Development, specification, anddiversity of callosal projection neuronsRyann M. Fame1,2,3*, Jessica L. MacDonald1,2,3* and Jeffrey D. Macklis1,2,3

1 MGH-HMS Center for Nervous System Repair, Departments of Neurosurgery and Neurology, and Program in Neuroscience,

Harvard Medical School, Boston, MA 02114, USA2 Nayef Al-Rodhan Laboratories, Massachusetts General Hospital, Boston, MA 02114, USA3 Department of Stem Cell and Regenerative Biology and Harvard Stem Cell Institute, Harvard University, Cambridge, MA 02138, USA

Review

Callosal projection neurons (CPN) are a diverse popula-tion of neocortical projection neurons that connect thetwo hemispheres of the cerebral cortex via the corpuscallosum. They play key roles in high-level associativeconnectivity, and have been implicated in cognitive syn-dromes of high-level associative dysfunction, such asautism spectrum disorders. CPN evolved relatively re-cently compared to other cortical neuron populations,and have undergone disproportionately large expansionfrom mouse to human. While much is known about theanatomical trajectory of developing CPN axons, andprogress has been made in identifying cellular and mo-lecular controls over midline crossing, only recently havemolecular-genetic controls been identified that specifyCPN populations, and help define CPN subpopulations.In this review, we discuss the development, diversityand evolution of CPN.

IntroductionThe cerebral cortex, and neocortex in particular, containshundreds of distinct neuronal subtypes that enable it toperform incredibly complex tasks. Complexity of cognitionandassociativebehaviorexpandswith increasedabundanceand diversity of unique, yet precise connectivity amongthese neuronal subtypes [1]. Callosal projection neurons(CPN) play a key role in this high-level associative complex-ity. They are interhemispheric commissural pyramidal neu-rons, whosemyelinatedaxonsmakeup the corpus callosum,the largest white-matter tract in the placental mammalianbrain,which connects the two cerebralhemispheres [2].Thisreview will discuss recent studies that have advanced ourunderstanding of the development, diversity and evolutionof CPN.

Development of the neocortexDuring neocortical development, initial populations of par-tially fate-restricted progenitors sequentially give rise tothe wide variety of diverse mature neural cells: neurons,astroglia and oligodendroglia [3]. The diverse neocorticalneuronal population can be broadly divided into two maindivisions: (i) excitatory glutamatergic projection neurons;and (ii) inhibitory GABAergic interneurons. Excitatoryneocortical projection neurons arise primarily from apicaland early basal intermediate progenitors in the dorsal(pallial) ventricular zone (VZ) and later from an interme-

Corresponding author: Macklis, J.D. (jeffrey_macklis@hms.harvard.edu)* These two authors contributed equally to this work.

0166-2236/$ – see front matter � 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.tins.2010.

diate population of basal progenitors in the subventricularzone (SVZ) [4,5], while interneurons arise from progenitorsin the ventral (subpallial) ganglionic eminences [6]. Thesix-layered neocortex progressively forms in an inside-outfashion during embryonic development, with later-bornsuperficial-layer neurons migrating past the earlier-borndeep-layer neurons. Superficial neocortical layers arise pri-marily from later-born intermediatebasalprogenitors of theSVZ, a type of transit-amplifying progenitor generated fromradial glia progenitors [7]. Neurons with the same laminarlocation share a common birthdate [8,9]; however, evenwithin a layer, many anatomically and molecularly distinctneuronal subtypes coexist. Thus, substantial neuronal di-versity exists both between and within layers, and laminarposition is not nearly sufficient to define neuronal subtypeidentity and function. While broad controls governing corti-cal patterning are well studied, only recently have neuronsubtype-specific controls begun to be elucidated [10–21].

Threebroadsubclassesexistwithin the classof excitatorycortical projection neurons: (i) corticofugal projection neu-rons (CFuPN); (ii) CPN; and (iii) ipsilateral circuit connec-tion neurons. CFuPN project away from the cortex; theyrespect the midline, are earlier born and reside in the deeplayers (as is discussed later in this review, are also evolu-tionarily older). CFuPN project both subcortically to deeperbrain areas, and subcerebrally to brainstem and spinal cordtargets. In contrast, CPN cross the midline in the brain,project interhemispherically, and function to integrate in-formation between the two cerebral hemispheres. CPN aremore abundant than CFuPN and comprise the largest classof commissural neurons in placentalmammals [2]; their cellbodies principally reside in cortical layers II/III (approxi-mately 80% in rodents), layer V (approximately 20% inrodents) and, to a lesser extent, layer VI.

Abnormalities of CPN are pathological. Absence of CPNconnectivity in humans is associated with defects in ab-stract reasoning, problem solving and generalization [22].CPN dysgenesis is one of only a few reproducibly identifiedpathologies in autism spectrum disorders (ASD), withreduced corpus callosum connectivity relative to overallbrain volume observed in affected individuals, often with asmaller corpus callosum [23–28]. In addition, complete orpartial agenesis of the corpus callosum, as well as surgicalcallosotomy, result in cognitive deficits that differ depend-ing on when in development CPN connections are lost, howmuch of the corpus callosum is disturbed and the extentof secondary effects, such as aberrant connectivity [22].

10.002 Trends in Neurosciences, January 2011, Vol. 34, No. 1 41

[()TD$FIG]

(a)

(b)

CPIZ

SVZVZ

IGGGW

SCSMZG

I - IV V

VI

Callosal neuron (layer II/III (~80%), V (~20%), VI (few%))Callosal neuron with striatal projection (medial Va)

Callosal neuron with ipsilateral forward projection(lateral V)Callosal neuron with ipsilateral backward projection(medial)

Premotor cortex

Cingulate cortex (“pioneer”) callosal neurons

Neocortical callosal neurons

Embryonic

Adult

Key:

Key:

TRENDS in Neurosciences

Figure 1. CPN development and diversity. (a) During development, callosal axons

(red) turn toward the midline. Multiple glial populations (blue) and mixed

neuronal/glial populations (purple) play critical roles in CPN axon guidance and

midline crossing. Pioneering axons (brown) from neurons of the cingulate cortex

begin the process of midline crossing. This schematic represents processes that

occur across multiple embryonic times during mouse CPN development.

Abbreviations: CP, cortical plate; GW, glial wedge; IGG, indusium griseum glia;

IZ, intermediate zone; MZG, midline zipper glia; SCS, subcallosal sling. (b) At least

four major types of adult CPN can be classified based on projection patterns. These

include: single projections to the contralateral cortex (red); dual projections to the

contralateral cortex and ipsilateral or contralateral striatum (green); dual

projections to the contralateral cortex and ipsilateral premotor cortex (blue); or

dual projections to the contralateral cortex and ipsilateral sensorimotor cortex

(purple).

