Post-transcriptional regulation in corticogenesis: how RNA

Advanced Review

Post-transcriptional regulationin corticogenesis: howRNA-binding proteins help buildthe brainLouis-Jan Pilaz1 and Debra L. Silver1,2,3,4∗

The cerebral cortex, the brain structure responsible for our higher cognitive func-tions, is built during embryonic development in a process called corticogenesis.During corticogenesis, neural stem cells generate distinct populations of progen-itors and excitatory neurons. These new neurons migrate radially in the cortex,eventually forming neuronal layers and establishing synaptic connections withother neurons both within and outside the cortex. Perturbations to corticogene-sis can result in severe neurodevelopmental disorders, thus emphasizing the needto better understand molecular regulation of brain development. Recent stud-ies in both model organisms and humans have collectively highlighted roles forpost-transcriptional regulation in virtually all steps of corticogenesis. Genomicapproaches have revealed global RNA changes associated with spatial and tem-poral regulation of cortical development. Additionally, genetic studies have uncov-ered RNA-binding proteins (RBPs) critical for cell proliferation, differentiation, andmigration within the developing neocortex. Many of these same RBPs play causalroles in neurodevelopmental pathologies. In the developing neocortex, RBPs influ-ence diverse steps of mRNA metabolism, including splicing, stability, translation,and localization. With the advent of new technologies, researchers have begun touncover key transcripts regulated by these RBPs. Given the complexity of the devel-oping mammalian cortex, a major challenge for the future will be to understandhow dynamic RNA regulation occurs within heterogeneous cell populations, acrossspace and time. In sum, post-transcriptional regulation has emerged as a criticalmechanism for driving corticogenesis and exciting direction of future research.© 2015 Wiley Periodicals, Inc.

How to cite this article:WIREs RNA 2015. doi: 10.1002/wrna.1289

∗Correspondence to: [email protected] of Molecular Genetics and Microbiology, Duke Uni-versity Medical Center, Durham, NC, USA2Department of Cell Biology, Duke University Medical Center,Durham, NC, USA3Department of Neurobiology, Duke University Medical Center,Durham, NC, USA4Duke Institute for Brain Sciences, Duke University Medical Center,Durham, NC, USA

Conflict of interest: The authors have declared no conflicts of interestfor this article.

INTRODUCTION

The cerebral cortex is the most complex biological“machine” known to man. Part of this complexity

resides in the web of coordinated functional units, thecortical areas. Cortical areas are radially organizedwithin layers, each of which contain neurons with sim-ilar molecular, electrophysiological, and connectivitycharacteristics.1 The cytoarchitecture of an area andthus the number of neurons in each layer is paramountto specify its post-natal function. Additionally, glialcells (astrocytes, oligodendrocytes, and microglia)

© 2015 Wiley Per iodica ls , Inc.

Advanced Review wires.wiley.com/rna

FIGURE 1 | Schematic representation of cortical development. Shown are three different progenitor populations (neuroepithelial cells, radial glialcells, and intermediate progenitors) and neurons (both migrating and differentiating). Progenitors residing within the VZ undergo self-renewaldivisions to generate new progenitors (curved arrow) as well as divisions to generate either neurons or progenitors (straight arrows). Ascorticogenesis proceeds, progenitors initially expand their population, shift to neuron, and intermediate progenitor production. Intermediateprogenitors within the SVZ also generate neurons. Neurons migrate through the IZ to the CP to form layers of the cerebral cortex. MZ, marginal zone;CP, cortical plate; IZ, intermediate zone; SVZ, sub-ventricular zone; VZ, ventricular zone

play a key role in the homeostasis of the cortex.Defects in cortical development can cause acute neuro-logical disorders affecting brain size and function suchas microcephaly or lissencephaly. Therefore, the devel-opmental mechanisms that regulate neuronal numberand positioning together with glial cells are crucial tobuild a healthy brain. This review will focus on themolecular regulation of neuronal generation and posi-tioning during embryonic neocortical development.

During embryonic development, excitatory neu-rons are generated from neural progenitor populationsin a process termed neurogenesis.2 The germinal zonesinclude the ventricular zone (VZ), located at the bor-der of the cerebral ventricles, and the subventricularzone (SVZ) located beside the VZ (Figure 1). Dur-ing early cortical development the predominant neuralprogenitors are neuroepithelial cells (NE cells), whichmainly undergo symmetric proliferative divisions toself-renew. NE cells are later replaced by radial glialcells (RGCs), which primarily undergo asymmetricdivisions to generate a new RGC and a more differ-entiated cell, either a neuron or an intermediate pro-genitor (IP)3,4. Both NEs and RGCs extend processes

from the ventricular border to the pial surface whiletheir cell body (nucleus) resides in the VZ. IPs arelineage-restricted multipolar progenitors which dividein the SVZ to amplify the neuronal population.5–7

Hence neurons are directly generated by both RGCsand IPs. In mice, the most widely utilized model forstudying corticogenesis, the proliferative period beginsaround embryonic day (E) 10.0, and the neurogenicperiod begins about E11.5 and continues to E18.5.Neurons of different layers are born in a sequentialfashion, with deep layer neurons born between E11.5and E14.5 and superficial layer neurons born betweenE14.5 and E18.5.8,9

