revealed the complexity of mirna - expression patterns among different cell types and have allowed identification of a number of candidate mirnas that appear to be enriched in cultured nscs . however , the technical difficulties of purifying populations of nscs and progenitors from in vivo material , using for example fluorescence activated cell sorting , make these approaches problematic to transfer to the in vivo setting ( see table 1 ) .
histological approaches to study mirna expression in brain tissue have to a great extent relied on in situ hybridization ( ish ) techniques . due to the small size of the mirna
it is not possible to use standard ish protocols ; an additional fixation step of the mirna is needed and probe hybridization must be optimal ( pena et al . , 2009 ) .
locked nucleic acid ( lna ) modified oligonucleotides is preferable to use , since the melting temperature of the lna probe / mirna duplex is increased , resulting in stringent hybridization conditions , which in turn increases specificity and sensitivity ( reviewed in obernosterer et al . , 2007 ; wheeler et al . , 2007 ) .
to do so , additional probes that target all the various precursor transcripts need to be used ( obernosterer et al . ,
however , this can be technically challenging when analyzing mirnas with multiple precursor transcripts ( such as mir-9 or mir-124 ) .
furthermore , the results from this method are of limited resolution , thereby making it difficult to distinguish between two adjacent cells . in addition , ish is also problematic to use in combination with other labeling techniques that are routinely used in nsc - research .
we have in our lab not been able to adopt protocols that allow the use of mirna  ish in combination with , for example brdu - labeling , which is widely used in this field .
this is primarily due to the stringent treatment of the tissue that is necessary for ish , which is incompatible with the tissue treatments for brdu - labeling .
the problem of in situ mirna - expression analysis is a major concern for the study of mirna in the nervous system where it is essential to understand the cellular localization with regards to functionality .
more recently , mirna reporter or sensor vectors have been used to visualize the expression pattern of endogenous mirna in cells .
these are gene transfer vectors that contain a reporter gene ( i.e. , gfp ) along with binding sites for specific mirna ( mansfield et al .
. in the case that a cell is actively expressing the specific mirna , the expression of the reporter gene will be suppressed by the binding of the mirna to the complimentary binding sites .
thus , this system reports the absence of the target mirna , and cells that do not express it will be gfp - positive .
this technique is highly specific , simple , and robust and makes it possible to study mirna expression as cells differentiate .
injection of a mir-124 reporter vector into the brain will separate reporter gene expression between different cell types such as neurons and astrocytes ( colin et al . , 2009 ) . the system has been used to segregate differentiated neural cells in pluripotent cell cultures , based on the expression of mir-292 that is expressed in embryonic stem cells but not in nscs ( sachdeva et al . , 2010 ) , as well as the opposite where a mirna let-7a reporter was used to select undifferentiated cells from more differentiated cells ( di stefano et al . , 2011 ) .
in nscs in vivo , sensor vectors have been used to track the expression of mir-132 in the dg ( luikart et al .
the use of sensor vectors has the advantage that they measure the activity of the mirna rather than expression per se allowing easy determination if the mature mirna is present .
also , the use of a fluorescent reporter gene allows excellent morphological analysis of cells in vivo .
still , the technique is time - consuming including the generation of viral vectors followed by experimentation in cell culture or in vivo models .
however , the versatility of the technique opens up the possibility of generating transgenic reporter animals , making it possible to visualize the expression pattern of a specific mirna throughout an organism over time ( see , e.g. , gentner et al . , 2010 ) .
although this approach remains to be tested in the cns , it may be an attractive alternative in order to achieve sensitive , high - quality expression analysis of mirnas in vivo .
as mentioned above , generation of ko mice for individual mirnas is often complicated since many mirnas are present in several copies or in clusters while some are present within introns of genes . to circumvent this issue several knockdown or inhibition approaches
an early approach to analyze specific mirnas was by using anti - mirna oligonucleotide ( amo ) which are nucleic acids that are antisense to the mirna , thus hindering the interaction between the mirna and target mrna .
this technique was first used to inactivate mir-2 and mir-13 in drosophila , in the search for mirna - target genes ( boutla et al . , 2003 ) .
it was soon found that unmodified oligonucleotides are ineffective since the cellular machinery degrades them .
