Patterning and Cell Type Specification in the Developing Cns and Pns || Area Patterning of the...

Patterning and Cell Type Specification in the Developing CNS and PN

Developmental Neuroscience, Volume 1 http://dx.doi.org/10.1016/B

C H A P T E R

4

Area Patterning of the Mammalian CortexD.D.M. O’Leary, A.M. Stocker, A. Zembrzycki

The Salk Institute, La Jolla, CA, USA

O U T L I N E

4.1 Introduction 61

4.2 Cortical Divisions and Components 624.2.1 Cerebral Cortex Divisions 624.2.2 Neocortical Areas 634.2.3 Thalamocortical Relationships 634.2.4 Origins of General Classes of Cortical

Neurons 64

4.3 Naturally Occurring Differences in AreaPatterning 654.3.1 Phylogenic Differences in Area Patterning 654.3.2 Differences in Area Patterning within

a Species 66

4.4 Extrinsic Influences on Area Patterning 664.4.1 Late Development of Area Patterning from an

Early More Uniform CP 664.4.2 Development and Plasticity in Area-Specific

Output Projections Related to AreaPatterning 67

4.4.3 Area-Specific TCA Input and Potential Rolesin Area Patterning 68

4.4.4 Sensory Periphery-Mediated Plasticity in AreaPatterning 68

4.5 Intrinsic Genetic Mechanisms RegulatingArealization 694.5.1 Telencephalic Patterning Centers

in Arealization 70

4.5.1.1 CoP:AnAnteriorPatterningCenter 70

4.5.1.2 Cortical Hem: A Dorsal–Caudal

Patterning Center 71

4.5.2 TF Expression by Cortical ProgenitorsRegulates Area Patterning 714.5.2.1 Emx2 71

4.5.2.2 Pax6 72

4.5.2.3 Sp8 73

4.5.2.4 COUP-TFI 73

4.5.2.5 Interactions Between TFs to Regulate

Area Patterning 75

4.6 Extent of Genetic Specification of Area-SpecificProperties 764.6.1 Do Area-Unique Genes Exist? 764.6.2 What Is ‘Area Identity?’ 764.6.3 Genetic Determination of Cortical Projection

Neurons Related to Arealization 774.6.4 Candidate Targets of TFs that Regulate Area

Patterning 774.6.5 Translating Gradients of TFs into Sharp

Borders 78

4.7 Regional Patterning of the Cerebral Cortex 80

4.8 Conclusions 81

Acknowledgments 81

References 81

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4.1 INTRODUCTION

Although a prominent role for intrinsic genetic specifi-cationofarea identity in corticalprogenitorswasproposeda quarter of a century ago (Rakic, 1988), the first direct ev-idence for thismodel, came just over adecade agowith thereports that the transcription factors (TFs) Emx2 (Bishop

et al., 2000; Mallamaci et al., 2000) and Pax6 (Bishopetal., 2000) regulated arealization through their gradedex-pression in progenitors in the cortical ventricular zone(VZ). Prior to those initial reports, the field and modelsofmechanismsof arealizationweredominatedby theverylarge body of evidence accrued over many decades onroles for thalamocortical axon (TCA) input in

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62 4. AREA PATTERNING OF THE MAMMALIAN CORTEX

differentiation and plasticity in area patterning (O’Leary,1989). It is now widely accepted that the specificationand differentiation of neocortical areas is controlled byan interplay between genetic regulation intrinsic to theneocortex – characterized by TFs expressed by corticalprogenitors – and extra-genetic influences that arise sub-cortically, predominantly sensory input relayed by TCAinput from the principal sensory nuclei of the dorsal thal-amus to the primary cortical areas (Figure 4.1; O’Leary,1989; O’Leary and Nakagawa, 2002; O’Leary et al., 2007;Rakic, 1988; Rakic et al., 2009; Rash and Grove, 2006; Surand Rubenstein, 2005). Here we summarize our currentunderstanding of the process of neocortical arealization,focusing on genetic regulation intrinsic to the neocortex,but also touching upon related issues including plasticityin area patterning and regionalization of the cerebralcortex.

4.2 CORTICAL DIVISIONS ANDCOMPONENTS

4.2.1 Cerebral Cortex Divisions

The cerebral cortex is the largest and most complexcomponent of the mammalian brain (Purves, 1988;Rakic, 2009). Early in embryonic development of mam-mals, the neural plate differentiates into the neural tube,

Signaling molecules (patterning centers)

Fgf8, Fgf17 (ANR, CoP)

Wnts, Bmps (hem)

Shh

Anti-hemGraded gene expression in VZ (dTel)

Ime

P

M

A

L

Graded geneexpression

in VZ and CP

PA

CP

VZ

Pax6Sp8

Emx2COUP-TFI

FIGURE 4.1 Patterning centers and graded transcription factors drivescription factors (TFs) in the ventricular zone (VZ) are established by sigcenters, such as Fgf8 and Fgf17 from the anterior neural ridge (ANR), whicthe cortical hem. The antihem is a putative patterning center identified baSfrp2, as well as Neurogulin 1 and 3) with known patterning functions. Amains of Sonic Hedgehog (Shh) in ventral telencephalon, but it does not hexpression of certain TFs, such as Pax6, Emx2, COUP-TFI, and Sp8, impartstheir neuronal progeny that form the cortical plate (CP). The CP also initiadistinct patternswith sharp borders. Coincident with this process, distinctseen in the adult (M1, S1, A1, V1), differentiate from the CP. Genes that are dpatterns in different layers, suggesting that area-specific regulation of sudefinition of area identity. Although the initial establishment of the gradintrinsic to the telencephalon, the more complex differentiation patterns enisms, for example, TCA input and the sensory activity that it relays from(2002) Patterning centers, regulatory genes and extrinsic mechanisms controlling

I. INDUCTION AND PATTERN

and during this process, the rostral end of the neural tubedevelops three vesicles – the forebrain, midbrain, andhindbrain – that ultimately give rise to the brain. Soonthereafter, the dorsolateral aspects of the forebrain evagi-nate to generate the telencephalic vesicles, and the re-mainder of the forebrain becomes the diencephalon (seeChapter 1 and 8). The expression of individual TFs orcombinations of TFs correlates with morphologic bound-aries within the telencephalon and plays an importantrole in its patterning into major subdivisions: the ventraltelencephalon, which gives rise to the striatum and basalganglia, and the dorsal telencephalon (dTel), which givesrise to the cerebral cortex (Figure 4.2; Rubenstein et al.,1998) (see Chapter 24). The diencephalon differentiatesinto several components, the major ones including thedorsally positioned epithalamus (habenula) and dorsalthalamus, which reciprocally connect with the neocor-tex, and the ventrally positioned hypothalamus (seeChapter 8).

The cerebral cortex itself is divided into regions. Theneocortex is the largest region, and is positioned betweentwo other regions of the cerebral cortex: the archicortex(including entorhinal cortex, retrosplenial, subiculum,and hippocampus) and the paleocortex (olfactory,i.e., piriform, cortex). In addition, the neocortex accountsformuchof the increase in size andcomplexity inmoread-vanced species, as well as arguably the most distinct

ntrinsicchanisms Anatomically

and functionallydistinct areas

A

ML

P

Discontinuous,sharply borderedgene expression

2/3

4

5

6

F/M

S1

A1

V1?

Extrinsicmechanisms

arealization of the neocortex. The initial, tangential gradients of tran-naling molecules/morphogens secreted from telencephalic patterningh later becomes the commissural plate (CoP), andWnts and Bmps fromsed on its expression of secreted signaling molecules (e.g., Tgfa, Fgf7,fourth telencephalic patterning center is defined by the expression do-ave defined roles in dorsal telencephalic (dTel) patterning. The gradedpositional or area identities to cortical progenitors,which is imparted tolly exhibits gradients of gene expression that are gradually converted tocortical layers (2–6), and the anatomically and functionally distinct areasifferentially expressed across the cortex are often expressed in differentch genes is modulated by layer-specific properties, and questions theed gene expression in the embryonic CP is controlled by mechanismsstablished postnatally might be controlled in part by extrinsic mecha-the periphery to the cortex.Modified from O’Leary DDM and Nakagawa Y

arealization of the neocortex. Current Opinion in Neurobiology 12: 14–25.

ING OF THE CNS AND PNS

Glu

GABA

ACh

dTel

MGE

LGE

AEP/POa

Hp

Nctx

OlfCtx Str

(a) (b)

(Emx1)

FIGURE 4.2 Regionalization and migration in the forebrain. (a andb) Drawings of a coronal section through the embryonic forebrain, me-dial is to the right. Progenitors in the VZ (green domain) of the dTel ex-press Emx1 and generate neocortical glutamatergic (Glu) projectionneurons that migrate radially (green arrows in b) to form the CP. A sub-population of postmitotic neurons of the Emx1-lineage born in the lateraldTel migrates not radially but ventrally to populate the olfactory cortex(Olf Ctx). The ventral telencephalon consists of threemolecularly distinctprogenitor domains: lateral ganglionic eminence (LGE), medial gangli-onic eminence (MGE), and preoptic area (AEP/POa). LGE and MGEprogenitors (blue domain) generate GABAergic (GABA) interneuronsthat migrate tangentially (blue arrows in b) in different routes to inte-grate into the striatum (Str), Olf Ctx, and neocortex (Nctx). Neurons bornin the AEP/POa (red domain) are cholinergic (Ach) (see Chapters 26and 24). Adapted fromMarin, O., Rubenstein, J.L. (2001). A long, remarkablejourney: tangential migration in the telencephalon. Nature ReviewsNeuroscience 2, 780–790.

634.2 CORTICAL DIVISIONS AND COMPONENTS

phylogenetic specializations (Krubitzer and Kaas, 2005).Among the many features that distinguish the neocortexfrom other regions of the cerebral cortex is its laminar pat-terning characterized by six major, radially organized,layers, which themselves are often substratified, each con-taining a heterogeneous population of neurons that aremorphologically, connectionally, and functionallydistinctfrom those of other layers. In its tangential dimension, theneocortex is organized into ‘areas’; these are functionallyunique subdivisions distinguished from one anotherby differences in cytoarchitecture and chemoarchitecture,input and output connections, and patterns of geneexpression (Figure 4.3; O’Leary and Nakagawa, 2002;O’Leary et al., 2007; Rash and Grove, 2006; Sur andRubenstein, 2005).

4.2.2 Neocortical Areas

The neocortex has four ‘primary’ areas, each servingas the anchor of surrounding clusters of functionally re-lated, ‘higher order’ areas, which are prominently inter-connected (Figure 4.3). Three of the primary areas aresensory: the primary visual (V1), somatosensory (S1),and auditory (A1), which process primary informationreceived from the eye/retina (vision), body (somatosen-sation), and inner ear/cochlea (audition), respectively.The only major sense not processed by the neocortex issmell. Distinct odors are sensed by olfactory and


vomeronasal receptors in the nose and related periph-eral organs and are processed in the olfactory cortex po-sitioned rostrally within the cerebral cortex, ventral toanterior parts of the neocortex (e.g., frontal areas). Thefourth primary area of the neocortex is the motor (M1)area, which controls voluntary movement of bodyparts. Historically, areas, or functional fields, of thecortex have also been related to the skull bones thatcover them, and the primary areas make up a large pro-portion of these fields, particularly in lower mammalianorders (e.g., Rodentia, gnawing mammals such as miceand rats; Marsupialia, pouched mammals such as opos-sums; and Monotremata, egg-laying mammals includ-ing platypuses (Krubitzer, 2007; Krubitzer and Kaas,2005). From anterior (A) to posterior (P), these relation-ships in mice are as follows: M1 is covered by the frontalbone and is part of the frontal cortex; S1 is covered by theparietal bone and is part of the parietal cortex; and V1 iscovered by the occipital bone and is part of the cortex.

Theneocortex, particularly in the brains of higher ordermammals (e.g., Primates, including humans, gorillas,chimpanzees, orangutans, and monkeys; Cetaceans, in-cluding dolphins and whales; and Proboscidea, such aselephants), is dominated by a much greater number ofhigher order areas positioned between the primary areas,serving as higher order processing centers focusedon spe-cific features of a particular modality, for example, higherorder visual areasmight be primarily concernedwith spe-cific features of vision such as movement or attention re-lated to the visual field; many higher order areas aremultimodal (Krubitzer, 2007; Krubitzer and Kaas, 2005).In the adult, the transition from one neocortical area to an-other is typically abrupt, with borders that can be sharplydefinedbyareadifferences inarchitecture (Figure4.3), andin some instances by the distributions of projection neu-rons, input projections, or gene expressionpatterns. Theseproperties determine the functional specializations thatcharacterize and distinguish areas in the adult.