After their generation, newborn neurons migratetoward the pial surface of the cortex, using the basalprocess of RGCs as their scaffold. Their route passesacross the intermediate zone (IZ) in the middle of thecortex and ends in the cortical plate (CP), the finallocation of neuronal layers (Figure 1). During normaldevelopment young neurons migrate up to the pial sur-face, bypassing neurons born earlier.10,11 Thus, deeplayer neurons born earlier in development eventuallyreside closer to the ventricle, whereas superficial layer


WIREs RNA Post-transcriptional regulation in corticogenesis

TABLE 1 RNA-binding Proteins Required for Corticogenesis

RNA-binding

Protein Cortical Function Cortical Expression RNA Function Key RNA Targets

Associated

Neuro-diseases References

NOVA2 Neuronal migration Neuron specific Alternative splicing Dab1 Paraneoplasticneurologic disorder

12,13

TRA2B Neuronal and progenitorsurvival

Progenitors andneurons

SR pre-mRNAsplicing

? 14,15

PTBP2 Progenitor polarity,proliferation, neuronalmaturation

Neuron specific Alternative splicing,exon inclusion

Psd-95 16–18

MAGOH Progenitor proliferation Ubiquitousprogenitorenriched

splicing, RNAlocalizationNMD, translation

Lis1 19

RBM8A Progenitor proliferation Ubiquitousprogenitorenriched

splicing, RNAlocalization,NMD, translation

? TAR syndrome 20

HuR Progenitor proliferation,neuronal identify andmaturation

Neuron specific Translation Dll1 21,22

MSI1,2 Progenitor proliferation Ubiquitous,enriched inprogenitors

Translation Numb, Jag1,Prfpf3, Kirrel3,Rbm22, Dhx37

23–25

FMRP Progenitor maintenance Progenitors andneurons

Translation Pfn1, Cdh2,NOS1

Fragile X 26–30

Eif4E/4E-T Progenitor maintenance Progenitors andneurons

Translation Neurog1, Neurog2,Neurod1

31

STAU2 Radial glia maintenance Progenitors andneurons

RNA localization Prox1, Bsb2,Trim32

32,33

neurons are ultimately found near the pial surface.Upon reaching their final position within the cortex,the excitatory neurons then establish connections withother neurons both within and outside of the cortex.Hence the fate and final function of projection neu-rons is ultimately defined by their birth and subsequentmigration to distinct layers of the brain.

Although much is known about transcriptionfactors and signaling molecules in corticogenesis, onlyrecently have we begun to appreciate the widespreadroles of RNA-binding proteins (RBPs) in neocorticaldevelopment. Specific RBPs are expressed acrossdifferent developmental stages of the developingneocortex as evidenced by quantitative RT-PCRanalyses, in situ hybridization and transcriptomeanalyses.34–36 However only a small number of RBPshave actually been tested for a functional impact uponcorticogenesis (Table 1). Those RBPs important forneocortical development impact diverse steps of RNAmetabolism, and thus collectively reveal that modu-lation of all stages of the RNA life cycle is necessaryfor cortical development (Figure 2). In this review wehighlight critical RBPs implicated in embryonic cor-ticogenesis, including the production, differentiation

and migration of excitatory neurons. We describetheir known functions in RNA regulation, corticaldevelopment, and in relevant cases disease patho-genesis. These include both RBPs strictly expressedin the developing cortex and ubiquitous RBPs withenriched neocortical expression. We organize ourreview by discussing neocortical RBPs relevant foreach major step of posttranscriptional regulation:alternative splicing, nucleo-cytoplasmic transport,RNA stability and translation, and localization(Figures 2 and 3).

ALTERNATIVE SPLICINGAlternative splicing (AS) is a powerful mechanism toamplify the output diversity of the genome throughthe editing of primary transcripts (Figure 2). The exci-sion or inclusion of intronic and exonic sequencesof pre-mRNA produces distinct transcripts that maybe translated into biochemically diverse proteins.AS of the 5’ and 3’ UTR, or coding regions ofa pre-mRNA can also impact downstream steps ofmRNA metabolism including stability, nuclear export,



FIGURE 2 | Cartoon depicting various stages of mRNA life cycle when RBPs function. Different stages of posttranscriptional regulation are shownalong with their nuclear-cytoplasmic location. This review discusses roles for RNA-binding proteins (shown as geometric shapes) in these variousaspects of mRNA metabolism.

nonsense mediated decay (NMD), and RNA local-ization, largely by exposing binding sequences forRBPs or miRNAs. Hence, AS is a powerful mechanismto differentially manipulate gene expression betweencells, and recent studies reviewed below underscore itsrelevance for cortical development.

