Posttranscriptional control of neuronaldevelopment by microRNA networksFen-Biao Gao

Gladstone Institute of Neurological Disease, and Department of Neurology, Neuroscience Graduate Program, University of

California, 1650 Owens Street, San Francisco, CA 94158, USA

Review TRENDS in Neurosciences Vol.31 No.1

The proper development of the nervous system requiresprecise spatial and temporal control of gene expressionat both the transcriptional and translational levels.In different experimental model systems, microRNAs(miRNAs) – a class of small, endogenous, noncodingRNAs that control the translation and stability of manymRNAs – are emerging as important regulators of var-ious aspects of neuronal development. Further dissec-tion of the in vivo physiological functions of individualmiRNAs promises to offer novel mechanistic insightsinto the gene regulatory networks that ensure the pre-cise assembly of a functional nervous system.

IntroductionRecent progress in understanding the different classes ofsmall, noncoding RNAs has led to the discovery of novelgene regulatory mechanisms. This knowledge greatlyenhances our ability to decipher the complex molecularnetworks that control the precise spatial and temporalpatterns of gene expression crucial for various aspects ofanimal development. One class of regulatory RNAs ismicroRNAs (miRNAs), small RNAs of �22 nucleotides(nt) processed from genome-encoded transcripts of 70–80nt [1–5]. The first two miRNAs shown to be involved in thecontrol of developmental timing in Caenorhabditis eleganswere lin-4, discovered in 1993 [6], and the evolutionarilyconserved let-7, discovered in 2000 [7]. Since then, hun-dreds of miRNAs have been cloned from different species,and a dozen or so have been found to regulate severaldevelopmental processes by inhibiting translation ordestabilizing target mRNAs [8–11]. Moreover, eachmiRNA is predicted to target hundreds of mRNAs,suggesting that many protein-coding genes are potentiallyregulated by this pathway [8–11].

Many miRNAs are developmentally regulated and showtissue-specific expression patterns, including dozensexpressed only in the nervous system [12–15]. These miR-NAs may play important roles in neuronal development,neuronal function, or both [16,17]. This review summarizesrecent excitingfindings about the roles ofmiRNAs and somemiRNA pathway proteins in neuronal development, fromearly neurogenesis and cell-fate specification to neuronaldifferentiation and synaptic development of postmitoticneurons. The potential link between the miRNA pathwayand human neurological disorders is also discussed.

Corresponding author: Gao, F.-B. (fgao@gladstone.ucsf.edu).Available online 3 December 2007.

www.sciencedirect.com 0166-2236/$ – see front matter � 2007 Elsevier Ltd. All rights reserve

Dicer in neuronal developmentOne of the key enzymes in miRNA biogenesis is Dicer, amember of the RNase III family of nucleases that cleavedouble-stranded RNAs [18]. In the canonical miRNA path-way, primary miRNAs (pri-miRNAs) are mostly tran-scribed by RNA polymerase II and contain 50 capstructures and poly(A) tails. Pri-miRNAs are processedby the RNase III Drosha in the nucleus to produce �70nt precursor miRNAs (pre-miRNAs) with a hairpin struc-ture [19] which are transported to the cytoplasm by expor-tin-5 in a Ran-GTP-dependent manner [20]. Dicer isrequired to cleave pre-miRNAs and generate an �22 ntmiRNA duplex that is incorporated into the RNA-inducedsilencing complex (RISC) [18,21,22]. In the absence ofDicer, most, if not all, miRNAs are not produced properly.Besides its functions in many other developmental pro-cesses [23–26], Dicer is essential for proper brain morpho-genesis: maternal-zygotic dicer zebrafish mutant embryosfail to produce mature miRNAs and exhibit gross morpho-logical defects in the nervous system. Remarkably, thesedefects were largely rescued by injection of microRNA-430(miR-430) alone [27]. Although this study did not detectany dramatic disruption of major signaling pathwaysinvolved in early patterning, subtle defects remain to beexamined in detail.

A more informative approach is to remove Dicer activityin specific neuronal cell types. In mice, for instance,deletion of Dicer in postmitotic midbrain dopamineneurons causes a progressive loss of those cells, suggestingan essential role of miRNAs in the differentiation or main-tenance of dopamine neurons [28]. A good candidate forthis process is miR-133b, which is enriched in midbrainand deficient in patients with Parkinson’s disease and inanimal models of that disorder. Loss of miR-133b activityseems to increase dopamine release slightly in cell cultures[28], suggesting a subtle role in dopamine neuron matu-ration and function.

The essential role of Dicer in the maintenance of themature nervous system is also demonstrated by the findingthat loss of Dicer-1 in Drosophila dramatically enhancesneurodegeneration caused by a mutant form of the spino-cerebellar ataxia type 3 (SCA3) protein; this effect may beexplained, at least in part, by the absence of bantam, anmiRNA that prevents apoptosis during development bysuppressing the proapoptotic gene hid [29]. Consistentwith this notion, selective genetic ablation of Dicer inPurkinje cells leads to cerebellar degeneration and ataxia[30]. Moreover, in miR-8 mutant flies, the increased

d. doi:10.1016/j.tins.2007.10.004

Review TRENDS in Neurosciences Vol.31 No.1 21

expression of the transcriptional regulator Atrophinincreases apoptosis in the nervous system [31]. Thus,miRNAs are important in maintaining the structuralintegrity of mature neurons through fine-tuning theexpression levels of key target genes.