4.2.3 Thalamocortical Relationships

The relationship between primary cortical areas andnuclei in the dorsal thalamus are critical for both adultfunction and the developmental differentiation of areas.The primary sensory areas receive their major sensoryinputs from dorsal thalamic nuclei that define an area’sfunctional modality. The principal sensory thalamicnuclei receive modality-specific sensory information ei-ther directly or indirectly from peripheral sense organsor receptors. The dorsal thalamus has four principal tha-lamic nuclei that functionally and connectionally paral-lel the four primary cortical areas (Jones, 2007). Eachprimary cortical area receives TCA input from a princi-pal thalamic nucleus that terminates primarily in layer 4,and sends outputs from layer 6 neurons to the same


VZ

MZ

CP

SP

IZ

VZVZVZ

Migratingneurons

Radial glia/progenitors

IZSP SP SP

WM

MZ

1

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PP

CP

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IZ

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DCP

2/3

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(a)

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3811

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V1V2

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123

4a4b4c56

1 2

34

MGE

LGEOB

CGE

FIGURE 4.3 Basics of corticogenesis. (a) Organization of the neocortex into areas. (1) Areas of the human cerebral cortex as defined by Brodmann(1909). View of the lateral surface of the human cerebral hemisphere shows a number of cytoarchitectonically distinct areas, such as the primary so-matosensory area (area 3, also termed S1) in the middle, the primarymotor area (area 4, also termedM1) just rostral to it, and the primary visual area(area 17, also termedV1) at the caudal pole. (2) Selected areas of the rat neocortex fromKaas and colleagues showing sensory areas (V1, primary visual;V2, secondary visual; S1, primary somatosensory; S2, secondary somatosensory; A1, primary auditory) as well as the primary and secondary motorareas (M1, M2). OB, olfactory bulb. (3) An example of abrupt borders between areas. The V1–V2 cytoarchitectonic border in an 8-month human fetusrevealed using a Nissl stain is pictured. Each area has six primary layers, but their architecture and internal sublayering differs. (b) Generation, mi-gration, and lamination of neocortical neurons. (1) Most neocortical neurons, including all glutamatergic and projection neurons, are generated in theneocortical VZ and later in the subventricular zone (SVZ; not shown).Most CP neuronsmigrate radially on radial glial fibers from the VZ, through theintermediate zone (IZ) and aggregate in the CP. The radial glia are also the progenitors in the VZ that give rise to cortical neurons. (2) Layer devel-opment of the rodent neocortex. The first neurons generated in the VZ, as well as neurons that migrate tangentially into themarginal zone (MZ) fromexternal germinal zones such as the cortical hem, aggregate on top of the VZ, and form the preplate (PP), which is later split intoMZ and subplate (SP)by the later generatedCPneurons. (3)Most neocortical interneurons (INs), that is, GABAergic INs of various peptide phenotypes, are generated in theMGE (pathway 1) and caudal ganglionic eminence (CGE, pathway 2), and migrate tangentially through the IZ and MZ to distribute across the neo-cortex, and then turnperpendicular to their tangential path andmigrate radially into theCP. The LGE is the source of GABAergic INs thatmigrate intothe piriform cortex (pathway 3) and to the OB (pathway 4). CP neurons are generated in an inside-out fashion, and layers differentiate from the CP inthe same pattern: earlier-born neurons form the deeper layers, while later-born neurons migrate past them and form the more superficial layers.Interestingly, INs generated external to the cortex also become distributed in an inside-out gradient depending upon their birth date (see Chapter 26).Adapted from: O’Leary, D.D., Nakagawa, Y. (2002). Patterning centers, regulatory genes and extrinsic mechanisms controlling arealization of the neocortex. Cur-rent Opinions in Neurobiology 12, 14–25.


nucleus, thereby generating the reciprocal area-/nuclei-specific relationships between cortex and thalamus: theventrolateral (VL) nucleus with M1, the ventroposterior(VP) nucleuswith S1, the dorsolateral geniculate nucleus(dLGN) with V1, and the ventral part of the medialgeniculate nucleus (MGv) with A1.

4.2.4 Origins of General Classes of CorticalNeurons

Most neocortical neurons, including all glutamatergicneuronsof the corticalplate (CP), fromwhich the six layersof the neocortex will differentiate, and the subplate (SP),are of anEmx1 lineage (Gorski et al., 2002). These glutama-tergic neurons of the SP and deeper layers of the CP, in-cluding layer 6 and layer 5 projection neurons, aregenerated in the VZ of the dorsal aspect of the lateral


ventricle,whereasglutamatergicneuronsof thesuperficialCPlayersaregenerated later inasecondgerminalzone, thesubventricular zone (SVZ), established by basal progeni-tors that themselves are generated by VZ progenitors(Figures 4.2 and 4.3; Gorski et al., 2002; Kriegstein andNoctor, 2004; Mione et al., 1994; Molyneaux et al., 2007).The SVZ is substantially larger in primates than in othermammals, and differences in proliferation in the posterioroccipital cortex have been reported to contribute to themajor increase in the numbers of superficial-layer neuronsin V1 compared to adjacent higher order visual areas(e.g., V2; Dehay and Kennedy, 2007).

Incontrast,neocorticalGABAergic interneurons,whichaccount forabout20%ofall corticalneurons, are froma lin-eage distinct from the Emx1 lineage of dTel progenitorsthat generates glutamatergic cortical neurons, and insteadare primarily produced by progenitors in the medial and


S1S1

S1

A1A1

A

M

L

654.3 NATURALLY OCCURRING DIFFERENCES IN AREA PATTERNING

caudal ganglionic eminences of the ventral telencephalonand migrate along multiple pathways to reach the cortex(Figure 4.3; Ang et al., 2003; Marin and Rubenstein, 2003;Nery et al., 2002). Once within the cortex, the GABAergicinterneuronsmigratealong tangentiallyalignedpathwaysin themarginal zone (MZ) and the intermediate zone, andeventually turn and migrate radially into the CP, perpen-dicular to their original tangential path (Nadarajah andParnavelas, 2002). In primates, a significant number of in-terneurons are generated within the cortical VZ (Letinicet al., 2002). GABAergic interneurons that populate theneocortex typically express a distinct neuropeptide thathelps define their multiple subclasses (Cherubini andConti, 2001; Kawaguchi and Kondo, 2002; Krimer andGoldman-Rakic, 2001) (see Chapter 26).

A third and proportionally very small general categoryof neocortical neurons, the Cajal–Retzius neurons thatpopulate the MZ (layer 1), are also generated external tothe corticalVZ,primarilywithin the corticalhembut inad-dition at other sites in the subpallium and septum (Bielleet al., 2005; Yamazaki et al., 2004; Yoshida et al., 2006;Zhao et al., 2006). Cajal–Retzius neurons express Reelin,a large secreted protein involved in establishing appropri-ate cortical layering by influencing the radial migrationand patterning of cortical neurons (Feng and Walsh,2001; Ross and Walsh, 2001; Tissir and Goffinet, 2003).

The distinct origins of these three general classes ofcortical neurons have implications for the extent towhich they exhibit an area identity that is determinedwithin their progenitors. It is most likely that only thoseneurons generated by the Emx1 lineage of progenitorsin dTel inherit any information relative to their ‘areaidentity’ from their progenitors, which suggests thatGABAergic interneurons and Cajal–Retzius neuronsdepend upon different sources of information to deter-mine their distributions and integration into corticalcircuits.

Short-tailedopossum

Mouse Ghost bat

V1V1 V1

A1

P

FIGURE 4.4 Primary cortical area size in three different species withsimilar-sized cortices. Three species parcel a similar amount of corticalsheet in very different ways, and this allotment corresponds to how eachutilizes sensory information. For example, the short-tailed opossum(Monodelphis domestica) is a diurnal animal that relies heavily on vision,and as such devotes a large amount of cortex to visual areas; primary vi-sual area (V1) shown in red. Themouse (Musmusculus) uses its whiskersto relay tactile information in its dark natural habitat to the primary so-matosensoryarea (S1), shown ingreen. Finally, theprimaryauditoryarea(A1, in light orange) is expanded in the echolocating ghost bat(Macroderma gigas), while the other primary areas are relatively small.A–P (anterior–posterior) and M–L (medial–lateral) axes labeled in theshort-tailed opossum diagram. Adapted from: Krubitzer, L., Kahn, D.M.

(2003). Nature versus nurture revisited: an old idea with a new twist. Progressin Neurobiology 70, 33–52.

4.3 NATURALLY OCCURRINGDIFFERENCES IN AREA PATTERNING

4.3.1 Phylogenic Differences in Area Patterning

Although the number of higher order cortical areas var-ies across mammals, roughly paralleling overall corticalsize but also relative to the phylogenetic tree, the primarysensory and motor areas are conserved, as is the generalspatial relationship between them: V1 is positioned cau-dally, M1 rostrally, and S1 is located between them; A1 islocated caudolaterally to S1 (Figure 4.3). However, the sizeand the positioning of the primary cortical areas within aspecies or across a given species also have a remarkable ca-pacity for plasticity. The simplest example of plasticity inarea patterning that is likely driven primarily by genetic


mechanisms can be observed by looking at brain evolution(Rakic, 2009). Inmammals, the size and complexity of apri-mary sensory area in relation to the overall neocortex sur-face area reflects the importance of a particular sensorymodality for the behavior and lifestyle of a given species.Forexample,animalswithunusualor largeandatypicalpe-ripheral appendages/sense organs (e.g., the platypus’ billor the echo-location system in bats) have modificationson this general geometrical scheme of area patterningto reflect their sensory specializations (Krubitzer, 2007;Krubitzer and Kaas, 2005). Nocturnal squirrels, comparedto mice and rats, use more neocortical surface area for S1and somatosensation relative to a small V1 for processingof visual information. Diurnal squirrels that rely muchmore on vision devote more neocortical surface area toV1 and visual processing, reflecting a major difference inlifestyle, behavior, and biological niche (Krubitzer, 2007).Perhaps themost straightforward exampleofnatural selec-tion affecting area patterning (Krubitzer, 2009) comesfrom a comparison of area patterning in the mouse, ghostbat, and short-tailedopossum(Figure 4.4). Theoverall sizesof the cerebral cortex in these species are similar, but thesizes of the three primary sensory cortical areas (S1, V1,and A1) differ substantially between them, reflecting theirunique sensory specializations and needs (Krubitzer andKahn, 2003).



4.3.2 Differences in Area Patterningwithin a Species

Area patterning also varies substantially across indi-viduals of the same species. For example, the sizes of pri-mary areas in the human neocortex vary by two- tothreefold within the normal population, despite overallcortical volume varying only by about 30% (Doughertyet al., 2003; White et al., 1997). Mice that are essentiallygenetically identical, that is, isogenic inbred strains ofmice, such as C57Bl/6J and DBA/2J mice, do not havesignificant variation in overall cortical surface area orin the sizes of specific cortical areas, whereas compari-sons between the inbred strains that are genetically dis-tinct shows significant differences in sizes (Airey et al.,2005). These studies have focused primarily on sizedifferences of S1, particularly on the posteromedialbarrel subfield (PMBSF) of S1, as well as V1, delineatedin adults of the isogenic inbred strains C57Bl/6J andDBA/2J. The overall surface area of the neocortex is7% larger in the C57Bl/6J strain than in the DBA/2Jstrain of mice. However, after normalizing for this over-all size difference, V1 is 12% larger in the C57Bl/6J strainthan in the DBA/2J strain whereas PMBSF is 10% largerin the C57Bl/6J strain than in the DBA/2J strain (Aireyet al., 2005). Likewise, V1 size and barrel patterningdiffer in inbred laboratory rats versus wild caught rats(Campi and Krubitzer, 2010; Jan et al., 2008).