Genomic Analyses of AS in the DevelopingNeocortexWith the advent of transcriptome techniques, weare now beginning to appreciate the contribution ofgenome-wide RNA splicing for cortical development.Studies of both mouse and human cortical modelshave collectively revealed both spatial and temporalAS differences during cortical development (Table 2).One of the first studies to dissect AS came fromNenad Sestan’s group, who utilized whole-genomeexon microarrays to reveal region specific differ-ences in AS in the human brain at mid-gestation.37

This comprehensive study discovered that at mid-fetaldevelopment, 28% of expressed genes are alternativelyspliced between different human brain structures.Among those genes showing robust AS is ROBO1,which is involved in axon guidance and neural

progenitor proliferation, and is implicated in vari-ous neurodevelopmental disorders.38,39 Distinct ASROBO1 transcripts might promote establishment ofconnectivity and/or the generation of the appropriatenumber of neurons in distinct layers of mature corticalareas.

A similar spatial analysis of AS was appliedwithin the developing embryonic mouse cortex atmid-gestation (E14.5) by Ayoub et al., who coupledRNA sequencing with laser-capture microdissec-tion to demonstrate the existence of differential ASbetween different embryonic cortical zones (VZ vsSVZ+ IZ vs CP).36 This study revealed that somegenes, such as Wdr61, show no significant differencein overall expression levels between zones but doexhibit differential expression of splice variants. Forother genes, such as Mfge8, differential expressionof just one splice variant across cortical zones canexplain overall shifts in expression. In addition forother classes of genes, such as Cugbp2 and Hes6,the relative ratio of spliceoform expression is similarbetween zones indicating AS may be less relevant.The three embryonic cortical zones assessed by Ayoubet al. contain largely different populations of pro-genitors and post-mitotic neurons. These spliceoform



FIGURE 3 | Summary of known RNA-binding proteins and the aspects of corticogenesis they regulate. Different aspects of neural progenitorfunction (cell cycle progression, cell fate decision, apoptosis) and neuronal function (migration, differentiation, maturation, apoptosis) are indicatedalong with the RBPs discussed in this review.

TABLE 2 Genomic Studies Highlighting Alternative Splicing in Cortical Development

Stage Organism Analysis References

Mid-gestation Human Spatial differences between brain structures 37

E14.5 Mouse Spatial differences in cortical layers(VZ/SVZ vs IZ vs CP) 36

E15.5-P1 Mouse Temporal and spatial differences in sorted excitatory neurons:callosal, subcerebral, corticothalamic/subplate neurons

40

E16-P30 Mouse Temporal differences in in vivo cerebral cortices 41

In vitro Human Temporal differences in in vitro differentiated ES cells 42

expression differences imply there are cell-specific ASwithin the developing mouse neocortex.

As embryonic development proceeds, the reper-toire of progenitors and neurons also changes. Henceit is not surprising that in addition to spatial differ-ences in AS, temporal differences in AS are evidentacross different stages of cortical development. Dill-man et al. compared cortical samples from embryonicday (e) 16 to those from postnatal day 30 in themouse, and discovered AS differences. Amongst thesethey noted that spliceforms more highly expressedpostnatally encode actin-related proteins.41 This find-ing is of interest as actin metabolism is paramount inmaturing neurons during early postnatal stages whenneuronal connections are being established.43 Tem-poral patterns of AS have also been discovered usingin vitro models of corticogenesis, in which human

embryonic stem cells are differentiated into neurons.42

This longitudinal analysis (termed Cortecon by theauthors) revealed widespread AS of 5017 genes dur-ing in vitro corticogenesis. Interestingly a significantfraction of these AS genes were associated with canceror nervous system diseases.

A significant limitation of the aforementionedanalyses is that most samples analyzed to date containheterogeneous cell populations, which collectivelymay contribute to AS differences, thus complicatinginterpretation. A recent comprehensive study byMolyneaux et al. significantly overcame this hurdle,using deep sequencing to probe transcriptome andAS changes in sorted excitatory neuronal popula-tions from various stages of corticogenesis.40 Thisgroup discovered 1181 genes with shifts in isoformexpression during corticogenesis. From transcriptome



analyses the authors identify genes showing uniformexpression at the gene-level but significant differencesat the isoform level, an observation also made byAyoub et al. as described above for Wdr61.36 Alto-gether, these analyses provide evidence that AS is atplay in the developing cerebral cortex across multipledimensions (tangential, radial, and temporal). Thecurrent studies collectively highlight new candidategenes that may regulate corticogenesis. Future studiesthat similarly apply cell sorting and/or single celltranscriptome analysis will be valuable for furtherdiscovery of AS differences in cortical development.

SPLICING FACTORS REGULATINGCORTICOGENESISThe spatio-temporal regulation of AS relies on thedifferential expression and function of trans-splicingfactors including RBPs. Both McKee et al. and DeBoeret al. uncovered splicing factors expressed within thedeveloping cortex.34,35 Several of these RBPs havebeen experimentally shown to be critical for corticaldevelopment.