Dendritic spines and postsynaptic densities areenriched in Dicer, raising the possibility that it also partici-pates in synaptic development and plasticity [32]. How-ever, the underlying mechanisms may be complicated.Besides the canonical miRNA pathway, Dicer is alsorequired to generate mirtrons—novel �22 nt small RNAsprocessed from short intronic hairpins [33,34]. UnlikemiRNAs processed by the canonical miRNA pathway,mirtrons are generated independently of Drosha. Theinvolvement of Dicer in other processes, such as hetero-chromatin assembly and processing of other endogenousdouble-stranded RNAs, also remains to be further eluci-dated in various model systems [35]. Therefore, neurode-velopmental defects as a result of the global loss of Diceractivity need to be interpreted with caution. For thisreason, using loss-of-function approaches to understandthe functions of specific miRNAs in different aspects ofneuronal development may prove to be more informative.

Fragile X mental retardation protein 1Another protein implicated in miRNA biogenesis is thefragile X mental retardation protein 1 (FMRP). Loss ofFMRP function causes fragile X syndrome, the most com-mon form of inherited mental retardation in humans [36].FMRP is an evolutionarily conserved RNA-binding proteinwith two ribonucleoprotein K homology (KH) domains andan arginine- and glycine-rich domain (RGG box) that pre-ferentially bind in vitro to tertiary RNA structures namedthe ‘kissing complex’ [37] and the ‘G quartet’ [38], respect-ively.AlthoughhundredsofmRNAsassociatepreferentiallywith FMRP-containing complexes [39,40], systematicidentification of theRNAspecies that directly bind toFMRPin the native environment in neurons remains a majorchallenge.

Although the exact function of FMRP at the molecularlevel still remains unknown, it appears to be associatedwith the miRNA pathway. In concurrent attempts toidentify new components in purified RISC [41] and in acomplex associated with the Drosophila homolog of theFMRP (dFMR1) [42], it was found that dFMR1 and theRISC subunit Argonaute 2 (Ago2) form a complex in Dro-sophila S2 cells. Ago2- and dFMR1-associated complexesalso contain Dicer and miR-2b [41,42]. Moreover, FMRP,the mammalian Argonaute family protein elF2C2 and 20nt small RNAs with unknown identity were found in theimmunoprecipitated mRNP complexes from human tumorcell lines [43]. These findings suggest a role for dFMR1 inthe miRNA pathway but dFMR1 does not appear to be anessential component in RISC [41,42].

Mutant mice or flies lacking FMRP or dFMR1 areviable and have a grossly normal nervous system. None-theless, the loss of these proteins causes various defectsin spine and synapse formation [44–47], axonal anddendritic growth and branching [48–51] and neurogen-esis [52]. Although FMRP is a multifunctional proteininvolved in several different stages of RNA metabolism,

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its effects on neuronal development are probablymediated at least in part through the miRNA pathway.Consistent with this notion, dFMR1 interacts withMel31B, a P-body protein required for miRNA-mediatedrepression of translation [53]. Further dissection of theexact role of dFMR1 and FMRP in RISC will help explainthe link between the miRNA pathway and human mentaldisorders.

miRNAs in early neurogenesisGenetic analysis of individual miRNAs has begun to shedlight on their specific functions in different aspects ofneuronal development, from early neurogenesis to synap-tic formation. TheDrosophila peripheral nervous system isan excellent model system for dissecting genetic programsunderlying early neurogenesis. Clusters of ectodermal cellsthat express proneural genes give rise to sensory organprecursors (SOPs) through lateral inhibition, a processthat requires the actions of the Notch signaling pathway(Figure 1) [54]. Enhancer of Split complex (E[spl]-C) andthe Bearded complex (Brd-C) are major Notch target geneswhose 30 UTRs contain potential target recognitionsequences for several miRNAs [55]. Indeed, ectopic expres-sion of miR-7 and a few other miRNAs increases SOPproduction, likely through downregulation of Notch targetgenes, although the effects of these miRNAs on SOP spe-cification have not been examined by loss-of-functionapproaches [56].

Drosophila miR-9a is 100% conserved at the nucleotidelevel with vertebrate miR-9a, which is specificallyexpressed in the brain [12–14], raising the possibility thatit may play an important role in brain development. Thephysiological function of miR-9a in neuronal developmentwas revealed in Drosophila by both loss- and gain-of-func-tion analyses [57]. miR-9a mutant flies are viable andfertile, but a small number of mutant embryos or adultsshow extra sensory neurons and sensory bristles as a resultof increased production of SOPs during early neurogenesis.Conversely, ectopic expression of the miR-9a precursorsuppressed SOP specification [57]. miR-9a is highlyexpressed in epithelial cells, including those adjacent toSOPs in proneural clusters, indicating that miR-9a inhi-bits neuronal fate in non-SOP cells to ensure the precisespecification of neuronal precursors during development(Figure 1).