4.4 EXTRINSIC INFLUENCES ON AREAPATTERNING

The specification and differentiation of neocorticalareas are controlled by an interplay between intrinsicmechanisms, that is, genetic mechanisms that operatewithin the cortex, andextrinsicmechanisms, for example,the sensoryperiphery, TCA input, or information relayedby it (Figure 4.1; O’Leary, 1989; O’Leary and Nakagawa,2002; O’Leary et al., 2007; Rakic 1988; Rakic et al., 2009;Sur and Rubenstein, 2005). Until recently, though, rolesfor extrinsic mechanisms in controlling area patterningwere emphasized, for various reasons. One reason is thatevidence for intrinsic genetic mechanisms was simplylacking, with the first direct evidence coming just overa decade ago with the demonstrations of roles for theTFs Emx2 (Bishop et al., 2000; Mallamaci et al., 2000)and Pax6 (Bishop et al., 2000) in specifying the tangential,positional (areal) identities of cortical progenitors. How-ever, much positive and compelling evidence for theaction of extrinsic mechanisms swayed the field, includ-ing demonstrations that the cortex is initially a more orless uniformstructure, thatmany area-specific propertiesdifferentiate in parallel spatially and temporally to thedevelopment of TCA input, and that area patterning


and functionexhibit considerableplasticitywhen the sen-soryperipheryorTCA input ismodifiedor followinghet-erotopic transplantation (Chenn et al., 1997; O’Leary andNakagawa, 2002; Sur andRubenstein, 2005).Wewill pro-vide a few examples for the action of extrinsic influencesin area patterning before discussing roles for intrinsicgenetic mechanisms.

4.4.1 Late Development of Area Patterning froman Early More Uniform CP

The properties that distinguish cortical areasgradually emerge during development, with variousarea-specific features becoming evident at differentdevelopmental stages (Chenn et al., 1997; O’Leary andKoester, 1993;O’Learyet al., 1994).ThenascentCP,beforeit acquires its mature functional abilities, lacks most ofthe anatomically based features that distinguish areasin the adult, even at a time after all CP neurons havebeen generated and layers begin to differentiate withinit. Across its tangential extent, the CP lacks cytoarchitec-tural distinctions thatmight presage future areas, and in-stead is uniform other than a smooth A–P and M–Ldecrease in its thickness. Also absent are the restricted,area-specific distributions of distinct types of projectionneurons characteristic of the functional specializationsof different cortical areas in adults. Instead, cortical pro-jection neurons have widespread distributions early onthat include parts of areas, and even entire areas, inwhichthey are not found in the adult; their restricted areal adultdistributionscomeaboutbytheeliminationof functionallyinappropriate axon segments and branches. As describedinthefollowingsection, thismechanismisusedtogeneratenot only the characteristic areal distributions of callosaland intracortical projection neurons, but in addition layer5 subcortical projection neurons (SPNs; also referred to assubcerebral projection neurons), which are the predomi-nant cortical output projection (Chenn et al., 1997;O’Leary and Koester, 1993; O’Leary et al., 1994).

Heterotopic transplant experiments show that thedevelopment of area-specific cytoarchitecture and func-tional modules is plastic during development. For exam-ple, transplants of embryonic occipital cortex, which willdifferentiate into visual areas, and intoPMBSFof S1 in theparietal cortex, receive TCA input appropriate for theirnew location, that is, from VP rather than dLGN, and de-velop cytoarchitecture and the patterned expression ofmarkers that define barrels and are characteristic of S1PMBSF (O’Leary et al., 1992; Schlaggar and O’Leary,1991). As detailed in the following section, considerable,extra-genetic plasticity is evident even the late selectionprocess that leads to degenerative axon elimination thatprunes the initially widespread distributions of projec-tion neurons to the restricted, functionally appropriatedistributions to match area patterning.


674.4 EXTRINSIC INFLUENCES ON AREA PATTERNING

4.4.2 Development and Plasticity inArea-Specific Output Projections Relatedto Area Patterning

The development of the adult patterns of callosal,intracortical, and subcortical projections of the mamma-lian neocortex is characterized by an initial widespreaddistribution of projection neurons, followed by the de-generative elimination of functionally inappropriateaxon projections, formed either by the distal segmentof the primary axon or primary collateral branches ex-tended from it, leading to the restriction in the distribu-tion of projection neurons to match area patterning(O’Leary and Koester, 1993). In the adult cortex, neuronsthat send an axon through the midline corpus callosumto the opposite cortical hemisphere, termed callosal pro-jection neurons (CPNs), have a limited, discontinuousdistribution that emerges from an early widespread,continuous distribution of CPNs through the eliminationof callosal axons without concomitant death of the par-ent neurons. A similar process operates during the de-velopment of intracortical projections to establish themature connections between cortical areas in the samehemisphere and in the refinement of horizontal connec-tions within an area.

The process of selective axon elimination in the devel-opment of callosal and intracortical projections is criticalfor establishing functionally appropriate connectivitywithin and between cortical areas. Perhaps the most dra-matic example of this phenomenon, and one that mostdirectly relates to the process of arealization per se, is se-lective axon elimination in the development of the pre-dominant cortical output projections formed by layer5 SPNs. There aremultiple subtypes of SPNs in the adult,named for their primary subcortical targets, includingcorticospinal projection neurons, the predominant out-put neuron of sensorimotor areas, and corticotectal pro-jection neurons, the predominant output neuron ofvisual areas. The adult distributions of these subtypesof SPNs match the functional specialization of corticalareas and therefore parallel area patterning. During de-velopment, though, the subtypes of SPNs aremuchmorebroadly distributed because SPNs in each area projectaxons to a larger set of layer 5 targets than are function-ally appropriate for their area – the functionally appro-priate patterns of layer 5 projections characteristic ofthe adult are later pruned from this initial widespreadpattern through selective degenerative axon eliminationof the functionally inappropriate axons, thereby generat-ing the distinct ‘area-specific’ subtypes of SPNs, for ex-ample, corticospinal and corticotectal. Recent studieshave reported that SPNs across all areas, independentof their adult subtype, are marked by the TF, Fezl (Fezf2)(Chen et al., 2005a; Hirata et al., 2004; Molyneaux et al.,2005). As discussed in detail in Section 4.7, the current


genetic evidence supports the notion that layer 5 SPNsacross the cortex are members of the same general classof projection neuron, and to date, TFs that might markthem uniquely or drive their subtype identity have notbeen reported.

The triggers and selection process that controlselective degenerative axon elimination and the finalpatterning of cortical output projections are not well un-derstood, but the available evidence for large-scalecortical pruning indicates a role for neural activity, spe-cifically the sensory information being relayed by TCAinput. The selectivity in the elimination of callosal axonsin visual or somatosensory areas is altered by a variety ofperipheral manipulations of either visual or somatosen-sory input, respectively, that alters either patterns ofneural activity (e.g., strabismus) or absolute levels of ac-tivity (e.g., dark-rearing, eyelid suture, or silencing ofretinal activitywith the sodium channel blocker, tetrodo-toxin). In these instances, callosal axon elimination is ab-normal, resulting in the retention of callosal connectionsin parts of the cortex that would normally lose them(O’Leary and Koester, 1993). Dramatic defects in callosalaxon elimination and termination patterns are alsofound in mice when neuronal excitation is suppressedin CPNs by overexpression of the inward rectifying po-tassium channel Kir2, or synaptic transmission isblocked by expression of the tetanus toxin light chain(Wang et al., 2007). Similar findings have been obtainedfor the development of layer 2/3 horizontal connections.Thus, sensory input and neuronal activation play an im-portant role in the selection process, driving selectiveaxon pruning to develop the adult pattern of callosaland intracortical connections.

Heterotopic transplant experiments in developing ratsshow that selective axon elimination by layer 5 SPNs isalso plastic during development. Late embryonic SPNstransplanted from visual (occipital) areas to motor (fron-tal) areas of newborns permanently retain their normallytransient spinal axon, whereas SPNs transplanted frommotor areas to visual areas lose their normally permanentspinal axon and retain their transient axon collateral to thesuperior colliculus (O’Leary and Stanfield, 1989; Stanfieldand O’Leary, 1985). Thus, the projections retained by thetransplanted SPNs are appropriate for the cortical area inwhich the transplanted neurons develop, not where theywere born. These transplants receive TCA input from tha-lamic nuclei appropriate for their new location in thedeveloping cortex (O’Leary et al., 1992; Schlaggar andO’Leary, 1991) that likely has a significant role in drivingtheir areal plasticity. The occipital (visual) to frontal(motor) transplants form permanent functional connec-tions to the spinal cord (Porter et al., 1987). These andotherexperimental manipulations reveal tremendous plastic-ity in the process of selective axon elimination by SPNsand the development of the area-specific SPN subtypes.



Further, these studies indicate that the development ofSPN subtypes (corticospinal, corticotectal) is driven bymechanisms that can be influenced by cortical localeand likely determined in part by afferent (TCA) input.Further, and as described in a later section, these findings,that is, the present lack of geneticmarkers for distinct SPNsubtypes, are consistent with a prominent role for extra-genetic mechanisms determining the distinct SPN sub-types rather than them being genetically specified.

4.4.3 Area-Specific TCA Input and PotentialRoles in Area Patterning

In contrast to the lack of adult areal specificity in theearly distribution of CPNs and SPNs, the reciprocal pro-jections formed between primary cortical areas and theprincipal sensory thalamic nuclei exhibit significantarea-specificity early on in their development prior tothe emergence of the sharp cytoarchitectonic borders be-tween areas that become evident later (O’Leary et al.,1994): both the projections of TCAs to the modality-specific primary areas and layer 6 neurons from thoseprimary areas back to their principal thalamic nuclei.As TCAs are the sole source of modality-specific sensoryinformation to the neocortex, the functional specializa-tions of the primary sensory areas are defined in largepart by, and are dependent upon, TCA input. In addi-tion, the differentiation of many anatomical features thatdistinguish cortical areas, including architecture anddistributions of output projection neurons, depend to alarge extent upon TCA input. This role for TCAs is con-sistent with them exhibiting area-specificity throughouttheir development, and that the gradual differentiationof areas within the CP parallels the elaboration of theTCA projection within it (Chenn et al., 1997). The plastic-ity in area-specific architecture and cortical outputprojections exhibited by heterotopic transplants as de-scribed earlier, as well as the plasticity in architectureand projections induced by peripheral manipulationsdescribed in the following section, demonstrate thatthe CP exhibits considerable plasticity in the develop-ment of area-specific features, and that diverse parts ofCP initially have similar potentials to develop featuresunique to a specific area. In addition, the functional plas-ticity exhibited by sensory cortical areas revealed byrewiring experiments that alter the modality of sensoryinput relayed by TCAs to the primary sensory areasfurther underscores the importance of this input in de-termining certain area-specific specializations and func-tions (Sur and Rubenstein, 2005).

The role of TCAs in shaping cortical architecture is notlimited to these later events in the differentiating CP.In vitro experiments using mouse tissue suggest thatTCAs release a diffusible mitogenic activity that pro-motes the production of both glia and neurons by


explants of the cortical VZ (Dehay and Kennedy,2007). If a similar mechanism operates in vivo, such anearly influence of TCAs on corticogenesis could contrib-ute to the reported areal differences in neuronal produc-tion in the SVZ in occipital visual areas (V1 versus V2) inthe monkey, and therefore, as described earlier, to thecytoarchitectural differences between areas that be-comes evident later in development (Dehay andKennedy, 2007; Lukaszewicz et al., 2005).

4.4.4 Sensory Periphery-Mediated Plasticityin Area Patterning

Input from sense organs in the periphery has a sub-stantial role in differentiating and maintaining properorganization and functioning of sensory area maps inthe neocortex. Complete disconnection of the neocortexfrom the periphery, called sensory deprivation as well asabnormal sensory input to the neocortex, results in con-siderable plasticity, which drives rearrangement of sen-sory map features like size, patterning, and synapticconnections that modify the functionality and acuity ofthe affected neocortical area map (Eysel et al., 1999;Hofer et al., 2006; Toldi et al., 1996). Perhaps the mostdramatic evidence for such developmental plasticity inarea-specific architecture and connectivity comes fromthe demonstration that mid-gestational bilateral eye re-moval in rhesus macaques results in a shift in the loca-tion of the border between V1 (area 17) and V2 (area18) (Dehay et al., 1989, 1991; Rakic, 1988; Rakic et al.,1991). In these animals, the dLG, which provides the ma-jor TCA to V1, contains only 30–50% of the normal num-ber of geniculocortical (TCA) projection neurons. Thesestudies find a corresponding reduction in the total areaof V1, but no significant deviations from normal in otherparameters, including cortical depth and number of neu-rons in a radial traverse. The reduction in the size of V1 isdue, at least in part, to a border shift, with the portion ofthe neocortex surrounding the reducedV1 exhibiting notonly the distinctive cytoarchitecture characteristic of theneighboring V2, but also its chemoarchitecture and evenits pattern of CPNs. These findings are consistent withthe interpretation that fields of the developing neocortexhave the capacity to develop features normally associ-ated with other areas, and strongly implicate a control-ling influence for TCAs in this plasticity. Predictably,then, peripheral manipulations done prior to TCA in-growth into the CP result in more substantial plasticitythan those done later. In monkeys, TCAs invade theCP of V1 around E80 (Kostovic and Rakic, 1984); bilateraleye removals done around or after this age affect the sizeof V1 much less than those done earlier (Dehay et al.,1991). Thus, lesion-induced areal compensation seemsto be related to the state of TCA ingrowth into the CPat the time of the lesion.