NOVA2NOVA1 and 2 are members of KH-domain RBPsand are among the best-characterized RBPs in thebrain. Nova proteins bind RNA via YCAY clustersand UCAU sequences, and regulate AS in vitro. Ofthese, Nova2 is highly expressed in the neocortex.44

Consistent with biochemistry data, Nova2 knockout mice contain a significant number of splicinganomalies in the postnatal brain.12 Interestingly, thevast majority of mis-spliced genes (34 of 40) encodeproteins localized to the synapse. During prenatalcortical development, NOVA2 binds to a large numberof transcripts (27,576).13 Yano et al. showed Nova2is necessary for the proper migration of upper-layerneurons toward the CP. The authors used HITS-CLIPof Nova2-deficient brains to identify key downstreamsplicing targets of Nova2. They focused on 20 genesof the Reelin pathway, because Reelin is a migrationcue secreted by early born Cajal Retzius (CR) neurons.Remarkably, the authors identified significant splicingchanges in only one transcript of this pathway, findingthat NOVA2 regulates excision of exons 7b and 7cof Dab1. Using in utero electroporation of minigeneconstructs, the authors then elegantly showed thatNova2 is essential for the proper expression of Dab1spliceforms and that this splicing mediates neuronalmigration in the neocortex. In the future it willbe of interest to identify additional splicing targetsgenome-wide that may also be regulated by Nova2 inthe developing cerebral cortex.

PTBP2Poly-pyrimidine tract-binding proteins (PTBPproteins) are involved in multiple steps of RNAmetabolism including splicing. Biochemical studiesof PTBP1 demonstrated this family of proteins bindsintrons (recognizing CU-repeats and UCUY-richelements).44 Depending on its relative positioningwith respect to certain exons (either upstream ordownstream), PTBP1 can either promote or inhibitexon inclusion.16 Although PTBP1 is minimallyexpressed in the brain, PTBP2 (also called nPTB)is highly expressed in the brain.16 In the developingmouse brain, PTBP2 binds thousands of RNAs.16

Among these mRNAs, splicing of Psd-95, whichencodes a synaptic protein, is repressed by PTBP1 andPTBP2 during development.17 To further understandthe role of PTBP2 in cortical development, two groupsrecently generated PTBP2-deficient mice. Licatolosiet al. discovered that E14.5 PTBP2 mutant brains haddefective neural progenitor polarity, accompanied bydefects in proliferation and neuronal differentiation.16

Li et al. observed only postnatal cortical defects intheir mutant mice, related to a role in neuronal differ-entiation, maturation, and survival.18 The phenotypicdifferences may be because of the nature of the mousemutation, as the former study used germline knock-outs and the latter study used conditional expressionwith Nestin-Cre and Emx1-Cre lines. Regardless,in both studies, PTBP2-deficiency was associatedwith significant alterations in AS, including mRNAsinvolved in regulation of the actin cytoskeleton,proliferation and cell fate. Although RNA-targets ofPTBP2 involved in the regulation of neurogenesis havenot yet been identified, these studies establish PTBP2as a key regulator of AS in both neural progenitorsand immature neurons.

TRA2BTRA2B splicing factor is a member of theSerine-/Arginine-Rich (SR) protein family, whichhave well-established roles in constitutive and AS.45

SR proteins recognize exonic splice enhancers, as wellas interact with other splicing factors to promotesplice site recognition. Once splicing is complete,SR proteins may or may not remain on the mRNA.This retention of SR proteins on the mature mRNAcan impact nuclear export and downstream RNAregulation. TRA2B regulates splicing of Tau andSmn2 mRNAs, involved in Alzheimer’s disease andspinal muscular atrophy, respectively.46,47 HencePTBP2 is relevant for human diseases of the nervoussystem. Constitutional loss of Tra2b in the mouseleads to embryonic lethality by E7.0, precluding the



use of this model for the study of its role in corti-cal development.48 However conditional knock-outembryos using either Emx1-Cre14 or Nestin-Cre15

point toward an essential role of Tra2b in the sur-vival of neural progenitors and neurons. Strikingly,conditional Tra2b knockout in the cerebral cortexleads to almost complete absence of the cortex atadulthood, following a massive wave of apoptosisduring embryonic cortical development. These studiesestablish the fundamental requirement of Tra2b insurvival of neural cells, and highlight the importanceof future studies to determine key mRNA targets ofTRA2B in the embryonic cortex. Altogether, thesedata suggest that AS regulation plays a critical role incortical development. Given the abundance of splicingfactors in the developing brain, clearly these studiesare just the tip of the iceberg.

From the Nucleus to the Cytoplasm: TheExon Junction ComplexesAs splicing proceeds, spliced transcripts becomedecorated by exon junction complexes (EJC), whichbind primarily at the junctions where introns areexcised.49,50 The EJC remains bound to the splicedmRNA as the RNA is exported into the cytoplasm.The core EJC is composed of the heterodimer, Magohand Rbm8a, the helicase, Eif4a3, and the cytoplas-mic component Casc3. This core complex interactstransiently with other proteins to mediate variousaspects of mRNA metabolism, including mRNAsplicing, localization, nonsense-mediated decay, andtranslation initiation.51–53

Magoh was recently shown to be essential forcorticogenesis in mice.19,54 Haploinsufficiency forMagoh causes a severe microcephaly, associated withdepletion of IPs and massive apoptosis of new neu-rons. Silver et al. showed that Magoh regulates propercell division of radial glia and hypothesized thatdefective mitosis induces aberrant production of pro-genitors and neurons.19 Microarray analysis of Magohhaploinsufficient cortices revealed only 147 transcriptswith significant differential expression. Given that theEJC decorates >80% of exon–exon junctions49 thisindicates Magoh haploinsufficiency may not globallyimpact RNA stability although this remains to be for-mally tested. The authors identified protein changesdownstream of Magoh, including one physiologicallyrelevant target, Lis1, a microtubule-associated proteinalso involved in brain development. Future studieswill be useful to assess how Magoh impacts radialglia divisions either via translation and/or some otherstep in mRNA metabolism.