Review Trends in Neurosciences January 2011, Vol. 34, No. 1

The potential for identifying roles of unique populations ofneurons specifically affected in diseases, such as ASD, willbe enhanced by understanding neuronal diversity of pro-jection neurons themselves within the neocortex. Molecu-lar diversity underlies this stark anatomical andfunctional dichotomy between the two broad subclassesof CFuPN and CPN, as well as diversity within them.

Callosal projection neuronsEarly development and axon guidance

CPN maturation follows a set of discrete sequential stepsas CPN establish correct circuitry and synaptic connec-tions with their targets in the contralateral hemisphere.CPN are born throughout corticogenesis at the same timesin development as other neurons with cell bodies residingin their same cortical layers: in the mouse, layer VI CPNare born at approximately embryonic day 12.5 (E12.5)along with corticothalamic projection neurons (CThPN);layer V CPN are born around E13.5 along with corticosp-inal motor neurons (CSMN); and superficial-layer CPN areborn from approximately E15.5 to E17.5 [14,29]. As CPNare born, the two telencephalic hemispheres begin fusing,aided, in part, by two populations of glia (midline zipperglia and indusium griseum glia) [30–33] (Figure 1a). Whilecellular and molecular mechanisms of midline fusion havenot been completely identified, it is clear that, if themidline is not fully fused, callosal axons have no substrateand cannot cross to the contralateral hemisphere. Manymidline fusion and glial defects cause partial or completeagenesis of the corpus callosum, independent from abnor-malities of CPN themselves (reviewed in [34]).

As the hemispheres continue to fuse, populations of gliaand local neurons form the transient bridge-like subcallosalsling across the midline [30,35,36]. CPN send axons ven-trally toward the intermediate zone guided, in part, bysignals from indusium griseum glia dorsally at the midlineand glial wedge and subcallosal sling populations ventrally[32,33,36]. Upon reaching the intermediate zone, callosalaxons turn toward the midline to cross at the corticoseptalboundary, rather than projecting laterally as CFuPN do.Upon encountering the contralateral glial wedge, CPNaxons turn dorsally and extend into the neocortex towardhomotopic targets. Mechanisms of precise CPN targeting tocontralateral homotopic regions are still largely unknown,but callosalaxonshavebeenshownto follow the trajectory ofradial glia in the contralateral hemisphere as they extendtheir axons to appropriate targets [37]. During development(at E17 in mouse), axons from neurons of the cingulatecortex begin the process of midline crossing [30,38–40]and might act as pioneers for neocortical CPN [41](Figure 1a), which begin to cross one day later [30]. First-born, deep-layer cingulatepioneers andneocorticalCPNarethe first of each respective population to cross the midlineand, therefore, reach final targets before superficial-layerCPN.

Processes of midline crossing and targeting are mediat-ed by a large number of long-range and short-range signalsthat, while highly studied, are not completely known. Keymolecular regulators controlling midline crossing and tar-geting are best studied for deep-layer, early-crossing CPN;it is not evident whether or not superficial-layer, later-born

42

CPN employ the same mechanisms. These processes havebeen extensively reviewed elsewhere [31,32,36,38] and wewill only briefly summarize that body of work here.

Studies over the past decade have uncovered somemidline crossing and targeting controls that operate atseveral different levels of CPN function. At the level ofgrowth cone dynamics in callosal axons themselves, Mam-

Review Trends in Neurosciences January 2011, Vol. 34, No. 1

malian Enabled (Mena) plays a role in actin cytoskeletaldynamics in neurons of neocortical layers II/III and V, andis required for proper formation of the corpus callosum aswell as the hippocampal commissure [42]. Long-rangeguidance molecules, such as members of the Slit/Robo,Wnt and Netrin families, also play active roles in axonguidance across the corpus callosum. Slits, including Slit2,are enriched along the midline, surrounding the areathrough which CPN axons pass; these axons express theSlit receptor Robo1 [31,43–47]. Wnts, particularly Wnt5a,are necessary for formation of all forebrain commissures,both through canonical, Frizzled3-mediated [48] and non-canonical, Receptor-like Tyrosine kinase (Ryk)-mediated[49,50], receptor transduction pathways [31]. In addition toSlits and Wnts, Netrin1 and its receptor, deleted in colo-rectal cancer (DCC), are also required for all forebraincommissure formation [31,51–53]; however, althoughCPN express DCC, there is no evidence that DCC-mediat-ed mechanisms of guidance are the same in the corpuscallosum as they are for Netrin1 in commissure formationof the spinal cord [51,54]. At least some guidance roles ofthe subcallosal sling are mediated by Semaphorin-3C(Sema3C) attraction through the Neuropilin1 receptoron CPN [36,55]. In addition to long-range signals, short-range, local interactors [in particular ephrins and theirreceptors (EphA5, EphB1 and EphrinB3)] are essential forcorpus callosum formation [31,56,57]. Notably, it appearsrare for guidance defects in corpus callosum formation tobe callosum specific; rather, they typically affect broaderpopulations of commissures.

As a broad population, CPN extend exuberant projec-tions throughout development, with the maximal numberof CPN with dual projections occurring at approximatelypostnatal day 8 (P8) in mice [58,59]. Dual projections areprogressively refined until approximately P21, when theadult projection pattern for CPN is established; this pro-cess is thought to occur largely through activity-dependantHebbian mechanisms [59–62]. Interestingly, laterally lo-catedCPN that are furthest from themidline and themanysignaling molecules present there, extend a bifurcatedaxon early in development that projects toward both themidline and the internal capsule. Only later (approximate-ly P11 in mice) do lateral developing CPN retract axonalsegments projecting to the internal capsule [63].

Anatomical diversity

CPN extend an axon to the homotopic region of the con-tralateral neocortex; thus, the location of a CPNwithin thecortex defines the target of its callosal axon [64]. Thecorpus callosum is often broadly categorized in six regionsfrom rostral to caudal, named for homotopic regions theseaxons connect: frontal, motor, somatosensory, auditory,temporoparietal and visual. Callosal fibers vary in densityand diameter across these regions, both in rodents andhumans [2].