During SOP specification, expression of the zinc fingertranscription factor Senseless (Sens) must be downregu-lated in non-SOP cells in the proneural cluster [58]. Thesens 30 UTR contains three computationally predictedmiR-9a binding sites [59]. Indeed, in transfected HEK 293 cells,reporter gene expression was suppressed by miR-9athrough the sens 30 UTR [57]. More importantly, miR-9aand sens showed strong genetic interactions. For instance,loss of one copy of sens significantly rescued the SOPdefects in miR-9a mutants, indicating that the sens 30

UTR is a key, physiologically relevant in vivo target ofmiR-9a in non-SOP cells during early neurogenesis [57].Although each miRNA is predicted to target hundreds ofmRNAs, it is possible that only the alterations in theexpression levels of a few key target mRNAs are relevantto a specific biological process.

Figure 2. Schematic representation of the double negative feedback loops

between miRNAs and transcription factors. (a) In ASEL sensory neurons in

C. elegans, a high level of lsy-6 suppresses Cog-1, which controls the expression of

miR-273. (b) In ASER, a high level of miR-273 suppresses Die-1, a transcription

factor required for lsy-6 expression.

Figure 1. The roles of miRNAs in the specification of SOPs. In non-SOP cells in the proneural cluster, enhanced Notch signaling leads to the association between Su(H) and

Notch intracellular domain (NIntra), which in turn activates the transcription of E(spl). E(spl) suppresses the expression of Sens and proneural genes. To ensure a low level of

Sens expression in non-SOP cells, miR-9a suppresses Sens through its 30 UTR. In SOPs, the lack of Notch signaling leads to the formation of a repressor complex containing

Su(H), which inhibits E(spl) expression. Sens expression is high and maintains proneural gene expression that endows the SOP fate. The absence of miR-9a in SOPs is

partially responsible for the high level of Sens expression. miR-7 and other miRNAs may be involved in the suppression of E(spl).

22 Review TRENDS in Neurosciences Vol.31 No.1

It is likely thatmiR-9a also regulates early neurogenesisin vertebrates, as it is specifically expressed in proliferatingneural precursors in zebrafish [14] and in mouse embryosand adultmice [60,61]. In fact,miR-9a appears to contributeto the in vitro differentiation of embryonic stem cells [62].Whether miR-9a also functions to suppress the randomactivation of neurogenic genes during mammalian neuro-genesis is unknown. Loss-of-function studies in geneticallyaltered mice should offer novel insights into this importantquestion. miR-9a is 100% conserved at the nucleotide levelfromflies to humans, but themechanismof its action and itstargets may not be evolutionarily conserved. Indeed, themicroRNA let-7 regulates different genetic pathways indifferent organisms, probably through distinct downstreamtarget mRNAs [63]. The key target mRNAs of miR-9a inmammalian neurogenesis are determined.

Lsy-6 and miR-273 in cell-fate specificationA remarkable feature of the nervous system is the diversityof its neurons, which differ in dendritic morphologies,axonal targeting specificities, neurotransmitters and othercell-fate-specific characteristics. Although transcriptionalcontrol of neuronal cell identity is well established, miR-NAs are also involved in this important step of neuronaldevelopment.

In C. elegans, for example, two morphologically similarchemosensory neurons – ASE left (ASEL) and ASE right(ASER) – express different chemoreceptors that correlatewith the functional differences between the two neurons[64,65]. lsy-6, the first miRNA shown to be involved inneuronal development, is expressed in ASEL but not inASER (Figure 2). Genetic analysis showed that lsy-6 isrequired to specify ASEL identity: loss of lsy-6 leads to lossof the ASEL-specific chemoreceptor Gcy-7 and ectopicexpression of the ASER-specific chemoreceptor Gcy-5 inASEL neurons [66]. lsy-6 exerts its effects by binding to the30 UTR of cog-1, an Nkx-type homeobox gene, which leadsto downregulation of Cog-1 expression [66]. The specificexpression of lsy-6 in ASEL but not in ASER neurons iscontrolled by the zinc finger transcription factors Lsy-2 [67]

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and Die-1 [68]. Interestingly, miR-273 is expressed at amuch higher level in ASER than in ASEL neurons, andoverexpression of miR-273 in ASEL suppresses Die-1expression, although the effects of loss of miR-273 havenot been examined yet [68]. Thus, downregulation of Die-1in ASER is probably a result of the action of miR-273.Moreover, miR-273 expression is activated by Cog-1. Sup-pression of Cog-1 expression in ASEL by lsy-6 accounts forthe lower expression of miR-273 in those neurons than inASER neurons [69]. These findings indicate that specificmiRNAs and transcription factors form a double-negativefeedback loop to maintain the cellular identities of ASELand ASER neurons (Figure 2) [70]. In this case, the directeffect ofmiRNAs on target gene expression does not have tobe dramatic, but they can still function as developmentalswitches as a result of their involvement in feedback loopsthat can amplify and maintain different expression levelsof key transcription factors in these two types of neurons.

miR-124a in neuronal differentiationmiR-124a is 100% conserved at the nucleotide level fromworms to humans and is expressed throughout the embryo-nic and adult central nervous systems of different species

Review TRENDS in Neurosciences Vol.31 No.1 23

[12–15]. It is estimated that miR-124a is the mostabundant miR in the brain, accounting for 25%–48% ofall brain miRNAs [12]. Thus, it may play an important rolein neuronal differentiation or function.