The same group recently probed whether hap-loinsufficiency for the Magoh heterodimer, Rbm8a,

impacts corticogenesis.20 Similar to Magoh, Rbm8ais also highly expressed in the developing cortex.Conditional haploinsufficiency for Rbm8a inducedmicrocephaly, even more severe than Magoh loss. Thisphenotype was associated with depletion of progen-itors and dramatic apoptosis especially of neurons.Rbm8a mutant embryos showed precocious neuronproduction and faster cell cycle exit of progenitors.Thee phenotypic similarities induced by Magoh andRbm8a haploinsufficiency support a model whereMagoh and Rbm8a act together as part of the EJC toregulate corticogenesis. The observed differences inseverity of phenotypes could reflect distinct functionsoutside of the EJC or redundancies with other pro-teins, such as MagohB.55 In addition to these roles ofcore EJC components in the brain, the peripheral EJCcomponent involved in NMD, Upf1, is also expressedin the developing neocortex and promotes a stem cellstate in primary cells.56 Future genetic and molecularstudies of these mutants will help establish whichaspect(s) of EJC regulation are critical to developmentof the brain.

Recent mouse and human genetic studies havecollectively implicated EJC dosage in neurodevelop-mental pathologies associated with aberrant corticaldevelopment, including autism, schizophrenia, andintellectual disability.57–59 UPF3B, an EJC componentrequired for nonsense-mediated decay, is mutatedin X-linked intellectual disability, schizophrenia,and autism. Copy number variations in several EJCcomponents, including UPF3B, EIF4A3, RBM8A,and MAGOH, are found in patients with intel-lectual disability frequently accompanied by brainmalformations.57 RBM8A is within the proximal1q21.1 microdeletion/duplication associated withmicrocephaly.58 EIF4A3 is mutated in Richeiri-CostaSyndrome, a developmental disorder which can alsobe associated with brain malformations.60 Continuinggenetic studies in model organisms will help establishif roles for these EJC components in corticogenesisare the root causes for these neurodevelopmentaldisorders.

RNA Stability and Translational ControlOutside of the nucleus, RNA stability and transla-tional regulation offer yet another layer of controlfor gene expression. The role for RNA stability incorticogenesis is poorly defined. A number of RNAshave been shown to have short-half lives but so farthis has been attributed to oscillations in transcrip-tion. Translational control can be exerted at differ-ent steps: initiation, elongation, or termination. Thesesteps are regulated by the coordination between ribo-somal complexes and a vast set of RBPs. RBPs which



impact translation control have recently been shownto influence development of the cerebral cortex.

HuRHuR is a well-characterized RBP, part of a fam-ily of Hu-related proteins that preferentially bindthe 3’UTR of its RNA targets to influence multipleaspects of RNA metabolism including RNA stabil-ity and translation.61,62 While several Hu proteinshave been implicated in neuron differentiation andpost-mitotic function,63,64 so far only HuR has beenformally shown to regulate normal embryonic cor-ticogenesis. HuR is expressed early in neuroepithelialcells, when these progenitors are undergoing primar-ily proliferative divisions.21 Garcia-Dominguez et al.postulated HuR influences the Notch pathway byregulating mRNA levels of the ligand Delta.21 Theexpression of Delta ligand promotes proliferationand prevents differentiation in neighboring cells viaa mechanism called lateral-inhibition.65 The authorsdiscovered that HuR interacts with Dll1 mRNA. HuRdepletion in neural precursors leads to reduced DllmRNA levels and less differentiation. This differenti-ation phenotype can be rescued by overexpression ofDll1. Hence the authors propose that HuR regulatesDll1 stability to promote lateral inhibition and thusinfluence early cell fates in the cortex.

Later in development when neuroepithelial cellshave been replaced by radial glial progenitors, HuR isexpressed in radial glia, IPs, and newborn neurons.22

In an elegant study evaluating translation, Krausharet al. used genome-wide polysome-profile analysis inconditional HuR knockout murine cortices at E13and P0 to uncover a large pool of HuR-regulatedmRNAs redistributed to different polysomal fractionsduring development. HuR-dependent RNAs wereenriched for regulators of transcription, translationand layer specific pathways. The transcripts regulatedby HuR were dramatically different in E13 and P0brains, perhaps reflecting different biological pro-cesses occurring at these distinct ages. The authorsalso make the novel discovery that HuR interacts withthe Eif2 kinase, Eif2ak4, which regulates the presenceof distinct ribosomal proteins in active sites of trans-lation at polysomes. The authors argue that HuRcoordinates the translation of a network of mRNAsencoding proteins that share common functions, akinto the RNA regulon model first proposed by JackKeene.62 Consistent with a functional requirement ofHuR in cortical development, phenotypic anatomi-cal analyses of P0 HuR conditional knockout micerevealed that HuR regulates the position, identity andmaturation of post-mitotic glutamatergic neurons.Future work will be valuable to further identify the

molecular mechanisms by which HuR regulates thesedevelopmental processes. Moreover this study sets thestage for future identification of signals that influencetemporal control of mRNA translation.