2-o - methyl amo is a simple chemical modification where the methyl group reduces the chances of endonucleolytic cleavage , and improves binding affinity to the mirna ( weiler et al . , 2006 ) .
another variant is 2-o - methoxyethyl amo , which are fully modified oligonucleotides , with higher affinity and specificity than 2-o - methyl amo variants ( davis et al . , 2006 ; esau et al . ,
recently , lna ( locked nucleic acid ) modified oligonucleotides allow for even further stabilization of the mirna / target duplex structure improving silencing and also making it possible to the use small oligonucleotides that enables targeting of entire mirna - families ( vester and wengel , 2004 ; obad et al . , 2011 ) .
anti - mirna oligonucleotide targets single mirnas with high specificity , as they are completely complementary to the mature sequence of the mirna ( boutla et al . , 2003 ) . despite the successful knockdown of mirna in vitro and in vivo using this method , it has several limitations .
first , a direct measurement of the down regulation of mirna is difficult , because amo binds to the mirna and sequesters it from its target rather than inducing its degradation ( krutzfeldt et al . , 2005 ; davis et al . , 2009 ) .
therefore the only possible way to confirm the decrease of mirna is to use indirect methods , whereby one can measure the level of expression of a reporter gene containing a target sequence of the mirna or analyzing upregulation of endogenous target genes .
secondly it is not possible to identify the cells in which the amos are active as they do not carry a reporter . on top of that
, the level of amo should preferably be kept constant to allow a continuous sequestration of the mirna .
oligonucleotide antagomirs are chemically modified ; cholesterol conjugated single stranded rna analogs complementary to a target mirna ( krutzfeldt et al . , 2005 ) .
the modification includes a partial phosphorothioate backbone in addition to 2-o - methoxyethyl to inhibit ago-2-mediated cleavage . in vivo
, antagomirs have been given through intravenous injection where they appear to efficiently target mirna in various tissues .
however , antagomirs do not cross the blood brain barrier , which means that in the brain , antagomirs have the same limitations as amos have .
microrna sponges are transcripts expressed from strong promoters , containing multiple , tandem binding sites to a selected member of the mirna seed family of interest ( ebert et al . , 2007 ) .
the binding site is imperfect , containing a bulge , for preventing rna interference cleavage and degradation of the sponge rna through endonucleolytic cleavage by ago-2 .
the main advantage of mirna sponges is the possibility to achieve stable expression from integrated transgenes in vivo ( gentner et al . , 2009 ) .
this can be used for studying long - term effects of mirna loss - of - function and also allows for stably expressing cell lines to be generated .
the use of vector - mediated delivery also enables the incorporation of a reporter gene in order to identify the modified cells .
another advantage is that sponges complementarily bind to the seed sequence of the mirna , which means that one sponge can target an entire family of mirnas . in summary , these features make sponge vectors an attractive approach to study mirna function in vivo in nscs . recently ; this technology was employed to demonstrate that mir-132 affects the integration of new - born neurons in the adult hippocampus ( luikart et al . , 2011 ) .
a previous report used a retrovirus expressing the cre - recombinase to delete a floxed mir-132 gene allowing a side - by - side comparison of sponge vs. conditional ko ( magill et al . , 2010 ; luikart et al . , 2011 ) .
the sponge recapitulates some , but not all , defects detected following a complete deletion of mir-132 suggesting that the use of a sponge reduces rather than eliminates levels of mir-132 ( magill et al .
, 2010 ; luikart et al . , 2011 ) . as with the use of amos ,
the sponge vectors also have other limitations , including the difficulty of validating the down regulation of a specific mirna and the only possible way to confirm the decrease of mirna is to use indirect methods as described above .
taken together , it is evident that is technically challenging to perform loss - of - function studies of mirnas in vivo in nscs .
the problem of validating the inhibition , the use of transient systems together with the appearance of potential  off - target  effects makes the interpretation of several studies challenging .
an example of this are two studies performed in the developing chick , one report suggested that mir-124 plays no role in neurogenesis while another found that mir-124 modestly promotes neurogenesis ( cao et al . , 2007 ; visvanathan et al . , 2007 ) .