In mouse brain, miR-124a seems to be largely restrictedto differentiating and mature neurons, with much lessexpression in neural progenitors [60]. This expressionprofile seems to be controlled by RE1 silencing transcrip-tion factor (REST), a transcription repressor that inhibitsmiR-124a expression in nonneuronal cells and neural pro-genitors but is absent from the miR-124a locus in matureneurons [71].

Ectopic expression of miR-124a in HeLa cells leads tothe suppression of a large number of nonneuronal tran-scripts [72]. Some of these transcripts are elevated incortical neurons treated with antisense 20-O-methyl oligo-nucleotides complementary to miR-124a [71], suggestingthat these mRNAs are endogenous targets of miR-124a.Therefore, neuronal differentiation may require both dere-pression of REST and downregulation of some mRNAs bymiR-124a. As themost abundantmiRNA in the brain,miR-124a likely regulates many target mRNAs and thereforemay even play a role in maintaining the homeostasis ofdifferentiated neurons. For instance, target mRNAs encod-ing small C-terminal domain phosphatase 1 (SCP1) [73],laminin g1 and integrin b1 [74] are downregulated indifferentiated neurons (Figure 3). Moreover, downregula-tion of the RNA-binding protein PTBP1 by miR-124aduring neuronal differentiation leads to a global neuron-specific alternative splicing pattern [75].

The exact developmental consequences of the loss ofendogenous miR-124a in differentiating neurons areunclear. In the chick neural tube, neither inhibition nor

Figure 3. The role of miR-124a in neuronal development and its regulation by

REST. The upregulation of miR-124a expression during neuronal differentiation

requires the derepression by the REST-SCP1 complex. As the most abundant

miRNA in the brain, miR-124a regulates the expression of many target mRNAs.

Yet, the developmental consequences of lack of miR-124a in vivo remain to be

further examined.

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overexpression of miR-124a altered neuronal fate, asassessed with cell-specific markers [74]. However, anotherstudy reported a seemingly opposite result from the sameassay system [73]. The reason for this discrepancy isunknown. Further investigation will be required to deter-mine whether endogenous miR-124a affects other aspectsof neuronal differentiation, such as dendritic/axonalgrowth as in the case of miR-132 [76], or synaptogenesis,as in the case of miR-134 [77].

miR-7 in photoreceptor differentiationAnother well-established system for studying cell differ-entiation is the Drosophila eye, which consists of �800ommatidia. In each ommatidium, the R8 photoreceptorneuron differentiates first and recruits other progenitorcells to differentiate into seven other photoreceptorneurons and support cells. A key factor that ensures thetimely differentiation of retinal cells is the ETS-domaintranscription repressor Yan, which is expressed in pro-genitor cells and suppresses their differentiation intophotoreceptors [78]. Signaling through the epidermalgrowth factor leads to activation of the RAS-ERK pathwayand rapid degradation of Yan [78]. The absence of Yan indifferentiating cells is essential for the specification ofphotoreceptor neuronal fate.

Yan controls miR-7 transcription, and high-level Yanexpression in progenitor cells represses miR-7 expression.In differentiating cells, miR-7 expression is elevated as aconsequence of Yan degradation, and miR-7 furtherrepresses Yan expression by binding to sequences in itsmRNA 30 UTR. Thus, miR-7 and Yan form a reciprocalnegative feedback loop and show a mutually exclusiveexpression pattern (Figure 4). Indeed, ectopic expressionof miR-7 promotes photoreceptor neuron differentiation[79]. Interestingly, miR-7 loss-of-function mutants haveno obvious defects in eye development [79], suggestingthat in this particular case, miR-7 does not function asan absolute switch in the feedback loop. ERK-mediatedphosphorylation and degradation likely play a major rolein downregulating Yan in differentiating photoreceptorneurons, whereas miR-7 ensures its complete depletion.

Figure 4. A negative feedback loop between an miRNA and a transcription factor in

Drosophila. (a) In progenitor cells in the Drosophila eye, high-level expression of

the transcription factor Yan suppresses miR-7 expression. (b) During

photoreceptor differentiation, transient activation of the epidermal growth factor

receptor (EGFR) signaling pathway leads to the degradation of Yan and the

expression of miR-7, which further downregulates the level of Yan through binding

to its 30 UTRs.