MusashiThe translational regulators, Musashi 1 and 2 (Msi-1and Msi-2), are highly expressed in NSCs throughoutthe central nervous system, including the mammaliancortex.66 Cortical Msi-1−/− dissociated NSCs trans-fected with antisense peptide-nucleic acid againstMsi-2 showed decreased neurosphere formation andproliferative capacity, perhaps linked to impairedcell-cycle progression.23 Hence Msi-1 and Msi-2 haveredundant functions in neural stem cells. Musashis(Msis) are reported to act as both positive and negativeregulators of translation, effects that are mediatedthrough binding to the 3’UTRs of target mRNAs,including mammalian Numb.24 Although this transla-tional relationship between Msi and Numb has so farbeen shown in fibroblasts it is tempting to speculatethat it may also hold true in NSCs, where numbis important for influencing neurogenesis.67,68 Msitargets have not yet been identified in NSCs; however,a recent study discovered translational targets fromprimary NSCs overexpressing Msi, using Ribosomeprofiling and RNA-seq.25 Among several transcriptswith reduced translation efficiency was Jag1, a ligandfor Notch receptors, as well as a number of RBPsincluding Prpf3, Kirrel3, Rbm22, and Dhx37. Inter-estingly these targets have abundant Msi-binding sitesin their 3’ UTRs, thus Msi is thought to directly bindthese targets. Katz et al. also demonstrated that Msioverexpression impacted AS while not perturbingoverall RNA levels extensively.25 Because Msi is pri-marily cytoplasmic, these changes are thought to bea secondary consequence of translational regulationof splicing factors, and not because of a direct role inAS or RNA stability per se. With identification of Msitranslational targets, it will be of interest in upcomingstudies to assess the role of these Msi targets uponNSC behavior in the cortex.

FMRPFMRP (Fragile-X mental retardation protein) is anRBP encoded by the Fmr1 gene. FMRP has beenlargely characterized as a translational inhibitor. Fmr1null mutations result in the Fragile-X syndrome (FXS)in humans, which is the most prevalent intellectualdisorder caused by mutations in one single gene.69,70

Postnatally, FMRP localizes at the synapses betweenneurons, where it inhibits the translation of a sub-set of localized mRNAs encoding proteins involved



in synaptic plasticity.69,71 In response to neuronalactivity, FMRP translational inhibition can be allevi-ated to allow for local, fast and massive, productionof proteins necessary for structural modifications ofthe postsynaptic compartment.69,72,73

Recent studies of humans and mice support arole for FMRP in regulation of prenatal cortical devel-opment. An analysis of brain region volumes of 1-to 3-year-old boys with FXS showed that severalcortical areas display enlarged gray matter volume,suggesting a possible regional increase in neurons.74

Indeed, analysis of P5 Fmr1−/− mouse pups revealedincreased neuronal density in the somatosensory cor-tical area.26 Moreover cultured NSCs derived fromeither pre- or postnatal Fmr1−/− mice generate moreneurons than those derived from comparably aged WTmice.27 Interestingly, the density of Tbr2+ IPs is higherin Fmr1-KO cortices, compared to controls, indicatingincreased neurons could be produced by more IPs.26

Hence FMRP may control neuron production eitherby regulating IP differentiation or IP generation fromradial glial cells. In support of the latter possibility,Saffary et al. used in utero electroporation to knock-down Fmr1 in the developing cortex and demonstrateFMRP is required for IP generation.28 The authorsidentified the candidate mRNA-target Profilin1 (Pfn1)as a mediator of this process, finding that Pfn1 over-expression rescues the overproduction of IPs in Fmr1mutant brains.

Another recent study revealed a role for FRMP inneuronal migration in the cortex. In a study by La Fataet al., newborn neurons labeled by in utero electro-poration in Fmr1 knockout brains showed defectiveneuronal migration.29 These defects eventually lead toabnormal neuronal networks in the postnatal brain,which could be rescued by the overexpression ofN-Cadherin, an mRNA target of FMRP. In additionto its well-established role in translational repression,FMRP has also been implicated as a pro-translationregulator in young neurons of the human neocortex.30

Kwan et al. showed that FMRP expression enhancesthe translation of NOS1, an important regulatorof synapse formation and spine maintenance.30,75

Interestingly FMRP-mediated regulation of NOS1translation was not evident in mouse projectionneurons, highlighting potentially interesting evolu-tionary differences in FMRP function. Altogetherthese two studies suggest that defective neuronalcircuits induced by defects in immature neurons couldbe at the origin of Fragile-X pathology in the adult.