694.5 INTRINSIC GENETIC MECHANISMS REGULATING AREALIZATION

Due to its readily defined anatomical patterning thatparallels its functional organization, the whisker to bar-rel projection of the trigeminal system is an excellentmodel system widely used for investigating sensorymap plasticity in mice (Feldman, 2009). Ablation of pe-ripheral input to S1 during a critical period by lesioningfacial whiskers results in the elimination of the corre-sponding whisker representation from the sensory mapsin the brain including the neocortex in a bottom-up fash-ion that results in amapwith decreased complexity (Vander Loos and Woolsey, 1973). Sensory map plasticitydriven by changed peripheral input is also capable of in-creasing map complexity in the neocortex. Ectopic or su-pernumerary whisker follicles will likewise beectopically represented and added to the map of S1 inthe neocortex (Ohsaki and Nakamura, 2006; Van derLoos et al., 1986). Analogous to S1 altered peripheraldrive in the visual system by enucleation of one or botheyes or dark rearing elicits drastic plasticity that changesneocortical organization, neuronal connectivity, as wellas normal gene expression, resulting in abnormal prop-erties and processing of visual information in V1 (Fellerand Scanziani, 2005; Huberman, 2007; Majewska andSur, 2006; Wiesel and Hubel, 1963). Several decades ofintense research has shown that during developmentand in the adult, cortical sensory maps are very sensitiveto changes that set up improper connectivity or alteredconnection properties between the sensory peripheryand the neocortex, and thus proper neuronal wiringand synaptic transmission including (spontaneous aswell as stimulus evoked) ordered electrical activityand normal sensory experience is a prerequisite for theproper establishment and maintenance of sensory neo-cortical maps that safeguard sensory acuity and properbehavior.

However, remarkable sensorymap plasticity also ex-ists across the different primary sensory areas. Thisphenomenon is known as cross-modal plasticity andis best described by plastic changes in the neocortex,which is provoked the most in congenitally sensory-deprived animals. It has been showed that while keep-ing its normal molecular organization, V1 of congeni-tally blind individuals in the same way as V1 infunctionally blind species like the naked mole rat dis-play a remarkable degree of plasticity and are able torespond and process auditory stimuli (Doron andWollberg, 1994; Frasnelli et al., 2011; Merabet andPascual-Leone, 2010). Cross-modal plasticity is there-fore one of the most extreme examples demonstratingthe capacity of sensory maps to adapt to changes thatare intrinsic or extrinsic to the neocortex.

The extent to which the forms of plasticity in area pat-terning described earlier is dependent upon genetic in-formation intrinsic to cortex or influences of TCAinput will differ between examples. Some forms, for


example, addition of extra barrels within PMBSF of S1,can be directly related to TCA input and sensory inner-vation of supernumerary vibrissae, whereas other exam-ples, for example, variations in area size or position,might be due to differences in TF expression in the cortexand/or changes in TCA input due to influences affectedsubcortically that are subsequently manifested withinthe cortex.

4.5 INTRINSIC GENETIC MECHANISMSREGULATING AREALIZATION

The initial evidence for intrinsic genetic mechanismsregulating arealization was indirect, and was basedupon demonstrations that many of the differential geneexpression patterns evident in embryonic mouse cortexemerged either prior to the development of TCA input,or in Gbx2 or Mash1 mutants that lacked TCAprojections to the cortex (Miyashita-Lin et al., 1999;Nakagawa et al., 1999; Sestan et al., 2001). Thus, thesedifferential patterns of gene expression develop throughmechanisms independent of TCA input, and thereforeare likely intrinsic to the dTel. However, shortly thereaf-ter, as discussed in detail later, several TFs were shownto have direct and significant functions in arealization(Figure 4.5). The most basic criteria required for candi-date genes that specify area identities are straightfor-ward: (a) these genes need to function as regulatorygenes, for example, as TFs, and (b) these TFs need tofunction differentially across the cortical axes, in a fash-ion required to impart positional, or areal, identities.This could be achieved by their differential expressionacross the A–P and M–L cortical axes by progenitors inthe VZ and/or SVZ, and/or could be achieved by theexpression of cofactors or other mechanisms that differ-entially influence TF function. TFs that regulate arealiza-tion in principle could have a range of functions; forexample, they could confer the complete set or a subsetof properties that comprise the area identity of a corticalneuron, and they could regulate the expression of axonguidance molecules that control the area-specific target-ing of TCAs. Scores of TFs meet the basic criteria for reg-ulation of arealization, and thus far, four have beenshown to have prominent functions in arealization:Emx2, Pax6, COUP-TFI, and Sp8 (Figure 4.5). Thesefour TFs have strongly graded expression patterns incortical progenitors in mice that allow for the uniqueencoding of position along the cortical axes, and there-fore in principal area identity. The morphogens and sig-naling molecules secreted by the patterning centersdescribed in the following section have a prominent rolein establishing the graded expression of these TFs incortical progenitors (Figure 4.1).


A

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M

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Emx1-Cre)

(sey/sey [Pax6]E18.5 KO)

(Emx2 KOE18.5)

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het null)(Nestin-Emx2

transgenic)

(Yac-Pax6 transgenic)

(conditional COUP-TF1 x

Emx1-Cre)

(conditional Sp8 x BF1-Cre E18.5)

(a)

(b)

FIGURE 4.5 Summary of graded expression of transcription factorsimplicated inarealizationandresults frommousemutants. (a)Gradedex-pression in cortical progenitors of the transcription factors directly impli-cated in arealization, Emx2, Pax6, Coup-TFI, and Sp8, along the anterior–posterior (A–P) and lateral–medial (L–M) axes of the cortex. (b) Summaryof reports of loss-of-function or gain-of-functionmutant mice of TFs thatexhibit changes in area patterning.Micewith a targeted deletion of Emx2die at birth, but late embryonic analyses suggest substantial changes inarealizationas indicated in thecartoon,witha reduction inposteriorareasand an expansion and posterior shift of anterior areas. Reducing Emx2levels in the cortex of the heterozygote mutant mice (Emx2 KO het null)results in posterior shifts of areaswith shrinkageofV1,while overexpres-sion of Emx2 under the control of nestin promoter (nestin-Emx2 trans-genic) shifts areas anteriorly. Small eye mutant mice (sey/sey), whichlack functional Pax6 protein, die at birth, but marker analyses suggest areduction in anterior areas and an expansion and anterior shift of poste-rior areas. Conditional deletion of Pax6 knockout (KO)mice crossedwithanEmx1-Cre line results in a reduction in anterior areas andanterior shiftand expansion of posterior areas, though less pronounced than the sey/sey mutants. However, YAC transgenic mice of Pax6 do not show areachanges other than a slight, but significant, reduction in the size of S1 (as-terisk). Selective deletion of COUP-TFI in conditional KO mice crossedwith an Emx1-Cre line results in a massive expansion of frontal/motorareas and a substantial reduction of the primary sensory areas that shiftposteriorly to the posterior cortical margin. Analyses of conditional KOmiceof Sp8 crossed toaBF1 (Foxg1)-Cre line showsanterior shifts of genemarkers at late embryonic ages, a phenotype similar to that reported forFgf8 hypomorphic mice. The BF1-Cre line deletes Sp8 not only from cor-tical progenitors but also from the CoP, resulting in diminished expres-sion of Fgf8 in the CoP. See text for details and references. Adapted from:

O’Leary, D.D., Sahara, S. (2008). Genetic regulation of arealization of theneocortex. Current Opinions in Neurobiology 18, 90–100.



4.5.1 Telencephalic Patterning Centersin Arealization

Three dorsal telencephalic patterning centers posi-tioned at the perimeter of dTel are likely involved di-rectly or indirectly in establishing the framework forarealization (Figure 4.1). Morphogens secreted fromthese patterning centers function in part to generatethe differential expression of TFs that determine the areaidentity of progenitors that is inherited by their neuronalprogeny that form the CP. The two patterning centersmost directly implicated in arealization are the commis-sural plate (CoP), which expresses fibroblast growthfactors (Fgfs), and the cortical hem, which expressesbone morphogenetic proteins (Bmps) and vertebrateorthologs of Drosophila wingless referred to as Wnts. Athird, albeit putative, patterning center is the antihem,identified by its expression of multiple signalingmolecules, including Tgfa, Neuregulin1, Neuregulin3,Fgf7, and the Wnt antagonist, secreted frizzled relatedprotein Sfrp2 (Assimacopoulos et al., 2003). The antihemis located in the neuroepithelium near the boundarybetween VL neocortex and the lateral ganglionic emi-nence of the ventral telencephalon, and forms a narrowstripe of expression extending along the entire anterior–posterior (A–P) axis of the telencephalon. The corticalhem and antihem are suggested to cooperate with theCoP to establish identities along the A–P and medial–lateral (M–L) axes of the developing cortex. Althoughno function has been defined for the antihem, it is essen-tially absent in small eye (sey) mutant mice, which lackfunctional Pax6 protein, and therefore some of the majordefects in telencephalic patterning observed in small eyemutants might be due to the loss of antihem function(Assimacopoulos et al., 2003) (see Chapters 1 and 2).

4.5.1.1 CoP: An Anterior Patterning Center

The CoP, an anterior patterning center for arealization(Figure 4.1), is the differentiated form of the anterior neu-ral ridge (ANR). The ANR is found at the anterior junc-tion between neural and nonneural ectoderm, and laterthrough fusion of the neural plate folds at the anteriormargin of the forebrain, becomes the CoP (CrossleyandMartin, 1995). The ANR/CoP is defined by the over-lapping expression domains of Fgf8, 17, and 18. Of these,Fgf8, and to a lesser degree Fgf17, has been most studiedin arealization. They locally induce members of the ETSfamily of TFs and establish the gradients of Emx2 andCOUP-TFI within cortical progenitors by repressingtheir expression anteriorly in a dose-dependent fashion(Garel et al., 2003; Storm et al., 2006). Altering levels ofFgf8 or 17 has substantial effects on area patterning,likely through repression of Emx2, COUP-TFI, and pos-sibly other TFs expressed by cortical progenitors(Fukuchi-Shimogori and Grove, 2001; Garel et al., 2003;



Storm et al., 2006). Recent studies show that Fgf8 andFgf17 have distinct roles in the patterning of frontal cor-tical areas: Fgf8 controls the size of both the dorsal fron-tal cortex and the ventral/orbital frontal cortex, whileFgf17 selectively controls the size of the dorsal frontalcortex (Cholfin and Rubenstein, 2007).

Fgf expression independent of that expressed in theCoP can also influence cortical area patterning by regu-lating the differentiation and proliferation properties ofcortical progenitors. For example, Fgf10 is expressed bycortical progenitors at their neuroepithelial cell state andcontrols the timing of their differentiation into neuro-genic radial glia; deletion of Fgf10 results in a delay inthe transitioning of progenitors into their neurogenicphase and extends their symmetric division phase, spe-cifically in the rostral cortex, resulting in an expansion inthe pool of rostral progenitors and subsequently the sizeof frontal/motor areas (Sahara and O’Leary, 2009).Manipulation of Fgf receptors can have complimentaryeffects to Fgf10. For example, overexpression of Fgfr3results in a selective increase in the size of the occipital(caudal) neocortex (Thomson et al., 2009) (see Chapters 1and 2).

4.5.1.2 Cortical Hem: A Dorsal–Caudal PatterningCenter

Thecortical hem isneuroepithelial tissueadjacent to thedorsal midline in the medial cortical wall, defined by itsexpression of multiple Bmps and Wnts (Figure 4.1;Furuta et al., 1997; Grove et al., 1998). The distributionand timing of Bmp/Wnt expression in the cortical hemand their receptors in the cortex suggest that the corticalhem is involved in cortical patterning (e.g., Kim andPleasure, 2003). It is likely that morphogens secreted bythe hem influence the graded expression of TFs in corticalprogenitors. For example, ectopic expression of BMP4 inthe dTel of embryonic chicks appears to enhance theexpression of Emx2, either directly or through downregu-lationofFGF8 (Ohkuboet al., 2002).However, the functionof the cortical hem in neocortical arealization remainsvague. Genetic ablation of the hem does not have a signif-icant effect onarealization, andalthough it results in a sub-stantial loss of Cajal–Retzius neurons, and thereby thepredominant source of Reelin in the cortical MZ, it doesnot lead to significant defects in cortical lamination(Yoshida et al., 2006) believed to be controlled by Reelin(Tissir and Goffinet, 2003) (see Chapters 1 and 2).