24 Review TRENDS in Neurosciences Vol.31 No.1

Such a negative feedback loop also operates betweenmiR-133b and the paired-like homeodomain transcriptionfactor Pitx3 during the maturation of midbrain dopamineneurons [28], raising the possibility that it is a commonmodule in gene regulatory networks.

miR-134 in synaptogenesisSince the discovery of polyribosomes near spines in distaldendrites of dentate granule neurons in 1982 [80], a largenumber of mRNAs have been found in dendrites [81]. Thefunctional significance of local protein synthesis in den-drites was demonstrated by its requirement in brain-derived neurotrophic factor (BDNF)-induced synapticplasticity in the hippocampus and by experiments in differ-ent experimental systems [81]. Because miRNAs, as wellas several protein factors that either positively or nega-tively influence mRNA translation and stability, are oftenassociated with active polyribosomes, some miRNAs areexpected to be present in dendrites and help control localprotein synthesis. Therefore, these miRNAs may contrib-ute to synapse formation and synaptic function by regulat-ing the local translation of their target mRNAs.

Indeed, miR-134, a brain-specific miRNA, localizes nearsynaptic sites in dendrites of hippocampal neurons andregulates the size of dendritic spines [77]. miR-134, butnot let-7c, negatively regulates thewidth of dendritic spinesbut not their density or dendritic branching. Reduced miR-134 activitywith 20-O-methylated antisense oligonucleotidedecreased spine width by 7.6%. Considering the heterogen-eity and the dynamic nature of dendritic spines on culturedneurons, this phenotype is relatively subtle and suggests amodulatory role for miR-134 in spine formation. Futuregenetic knockout of miR-134 will no doubt reveal the fullextent of miR-134 function in this important process.

How does miR-134 exert its effect on spine shape? The30 UTR of Limk1, one of the BDNF-induced genes thatregulate actin polymerization and microtubule disassem-bly [82], contains one miR-134 binding site and acts as themajor downstream mediator of miR-134 function. miR-134 negatively regulates the translation of Limk1 mRNA

Table 1. The functions of different miRNAs in the development an

miRNAs Species Approaches Functions

lsy-6 C. elegans LOF and GOF in vivo Required to specify A

miR-273 C. elegans GOF in vivo Expressed in ASER a

miR-7 Drosophila LOF and GOF in vivo Ensures complete de

differentiation

miR-430 Zebrafish Genetic rescue Required for clearanc

morphogenesis

miR-134 Rodent LOF and GOF in culture Modulates the size of

miR-9a Drosophila LOF and GOF in vivo Ensures the precise s

miR-124a Vertebrates LOF and GOF in vivoa and

in culture

Promotes neuronal d

miR-132 Rodent LOF and GOF in culture Regulates neuronal m

miR-9a Rodent LOF and GOF in culture Involved in neural lin

stem cells

miR-133b Rodent LOF and GOF in culture Regulates the matura

neurons

Bantam Drosophila LOF and GOF in vivo Prevents neurodegen

miR-8 Drosophila LOF and GOF in vivo Required for neurona

miR-219 Rodent LOF in vivob Regulates circadian p

Abbreviations: GOF, gain of function; LOF, loss of function; SCA3, spinocerebellar ataxaAntisense 20-O-methyl oligonucleotides were used to inhibit the activity of endogenoubCholesterol-modified oligonucleotides (antagomirs) were used to repress miR-219 in v

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in dendrites through its 30 UTR in a manner that isdependent on the miR-134 binding site. Moreover, theeffect of miR-134 overexpression on spine shape can berescued by overexpression of Limk1, whose mRNA is notregulated by miR-134. Interestingly, BDNF treatmentrelieves miR-134-dependent inhibition of Limk1 trans-lation, which seems to be mediated by the mammaliantarget of rapamycin (mTOR) pathway [77]. Exactly howBDNF does so and howmiRNAs regulate local translationin dendrites remain to be determined [83]. It is largelyunknown what regulates the association and dissociationof miRNAs and their target 30 UTRs in neurons. However,recent advances in our understanding of the actions ofmiRNAs in other cell types [84–87] raise the possibilitythat regulation of translation initiation, elongation, poly-adenylation or mRNA stability by different miRNAs inresponse to extrinsic factors or neuronal activity mayoperate locally near synapses as well.

Concluding remarksIt has become increasingly clear that miRNAs modulategene expression levels during multiple steps of neuronaldevelopment in diverse organisms, from early neurogen-esis to synaptogenesis. In a few cases, miRNAs areinvolved in feedback loops with some key transcriptionfactors and seem to function as molecular switches inneuronal development. In many other cases, the effectsof a specific miRNA are relatively subtle, suggesting thatmiRNAs ensure the precision of gene expression and theaccuracy of these neurodevelopmental events. This uniquefunction is no less important than other molecular regu-lators whose misexpression often leads to robust develop-mental defects. However, our current understanding ofmiRNA function in the nervous system is still in itsinfancy, and the number of miRNAs that have been ana-lyzed by loss-of-function approaches remains very small(Table 1). Additional genetic analysis with more sensitivefunctional assays will undoubtedly reveal the full extent ofmiRNA function in neuronal development and may offernovel insights into human neurological disorders as well.

d maintenance of the nervous system

Targets Refs

SEL sensory neuron identity Cog-1 [66,70]

nd suppresses ASEL identity Die-1 [68–70]

pletion of Yan photoreceptor Yan [79]

e of maternal mRNAs and brain ?? [27]

dendritic spines in cultured neurons LimK1 [77]

pecification of SOPs in Drosophila Senseless [57]

ifferentiation (?) Laminin g1, integrin

b1, SCP1, PTBP1

[71–75]

orphogenesis and circadian clock P250GAP, etc. [76,88]

eage differentiation from embryonic ?? [62]

tion/function of midbrain dopamine Pitx3 [28]

eration in a Drosophila model of SCA3 ?? [29]

l survival Atrophin [31]

eriod length in mice SCOP, etc. [88]

ia type 3.

s miR-124a in the developing chick neural tube.

ivo.