Pfn1, NOS1, and N-Cadherin are likely partof a vast FMRP-regulated mRNA network involvedin the regulation of the NSC-to-IP transition, earlyneuronal differentiation, and migration. Although

FMRP targets have been elucidated in adult brains,76

it still remains an outstanding question which RNAsare FMRP targets in neural stem cells of the developingneocortex and whether their translation is repressed oractivated by FMRP. Future work is needed to identifythose potential targets, and to assess their contributionto behavior of NSCs and neurons.

Eif4E/4E-T ComplexThe EIF4E protein family, composed of Eif4E1, 2,and 3 (Eukaryotic Initiation Factor 4E), is part of asupercomplex docked to the 5’ cap of mRNAs.77 Oncebound to mRNAs this complex can either promoteor inhibit translation, depending on its composi-tion. These functions are mediated via interactionswith additional translation factors. For example,EIF4E1 association with EIF4G initiates translationwhereas EIF4E1 binding to 4E-T blocks translationor promotes mRNA decay by targeting mRNAs to Pbodies.78

A role for translational regulators in corticoge-nesis was recently revealed using in utero knockdownin embryonic brains.31 Yang et al. discovered thatdecreased levels of either Eif4e1 or 4E-T in neuralprogenitors lead to more neurons and fewer neuralprogenitors. This is accompanied by an increase inthe number of cells with high protein levels for Ngn1,Ngn2, and Neurod1, basic-Helix-Loop-Helix (bHLH)pro-neuronal transcription factors. The authors dis-covered that in neural progenitors, EIF4E1 bindsto Neurog1 and Neurog2 and NeuroD1 mRNAs.These biochemical results, along with rescue experi-ments using constructs deficient in RNA binding orprotein–protein interactions, collectively revealed theEif4e1/4E-T complex may repress translation of keyneurogenic transcripts. Conversely, knockdown ofEif4G in neural progenitors (a positive regulator oftranslation when associated with EIF4E1) promotesfewer neurons and more progenitors. Altogether,these results suggest that certain neural progenitorsare predisposed to the generation of neurons throughthe transcription of proneural bHLH transcriptionfactors, but are stalled in a proliferating state byEIF4E1/4E-T-mediated translational repression ofthese target mRNAs. The authors speculate that NSCsare preloaded with mRNAs encoding prodifferenti-ation factors; however, translation of these mRNAsis repressed by Eif4E1 binding. This is a compellinghypothesis and it will be exciting in the future to testthis model. It will also be valuable to demonstratethe direct role of Eif4E1/4E-T on translation of keymRNAs in NSCs, to rule out potential roles in nuclearexport or sequestration of mRNAs in RNA-processingbodies.



RNA LocalizationRNA localization plays a critical role in neuronsboth pre- and postnatally. When paired with transla-tional regulation, RNA localization allows for localprotein synthesis in the cytoplasm in response tointra- or extracellular signals (see Buxbaum et al., fora recent comprehensive review on this topic).79 Inimmature neurons, RNA localization and translation,in response to extracellular guidance molecules, isparamount for axon guidance and synaptic function.80

In mature neurons, RNA localization at the synapsemay be involved in the precise, fast response of cellsto integrate signals from other neurons, in order toconsolidate or suppress memories.72,81 Recent studieshave now highlighted roles for mRNA localization inmammalian neural progenitors, within both the cellbody and basal process.

RNA Localization to the Basal EndfeetRecently, it was shown that mRNAs encodingCyclinD2 accumulate in structures called basal end-feet, located at the end of basal process.82 CyclinD2 isan outstanding candidate for the maintenance of neu-ral progenitor proliferation as it is a well-characterizedG1-phase regulator, and G1 phase is strongly linkedto neural progenitor proliferation (Figure 1).83,84

Osumi’s group identified a region in the 3’UTR ofthe Cyclin D2 mRNA that is sufficient for its translo-cation to the basal endfeet. They also used a GFPreporter construct to argue that these mRNAs arelocally translated, although diffusion of GFP proteinsfrom the cell body could not be ruled out from thisexperiment. During asymmetric division of NSCs,the daughter cell which adopts proliferative behav-ior inherits the basal process, whereas the daughtercell that does not inherit the basal process proceedstoward differentiation.85 This led to the hypothesisthat following cell division, Cyclin D2 mRNA islocally translated and newly generated proteins sub-sequently migrate back to the soma through the basalprocess to promote proliferation. Identification of thetransmachinery, including RBPs that bind CyclinD2will be useful for understanding why it is asymmetri-cally localized in NSCs, and for identifying additionallocalized RNAs.