In addition to homotopic, interhemispheric projectionsextended by all CPN, subpopulations of CPN can be de-fined by the variety of long-range dual axonal projectionsthey extend. Subpopulations of CPN send dual projectionsto contra- or ipsilateral striatum (here, referred to asCStrPNi) [65], caudally to contra- and/or ipsilateral pri-

mary somatosensory cortex (here, referred to as BPN)[58,66], or rostrally to contra- or ipsilateral frontal areas(here, referred to as FPN) [58] (Figure 1b). CPN with dualprojections reside preferentially in the deep layers of theneocortex. For example, in adult mice, only about 4% oflayer II/III CPN extend dual axonal projections to thefrontal premotor cortex, while approximately 40% of layerV CPN do [58]. CStrPNi reside almost exclusively in layerVa [65], which supports the hypothesis that CPNwith dualprojections reside in evolutionarily older deep layers and,therefore, are more likely to have been evolutionarily co-opted from existing CFuPN. Additionally, deep-layer CPN(layers V and VI) provide about 80% of the collateralsconnecting primary motor cortex to primary somatosenso-ry cortex, and some deep-layer CPN have also been shownto project to secondary somatosensory cortex and theclaustrum, in addition to the striatum [67]. In patientswith partial agenesis of the corpus callosum, diffusiontensor imaging detects heterotopic axonal projections thatare not detectable in healthy subjects [68]. It is possiblethat, in healthy subjects, these heterotopic projections arestill present, but undetectable over overwhelming signalfrom intact, homotopic axons. If this is the case, theremight be much more diversity of connectivity within thehuman corpus callosum than investigators have been ableto detect with current technology.

While deep-layer CPN have long-distance dual project-ing axons, superficial-layer CPN participate in local cir-cuitry within cortical columns. Ipsilaterally, superficial-layer CPN send collaterals to pyramidal neurons withinlayers II/III, and even more strongly to layer V. They alsosend collaterals to pyramidal and stellate neurons in layerVI. In addition to participating in column circuitry locally,superficially located CPNalso extend collaterals within thecontralateral cortex, as their axons project radially intothe neocortex after crossing through the corpus callosum[69]. Thus, in addition to their role in integrating twohomotopic regions of the neocortical hemispheres, CPNare responsible for association and integration amongdifferent neuronal types in ipsilateral and contralateralcortical hemispheres.

Such laminar, anatomical and connectivity diversitywithin the broad population of CPN demonstrate that itis not a homogenous population of projection neurons.Rather, CPN make up a strikingly diverse set of subpopu-lations requiring precise control of their neuronal diversityby a complex and interactive set of molecular–geneticcontrols.

Molecular–genetic controls over CPN development

and diversity

Although, as discussed earlier, much progress has beenmade in beginning to understand the anatomical trajectoryof developing CPN axons and cellular and molecular con-trols over midline crossing [30–32,36–38,42,44–46,54,70–

72], considerably less is known about molecular–geneticcontrols that specify CPN subtype identity and control thisprecise development. Because themajority of CPN reside insuperficial layers, thefirst identifiedmolecular controls overCPN generation and development were identified as lami-nar-specific genes (Online Supplementary Material Table

43

Review Trends in Neurosciences January 2011, Vol. 34, No. 1

S1). In 2002, two POU-domain transcriptional regulators,Brn1 and Brn2, were identified as expressed in superficialcortical layers, and as necessary for correct cortical lamina-tion and neuronal migration [73,74]. When both Brn1 andBrn2 are deleted, superficial-layer pyramidal neurons arenot generated [74]. Inaddition, transcription factors cut-likehomeobox 1 and2 (Cux1 andCux2)are expressed in the SVZand selectively in superficial neocortical layers [75], withCux2 function being necessary for SVZ formation [76]. In2008, the first critical molecular regulator of broad CPNspecification, special AT-rich sequence-binding protein 2(SATB2), was identified and characterized as a DNA-bind-ing transcription factor expressed in CPN. SATB2 is neces-sary for specification of CPN through repression of COUP-TF interacting protein 2 (CTIP2) [16,17], a transcriptionfactor critical for CSMN axon outgrowth and fasciculation[10,11,14,77,78]. In the absence of SATB2 function, neuronsthat would have extended axons across the corpus callosuminstead project subcortically through the internal capsuleand take on some molecular characteristics of CFuPN.Identification of SATB2 as a molecular regulator of CPNidentity across all layers, especially axonal connectivitythrough inhibition of CTIP2, significantly advanced thecharacterization of CPN at a molecular level. However,many interesting questions about instructive molecularsignals responsible for midline crossing and precise homo-and heterotopic connections remain to be answered. Impor-tantly, mechanisms by which this and other still-unchar-acterized signals govern general CPN development are stilllargely unknown.

Recently, molecular–genetic controls that act specificallyin subclasses of CPN have begun to be identified. Thetranscription factor activator enhancing binding protein 2gamma (AP2g) acts specifically in a subset of radial gliacortical progenitors to specify SVZ intermediate progenitorsand enable the switch from proliferative to neurogenicdivision, and to generate a specific subpopulation of super-ficial-layer CPN in visual cortex [79]. Interestingly, whilethe action of AP2g is highly area specific, the expression ofthe gene encoding AP2g is not, suggesting an areally re-stricted partner or compensatory activity. In addition,Cux1and Cux2, previously discussed as layer-specific identifiers,regulate dendrite branching, spine development and syn-apse formation specifically in layer II/III CPN [80]. Thesesubtype-specific controls are important for understandingthe diversity that exists within, and is integral to, the broadCPN population.

Multiple approaches have been used successfully toidentify molecular controls over temporal and/or laminarstages of neocortical projection neuron development. Inves-tigators have screened gene expression databases for tran-scriptional regulators expressed in relevant laminae orprogenitor zones of the neocortex, or have investigatedfunctions of guidancemolecules known to play critical rolesin guiding neuronal populations in other regions of thenervous system. However, in order to identify molecularcontrols over development of specific, individual popula-tions of neocortical projection neurons in a more direct andunbiased manner, approaches to isolate and purify indi-vidual neuronal populations have provided substantialpower and sensitivity. One approach that has proven

44

useful for isolating distinct populations of cortical projec-tion neurons has been to first retrogradely label them fromtheir developmental axonal trajectories and final axonaltargets, and then to purify them using fluorescence acti-vated cell sorting (FACS; Figure 2). For a variety of reasonsdiscussed elsewhere [10,11,14], this approach was firstused to label and isolate CPN [10,81–83], CSMN[10,11,84], corticotectal projection neurons (CTPN [10]),CThPN, CStrPN and segmentally specific cervical or lum-bar CSMN. These purified neurons were submitted tocomparative microarray analysis to identify genes differ-entially expressed by each population at four key embry-onic and postnatal developmental stages (E18.5, P3, P6and P14). Other purification methods have also provenfruitful for isolating distinct populations of cortical projec-tion neurons [85,86].