Review TRENDS in Neurosciences Vol.31 No.1 25

AcknowledgementsI thank J. Carroll for help with graphics, S. Ordway for editorialassistance, E. Pierce for administrative assistance, and laboratorymembers for discussions over the years. This work is supported by theFRAXA Foundation and the National Institutes of Health (F-B.G.).

References1 Ambros, V. (2001) microRNAs: tiny regulators with great potential.

Cell 107, 823–8262 Bartel, D.P. (2004) MicroRNAs: genomics, biogenesis, mechanism, and

function. Cell 116, 281–2973 Meister, G. and Tuschl, T. (2004) Mechanisms of gene silencing by

double-stranded RNA. Nature 431, 343–3494 Du, T. and Zamore, P.D. (2005) microPrimer: the biogenesis and

function of microRNA. Development 132, 4645–46525 Lee, Y. et al. (2006) Drosha in primary microRNA processing. Cold

Spring Harb. Symp. Quant. Biol. 71, 51–576 Lee, R.C. et al. (1993) The C. elegans heterochronic gene lin-4 encodes

small RNAswith antisense complementarity to lin-14.Cell 75, 843–8547 Reinhart, B.J. et al. (2000) The 21-nucleotide let-7 RNA regulates

developmental timing in Caenorhabditis elegans.Nature 403, 901–9068 Kloosterman, W.P. and Plasterk, R.H. (2006) The diverse functions of

microRNAs in animal development and disease. Dev. Cell 11, 441–4509 Zhao, Y. and Srivastava, D. (2007) A developmental view of microRNA

function. Trends Biochem. Sci. 32, 189–19710 Bushati, N. and Cohen, S.M. (2007) microRNA functions. Annu. Rev.

Cell Dev. Biol. 23, 175–20511 Chang, T.C. and Mendell, J.T. (2007) The roles of microRNAs in

vertebrate physiology and human disease. Annu. Rev. GenomicsHum. Genet. 8, 215–239

12 Lagos-Quintana, M. et al. (2002) Identification of tissue-specificmicroRNAs from mouse. Curr. Biol. 12, 735–739

13 Kim, J. et al. (2004) Identification of many microRNAs that copurifywith polyribosomes in mammalian neurons. Proc. Natl. Acad. Sci.U. S. A. 101, 360–365

14 Wienholds, E. et al. (2005) MicroRNA expression in zebrafishembryonic development. Science 309, 310–311

15 Aboobaker, A.A. et al. (2005) Drosophila microRNAs exhibit diversespatial expression patterns during embryonic development. Proc. Natl.Acad. Sci. U. S. A. 102, 18017–18022

16 Cao, X. et al. (2006) Noncoding RNAs in the mammalian centralnervous system. Annu. Rev. Neurosci. 29, 77–103

17 Kosik, K.S. (2006) The neuronal microRNA system.Nat. Rev. Neurosci.7, 911–920

18 Bernstein, E. et al. (2001) Role for a bidentate ribonuclease in theinitiation step of RNA interference. Nature 409, 363–366

19 Lee, Y. et al. (2003) The nuclear RNase III Drosha initiates microRNAprocessing. Nature 425, 415–419

20 Yi, R. et al. (2003) Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs. Genes Dev. 17, 3011–3016

21 Hutvagner, G. et al. (2001) A cellular function for the RNA-interferenceenzyme Dicer in the maturation of the let-7 small temporal RNA.Science 293, 834–838

22 Grishok, A. et al. (2001) Genes and mechanisms related to RNAinterference regulate expression of the small temporal RNAs thatcontrol C. elegans developmental timing. Cell 106, 23–34

23 Wienholds, E. et al. (2003) The microRNA-producing enzyme Dicer1 isessential for zebrafish development. Nat. Genet. 35, 217–218

24 Hatfield, S.D. et al. (2005) Stem cell division is regulated by themicroRNA pathway. Nature 435, 974–978

25 Harfe, B.D. et al. (2005) The RNaseIII enzyme Dicer is required formorphogenesis but not patterning of the vertebrate limb. Proc. Natl.Acad. Sci. U. S. A. 102, 10898–10903

26 Murchison, E.P. et al. (2007) Critical roles for Dicer in the femalegermline. Genes Dev. 21, 682–693

27 Giraldez, A.J. et al. (2005)MicroRNAs regulate brainmorphogenesis inzebrafish. Science 308, 833–838

28 Kim, J. et al. (2007) A microRNA feedback circuit in midbraindopamine neurons. Science 317, 1220–1224

29 Bilen, J. et al. (2006) MicroRNA pathways modulate polyglutamine-induced neurodegeneration. Mol. Cell 24, 157–163

30 Schaefer, A. et al. (2007) Cerebellar neurodegeneration in the absenceof microRNAs. J. Exp. Med. 204, 1553–1558

www.sciencedirect.com

31 Karres, J.S. et al. (2007) The conserved microRNA miR-8 tunesatrophin levels to prevent neurodegeneration in Drosophila. Cell131, 136–145