4.5.2 TF Expression by Cortical ProgenitorsRegulates Area Patterning

Asmentioned earlier, of the scores of TFs with expres-sion patterns consistent with roles in controlling areali-zation, to date only four have been shown throughthe analyses of mouse mutants to have prominent func-tions in this process: Emx2, Pax6, COUP-TFI, and Sp8


(Figure 4.5). In addition to controlling arealization, eachof these TFs has other roles in forebrain development.For example, Pax6 regulates D–V regional patterningof the telencephalon and maintains dorsal telencephalicfate of cortical progenitors (Kroll and O’Leary, 2005;Stoykova et al., 1996; Toresson et al., 2000; Yun et al.,2001) and Sp8 is a direct transcriptional activator ofFgf8 in the CoP (Sahara et al., 2007). However, here wefocus on their roles in arealization studied in mouse mu-tants, which have implicated to varying degrees each ofthese TFs in area patterning (Figure 4.5). Because theseTFs appear to have similar graded expression in humanembryos (Bayatti et al., 2008; Ip et al., 2010) it is feasiblethat their function in arealization is conserved frommouse to human. In the following sections, we summa-rize the reported functions for each in arealization.

4.5.2.1 Emx2

Roles forEmx2 inarealizationhavebeen themost stud-ied for any TF. Emx2, a homeodomain TF related toDrosophila empty spiracles (ems), is expressed most inprogenitors that generate posterior–medial areas of theneocortex, such asV1, and least in progenitors that gener-ate anterior-lateral areas, such as frontal and motor(Figure 4.5; Simeone et al., 1992). The initial studies,and the first to showa role forTFs in areapatterning,wereloss of function (LOF) performed on Emx2 constitutiveknockout (KO) mice (Figure 4.5; Bishop et al., 2000;Mallamaci et al., 2000). Changes in patterns of markergene expression suggest that rostral–lateral (frontal/mo-tor) areas are expanded, whereas caudal–medial (visual)areas are reduced in the Emx2 nullmutant. Alterations inthe organization of area-specific TCAprojections are alsoconsistent with this interpretation. Retrograde labelingfrom the neocortex of Emx2 mutants indicates anorderly expansion and caudal shift of the topographicTCA projection of VP and contraction of the TCA projec-tion fromdLG, indicative of an expansionof S1 and a cau-dal shift of its border, and a contraction of V1. Emx2 KOmice, however, die at birth, well before cortical areas dif-ferentiate, limiting these studies to marker analyses andpatterning of area-specific TCA projections. However,subsequent analyses of nestin-Emx2 transgenic mice,which use nestin promoter elements to drive elevatedlevels of Emx2 expression limited to progenitors, and ofheterozygous Emx2 constitutive KO mice, at postnatalages after areas emerge provide a more complete pictureof roles for Emx2 in arealization (Figures 4.5 and 4.6;Hamasaki et al., 2004). These genetic manipulations thatchange the levels of Emx2 expression in cortical progen-itors result in disproportionate changes in the sizes ofthe primary sensory and frontal/motor cortical areas,but have no effect on overall cortical size (Hamasakiet al., 2004). These analyses indicate that Emx2 operatesby a concentration-dependent mechanism in corticalprogenitors to specify disproportionately the sizes and


A1 A1V1 V1

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(a) (b) (c) (d)

Wild typeEmx2 +/- Ne-Emx2 het Ne-Emx2 homo

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FIGURE 4.6 Concentration-dependent changes of primary area sizes and locations controlled by Emx2. Serotonin immunostaining on tangen-tial sections of flattened cortex of P7 Emx2 heterozygous KO (Emx2þ/�, a), wild-type (b), heterozygous nestin-Emx2 transgenic (Ne-Emx2 het, c),and homozygous nestin-Emx2 transgenic (Ne-Emx2 homo, d) mice. Levels of Emx2 transcripts increase progressively in the genotypes from A toD, with the lowest level in the Emx2 þ/�mice and highest level in the homozygous nestin-Emx2 transgenic mice. As levels of Emx2 increase, V1expands, S1 is reduced in size and shifts anteriorly, and the domain remaining for frontal/motor areas (F/M) is reduced. For each genotype, thelevel of Emx2 in the embryonic cortex determined by real-time PCR (Leingartner et al., 2007), and the size of V1 (Hamasaki et al., 2004), relative tothe wild type (set at 100%) are indicated below each photo. Scale bar is 1 mm. The C3 barrel is marked with an asterisk (*), and the hind pawrepresentation in S1 is marked with a white ^, to provide references for the anterior shift of the body representation in S1. Modified from

Hamasaki T, Leingartner A, Ringstedt T, and O’Leary DDM (2004) EMX2 regulates sizes and positioning of the primary sensory and motor areas in neocortexby direct specification of cortical progenitors. Neuron 43: 359–372; Leingartner A, Thuret S, Kroll TT, et al. (2007) Cortical area size dictates performance at

modality-specific behaviors. Proceedings of the National Academy of Sciences of the United States of America 104: 4153–4158.


positioning of the primary cortical areas, and that higherlevels of Emx2 preferentially impart posterior-medialarea identities, such as those associated with V1(Figure 4.6).

Genetic rescue studies have validated that Emx2 con-trols arealization and that the levels of Emx2 expressionare a critical parameter (Leingartner et al., 2007). Thesestudies were done by crossing the nestin-Emx2 mice,which have about a 50% increase in Emx2 expressionin cortical progenitors, with Emx2 þ/� mice, whichhave about a 50% reduction in Emx2 expression. In theprogeny obtained from this cross, both Emx2 expressionin cortical progenitors and the size and positioning ofcortical areas are restored to wild type.

4.5.2.2 Pax6

Pax6 is a paired box domain TF expressed by corticalprogenitors in a low posterior-medial to high anterior-lateral gradient that opposes the pattern of Emx2 expres-sion (Figure 4.5; Bishop et al., 2000). Thus, Pax6 is mosthighly expressed in frontal/motor areas, consistent withthe conclusion from marker analyses of small eye (sey)mutant mice that are deficient in functional Pax6 protein,which implicated Pax6 in specifying anterior area identi-ties associated with frontal/motor areas (Figure 4.5;Bishop et al., 2000, 2002; Muzio et al., 2002). Again,analyses of the sey mutants are limited because they dieat birth, and have other major defects that preclude anal-ysis of TCA projections. Defects in area patterning in-ferred from marker analyses of the sey (Pax6) mutantshave also been reported in a more recent study of


postnatally viablemice using a cortex-specific conditionaldeletion of Pax6 from cortical progenitors (Pinon et al.,2008); for example, they observe a reduction in frontal/motor areas and a complimentary expansion of markersof caudal (visual/occipital) areas (Figure 4.5). Althoughthey also observe a sizable reduction in S1 (particularlyPMBSF), Pinon et al. (2008) report that it does not exhibita relative change in the conditional mutant compared towild type because of the pronounced reduction in overallcortical area. In contrast to these findings from LOF ana-lyses, a study that used a YAC transgenic gain-of-function(GOF) approach to overexpress Pax6 observed nochanges in area patterning other than a small but signifi-cant decrease in S1 size (Figure 4.5), even in lines inwhichPax6 is overexpressed in cortical progenitors by up to300% (Manuel et al., 2007).

Although the GOF study of Manuel et al. (2007) failsto reveal a prominent role for Pax6 in area patterning, thedramatic changes in genemarker expression observed inPax6 (sey) mutants (Bishop et al., 2000, 2002; Li et al.,2006) appear to exceed changes that could be explainedsolely by the reported preferential loss of rostral corticaltissue in thesemutants (Muzio et al., 2002). An appealingexplanation for this discrepancy is that another gene orset of genes normally represses the ability of Pax6 to im-part frontal/motor area identities in the cortical fieldsthat give rise to sensory areas and could conceivablyhave a similar action even in the face of Pax6 overexpres-sion. For example, COUP-TFI, which is expressed ro-bustly by progenitors in the parietal and occipitalcortex that generate the primary sensory areas and ex-hibit a steep decline in expression in the frontal cortex



(Liu et al., 2000), could have this function if above athreshold level of its expression, COUP-TFI can repressan ability of Pax6 to impart frontal/motor area identitiesto sensory area progenitors. Indeed, overexpression ofCoup-Tf1 using a D6 promoter in cortical progenitors re-presses Pax6 expression in the ectopic Coup-Tf1 expres-sion domain (Faedo et al., 2008).

4.5.2.3 Sp8

Sp8 is expressed by progenitors in a high anterior-medial to low posterior-lateral gradient (Figure 4.5).Two recent studies have reported roles for Sp8 in area-lization. One study employed in utero electroporation ofvarious constructs for GOF and LOF analyses of Sp8function in arealization (Sahara et al., 2007), and theother generated a conditional KO of Sp8 and by cross-ing with a BF1 (Foxg1)-Cre line deleted Sp8 from thetelencephalon, including Sp8 expressed in both theANR/CoP and progenitors in the cortical VZ(Figure 4.5; Zembrzycki et al., 2007). Because Sp8 andFgf8 reciprocally induce one another, and Fgf8 itselfhas potent effects on arealization by controlling thegraded expression of Emx2, COUP-TFI, and likely otherTFs, it is difficult to sort out from these studies the spe-cific role of Sp8 expression in cortical progenitors inarealization. Nonetheless, analyses of the conditionalSp8 KO mice at late embryonic ages show an anteriorshift of cortical markers, suggesting that Sp8 preferen-tially specifies identities associated with frontal/motorareas (Hamasaki et al., 2004). However, the use of theBF1-Cre line complicates analyses of roles for Sp8 inarealization because it results in the deletion of Sp8from progenitors in the cortical VZ as well as fromthe ANR/CoP. As described earlier, Sp8 is a direct tran-scriptional activator of Fgf8 (Sahara et al., 2007) and inaddition is required for its maintained expression in theCoP (Sahara et al., 2007; Zembrzycki et al., 2007). There-fore, because Fgf8 helps establish the graded expressionof Emx2 and COUP-TFI in cortical progenitors throughrepression, and altering Fgf8 expression has prominenteffects on area patterning, the marker shifts observed inthe BF1-Cre-mediated conditional deletion of Sp8 isconsistent with either the diminished expression ofFgf8 in the CoP or a direct role for Sp8 in specifying areaidentities of cortical progenitors.

A question relevant for arealization is why does Sp8not induce Fgf8 within cortical progenitors? In vitro as-says show that Emx2, which is coexpressed with Sp8in cortical progenitors but not in the CoP, represses theability of Sp8 to bind regulatory elements of Fgf8 and in-duce its expression (Sahara et al., 2007). Thus, in vivo,Emx2 likely suppresses the Sp8 transcriptional activa-tion of Fgf8 in cortical progenitors, thereby restrictingFgf8 expression to the CoP.


4.5.2.4 COUP-TFI

COUP-TFI is an orphan nuclear receptor expressed ina high posterior-lateral to low anterior-medial expressiongradient by both progenitors andCP neurons (Figure 4.5).The initial evidence of a role for COUP-TFI in arealizationcame from studies of constitutive null mice, but again an-alyses were limited because most of the mice die within afew days after birth, and themajority of TCAs fail to reachthecortex (Zhouetal.,2001).Thesemiceexhibit substantialchanges in patterns of gene markers, with most markersreported to lose their differential expression along the cor-tical axes and instead be broadly expressed. In addition,TCAs are reported to exhibit aberrant targeting and lossof thehighdensity of neurons characteristic of theprimarysensory areas in layer 4.However, these findings and theirinterpretationsarecomplicatedby therobustexpressionofCOUP-TFI within forebrain structures, particularly theprincipal sensory nuclei in dorsal thalamus. Indeed, inCOUP-TFI constitutivenullmice, themajorityofTCAs failto reach the cortex, which compromises an analysis ofdefects in the targeting of TCAs and changes in layer 4,theprincipal target layerofTCAs (Zhouet al., 1999).None-theless, findings fromtheCOUP-TFI constitutivenullmicesuggest a role for COUP-TFI in arealization.