direct delivery of mirna - duplexes or the use of various plasmid based approaches and viral vectors have been effectively used to overexpress mirna .
there are several relatively simple designs of vectors enabling stable expression that all appear to work efficiently ( krutzfeldt et al . , 2006 ;
the statement that gain of function studies are easier to perform than loss of function studies is reflected in the literature and a great extent of the insight gained in the mirna field is from overexpression studies . in the last part of this review we discuss in detail three mirnas that have been functionally implicated in neurogenesis ; mir-9 , mir-124 , and the let-7 family .
we give an overview of the current understanding of how these mirnas influence neurogenesis and also highlight the technical shortcomings that still prevent a full understanding of the role of these mirnas in vivo .
in addition to the three above - mentioned mirna , there is a growing literature of other mirnas , including for example mir-125b , mir-132 , mir-137 , and mir-184 that influence neurogenesis ( le et al . , 2009 ; liu et al . , 2010 ; magill et al .
, 2010 ; szulwach et al . , 2010 ; luikart et al . , 2011 ; sun et al . , 2011 ) .
several of the technical problems that limit our understanding of mir-9 , mir-124 , and the let-7 family also hold true for other mirnas .
mir-124 is perhaps the best characterized brain - specific mirna , and accounts for 2548% of all brain mirna ( lagos - quintana et al . , 2002 ) .
mir-124 is expressed in neurons and have been proposed to suppress non - neural transcripts to promote neural identity ( visvanathan et al .
, 2007 ; de pietri tonelli et al . , 2008 ; maiorano and mallamaci , 2009 ; farrell et al . , 2011 ; liu et al . , 2011a ; yoo et al . , 2011 )
several studies suggest that mir-124 is not expressed in other cells of the cns such as astrocytes ( see , e.g. , smirnova et al . , 2005 ) while ponomarev et al .
( 2010 ) also found it to be expressed in microglia and downregulated in activated microglia , being a key regulator of microglia quiescence .
mir-124 is not expressed in nscs but is suggested to regulate the temporal progression of neurogenesis in svz .
it is upregulated in the transition between type - c and type - a cells and further upregulated as the neuroblasts exit the cell cycle ( cheng et al . , 2009 ) .
delivery of mir-124 to mouse or human cells in vitro causes the global expression profile of mrna to shift toward that of the brain .
( 2008 ) used a dna plasmid with primary mir-124 transcript whereas yoo et al .
( 2011 ) delivered mir-124 with a lentiviral vector harboring a mir-124 precursor , all resulting in promoting a neural phenotype .
in addition , transfection of mir-124 duplexes into glioblastoma cells inhibits proliferation and induces differentiation ( silber et al . , 2008 ) .
in vitro loss of function , by using antisense 2-o - methyl amo , results in delayed neurite outgrowth ( yu et al . , 2008 ) and upregulation of non - neural transcripts ( conaco et al . , 2006 ) . blocking mir-124 in svz cell populations in vivo by delivering antisense 2-o - methyl amo by a micro - osmotic pump into the ventricle ,
maintains neural progenitors as dividing precursors ( cheng et al . , 2009 ) . on the contrary ,
injecting a retrovirus overexpressing mir-124 into the svz promotes precocious neural maturation ( cheng et al . , 2009 ) .
although mir-124 has been reported to be an important regulator of neurogenesis both in the developing and the adult brain , contradictory findings have been published suggesting mir-124 to be less important for neurogenesis in the developing spinal cord ( cao et al .
based on in vitro mir-124 suppression or overexpression experiments , numerous mir-124 target genes have been found and validated .
mir-124 has been shown to suppress several components of the re1 silencing transcription factor ( rest ) pathway ( conaco et al . , 2006 ;
rest is a master regulator of neuronal phenotype ( lunyak and rosenfeld , 2005 ) and together with co - repressors it recruits histone deacetylases to suppress non - neural genes . mir-124 and rest act reciprocally ; mir-124 represses rest in neurons to promote expression of neural genes , whereas rest downregulates mir-124 in non - neural cells to inhibit expression of neural genes ( conaco et al . , 2006 ) .
another mir-124 target is ptbp1 ( makeyev et al . , 2007 ) , a repressor of alternative splicing in non - neural cells demonstrating that mir-124 promotes a neuronal transcriptome by altering splicing .