Purification of specific neuronal populations, followedby comparative gene expression analysis, has not only ledto the identification of genes expressed by each populationat distinct stages in development, but has also enriched forcritical subtype-specific molecular controls by comparinggene expression between very closely related cortical pro-jection neuron populations. This work has already identi-fied a set of genes that, in combination, define aprogressively restricting program of molecular–geneticcontrols (or a ‘molecular–logic’) over development of impor-tant populations of cortical projection neurons includingCPN [10,11,15,18,21,81,87]. These data were collected assymmetric with regard to development of the neocorticalprojection neuron populations compared, and providesequivalent (but not yet functionally investigated) informa-tion regarding molecular–genetic controls over CPN devel-opment both as a broad population and in specificsubpopulations [81] (Figure 3, Online Supplementary Ma-terial Table S1).

CPN genes identified in this way have been analyzedbased on their laminar- and sublaminar-specific distribu-tions across different stages of maturation [81] (Figure 3,Online Supplementary Material Table S1). Temporal infor-mation from these analyses identifies distinct molecularstages of CPN development that likely reflect known pro-cesses occurring during CPN maturation. Molecular con-trols expressed most highly early in CPN development (i.e.at or before E18.5 in the mouse, such as Inhba, Btg1,Frmd4b,Epha3 andPtn) likely act during neuronal subtypespecification, refinement of differentiation, migration orinitial axonal extension. Genes whose expression sharplyrises and falls (i.e. are specifically expressed only during themid-stages of CPN development, such as Cpne4, Tmtc4,Nnmt, Cav1, Nectin-3 and Chn2) might be hypothesizedto functionwhenCPNhave already crossed themidline andare extending toward their specific targets.Genes expressedspecifically late in CPN development (e.g. Plexin-D1,Gfra2,TcrB and Dkk3) might more likely function in final CPNmaturation and refinement of adult connectivity.

In addition to temporal gene expression data, this workidentifies differential subtype-specific laminar gene expres-sion. A subset of the identified CPN genes appear specific toCPN in all layers in which CPN reside (i.e. layers II/III andV,VI),while othersdiscriminatebetweenCPNofdeep layersand those of superficial layers (Figure 3). Further, several

[()TD$FIG]

1. Retrograde labeling of projection neuron subtypes

2. FACS purification of labeled projection neurons3. Comparative microarray analysis

I - IVV

VI

OB

I - IVVVI

IC

Th

Po

SC

Crb

Corticothalamic projection neuron (layer VI)

Corticospinal motor neuron (layer V)

Corticotectal projection neuron (layer V)

Callosal projection neuron (layers II/III (~80%), V (~20%), VI (few%))

Key:

Key:

TRENDS in Neurosciences

Figure 2. Schematic representation of an experimental approach used to identify CPN-specific genes. (1) CPN (red), CThPN (purple), CSMN (green) and CTPN (blue) were

retrogradely labeled at distinct stages of development from the contralateral hemisphere, the thalamus, the spinal cord and the superior colliculus, respectively. (2) Labeled

neurons were dissociated, purified using fluorescence activated cell sorting (FACS), and (3) followed by comparative microarray genetic expression analysis [10,14,81–84].

Adapted, with permission, from [81].

Review Trends in Neurosciences January 2011, Vol. 34, No. 1

genes finely subdivide CPN within individual layers, andappear to label discrete CPN subpopulations that have notbeen previously described using anatomical or morphologi-cal criteria [58,81]. Interestingly, while a number of thegenes expressed specifically in superficial-layer CPN areexpressed throughout the entirety of layers II/III, somegenes are restricted to only the most superficial portion oflayers II/III, while other genes are restricted to the deeperportion of superficial layers (Figure 3).

In isolation, differences in laminar expression mightmerely reflect major orminor birthdate differences. Howev-er, in light of being specific to CPN versus other neocorticalprojection neurons, and considering the existence of diversehodological CPN subpopulations already identified, thesedifferentially expressed genes might be hypothesized to be

molecular controls and/or functional hallmarks of thesepreviously identified subpopulations, as well as of novel,as-of-yet unidentified subpopulations of CPN. Molecular–genetic controls expressed in all laminae where CPN resideare more likely to control top-level, unifying properties ofCPN, such asmidline crossing and avoidance of corticofugalfate and connectivity. Alternatively, these broadlyexpressed genes might have more restricted functions dueto combinatorial intersectionwith areally restrictedbindingpartners and/or co-factors (such precedents exist for a num-ber of broadly expressed cortical genes, including Bhlhb5[18], Clim and Lmo4 [19], and AP2g [79]).

Since superficial layers have undergone substantialevolutionary expansion in comparison to deep layers(as discussed in more detail below), genes with expression

45

[()TD$FIG]

Figure 3. Spatially restricted genes identify novel CPN subpopulations. Schematic representation of neocortical layers depicting laminar-specific expression of 20 selected,

representative genes expressed by early postnatal CPN within the neocortex [81]. Dark-blue and light-blue bands indicate high and low levels of expression, respectively.

Grey oblique stripes demarcate layers in which CPN reside. Most of these genes have dynamic patterns of expression through development; therefore, developmental

stage must be considered when using these genes to identify specific populations of CPN. Representative genes are depicted with multiple patterns of laminar expression:

(a) most cortical layers and deep cortical layers only; (b) superficial cortical layers only; and (c) subdivisions of superficial layers. See main text and Online Supplementary

Material Table S1 for more detailed expression and references. Roman numerals indicate neocortical layers (I–VI). Abbreviation: SP, subplate.

Review Trends in Neurosciences January 2011, Vol. 34, No. 1

restricted to deep layers might reflect transcriptionalchanges that allowed CPN to arise from evolutionarilyolder CFuPN populations [15,19,88]. Deep cortical layersalso contain the overwhelming majority of CPN with dual-projecting axons [58,65], so genes expressed in subpopula-tions of deep-layer CPN might, for example, identify andcontrol development of specific dual-projecting popula-tions. In contrast, genes restricted to superficial-layerCPNmight serve as controls over development of columnarcollaterals. Alternatively, CPN expressing superficial-lay-er-restricted genes might represent novel CPN subpopula-tions that evolved later and are born later in corticaldevelopment than deep-layer CPN. In addition, differen-tial combinations of these genes identify even more sub-laminae than are observed by examining single genes inisolation. Thus, it is evident that there is striking molecu-lar, and likely hodological and functional, diversity withinthe broad population of CPN. Investigation of functions ofthese combinatorial molecular–genetic controls will allowfor better characterization and understanding of functionsand clinical relevance of these and other unique subpopu-lations of CPN.