However, these complications have been overcome bythe recent analyses of conditional COUP-TFI KO mice inwhich COUP-TF1 is selectively deleted from the cortexat E10 by crosses to an Emx1-Cre line (Armentano et al.,2007). Cortical deletion of COUP-TFI results in a massiveexpansion of frontal/motor areas to occupy most of theparietal and occipital cortex, which in wild-type mice areoccupied by somatosensory and visual areas (Liu et al.,2000) respectively (Figures 4.5 and 4.7). This expansionof frontal/motor areas is paralleled by a substantial re-duction in the sizes of the three primary sensory areas,which are compressed to the caudal pole of the corticalhemisphere. Thus, COUP-TFI is required to balance thepatterning of the neocortex into frontal/motor areas andsensory areas (Armentano et al., 2007). These findings sug-gest that COUP-TFI functions predominantly by repres-sing the identities of frontal/motor cortical areas withinits expression domain in the parietal and occipital cortex,allowing for the appropriate specification of the sensorycortical areas and limiting frontal/motor areas to their an-terior domain,which has very low levels of COUP-TFI ex-pression (Figure 4.8).

Consistent with the findings and conclusions ofArmentano et al. (2007) that COUP-TFI acts to suppressmotor fates are subsequent marker analyses of COUP-TFI conditional KOmice by Tomassy et al. (2010) indicat-ing a premature production of corticospinal neuronsectopically located in the superficial portion of layer 6and aberrantly coexpressing both layer 5 and layer 6markers (e.g., Ctip2 and Tbr1). Complementing these


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FIGURE 4.7 Conditional deletion of COUP-TFI from Emx1-lineage results in themassive expansion of frontal/motor areas and posterior com-pression of primary sensory areas. Findings from Armentano et al. (2007) showing a prominent role for COUP-TFI in arealization. (a and b)Serotonin (5-HT) immunostaining on tangential sections through layer IV of flattened cortices of P7 control (COUP-TFI fl/þ) and conditional mu-tant (fl/fl; Emx1-Cre) cortices. Anterior is to the left, andmedial to the top. (a) Serotonin staining reveals primary sensory areas, including primarysomatosensory (S1), visual (V1), and auditory (A1) areas, by marking area-specific TCA axon terminations. (b) In COUP-TFI fl/fl; Emx1-Cre con-ditional mutant brains, the primary sensory areas aremuch smaller than in controls and are compressed to ectopic positions at the posterior pole ofthe cortical hemisphere. The barrelfield of the ectopic S1 retains its characteristic patterning but is substantially reduced in size and caudallyshifted, while a reduced V1 is located medial and a reduced A1 lateral to the miniature S1 barrelfield. (c and d) In situ hybridization for Cad8on whole mounts of P7 wild-type (þ/þ; Emx1-Cre) and homozygous conditional mutant (COUP-TFI fl/fl; Emx1-Cre) brains uniquely marksthe frontal/motor areas (F/M). The F/M areas substantially expand following selective deletion of COUP-TFI from cortex. The reduced ectopicprimary sensory areas (V1, S1) can be identified by small domains of diminished cad8 expression in posterior cortex. (e–j) Serotonin (5-HT) immu-nostaining (e and f) MDGA1 (g and h) and RORb (i and j) in situ hybridization on serial sagittal sections of P7 control (COUP-TFI fl/þ) and con-ditional mutant (fl/fl; Emx1-Cre) cortices. Anterior is to the left, and dorsal to the top. Serotonin immunostaining reveals area-specific TCAterminations in layer 4 of S1 and V1. In conditional mutant cortex, both S1 and V1 are reduced in size and are ectopically positioned at the posteriorpole of the cortical hemisphere (f). (g and h)MDGA1 selectivelymarks layers 4 and 6 of S1, and layer 2/3more broadly in the cortex. The S1-specificexpression ofMDGA1 in layers 4 and 6 confirms the reduced size and posterior shift of S1 in the COUP-TFI-deficient cortex, and that these changesoccur in parallel across cortical layers (i and j). Summary of the data from: Armentano,M., Chou, S.J., Tomassy, G.S., Leingartner, A., O’Leary, D.D., Studer,

M. (2007). COUP-TFI regulates the balance of cortical patterning between frontal/motor and sensory areas. Nature Neuroscience 10, 1277–1286.

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FIGURE 4.8 Rolesand interactionsbetweentranscription factors thatcontrol neocortical arealization. Sp8 and Pax6 have been implicated inpreferentially specifying the identities of frontal/motor (F/M) areas incortical progenitors and their progeny. Emx2 preferentially specifiesthe identities of posterior (P)/sensory (e.g., V1) areas in cortical progen-itors. COUP-TFI represses the phenotypic function of any TF that mayspecify F/M area identities, such as Pax6 and Sp8 and any other TF tobe identified,within itsmore robust expression domain, thereby limitingtheir action toanterior (A) corticalprogenitors that specifyF/Marea iden-tities.Wealso suggest, basedon current evidence, that TFs that specify F/M area identities are dominant over the TFs that specify caudal/sensoryareas and can phenotypically repress their function. Modified from:

O’Leary, D.D., Sahara, S. (2008). Genetic regulation of arealizations of the neo-cortex. Current Opinions in Neurobiology 18, 90–100.



findings, overexpressionofCOUP-TFI (mediatedby theD6promoter) reduces the number of subcerebral projectionneurons (e.g., corticospinal neurons) that express Fezf2within the ectopic expression domain (Faedo et al., 2008).

The presence of the primary sensory areas, particularlyV1, in the COUP-TFI conditional KO mice (Armentanoet al., 2007) is likelydue at least inpart to the retained func-tion of Emx2. However, the finding that heterozygousconditional KO mice exhibit an intermediate phenotypeindicates that COUP-TFI also has a role in specifying theidentities of sensory areas. Although Emx2 and COUP-TFI both exhibit a low to high expression gradient alongthe A–P axis (Figure 4.5), they differ substantially in func-tion: Emx2preferentially specifiesposterior area identitiesin the posterior cortex (Hamasaki et al., 2004), whereasCOUP-TFI predominantly represses anterior area iden-tities in the posterior cortex (Figures 4.5 and 4.8;



Armentano et al., 2007). This difference in function is per-haps best illustrated by the effect of changes in the expres-sion of these TFs on the size of S1 versus V1. Diminishingthe expressionofCOUP-TFI has a similar effect on bothV1and S1 (i.e., both are reduced in size) and an opposing ef-fect on frontal/motor areas (i.e., they are increased in size;Armentano et al., 2007),whereas altering the expression ofEmx2 has opposing effects on V1 and S1 (e.g., changes inEmx2 levels that increase V1 size decrease S1 size) and asimilar effect on both frontal/motor areas and S1 (bothare decreased in size; Hamasaki et al., 2004).

4.5.2.5 Interactions Between TFs to Regulate AreaPatterning

The four TFs expressed in progenitors and implicatedto have primary roles in arealization have inductive orrepressive effects upon one another that affect their func-tion and their level of expression (Figure 4.8). For exam-ple, Sp8 is a direct transcriptional activator of Fgf8, andSp8 induction of Fgf8 is repressed by Emx2 (Sahara et al.,2007), which itself binds Sp8 (Zembrzycki et al., 2007).This provides a mechanism to limit Fgf8 expression tothe CoP, and accounts for the finding of expandeddomains of Fgf8/17 in Emx2 mutants (Fukuchi-Shimogori and Grove, 2003). In addition, many of theseTFs influence the expression of one another (Figure 4.8).For example, Emx2 and COUP-TFI appear to repressPax6 (Armentano et al., 2007; Faedo et al., 2008;Hamasaki et al., 2004; Muzio et al., 2002). Other TFs,for example, Foxg1 (BF1), which has a role in

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FIGURE 4.9 Distinct modes of cell-type specification in spinal cord vetypes are specified by genetic determinants that in the preneurogenic phalater become restricted to gene expression domains with sharp borders in tduring the neurogenic phase exclusively specifies progenitors within thisthe domain of gene B that only gives rise to area 1 neurons (Shirasaki andmodel will generate a spinal cord where the corresponding neuronal progspecified and therefore completely absent. (b) The specification of neocortioperates very differently than the spinal cord model. In the neocortex, gradoverlapping domains of progenitors in the VZ during the preneurogeniccentration of a number of different regulatory genes expressed at a given loeny toward a given area fate versus another (Hamasaki et al., 2004). Corresspinal cord model, a hypothetical LOF of gene B in the neocortex will nottraction or shift of area 1 and simultaneously a concomitant expansion or


determining dTel identity, influences expression ofPax6, as well as Emx2, and its deletion results in defectsin S1 patterning (Eagleson et al., 2007; Manuel et al.,2011). Further, as described in a preceding section,Fgf8 influences the expression of many of these TFs. Insummary, the TFs that control arealization also regulateone another aswell as at least a subset of themorphogens(e.g., Fgf8) that establish their graded expression by pro-genitors in the cortical VZ/SVZ, through reciprocal in-duction or repression loops. This mechanism canmodify levels of expression and slopes of expressiongradients.

In the ventral spinal cord, distinct pools of progenitorsare defined by their expression of unique sets of TFs thatdistinguish the progenitor pools from one another andthe neuronal types they generate (see Chapter 7). Incontrast, in the cortex, TFs that control arealizationcooperate to generate area patterning, but these TFsdefine unique area identities in progenitors and theirprogeny by operating through what appears to be aconcentration-dependentmechanism (Figure 4.9). In thismodel, progenitors across the cortex express the same setof TFs but at different levels, and it is the level of TF ex-pression that distinguishes progenitors from one an-other and specifies their area identities. This concept,termed the ‘cooperative concentration’ model, was de-veloped largely on the basis of studies of the functionof Emx2 in arealization (Bishop et al., 2000; Hamasakiet al., 2004; Leingartner et al., 2007) described in the pre-ceding section. The analyses of LOF and GOF Emx2

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rsus neocortex. (a) In a simplified model of the spinal cord, distinct cellse are expressed in overlapping and/or nonoverlapping domains thathe VZ. The clustered expression domain of a specific gene (e.g., gene A)domain to generate only cell type 2 or area 2 neurons and vice versa forPfaff, 2002) (see Chapters 7 and 21). An idealized LOF of gene B in thisenitors for gene B and consequently neurons of cell type area 1 are notcal areas can be described as a cooperative concentration model, whichients and countergradients of regulatory genes are expressed in mostlyand neurogenic phase. Simultaneous expression and the absolute con-cation of the VZ restricts the corresponding progenitors and their prog-ponding to the cooperative concentration model, and in contrast to theresult in a complete loss of area 1 neurons but rather introduce a con-shift of area 2.



mutants indicate that Emx2 operates by a concentration-dependent mechanism in cortical progenitors to specifydisproportionately the sizes and positioning of the pri-mary cortical areas, and that higher levels of Emx2 pref-erentially impart posterior-medial area identities, suchas those associated with V1 (Figures 4.6 and 4.9).

4.6 EXTENT OF GENETICSPECIFICATION OF AREA-SPECIFIC

PROPERTIES

4.6.1 Do Area-Unique Genes Exist?

Although TFs that control area patterning exhibit mu-tual repression in the cortex (Figure 4.8), at no time dur-ing neurogenesis are sharply bordered patterns of TFsobserved in the cortical VZ; instead, each TF retains agraded pattern across the cortical VZ. Even in the CP,the expression of TFs and other gene families is initiallygraded before many of them acquire expression patternswith abrupt borders (see Figures 4.1 and 4.9). However,the expression of none of these genes is limited to a singlearea. To date, the only genetic marker restricted to onearea is the H-2Z1 transgene, which marks the granularparts of mouse S1 (Cohen-Tannoudji et al., 1994). Al-though H-2Z1 expression is not detected until afterTCAs from VP have invaded the S1 CP, in vitroexplantation and in vivo heterotopic transplantation in-dicate that the S1 expression of H-2Z1 is specified earlyin embryonic cortical development (Gitton et al., 1999a,b). However, the maintenance of H-2Z1 expressionin vivo requires TCA input, and the refinement in its ex-pression pattern parallels the differentiation of thecytoarchitectural features characteristic of S1, a processdriven by TCAs (Cohen-Tannoudji et al., 1994; Gittonet al., 1999a,b). Thus, if the expression of H-2Z1 mimicsthat of an endogenous gene, this endogenous gene doesnot drive arealization but instead is a product of it.

Numerous screens using different technologies andtissue sources have now been reported that by virtueof their designs had the potential to identify genes witharea-unique expression (Arai et al., 2005; Funatsu et al.,2004; Gangemi et al., 2006; Holm et al., 2007; Leameyet al., 2008; Li et al., 2006; Liu et al., 2000; Muhlfriedelet al., 2007; Sansom et al., 2005). However, none of thesescreens identified a gene that uniquely marked a corticalarea, nor a subdomain within the progenitor population.Therefore, area-specific genes per se either do not exist orare exceedingly rare, at the developmental stages whenarea identities are genetically being specified, and per-haps even later, when the anatomical features that dis-tinguish areas begin to differentiate within CP. Thus,in terms of gene expression, a neocortical area is not de-fined by the expression of a specific set of genes


restricted to that area. Instead, a neocortical area is de-fined by the expression of a unique subset of genes, eachof which is also expressed in other areas. Therefore, amajor issue that remains unsettled is the extent to whichareas are genetically distinct, which we will address fur-ther in the subsequent section.