these targets suggest that mir-124 expression maintains a neuronal transcriptome by repressing non - neuronal genes at several levels .
other targets include jagged1 in the notch signaling pathway ( cheng et al . , 2009 ; liu et al . ,
these targets suggest that mir-124 also plays a role in regulating the exit of a self - renewing state of nsc . however , these are only a few examples since computational algorithms suggest more than 1000 mir-124 targets ( griffiths - jones , 2006 ) .
the large number of mir-124 target genes together with the observation that overexpression of mir-124 can induce a neuronal gene program suggest that mir-124 plays a crucial role in establishing and maintaining a neuronal transcription network . in light of this , it is surprising that the in vivo phenotypes found when blocking mir-124 using antisense technology is subtle , characterized mainly by a delay in differentiation or without detectable malformations ( cao et al . , 2007 ;
however , a recent report using a classic ko strategy shows that deletion of mir-124 - 1 leads to major developmental phenotypes including small brain size , defective axonal outgrowth , and cell death confirming a crucial role for mir-124 in neurogenesis ( sanuki et al . , 2011 ) .
as mentioned above , it is clear that compensation of mir-124 - 2 and mir-124 - 3 influence the phenotype of the mir-124-ko mouse , which highlights the need for generation of a conditional triple - mir-124-ko mouse .
although this will be challenging and time consuming , such a strategy is necessary in order to understand the role of mir-124 during brain development and will assist the understanding of mir-124 in adult neurogenesis . however , since the use of retroviral or lentiviral vectors allow targeting of nscs in vivo , the application of stable inhibition vectors using for example mirna sponges to reassess the role of mir-124 in adult nscs is an interesting alternative .
another well - studied brain - specific mirna involved in neurogenesis is mir-9 that is expressed in nscs and upregulated upon neural differentiation ( krichevsky et al . , 2006 ; de pietri tonelli et al . , 2008 ; zhao et al . , 2008 ; laneve et al . ,
2010 ; bonev et al . , 2011 ; shibata et al . , 2011 ;
initial studies , done in zebrafish , showed that mir-9 directs late organizer activity of the midbrain hindbrain boundary ( mhb ; leucht et al . , 2008 ) .
the mhb is an organizing center in the vertebrate neural tube essential for proper development of the midbrain and anterior hindbrain ( wurst and bally - cuif , 2001 ) . in human cells
, mir-9 was found to have an important role in migration and proliferation of nscs .
knockdown experiments in neurospheres , using an lna antisense probe , led to reduced proliferation , and an increased migration ( delaloy et al . , 2010 ) .
in contrast , zhao et al . found reduced proliferation accompanied with increased differentiation of mouse nscs when overexpressing mir-9 by rna duplexes .
when they knocked down mir-9 with 2-o - methyl amo , proliferation increased ( zhao et al . , 2009 ) . in mouse , mir-9 has been shown to have a regional diversity along the anterior / posterior - axis ; knockdown in hindbrain leads to a failure of cell cycle , promoting proliferation of neural progenitor cells , whereas cells lacking mir-9 in the forebrain undergo p53-dependent apoptosis ( bonev et al . ,
, mir-9 has reciprocal actions with rest ; rest inhibits mir-9 - 2 in undifferentiated neuroblastoma cells , rest and creb inactivation triggers mir-9 - 2 activation ( laneve et al . , 2010 ) .
other mir-9 targets include stathmin that increases microtubule instability ( delaloy et al . , 2010 ) and tlx that regulate stem cell fate ( zhao et al . , 2009 ) .
tlx is suppressed by mir-9 to negatively regulate stem cell proliferation and accelerate neural differentiation ( zhao et al . , 2009 ) .
hairy1 has also been suggested to mediate the effects of mir-9 on proliferation ( bonev et al . , 2011 ) .
mir-9 expression pattern during mouse development has been investigated by ish ; it is expressed in the developing medial pallium although it is most abundant in cortex .
when a mir-9 amo was electroporated into e11.5 cerebral cortex , deficiencies in differentiation of cajal  retzius cells and early born neurons were seen , suggested to be due to the increased expression of the target gene foxg1 ( shibata et al . , 2008 ) .