Evolution

CPN and their associated axonal pathway in the corpuscallosum arose relatively recently in evolution; observedfirst in placental mammals [2]. The corpus callosum is not

46

unique in its ability to connect the two neocortical hemi-spheres, but is the only fiber tract devoted solely to inte-gration of information from the two cortical hemispheres.In non-placental mammals, such as marsupials, the domi-nant interhemispheric fiber tract in the brain is the muchsmaller anterior commissure, consisting of interhemispher-ic fibers from the amygdala, olfactory tract and temporallobes, as well as long-distance connections from the neocor-tex that take a convoluted route to project to the contralat-eralneocortex [2].Thehippocampal commissurealsoplaysarole, albeit much smaller than the anterior commissure, inenabling interhemispheric communication in animals lack-ing a corpus callosum [2,89]. Pre-existence of these othercommissural tracts might have more readily allowed forevolution and establishment of cortical commissural projec-tion neurons that became CPN. For example, hippocampalcommissure axons, in addition to cingulate axons, mightpioneer thepath thatneocorticalCPN later followacross thecorpus callosum, suggesting that these hippocampal com-missural neurons might have fasciculated with emergingearly CPN to enable their crossing of the midline barrier tofirstprojectacross the region that isnowthe corpus callosum[34,38,89].

The broad laminar distribution of CPN speaks notonly to a broad time window of CPN generation in devel-opment from diverse progenitor populations, but also sug-gests preferential evolutionary expansion of this neuronal

Review Trends in Neurosciences January 2011, Vol. 34, No. 1

population as the cortex expanded throughout evolution.The telencephalon of sauropsids (e.g. reptiles and birds) isa three-layered structure that is evolutionarily related tolayers I, V and VI in the mammalian neocortex, but isdevoid of CPN [88,90]. The evolutionarily novel corticallayers II/III, which are present in rodents, are greatlyexpanded in primates [88,91] (Figure 4), as is the volumeof white matter in the corpus callosum [1], with >190million axons in the human [92]. These mammalian neo-cortical superficial layers arise primarily from progenitorsof the SVZ [7], which is a distinct progenitor zone present inmammals and some sauropsids (including birds and cro-codilians, but not turtles), but not in amphibians [90]. TheSVZ itself has greatly expanded and diversified in primatesto include two distinct regions, the inner and the outerSVZ, which generate the expanded cortical diversity, par-ticularly in superficial layers, found in primates [91,93,94]

[()TD$FIG]

Figure 4. Comparison of developing and adult mammalian neocortex of mouse, macaq

layer expansion, and white matter expansion. Schematic comparison of histological sec

monkey and (c) adult human neocortex. Adult cross-sections are from visual cortex. The

layers II/III (�80%), V (�20%), and VI (few %) in the adult neocortex. There is a strong corr

thickness and neocortical white matter. Roman numerals denote neocortical layers (I–VI

IFL, inner fibrous layer; ISVZ, inner SVZ; MZ, marginal zone; OFL, outer fibrous layer; OSV

progenitors (yellow/ brown), immature neurons (blue), stellate neurons of layer IV (purp

(macaque) and with data from [1] and the Allen Brain Atlas (mouse: http://developingm

(Figure 4). The outer SVZ contains unique self-renewing,proliferative, radial glia-like progenitors that are distinctfrom those in the inner SVZ [93,94]. A number of molecularcontrols over SVZ populations have been identified, includ-ing T-box brain gene 2 (Tbr2; also known as Eomes) [95–

97], and subventricular-expressed transcript 1 (Svet1) [7].Given that themajority of CPN reside in the evolutionarilyexpanded superficial layers generated from this progres-sively expanded population of intermediate progenitors, itis logical to hypothesize that CPN have undergone exten-sive expansion throughout evolution of the cerebral cortex.Thus, compared to deep-layer-restricted CFuPN, CPNmight be predicted to serve especially important roles inprimate cognitive function.

Expansion of superficial neocortical layers in primateevolution far exceeds expansion of deep layers [88]. Alarge portion of CPN with known heterotopic long-range

ue, and human shows correlations between SVZ expansion, superficial neocortical

tions of (a) developing and adult mouse, (b) developing and adult rhesus macaque

thicknesses are represented relative to a common scale. CPN (red) reside mostly in

elation between the expansion of the SVZ and the expansion of the superficial-layer

). Abbreviations: CP, cortical plate; E, embryonic; PP, preplate; VZ, ventricular zone;

Z, outer SVZ; PP, preplate; SP, subplate; WM, white matter. Other colors represent:

le), white matter/ axons (black). Adapted and expanded with permission from [91]

ouse.brain-map.org and human: http://humancortex.alleninstitute.org).

47

Review Trends in Neurosciences January 2011, Vol. 34, No. 1

dual-projecting axons reside in deep neocortical layers, sug-gesting that deep-layerCPNmight have been evolutionarilyco-opted from existing populations of CFuPN to project notonly to subcortical targets, but also across the midline toconnect and integrate the two hemispheres of the neocortex.Once the early, deep population(s) of CPNwere established,their presence might have favored expansion of the neocor-tex, and addition of more subtypes of CPN to augment thefunctionally advantageous rapid and precise integration ofneocortical hemispheres, perhaps first in primary visualcortex [2] or newly evolvingmotor cortex [89]. Interestingly,there is a large group of CPN-enriched genes expressed insublaminae within superficial neocortical layers in mouse[81]. In the mouse, neocortical layers II and III are nottypically distinguished as distinct, but they are expandedand obviously distinct in primates. While developmentalexpression of thesesublaminarCPNgenes inprimate cortexis not currently known, some (e.g. Cux2, Nectin-3 andPlxnd1) display similar expression in adult human cortexas in mouse cortex ([98] and the Allen Brain Atlas [http://humancortex.alleninstitute.org]). It is interesting to specu-late that sublaminarly distributed genes might serve asevolutionarily early molecular identifiers of the furtherexpansion and specialization of superficial layers in pri-mates.