4.6.2 What Is ‘Area Identity?’

This discussion leads directly to the major issue of theextent to which areas are genetically distinct in the adultcortex. The available evidence indicates that in terms ofgene expression, a neocortical area is not defined by theexpression of a gene or set of genes restricted to that area.Instead, neocortical areas are defined by unique patternsof expression of sets of genes; these unique expressionpatterns are defined by subsets of genes that areexpressed in the area and subsets that are not expressedin the area – each gene that is a member of these setsmight also be a member of other unique sets that genet-ically define other areas through their inclusion or exclu-sion of expression.

The findings and issues discussed begs the question,‘what is area identity?’ and its corollary, ‘what is the ex-tent to which areas are genetically distinct?’ The answersto these important questions remain vague, yet they aretightly linked to defining themechanisms that determinearea patterning. Thus, in terms of gene expression, a neo-cortical area is not defined by the expression of a specificset of genes restricted to that area. Instead, a neocorticalarea is defined by the expression of a unique subset ofgenes, each of which is also expressed in other areas.

However, the actual scenario is evenmore complex aseach layer has a unique profile of gene expression. Eachgene differentially expressed in the neocortex, andexpressed in more than one layer, has different expres-sion patterns in each layer. Two distinct but excellent ex-amples are Id2 and MDGA1. Id2, a helix–loop–helix TF,has an abrupt border of expression in layer 5 that ap-pears to correspond to the border between S1 and M1,but in layer 2/3, Id2 has a graded expression that con-tinues across the tangential extent of the neocortex, beinghighest in rostral (motor), intermediate in S1, and lowestin caudal (visual) areas (Bishop et al., 2000, 2002).MDGA1 is an Ig cell adhesion molecule that is expressedby most layer 2/3 neurons throughout the neocortex,and is also expressed by neurons in layers 4 and 6 in adistribution limited to S1 (Takeuchi et al., 2007). Thus,MDGA1 has both layer and area-specific patterns of ex-pression, and can be used to define an area, S1, but itsexpression is not limited to that area. Thus, the term ‘areaidentity’ in the strictest sense might not truly exist forneurons across layers and uniquely mark neurons withthe specific identity of a given area.


774.6 EXTENT OF GENETIC SPECIFICATION OF AREA-SPECIFIC PROPERTIES

4.6.3 Genetic Determination of CorticalProjection Neurons Related to Arealization

An issue of great relevance to the discussion of geneticdetermination of area-specific properties is the mecha-nisms that determine the functionally appropriate out-put projections of cortical areas, which would includethe three major types: CPNs, which in mice are presentin all layers, but are predominantly found in layers2/3 and 5, and two classes of corticofugal projectionneurons, with SPNs in layer 5 (e.g., corticospinal andcorticotectal), and corticothalamic neurons in layer 6(O’Leary and Koester, 1993). Great strides have beenmade in the understanding of the specification of thesedistinct classes of CPNs (see reviews by Fame et al.,2011; Leone et al., 2008; Molyneaux et al., 2007) (seeChapters 18 and 25). However, much less is knownabout the mechanisms that underlie areal specificationof layer 5 neurons.

Recent studies have reported on the TFs that geneti-cally determine callosal neurons from subcortically pro-jecting layer 5 neurons. Some of the earliest evidence forthe molecular specification of CPNs came from analysesof genes differentially expressed in different corticallayers. The Pou domains TFs Brn1 and Brn2 areexpressed in superficial layers and control specificationof upper-layer glutamatergic pyramidal neurons(McEvilly et al., 2002; Sugitani et al., 2002). Similarly,the cut-like TF Cux2 is expressed in superficial neocorti-cal layers and in basal progenitors of the SVZ that gen-erate them. Loss of Cux2 disrupts SVZ formation andsubsequent generation of CPNs in upper neocorticallayers (Cubelos et al., 2008). Another molecular determi-nant, the TF Satb2, regulates specification of cortical pro-jection neurons by repressing activity of Ctip2. In theabsence of Satb2 function, CPNs that normally wouldproject through the corpus callosum aberrantly extendtheir axons subcortically through the internal capsuleand show other characteristics of SPNs (Alcamo et al.,2008; Britanova et al., 2008).

The TF Fezl (Fezf2) is expressed in all layer 5 SPNsand is crucial for their proper specification. In Fezf2KO mice, layers 5 and 6 are generated but projectionsto the spinal cord and brain stem fail to develop (Chenet al., 2005a; Hirata et al., 2004; Molyneaux et al., 2005).In contrast, the upper layers and their callosal projec-tions remain intact (Chen et al., 2005a; Molyneauxet al., 2005). Overexpression of Fezf2 is sufficient tospecify ectopic populations of neurons that project sub-cortically (Chen et al., 2005b; Molyneaux et al., 2005).Another TF, Ctip2, is important for later aspects of devel-opment of SPNs, mainly acting as a crucial regulator ofsubcerebral axon growth, and in Ctip2 KO mice, fewaxons reach their subcortical targets (Arlotta et al., 2005).


Recent studies have shown that the TF Bhlhb5, whichis broadly expressed in postmitotic neurons of layers 2through 5 throughout much of the neocortex, is requiredfor the proper development of a subset of layer 5 projec-tions to the spinal cord (Joshi et al., 2008). Further,Bhlhb5 KO mice exhibit a reduction in the expressionof markers for SPNs within their normal expressiondomain. However, Bhlhb5 does not selectively mark dis-tinct subtypes of SPNs, for example, corticotectal fromvisual areas versus corticospinal from sensorimotorareas; nor is it required in an area-specific fashion todrive the development of these subtypes of subcerebralprojections. Therefore, genes have yet to be identifiedthat selectively mark subtypes of SPNs, or selectivelyinfluence the development of their functionally appro-priate, area-specific projections. As described in a pre-ceding section, the distinct subtypes of SPNs aresculpted through degenerative axon pruning, a processthat limits the initially widespread ‘exuberant’ distribu-tions of these projection neurons to their more restrictedadult distributions, and extra-genetic mechanisms caninfluence this process (O’Leary andKoester, 1993). How-ever, the relative roles of intrinsic genetic specificationversus extra-genetic influences in driving the selectivedegenerative axon pruning of exuberant layer 5 subcer-ebral projections to generate the adult projections remainobscure.

4.6.4 Candidate Targets of TFs that RegulateArea Patterning

Defining the target genes of TFs that control areali-zation and determining how they function to generatearea specializations are some of the many major chal-lenges for the future. An initial step in this process is todo large-scale screens to define candidate target genes.Some screens have been designed to identify addi-tional genes that are differentially expressed withinthe cortex and that therefore might be involved in area-lization. The first reported screen of this type was adifferential display polymerase chain reaction (PCR)screen that compared RNAs derived from the frontaland occipital embryonic cortex, and identified scoresof known and novel genes, including, for example,the graded cortical expression of COUP-TFI and CloseHomolog of L1 (CHL1) (Liu et al., 2000), both of whichhave been subsequently shown to have significantfunctions in cortical development. More recently,others have used microarray technology to do similarsearches for genes differentially expressed along theaxes of developing mouse cortex (Funatsu et al.,2004; Leamey et al., 2008; Muhlfriedel et al., 2007). Adistinct series of recent screens have used a differentapproach, and were designed to identify genes thatare candidate targets of TFs or morphogens implicated



in arealization, such as Emx2 and Pax6 (Arai et al.,2005; Gangemi et al., 2006; Holm et al., 2007; Liet al., 2006) or Fgfs (Sansom et al., 2005). Each of thesescreens identified hundreds of candidate targets withincreased or decreased expression, and therefore werepotentially involved in cortical arealization as well asfunctions relevant to other prominent phenotypesexhibited by Emx2 and Pax6 (sey) mutants, as wellas Fgfr1 mutants, including proliferation, neuronal dif-ferentiation, migration, axon guidance, and regionalpatterning of the telencephalon.

One screen used a representational display analysisthat compared Emx2 null cortex to wild type, and viceversa, and among the many genes identified wasOdz4/Ten_m4, which, along with the other three mem-bers of this gene family, was analyzed (Li et al., 2006).The vertebrate Odz genes (also referred to as the Ten_mfamily inmouse) are orthologs of theDrosophila pair-rulepatterning gene, Odd Oz (Odz), which encodes a trans-membrane protein with structural domains similar totenascin and is involved in segmental patterning inDrosophila. In embryonic mice, Odz4 has an expressionpattern that parallels the graded expression of Emx2,but rather than being expressed in the VZ, Odz4 isexpressed in the CP throughout its development. Odz2and Odz3 have similar gradients of expression asOdz4 in the CP, whereas Odz1 has an opposing expres-sion gradient (Li et al., 2006). As described in a precedingsection for RORb, these graded Odz expression patternsrefine postnatally into more restricted patterns, withOdz2, 3, and 4 having patterns that relate to theposterior-medial positioned visual areas, and Odz1 tothe more anterior sensorimotor areas. The Odz genesalso have distinct laminar expression patterns (Li et al.,2006). Each Odz familymember exhibits an anterior shiftin cortical expression in Emx2 mutants and a posteriorshift in Pax6 (sey) mutants, consistent with the opposingarea-patterning functions of Emx2 and Pax6 and poten-tial roles for the Odz genes in arealization as targets ofEmx2 and Pax6 (Li et al., 2006). Odz3/Ten_m3 wasalso independently identified in a microarray screendesigned to identify genes differentially expressed in so-matosensory versus visual areas of developing mousecortex (Leamey et al., 2008). These investigators also findthe preferential expression of Odz3within visual corticalareas and provide evidence that Odz3 promotes homo-philic adhesion and neurite outgrowth by neurons thatexpress it (Leamey et al., 2008).

4.6.5 Translating Gradients of TFs into SharpBorders

A defining property of area patterning of the adultcortex is the abrupt transition in anatomical and func-tional characteristics as one moves from one area to


another, resulting in sharply defined borders. In addi-tion, many genes expressed in the CP have sharply bor-dered patterns of expression that often relate to bordersof cortical areas, especially as areas themselves emergeand become defined. An important process of arealiza-tion during development is translating the graded ex-pression of TFs expressed by cortical progenitors inthe VZ/SVZ and early on by their neuronal progenyin the CP into these abrupt, bordered patterns of expres-sion and other properties that relate to and define areas.Other TFs are likely involved as primary regulators ofarea patterning and cooperate with the four TFs de-scribed earlier to not only determine area identity butalso generate sharp areal borders. These would includeTFs that have primary roles in progenitors, as well asgenes that have secondary roles in area patterning, suchas LMO4, a TF that helps mediate the function of LIMhomeodomain genes and exhibits patterned expressionin postnatal cortex with sharp borders that relates tosensory areas (Armentano et al., 2007). Conditionaldeletion of LMO4 has subtle but reproducible effectson area patterning, with S1 exhibiting a small reductionin size and borders that appear less sharply defined(Huang et al., 2009).

Although little is known about the mechanisms thatgenerate sharp borders between areas in the developingcortex, studies in other systems suggest potential ones.Perhaps the most definitive examples from a mechanis-tic perspective come from studies of Drosophila embryosand the development of the sharply patterned expres-sion of the even-skipped gene expression, as well as ex-pression of targets of the regulatory protein, Dorsal(Figure 4.10). The graded distribution of Dorsal acrossthe embryo generates, through concentration-dependentdifferences in binding efficacy to promoter and repressorelements, expression patterns of downstream geneswith sharp borders that align with the boundaries ofdifferent embryonic tissues and related patterns of geneexpression (Rusch and Levine, 1996). The expression ofthe even-skipped gene, limited to multiple sharp stripesperpendicular to the A–P axis of the embryo, emergesthrough the combined action of multiple activatorsand repressors of its transcription; even-skipped isexpressed where expression of repressors is subthresh-old and that of activators is suprathreshold (Rusch andLevine, 1996; Small et al., 1996).