32 Lugli, G. et al. (2005) Dicer and eIF2c are enriched at postsynapticdensities in adult mouse brain and are modified by neuronal activity ina calpain-dependent manner. J. Neurochem. 94, 896–905

33 Okamura, K. et al. (2007) The mirtron pathway generates microRNA-class regulatory RNAs in Drosophila. Cell 130, 89–100

34 Ruby, J.G. et al. (2007) Intronic microRNA precursors that bypassDrosha processing. Nature 448, 83–86

35 Zofall, M. and Grewal, S.I. (2006) RNAi-mediated heterochromatinassembly in fission yeast. Cold Spring Harb. Symp. Quant. Biol. 71,487–496

36 Penagarikano, O. et al. (2007) The pathophysiology of fragile Xsyndrome. Annu. Rev. Genomics Hum. Genet. 8, 109–129

37 Darnell, J.C. et al. (2001) Fragile X mental retardation protein targetsG quartet mRNAs important for neuronal function. Cell 107, 489–499

38 Darnell, J.C. et al. (2005) Kissing complex RNAs mediate interactionbetween the fragile-X mental retardation protein KH2 domain andbrain polyribosomes. Genes Dev. 19, 903–918

39 Brown, V. et al. (2001) Microarray identification of FMRP-associatedbrain mRNAs and altered mRNA translational profiles in fragile Xsyndrome. Cell 107, 477–487

40 Miyashiro, K.Y. et al. (2003) RNA cargoes associating with FMRPreveal deficits in cellular functioning in Fmr1 null mice. Neuron 37,417–431

41 Caudy, A.A. et al. (2002) Fragile X related protein and VIG associatewith the RNA interference machinery. Genes Dev. 16, 2491–2496

42 Ishizuka, A. et al. (2002) A Drosophila fragile X protein interacts withcomponents of RNAi and ribosomal proteins.Genes Dev. 16, 2497–2508

43 Jin, P. et al. (2004) Biochemical and genetic interaction between thefragile X mental retardation protein and the microRNA pathway. Nat.Neurosci. 7, 113–117

44 Nimchinsky, E.A. et al. (2001) Abnormal development of dendriticspines in FMR1 knock-out mice. J. Neurosci. 21, 5139–5146

45 Zhang, Y. et al. (2001) Drosophila fragile X-related gene regulates theMAP1B homolog Futsch to control synaptic structure and function.Cell 107, 591–603

46 Grossman, A.W. et al. (2006) Hippocampal pyramidal cells in adultFmr1 knockout mice exhibit an immature-appearing profile ofdendritic spines. Brain Res. 1084, 158–164

47 Pfeiffer, B.E. and Huber, K.M. (2007) Fragile X mental retardationprotein induces synapse loss through acute postsynaptic translationalregulation. J. Neurosci. 27, 3120–3130

48 Dockendorff, T.C. et al. (2002) Drosophila lacking dfmr1 activity showdefects in circadian output and fail to maintain courtship interest.Neuron 34, 973–984

49 Morales, J. et al. (2002) Drosophila fragile X protein, dFXR, regulatesneuronal morphology and function in the brain. Neuron 34, 961–972

50 Lee, A. et al. (2003) Control of dendritic development by theDrosophilafragile X-related gene involves the small GTPase Rac1. Development130, 5543–5552

51 Michel, C.I. et al. (2004) Defective neuronal development in themushroom bodies of Drosophila fragile X mental retardation 1mutants. J. Neurosci. 24, 5798–5809

52 Castren, M. et al. (2005) Altered differentiation of neural stem cells infragile X syndrome. Proc. Natl. Acad. Sci. U. S. A. 102, 17834–17839

53 Barbee, S.A. et al. (2006) Staufen- and FMRP-containing neuronalRNPs are structurally and functionally related to somatic P bodies.Neuron 52, 997–1009

54 Modolell, J. (1997) Patterning of the adult peripheral nervous system ofDrosophila. Perspect. Dev. Neurobiol. 4, 285–296

55 Lai, E.C. (2002) Micro RNAs are complementary to 30 UTR sequencemotifs that mediate negative post-transcriptional regulation. Nat.Genet 30, 363–364

56 Lai, E.C. et al. (2005) Pervasive regulation of Drosophila Notch targetgenes by GY-box-, Brd-box-, and K-box-class microRNAs. Genes Dev.19, 1067–1080

57 Li, Y. et al. (2006) MicroRNA-9a ensures the precise specification ofsensory organ precursors in Drosophila. Genes Dev. 20, 2793–2805

58 Nolo, R. et al. (2000) Senseless, a Zn finger transcription factor, isnecessary and sufficient for sensory organ development in Drosophila.Cell 102, 349–362