ConclusionRecent evidence supports a growing understanding andnewappreciation of the striking diversity within the broad pop-ulation of CPN. CPN are defined by their homotopicallyprojecting axons across the corpus callosum, but some sub-populations of CPN have dual-projecting axons to long-distance ipsilateral and contralateral targets, while othersubpopulations participate in local column circuitry, bothipsilaterally and contralaterally. The progressive evolution-ary emergence of complexity and increased diversity ofconnectivity in the cerebral cortex also suggests diversityin the origin of CPN. Deep-layer CPN might have arisenfrom evolutionarily older CFuPN [15,19], while superficial-layer CPN expanded greatly with the expansion of the SVZin the progression from lower mammals to primates andhumans.

Currently, molecular–genetic controls over CPN as abroad population, and over specific subpopulations of CPN,are being discovered and functionally investigated. Thiswork elucidating the connectivity, guidance and molecularcharacteristics of CPN, both as a broad population and asdistinct hodological and functional subpopulations of CPN,will greatly contribute to a better understanding of CPNand complex associative cognitive functions in which theyplay critical roles. More in-depth understanding of thespecific functions of individual CPN subpopulations willenable more sophisticated understanding of cortical func-tion, and diagnosis of neurological abnormalities involvingCPN and the corpus callosum, including agenesis of thecorpus callosum, ASD and likely other syndromes of high-level dysfunction of associative connectivity.

AcknowledgementsWe thank L. Pasquina for superb artistic assistance. This work waspartially supported by the National Institutes of Health (Grants NS41590

48

and NS45523), the Harvard Stem Cell Institute, the Jane and Lee SeidmanFund for CNS Research, and the Emily and Robert Pearlstein Fund forNervous System Repair (J.D.M.), with additional infrastructure support byNational Institutes of Health Grant NS49553 (J.D.M). R.M.F. was partiallysupported by a National Science Foundation Graduate ResearchFellowship.

Appendix A. Supplementary dataSupplementary data associated with this article can befound, in the online version, at doi:10.1016/j.tins.2010.10.002.

References1 Schoenemann, P.T. et al. (2005) Prefrontal white matter volume is

disproportionately larger in humans than in other primates. Nat.Neurosci. 8, 242–252

2 Aboitiz, F. and Montiel, J. (2003) One hundred million years ofinterhemispheric communication: the history of the corpus callosum.Braz. J. Med. Biol. Res. 36, 409–420

3 Peters, A. and Jones, E.G., eds (1984) Cellular Components of theCerebral Cortex, Plenum Press

4 Kowalczyk, T. et al. (2009) Intermediate neuronal progenitors (basalprogenitors) produce pyramidal-projection neurons for all layers ofcerebral cortex. Cereb. Cortex 19, 2439–2450

5 Noctor, S.C. et al. (2004) Cortical neurons arise in symmetric andasymmetric division zones and migrate through specific phases. Nat.Neurosci. 7, 136–144

6 Hevner, R.F. (2007) Layer-specific markers as probes for neuron typeidentity in human neocortex and malformations of corticaldevelopment. J. Neuropathol. Exp. Neurol. 66, 101–109

7 Tarabykin, V. et al. (2001) Cortical upper layer neurons derive from thesubventricular zone as indicated by Svet1 gene expression.Development 128, 1983–1993

8 Bayer, S.A. and Altman, J. (1991) Neocortical Development, RavenPress

9 Rakic, P. (1974) Neurons in rhesus monkey visual cortex: systematicrelation between time of origin and eventual disposition. Science 183,425–427

10 Arlotta, P. et al. (2005) Neuronal subtype-specific genes that controlcorticospinal motor neuron development in vivo. Neuron 45, 207–221

11 Molyneaux, B.J. et al. (2005) Fezl is required for the birth andspecification of corticospinal motor neurons. Neuron 47, 817–831

12 Chen, B. et al. (2005) Fezl regulates the differentiation and axontargeting of layer 5 subcortical projection neurons in cerebral cortex.Proc. Natl. Acad. Sci. U. S. A. 102, 17184–17189

13 Chen, J.G. et al. (2005) Zfp312 is required for subcortical axonalprojections and dendritic morphology of deep-layer pyramidalneurons of the cerebral cortex. Proc. Natl. Acad. Sci. U. S. A. 102,17792–17797

14 Molyneaux, B.J. et al. (2007) Neuronal subtype specification in thecerebral cortex. Nat. Rev. Neurosci. 8, 427–437

15 Lai, T. et al. (2008) SOX5 controls the sequential generation of distinctcorticofugal neuron subtypes. Neuron 57, 232–247

16 Alcamo, E.A. et al. (2008) Satb2 regulates callosal projection neuronidentity in the developing cerebral cortex. Neuron 57, 364–377

17 Britanova, O. et al. (2008) Satb2 is a postmitotic determinant for upper-layer neuron specification in the neocortex. Neuron 57, 378–392

18 Joshi, P.S. et al. (2008) Bhlhb5 regulates the postmitotic acquisition ofarea identities in layers II–V of the developing neocortex. Neuron 60,258–272

19 Azim, E. et al. (2009) Lmo4 and Clim1 progressively delineate corticalprojection neuron subtypes during development. Cereb. Cortex 19(Suppl. 1), i62–i69

20 Azim, E. et al. (2009) SOX6 controls dorsal-ventral progenitorparcellation and interneuron diversity during development of theneocortex. Nat. Neurosci. 12, 1238–1247

21 Tomassy, G.S. et al. (2010) Area-specific temporal control ofcorticospinal motor neuron differentiation by COUP-TFI. Proc. Natl.Acad. Sci. U. S. A. 107, 3576–3581

22 Paul, L.K. et al. (2007) Agenesis of the corpus callosum: genetic,developmental and functional aspects of connectivity. Nat. Rev.Neurosci. 8, 287–299

Review Trends in Neurosciences January 2011, Vol. 34, No. 1

23 Minshew, N.J. and Williams, D.L. (2007) The new neurobiology ofautism: cortex, connectivity, and neuronal organization. Arch. Neurol.64, 945–950

24 Herbert, M.R. and Kenet, T. (2007) Brain abnormalities in languagedisorders and in autism. Pediatr. Clin. North Am. 54, 563–583 vii

25 Mcalonan, G.M. et al. (2009) Differential effects on white-mattersystems in high-functioning autism and Asperger’s syndrome.Psychol. Med. 39, 1885–1893

26 Freitag, C.M. et al. (2009) Total brain volume and corpus callosum sizein medication-naıve adolescents and young adults with autismspectrum disorder. Biol. Psychiatry 66, 316–319

27 Vidal, C.N. et al. (2006) Mapping corpus callosum deficits in autism: anindex of aberrant cortical connectivity. Biol. Psychiatry 60, 218–225