generation of a mir-9 - 2 and mir-9 - 3 double ko mouse , that is the two most abundant forms during telencephalon development , resulted in major phenotypic brain defects .
cortical layers and vz were reduced , lateral ventricles expanded , the proliferative zones hyperplasic and differentiated structures reduced .
in addition , mice suffered from growth retardation and died within 1  week , demonstrating the importance for mir-9 in neurogenesis ( shibata et al . , 2011 ) . these reports clearly demonstrate that mir-9 influence nscs , perhaps by regulating self - renewal and migration .
still , much remains unclear regarding the functional role of mir-9 in nscs in vivo . as with mir-124 , it will be interesting to follow the generation of a conditional triple - mir-9-ko mouse or the application of stable mirna sponges to study the functional role of mir-9 .
let-7 is one of the first mirna discovered in c. elegans , the first known human mirna and it is conserved over species .
there are 12 human let-7 genes encoding for nine distinct mature forms of the mirna , let-7a through to let-7i
. increased let-7 expression is seen in early neurogenesis and neural differentiation while it is decreased in the adult brain .
ish shows induction of let-7 already at e9.5 in the developing cns ( wulczyn et al . , 2007 ) .
let-7 expression closely resembles the expression of other brain - enriched mirnas ( wulczyn et al . , 2007 ) .
in utero electroporation of let-7b duplexes injected into the lateral ventricles of e13.5 mice causes a reduction in cell cycle progression in nscs ( zhao et al . ,
overexpression of let-7b in nscs causes reduced proliferation and an increase in neural differentiation ( zhao et al . , 2010 ) .
suppression of let-7 , using a mirna - sponge , causes an increase in levels of lin28 protein ( rybak et al . , 2008 ) .
lin28 is a protein that specifically binds and blocks processing of let-7 , thereby inducing pluripotency ( rybak et al . , 2008 ; balzer et al . , 2010 )
lin28 is expressed broadly throughout the neural tube early during development where neural differentiation has not begun ( balzer et al .
, 2010 ) , at this stage it co - localizes with sox2 , a maker for nscs .
it has also been shown that let-7b suppresses the expression of tlx ( zhao et al . , 2010 ) .
these reports suggest that the let-7-family serves as key regulators of nsc proliferation and accelerated neural differentiation ( wulczyn et al . , 2007 ;
. however , the large size of the let-7 family posses a technical hurdle for the generation of loss - of - function mutants , which limits our understanding of the role of let-7 . in the future it appears likely that the use of sponge - vectors allowing stable inhibition of the entire family may be the most feasible choice to study the functional role of let-7 in nscs in vivo .
it is likely that we have so far only begun to realize the complexity of mirna - mediated regulation of nscs .
the multitude of mirna complimentary targets in the genome implicates the complexity of mirna gene regulation
. therefore studies of mirna - target regulation in specific cell types at various developmental time points are essential .
other questions that need to be answered are ; can several mirnas act to suppress the same mrna simultaneously and do they have compensatory , collaborative or competitive effects ?
the development of new biotechnological tools such as mirna sponges and transgenic reporter systems will enable new types of studies that will clarify the functional properties of individual mirnas .
in the coming years it will be extremely interesting to follow this field as it matures and unravels the full role of mirnas in nscs .
the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .
 in adult mammals , neural stem cells ( nscs ) are found in two niches of the brain ; the subventricular zone by the lateral ventricles and the subgranular zone of the dentate gyrus in the hippocampus . 
 neurogenesis is a complex process that is tightly controlled on a molecular level . 
 recently , micrornas ( mirnas ) have been implicated to play a central role in the regulation of ncss . 
 mirnas are small , endogenously expressed rnas that regulate gene expression at the post - transcriptional level . however , functional studies of mirnas are complicated due to current technical limitations . in this review 
 we describe recent findings about mirnas in nscs looking closely at mir-124 , mir-9 , and let-7 . 
 in addition , we highlight technical strategies used to investigate mirna function , accentuating limitations , and potentials . 
Introduction
Expression Profiling of miRNA in Neural Stem Cells
Loss of Function Studies of miRNAs
miR-124
miR-9
The Let-7-Family
Conclusion
Conflict of Interest Statement