26 Review TRENDS in Neurosciences Vol.31 No.1

59 Stark, A. et al. (2005) Animal microRNAs confer robustness to geneexpression and have a significant impact on 30UTR evolution. Cell 123,1133–1146

60 Deo, M. et al. (2006) Detection of mammalian microRNA expression byin situ hybridization with RNA oligonucleotides. Dev. Dyn. 235, 2538–2548

61 Kloosterman, W.P. et al. (2006) In situ detection of miRNAs in animalembryos using LNA-modified oligonucleotide probes. Nat. Methods 3,27–29

62 Krichevsky, A.M. et al. (2006) Specific microRNAsmodulate embryonicstem cell-derived neurogenesis. Stem Cells 24, 857–864

63 Ambros, V. and Chen, X. (2007) The regulation of genes and genomesby small RNAs. Development 134, 1635–1641

64 Bargmann, C.I. and Horvitz, H.R. (1991) Chemosensory neurons withoverlapping functions direct chemotaxis to multiple chemicals inC. elegans. Neuron 7, 729–742

65 Yu, S. et al. (1997) Guanylyl cyclase expression in specific sensoryneurons: a new family of chemosensory receptors. Proc. Natl. Acad. Sci.U. S. A. 94, 3384–3387

66 Johnston, R.J. and Hobert, O. (2003) A microRNA controlling left/right neuronal asymmetry inCaenorhabditis elegans.Nature 426, 845–849

67 Johnston, R.J. and Hobert, O. (2005) A novel C. elegans zinc fingertranscription factor, lsy-2, required for the cell type-specific expressionof the lsy-6 microRNA. Development 132, 5451–5460

68 Chang, S. et al. (2004) MicroRNAs act sequentially and asymmetricallyto control chemosensory laterality in the nematode. Nature 430, 785–789

69 Johnston, R.J. et al. (2005) MicroRNAs acting in a double-negativefeedback loop to control a neuronal cell fate decision. Proc. Natl. Acad.Sci. U. S. A. 102, 12449–12454

70 Hobert, O. (2006) Architecture of a microRNA-controlled generegulatory network that diversifies neuronal cell fates. Cold SpringHarb. Symp. Quant. Biol. 71, 181–188

71 Conaco, C. et al. (2006) Reciprocal actions of REST and a microRNApromote neuronal identity. Proc. Natl. Acad. Sci. U. S. A. 103, 2422–2427

72 Lim, L.P. et al. (2005)Microarray analysis shows that somemicroRNAsdownregulate large numbers of target mRNAs. Nature 433, 769–773

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73 Visvanathan, J. et al. (2007) The microRNA miR-124 antagonizes theanti-neural REST/SCP1 pathway during embryonic CNS development.Genes Dev. 21, 744–749

74 Cao, X. et al. (2007) A functional study of miR-124 in the developingneural tube. Genes Dev. 21, 531–536

75 Makeyev, E.V. et al. (2007) The microRNAmiR-124 promotes neuronaldifferentiation by triggering brain-specific alternative pre-mRNAsplicing. Mol. Cell 27, 435–448

76 Vo, N. et al. (2005) A cAMP-response element binding protein-inducedmicroRNA regulates neuronal morphogenesis. Proc. Natl. Acad. Sci. U.S. A. 102, 16426–16431

77 Schratt, G.M. et al. (2006) A brain-specific microRNA regulatesdendritic spine development. Nature 439, 283–289

78 Voas, M.G. and Rebay, I. (2004) Signal integration during development:insights from the Drosophila eye. Dev. Dyn. 229, 162–175

79 Li, X. and Carthew, R.W. (2005) A microRNA mediates EGF receptorsignaling and promotes photoreceptor differentiation in theDrosophilaeye. Cell 123, 1267–1277

80 Steward, O. and Levy, W.B. (1982) Preferential localization ofpolyribosomes under the base of dendritic spines in granule cells ofthe dentate gyrus. J. Neurosci. 2, 284–291

81 Sutton, M.A. and Schuman, E.M. (2006) Dendritic protein synthesis,synaptic plasticity and memory. Cell 127, 49–58

82 Bernard, O. (2007) Lim kinases, regulators of actin dynamics. Int. J.Biochem. Cell Biol. 39, 1071–1076

83 Tai, H.C. and Schuman, E.M. (2006) MicroRNA: microRNAs reach outinto dendrites. Curr. Biol. 16, R121–R123

84 Suvendra, N. et al. (2006) Relief of microRNA-mediated translationalrepression in human cells subjected to stress. Cell 125, 1111–1124

85 Lytle, J.R. et al. (2007) Target mRNAs are repressed as efficiently bymicroRNA-binding sites in the 50UTR as in the 30UTR. Proc. Natl.Acad. Sci. U. S. A. 104, 9667–9672

86 Chendrimada, T.P. et al. (2007) MicroRNA silencing through RISCrecruitment of eIF6. Nature 447, 823–828

87 Mathonnet, G. et al. (2007) MicroRNA inhibition of translationinitiation in vitro by targeting the cap-binding complex eIF4F.Science 317, 1764–1767

88 Cheng, H.Y. et al. (2007) MicroRNA modulation of circadian-clockperiod and entrainment. Neuron 54, 813–829

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