28 Egaas, B. et al. (1995) Reduced size of corpus callosum in autism. Arch.Neurol. 52, 794–801

29 Angevine, J.B. and Sidman, R.L. (1961) Autoradiographic study of cellmigration during histogenesis of cerebral cortex in the mouse. Nature192, 766–768

30 Silver, J. et al. (1982) Axonal guidance during development of the greatcerebral commissures: descriptive and experimental studies, in vivo, onthe role of preformed glial pathways. J. Comp. Neurol. 210, 10–29

31 Lindwall, C. et al. (2007) Commissure formation in the mammalianforebrain. Curr. Opin. Neurobiol. 17, 3–14

32 Richards, L.J. et al. (2004) Mechanisms regulating the development ofthe corpus callosum and its agenesis in mouse and human.Clin. Genet.66, 276–289

33 Shu, T. et al. (2003) Development of midline glial populations at thecorticoseptal boundary. J. Neurobiol. 57, 81–94

34 Donahoo, A.L.S. and Richards, L.J. (2009) Understanding themechanisms of callosal development through the use of transgenicmouse models. Semin. Pediatr. Neurol. 16, 127–142

35 Shu, T. et al. (2003) The glial sling is a migratory population ofdeveloping neurons. Development 130, 2929–2937

36 Niquille, M. et al. (2009) Transient neuronal populations are required toguide callosal axons: a role for semaphorin 3C. PLoS Biol. 7, e1000230

37 Norris, C.R. and Kalil, K. (1991) Guidance of callosal axons by radialglia in the developing cerebral cortex. J. Neurosci. 11, 3481–3492

38 Rash, B.G. and Richards, L.J. (2001) A role for cingulate pioneeringaxons in the development of the corpus callosum. J. Comp. Neurol. 434,147–157

39 Koester, S.E. and O’Leary, D.D. (1994) Axons of early generatedneurons in cingulate cortex pioneer the corpus callosum. J.Neurosci. 14, 6608–6620

40 Ozaki, H.S. and Wahlsten, D. (1998) Timing and origin of the firstcortical axons to project through the corpus callosum and thesubsequent emergence of callosal projection cells in mouse. J. Comp.Neurol. 400, 197–206

41 Piper,M. et al. (2009) Neuropilin 1-sema signaling regulates crossing ofcingulate pioneering axons during development of the corpus callosum.Cereb. Cortex 19 (Suppl. 1), i11–i21

42 Lanier, L.M. et al. (1999) Mena is required for neurulation andcommissure formation. Neuron 22, 313–325

43 Shu, T. et al. (2003) Slit2 guides both precrossing and postcrossingcallosal axons at the midline in vivo. J. Neurosci. 23, 8176–8184

44 Lopez-Bendito, G. et al. (2007) Robo1 and Robo2 cooperate to controlthe guidance of major axonal tracts in the mammalian forebrain. J.Neurosci. 27, 3395–3407

45 Andrews, W. et al. (2006) Robo1 regulates the development of majoraxon tracts and interneuron migration in the forebrain. Development133, 2243–2252

46 Sundaresan, V. et al. (2004) Dynamic expression patterns of Robo(Robo1 and Robo2) in the developing murine central nervoussystem. J. Comp. Neurol. 468, 467–481

47 Bagri, A. et al. (2002) Slit proteins prevent midline crossing anddetermine the dorsoventral position of major axonal pathways inthe mammalian forebrain. Neuron 33, 233–248

48 Wang, Y. et al. (2006) Axonal growth and guidance defects in Frizzled3knock-out mice: a comparison of diffusion tensor magnetic resonanceimaging, neurofilament staining, and genetically directed cell labeling.J. Neurosci. 26, 355–364

49 Keeble, T.R. et al. (2006) The Wnt receptor Ryk is required for Wnt5a-mediated axon guidance on the contralateral side of the corpuscallosum. J. Neurosci. 26, 5840–5848

50 Li, L. et al. (2009)Wnt5a induces simultaneous cortical axon outgrowthand repulsive axon guidance through distinct signalingmechanisms. J.Neurosci. 29, 5873–5883

51 Serafini, T. et al. (1996) Netrin-1 is required for commissural axonguidance in the developing vertebrate nervous system. Cell 87, 1001–

101452 Ren, T. et al. (2007) Diffusion tensor magnetic resonance imaging and

tract-tracing analysis of Probst bundle structure in Netrin1- and DCC-deficient mice. J. Neurosci. 27, 10345–10349

53 Fazeli, A. et al. (1997) Phenotype of mice lacking functional Deleted incolorectal cancer (Dcc) gene. Nature 386, 796–804

54 Shu, T. et al. (2000) Expression of the netrin-1 receptor, deleted incolorectal cancer (DCC), is largely confined to projecting neurons in thedeveloping forebrain. J. Comp. Neurol. 416, 201–212

55 Gu, C. et al. (2003) Neuropilin-1 conveys semaphorin and VEGFsignaling during neural and cardiovascular development. Dev. Cell 5,45–57

56 Mendes, S.W. et al. (2006) Multiple Eph receptors and B-class ephrinsregulate midline crossing of corpus callosum fibers in the developingmouse forebrain. J. Neurosci. 26, 882–892

57 Hu, Z. et al. (2003) Corpus callosum deficiency in transgenic miceexpressing a truncated ephrin-A receptor. J. Neurosci. 23, 10963–10970

58 Mitchell, B.D. and Macklis, J.D. (2005) Large-scale maintenance ofdual projections by callosal and frontal cortical projection neurons inadult mice. J. Comp. Neurol. 482, 17–32

59 Innocenti, G.M. and Price, D.J. (2005) Exuberance in the developmentof cortical networks. Nat. Rev. Neurosci. 6, 955–965

60 Wang, C. et al. (2007) Activity-dependent development of callosalprojections in the somatosensory cortex. J. Neurosci. 27, 11334–11342

61 Mizuno, H. et al. (2007) Evidence for activity-dependent cortical wiring:formation of interhemispheric connections in neonatal mouse visualcortex requires projection neuron activity. J. Neurosci. 27, 6760–6770

62 Mizuno, H. et al. (2010) Pre-synaptic and post-synaptic neuronalactivity supports the axon development of callosal projectionneurons during different post-natal periods in the mouse cerebralcortex. Eur. J. Neurosci. 31, 410–424

63 Garcez, P.P. et al. (2007) Axons of callosal neurons bifurcate transientlyat the white matter before consolidating an interhemisphericprojection. Eur. J. Neurosci. 25, 1384–1394