Stau2Stau2 is a double-stranded RBP, which in neuronshas been well characterized as a translational repres-sor and a regulator of subcellular localization.86 Inneurons, Stau2-positive RNA granules aggregateto form heterogeneous RNA granules that subse-quently associate with motor proteins to translocate

along microtubules to distal regions. Inspired by theDrosophila literature which established a role forStau in neuroblasts, two independent groups recentlyshowed that Stau2 plays a key role in mammaliancortical development.32,33 In mitotic progenitors,Stau2 is enriched at one pole of the cell, and becomesasymmetrically localized to only one of the post-mitotic progeny. Kusek et al. showed Stau2 wasspecifically inherited by the daughter cell that differ-entiates into an intermediate precursor cell followingmitosis.33 Both groups showed that downregulationof Stau2 by shRNA knockdown leads to increaseddifferentiation and a depletion of radial glia both invitro and in vivo. Additionally, Vessey et al. showedthat Stau2 acts in coordination with at least twoother RBPs: the helicase Ddx1 and the translationalrepressor Pum2.32 RIPs were employed to identifyStau2 targets, with Sally Temple’s group identifyinggenome-wide targets in the entire cortex and FredaMiller’s group focussing on specific candidates. Geneontology (GO) analysis showed Stau2 RNA targetswere enriched in transcripts encoding regulators ofcell-cycle exit and cilia.33 Of note, several of thesetargets, Prox1, Bbs2 and Trim32, are asymmetricallylocalized in a Stau2-dependent fashion. Altogether,these results suggest that Stau2 plays a preponderantrole in the selective transmission of pro-differentiationmRNAs in progeny. In parallel, given other RNA reg-ulatory roles of Stau2, the RBP may additionallyrepress translation of pro-differentiation mRNAsin NSCs. The authors speculated that this transla-tional repression would be relieved in differentiateddaughter cells.

These studies indicate that RNA localizationmay serve as a cell fate determinant to help two daugh-ter cells adopt different fates. Utilizing the “RNAmedium” to segregate cell fate determinants representsa certain advantage. As translation is largely pausedduring mitosis87 the inheritance of select mRNAmolecules in daughter progeny might allow for therapid and massive synthesis of this determinant imme-diately after completion of mitosis. This mechanisminvolves a multistep process which includes: (1) theproduction of an mRNA and its associated RBPsprior to mitosis, (2) the translational repression of thismRNA until the completion of mitosis, (3) the pre-cise localization of this mRNA to a cellular regionwhich will be specifically inherited by one daughtercell, and (4) the derepression of translation followingmitosis. Future studies and identification of asymmet-rically localized mRNAs and RBPs in mitotic neuralprogenitors will help define whether this mechanism isbroadly used for cell fate determination in the mam-malian cortex.



CONCLUSIONThis review highlighted key players in posttranscrip-tional RNA regulation with fundamental roles in cor-ticogenesis. One theme that emerges from this reviewis that we have just scratched the surface in terms ofa comprehensive understanding of how RBPs influ-ence cortical development and which RBPs are impor-tant. A second theme that emerges is that virtuallyall aspects of posttranscriptional regulation are impli-cated in corticogenesis. Many fundamental questionsnow remain to be answered. How is RNA regula-tion coordinated within rapidly dividing populationsacross stages of embryonic development? What addi-tional RBPs influence corticogenesis, how do they doso, and what are their key targets? What role doesRNA stability play in modulating cell fate choices inthe developing brain? Answering these questions in acomplex tissue such as the embryonic mammalian cor-tex is challenging and will require multidisciplinaryapproaches encompassing bioinformatics, biochem-istry, and genetics.

Identifying RNA targets for RBPs is critical togain a mechanistic understanding of how these RBPshelp shape the developing brain. Techniques in RNAimmunoprecipitation have been critical for uncover-ing RNA targets for many RBPs within immortal-ized cells. With variations on RIP approaches, suchas HITs-CLIP, it has now become possible to identifyRNA targets within whole mouse brains. However,as noted throughout this review, approaches usingentire tissue only give a superficial understanding ofRBP targets, given the heterogeneous nature of the

developing brain over time and space. Future stud-ies which couple optimized RNA immunoprecipita-tion approaches with single cell resolution will beideal. These will inform our understanding of howRBPs function in progenitors versus postmitotic neu-rons, and in early versus late development. More-over, the use of Ribotag-sequencing approaches orribosomal profiling within neural stem cells will beextremely valuable to assess genome-wide transla-tion profiles for RBPs. On an individual transcriptbasis, the direct visualization of RNA targets in situ isnow also possible, using single-molecule FISH probes.Using the MS2-tagging approach, one can also nowfollow single RNA movements in the cell.79 Similarly,live-imaging can be used to evaluate translational tar-gets, by use of reporter constructs in which regulatorymRNA sequences are tethered to photo-convertibleprotein, such as Kaede or Dendra.88

Over 800 RBPs have now been bioinformat-ically and empirically identified, and among thesemany have annotated expression in the developingcerebral cortex.34,35,89 Yet it remains an outstandingquestion as to which of these RBPs are critical. Thesequestions can be addressed using traditional genetics,CRISPR/Cas9 approaches, or in utero electroporation,the latter of which allows one to rapidly manipulategene expression within the developing brain. More-over, the ability to utilize primary cell culture of pro-genitors and ex vivo embryonic brain slice culture pro-vide tools that make this developing organ amenableto testing candidates involved in posttranscriptionalregulation. The future is exciting for RNA regulationin corticogenesis as the advent of new technologies willlead to great advances in this field of research.

ACKNOWLEDGMENTSWe thank members of the Silver lab for helpful discussions. Work in this lab is supported by an R01NS0830897(to DLS). We apologize to all those whose work we could not include in this review.

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Post-transcriptional regulation in corticogenesis: how RNA