64 Yorke, C.H., Jr and Caviness, V.S., Jr (1975) Interhemisphericneocortical connections of the corpus callosum in the normal mouse:a study based on anterograde and retrograde methods. J. Comp.Neurol. 164, 233–245

65 Wilson, C.J. (1987) Morphology and synaptic connections of crossedcorticostriatal neurons in the rat. J. Comp. Neurol. 263, 567–580

66 Cauller, L.J. et al. (1998) Backward cortical projections to primarysomatosensory cortex in rats extend long horizontal axons in layer I. J.Comp. Neurol. 390, 297–310

67 Veinante, P. and Deschenes, M. (2003) Single-cell study of motor cortexprojections to the barrel field in rats. J. Comp. Neurol. 464, 98–103

68 Wahl, M. et al. (2008) Variability of homotopic and heterotopic callosalconnectivity in partial agenesis of the corpus callosum: a 3T diffusiontensor imaging and Q-Ball tractography study. AJNR Am. J.Neuroradiol. 30, 282–289

69 Petreanu, L. et al. (2007) Channelrhodopsin-2-assisted circuit mappingof long-range callosal projections. Nat. Neurosci. 10, 663–668

70 Plachez, C. et al. (2008) Nuclear factor I gene expression in thedeveloping forebrain. J. Comp. Neurol. 508, 385–401

71 Smith, K.M. et al. (2006) Midline radial glia translocation and corpuscallosum formation require FGF signaling. Nat. Neurosci. 9, 787–797

72 Tole, S. et al. (2006) Development ofmidline cell types and commissuralaxon tracts requires Fgfr1 in the cerebrum. Dev. Biol. 289, 141–151

73 McEvilly, R.J. et al. (2002) Transcriptional regulation of cortical neuronmigration by POU domain factors. Science 295, 1528–1532

74 Sugitani, Y. et al. (2002) Brn-1 and Brn-2 share crucial roles in theproduction and positioning of mouse neocortical neurons. Genes Dev.16, 1760–1765

75 Nieto, M. et al. (2004) Expression of Cux-1 and Cux-2 in thesubventricular zone and upper layers II-IV of the cerebral cortex. J.Comp. Neurol. 479, 168–180

76 Cubelos, B. et al. (2007) Cux-2 controls the proliferation of neuronalintermediate precursors of the cortical subventricular zone. Cereb.Cortex 18, 1758–1770

49

Review Trends in Neurosciences January 2011, Vol. 34, No. 1

77 Chen, B. et al. (2005) Fezl regulates the differentiation and axontargeting of layer 5 subcortical projection neurons in cerebral cortex.Proc. Natl. Acad. Sci. U. S. A. 102, 17184–17189

78 Chen, J.G. et al. (2005) Zfp312 is required for subcortical axonalprojections and dendritic morphology of deep-layer pyramidalneurons of the cerebral cortex. Proc. Natl. Acad. Sci. U. S. A. 102,17792–17797

79 Pinto, L. et al. (2009) AP2g regulates basal progenitor fate in a region-and layer-specific manner in the developing cortex. Nat. Neurosci. 12,1229–1237

80 Cubelos, B. et al. (2010) Cux1 and Cux2 regulate dendritic branching,spine morphology, and synapses of the upper layer neurons of thecortex. Neuron 66, 523–535

81 Molyneaux, B.J. et al. (2009) Novel subtype-specific genes identifydistinct subpopulations of callosal projection neurons. J. Neurosci.29, 12343–12354

82 Catapano, L.A. et al. (2004) Stage-specific and opposing roles of BDNF,NT-3 and bFGF in differentiation of purified callosal projectionneurons toward cellular repair of complex circuitry. Eur. J.Neurosci. 19, 2421–2434

83 Catapano, L.A. et al. (2001) Specific neurotrophic factors support thesurvival of cortical projection neurons at distinct stages ofdevelopment. J. Neurosci. 21, 8863–8872

84 Ozdinler, P.H. and Macklis, J.D. (2006) IGF-I specifically enhancesaxon outgrowth of corticospinal motor neurons.Nat. Neurosci. 9, 1371–

138185 Dugas, J. et al. (2008) A Novel purification method for CNS projection

neurons leads to the identification of brain vascular cells as a source oftrophic support for corticospinal motor neurons. J. Neurosci. 28, 8294–

830586 Barres, B.A. et al. (1988) Immunological, morphological, and

electrophysiological variation among retinal ganglion cells purifiedby panning. Neuron 1, 791–803

50

87 Arlotta, P. et al. (2008) Ctip2 controls the differentiation of mediumspiny neurons and the establishment of the cellular architecture of thestriatum. J. Neurosci. 28, 622–632

88 Molnar, Z. et al. (2006) Comparative aspects of cerebral corticaldevelopment. Eur. J. Neurosci. 23, 921–934

89 Mihrshahi, R. (2006) The corpus callosum as an evolutionaryinnovation. J. Exp. Zoolog. Part B. Mol. Dev. Evol. 306, 8–17

90 Charvet, C.J. et al. (2009) Phylogeny of the telencephalic subventricularzone in sauropsids: evidence for the sequential evolution of pallial andsubpallial subventricular zones. Brain Behav. Evol. 73, 285–294

91 Smart, I.H. et al. (2002) Unique morphological features of theproliferative zones and postmitotic compartments of the neuralepithelium giving rise to striate and extrastriate cortex in themonkey. Cereb. Cortex 12, 37–53

92 Tomasch, J. (1954) Size, distribution, and number of fibres in thehuman corpus callosum. Anat. Rec. 119, 119–135

93 Fietz, S.A. et al. (2010) OSVZ progenitors of human and ferretneocortex are epithelial-like and expand by integrin signaling. Nat.Neurosci. 13, 690–699

94 Hansen, D.V. et al. (2010) Neurogenic radial glia in the outersubventricular zone of human neocortex. Nature 464, 554–561

95 Baala, L. et al. (2007) Homozygous silencing of T-box transcriptionfactor EOMES leads to microcephaly with polymicrogyria and corpuscallosum agenesis. Nat. Genet. 39, 454–456

96 Arnold, S.J. et al. (2008) The T-box transcription factor Eomes/Tbr2regulates neurogenesis in the cortical subventricular zone. Genes Dev.22, 2479–2484

97 Sessa, A. et al. (2008) Tbr2 directs conversion of radial glia into basalprecursors and guides neuronal amplification by indirect neurogenesisin the developing neocortex. Neuron 60, 56–69

98 Arion, D. et al. (2007) Molecularmarkers distinguishing supragranularand infragranular layers in the human prefrontal cortex. Eur. J.Neurosci. 25, 1843–1854