Similar mechanisms appear to operate in the devel-oping vertebrate brain, for example, during the differ-entiation of rhombomeres in the hindbrain (Kieckerand Lumsden, 2005) and establishment of unique pro-genitor domains in the ventral spinal cord (Jessell, 2000;Shirasaki and Pfaff, 2002) (see Chapters 7 and 21). Inthe ventral spinal cord, Shh secreted by the notocordand floorplate represses or induces the expression of dif-ferent classes ofTFs in theVZofventral spinal cord,whichinitially are expressed in gradients over the D–V axis,


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FIGURE 4.10 Genetic mechanisms for generating patterns of gene expression. (a) A transcription factor gradient can generate regulatory net-works that establish a patterning event. The expression gradient of one transcription factor, Dorsal, is able to regulate dorsal–ventral patterning inthe Drosophila embryo through the regulation of target genes to establish expression patterns. High levels of Dorsal activate target genes in thepresumptive mesoderm with low-affinity binding sites, such as Snail (sna), sometimes with the aid of coactivators, such as CREB-binding pro-tein(CBP). The boundary between the presumptive mesoderm and the neurogenic ectoderm is generated through sna-mediated downregulationof neuroectoderm-specific genes in the mesoderm, many of which possess high-affinity Dorsal binding sites. Thus, along with the corepressorC-terminal binding protein (CtBP), sna is able to downregulate a neuroectoderm gene rhomboid (rho) in the mesoderm. Correspondingly, rhoexpression is promoted by Dorsal (and CBP) when its concentration is insufficient to drive sna activation. Dorsal can also activate or repress targetgenes across its entire expression gradient. Zerknullt (zen) possesses a high-affinity Dorsal binding site and proximal sequences that bind regu-latory proteins such as Cut (Ct) and Dead Ringer (Dri). The Dorsal-Ct/Dri complex then recruits the corepressor Groucho (Gro) to mediate therepression of zen throughout Dorsal’s expression gradient. Absence of Dorsal then leads to the disassembly of the repression complex and sub-sequent activation of zen. It is possible that similar mechanisms may be utilized by the transcription factors expressed in gradients within the VZand CP. Adapted from Stathopoulos A and Levine M (2002) Dorsal gradient networks in the Drosophila embryo. Developmental Biology 246: 57–67.(b) Combinatorial action of gene networks can generate patterns of expression with sharp borders. The different concentrations of transcriptionfactors encoded by gap genes hunchback, giant, and Kruppel specify the second stripe of even-skipped (eve) expression in parasegment 3 of theDrosophila embryo. The proteins bicoid and hunchback activate the gene in a broad domain, and the anterior and posterior borders are formedthrough repression by giant and Kruppel proteins, respectively. The striped expression pattern of eve appears gradually. It is initially expressed ata low level in all nuclei, then a single broad band of expression appears anteriorly and narrows as other stripes develop. Each stripe is initiallyfuzzy, but eventually acquires sharp borders. It is possible that similar patterning mechanisms operate in the formation of sharp borders of geneexpression in the CP. Adapted from Alberts B, Bray D, Lewis J, Raff M, Roberts K, and Watson J (1994)Molecular Biology of the Cell, 3rd edn. New York:

Garland Science.

794.6 EXTENT OF GENETIC SPECIFICATION OF AREA-SPECIFIC PROPERTIES

similar to the A–P andM–L graded expression of TFs thatcontrolsareapatterning in the cortex.However, incontrastto the cortex, in the ventral spinal cord, the graded expres-sion of these TFs is transformed through mutual repres-sion into sharply bordered expression patterns in the VZthat result in domains of genetically distinct progenitorsdefined by their expression of distinct subsets of TFs.The distinct domains give rise to different classes of spinalneurons (Figure 4.9). Aswedescribed in an earlier section,we propose that area identities in the cortex are specifiedthrough a cooperative concentration model. This model


posits that the sameset ofTFs,whichare expressedbypro-genitors across the entire cortex, cooperate to control area-lization, and, importantly, the level of expression of anindividual TF such as Emx2 is a defining parameter thatspecifies the area identity of a cortical progenitor and itsprogeny (Hamasaki et al., 2004). Although these TFsmay exhibit mutual repression, unlike in the developingventral spinal cord, in the cortex mutual repression, orany other mechanism, does not generate distinct pools ofprogenitors that express unique subsets of TFs. Deletionof one of the determining TFs from spinal progenitors


WT

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FIGURE 4.11 Conditional deletion of Lhx2 from telencephalic pro-genitors of the Emx1 lineage alters regional fate. (Top panels)Micewithfloxed alleles of the LIM homeodomain transcription factor Lhx2 weremade and crossed with Emx1-Cre to conditionally delete Lhx2 fromprogenitors of the Emx1 lineage that generate the majority of neuronsthat form the regions of cerebral cortex, including the neocortex (Nctx),the paleocortical piriform cortex (wild-type piriform or olfactory cor-tex, wtOlf Ctx), and the archicortical hippocampus (Hp). In embryonicmice, Lhx2 (blue) is expressed in a high dorsal (medial) to low ventral(lateral) gradient in the VZ of the dTel. Radial glia (RG) are the progen-itors of the Emx1 lineage, and generate neurons of the cerebral cortex aswell as provide a migrational guide for them with long processes thatthey extend from the VZ to the pial surface. wtOlf Ctx progenitors arelocated in the vicinity of the pallium–subpallium boundary (PSB) andtheir neuronal progeny migrate along a dense collection of RG pro-cesses originating from these progenitors at the PSB VZ to their far ven-tral destination. Crossing of floxed-Lhx2 and Emx1-Cre mice togenerate Lhx2 cKO mice results in the selective deletion of Lhx2 fromdTel progenitors of the Emx1 lineage, and results in the refating of pro-genitors that would normally generate lateral neocortex and insteadgenerate an ectopic Olf Ctx (eOlf Ctx). The wtOlf Ctx is also generatedin Lhx2 cKO mice but is subsequently eliminated. (Bottom panels) InWT adults, the wtOlf Ctx is located ventral to the rhinal fissure (RF),and the neocortex (Nctx) is positioned dorsal to it. In adult Lhx2cKO mice, the neocortex has two distinct architectures: dorsomediallythe neocortex has a six-layer architecture andmarker expression typicalfor the neocortex, albeit with some defects, whereas the lateral neocor-tex has a three-layer pattern and phenocopies the architecture, markerexpression, and connectivity of the Olf Ctx. This eOlf Ctx is generatedby progenitors of the Emx1 lineage that would normally generate thelateral neocortex. The eOlf Ctx is located dorsal to the RF, is signifi-cantly larger than wtOlf Ctx, and extends well caudal to it, parallelingthe entire normal rostral–caudal extent of the lateral neocortex. Thesefindings, and others detailed in Chou et al. (2009) and in the text, dem-onstrate that Lhx2 regulates a fate decision among dTel progenitors ofthe Emx1 lineage to generate phylogenetically distinct telencephalicregions, lateral neocortex or paleocortical Olf Ctx, and is required for



leads to the production of defective neuronal types,whereas we predict from the cooperative concentrationmodel that deletion of a determining TF from cortical pro-genitors, for example, Emx2, would result in the produc-tion of neurons with normal properties, but refated intoadifferent area identity (Figure 4.9).Nonetheless, it is clearthat theprocess of arealization ismuchmore complex thanthis simplemodel, andmanydetailsneedtobeworkedoutboth for the intrinsic genetic contributions operating withprogenitors and their progeny, and the extra-genetic ones,for example, influences contributedbyTCA input, operat-ing within the intrinsic genetic constraints.

4.7 REGIONAL PATTERNING OF THECEREBRAL CORTEX

The mammalian cerebral cortex is comprised of sev-eral major regions, including a six-layer neocortex, andarchitecturally simpler and phylogenetically older corti-ces, including a three-layer olfactory (piriform) cortexand archicortex, which is predominantly hippocampalformation (Sanides, 1969) (see Chapter 10). The greatmajority of neurons that form each region, includingall glutamatergic and projection neurons, arise from pro-genitors within the VZ of dTel of a lineage defined by ex-pression of Emx1, a homeodomain TF expressed by allprogenitors within the dTel VZ (Gorski et al., 2002).For TFs that exhibit low to high A–P graded expressionpatterns in the neocortex, their graded expression pat-terns often continue beyond the posterior extent of theneocortex and into other regions of the cerebral hemi-sphere and through the hippocampal fields. Examplesof TFs that function in neocortical arealization and ex-hibit this form of continuing graded expression includeEmx2, COUP-TFI, and Lhx2. These continuous, gradedexpression patterns suggest a relationship between area-lization of the neocortex and arealization of other regionsof the cerebral cortex, as well as a relationship betweenarealization and regionalization of the cerebral cortex.

Although expression of Emx1 is a defining character-istic of dTel progenitors, Emx1 does not determineregional fate of progenitors within the Emx1 lineage.Nor has the Emx1 progenitor lineage been subdividedinto distinct populations, or sublineages, that generatespecific regions of cerebral cortex by their expressionof a distinct TF, and such a relationship between a line-age and a specific region of cerebral cortex might not

progenitors of the lateral neocortex and their progeny to acquire a neo-cortical fate. These findings establish a genetic mechanism for deter-mining the regional fate of dTel progenitors of the Emx1 lineage thatgenerate the cerebral cortex.Adapted fromChou SJ, Perez-Garcia CG, KrollTT, and O’Leary DDM (2009) Lhx2 specifies regional fate in Emx1 lineage of

telencephalic progenitors generating cerebral cortex. Nature Neuroscience12: 1381–1389.


814.8 CONCLUSIONS

exist. However, unique subpopulations of progenitors ofthe Emx1 lineage must generate distinct regions of thecerebral cortex and must be specified by their uniqueexpression of one or more TFs. One of these is the LIMhomeodomain TF Lhx2, which is expressed in all dTelprogenitors of the Emx1 lineage in a high to low caudo-medial to rostrolateral graded pattern across the dTel VZ(Monuki et al., 2001). The cerebral cortex largely fails todevelop in the Lhx2 constitutive KO because VZ progen-itors become quiescent early in corticogenesis and thecortical hem dramatically expands (Bulchand et al.,2001; Monuki et al., 2001; Porter et al., 1997).

Roles for Lhx2 in dTel patterning have been substan-tially advanced by use of a conditional KO of Lhx2 andgenetic mosaics in chimeric mice comprised of Lhx2 nulland wild-type cells (Mangale et al., 2008). These studiesprovide further evidence that Lhx2 specifies corticalidentity in a cell-autonomous fashion and acts to sup-press hem fates in the medial cortex, and in a comple-mentary fashion, to suppress antihem fates in thelateral cortex. These studies demonstrate that Lhx2 is aclassic selector gene in regional fate determinationwithin dTel, being required to define cortical identity,and that the cortical hem is a hippocampal organizer(Mangale et al., 2008).

Analyses of conditional deletions of Lhx2 that arepostnatal-viable show that Lhx2 specifies within theEmx1 lineage the fate decision to produce the neocortexor olfactory (piriform) cortex (Figure 4.11; Chou et al.,2009). Deletion of Lhx2 specifically from progenitors ofthe Emx1-lineage results in lateral neocortex transfatinginto an ectopic olfactory cortex that exhibits the cytoarch-itecture, connections, and marker expression of thethree-layer olfactory cortex rather than the six-layer neo-cortex. Lhx2 regulates this regional fate within dTel pro-genitors of the Emx1 lineage during a critical period thatcloses coincident with the differentiation of cortical pro-genitors from stem-cell-like neuroepithelial cells to neu-rogenic radial glia and the onset of cortical neurogenesis(Chou et al., 2009). The timing of the critical period couldreflect the action of distinct mechanisms; for example,the olfactory cortex could be the default regional fatefor lateral neocortical progenitors following early butnot later deletion of Lhx2, or this regional fate decisionby progenitors of the Emx1 lineage might be determinedin part by their ‘exposure’ to Lhx2, which is the productof exposure time and expression level.

4.8 CONCLUSIONS

The mechanisms that control arealization of the neo-cortex have received considerable attention over the pasttwo decades (O’Leary, 1989; O’Leary et al., 2007; Rakic,1988; Rakic et al., 2009). Several TFs, morphogens, and


signaling molecules have been defined to have roles inarealization, and headway is being made in understand-ing their interactions. Yet, much work needs to be doneto better characterize roles in arealization for the TFs cur-rently identified and define additional players near thetop of the genetic hierarchy, as well as determine theirtargets and mode of action. In addition, roles of extra-genetic mechanisms and TCAs in arealization, whichare currently vague and primarily defined only at a phe-nomenological level, need to be better understood in partbecause they are likely a significant source of corticalplasticity, both at the level of overall area patterningand patterning within an area. These efforts will be crit-ical to understand how a set of TFs expressed at varyinglevels in cortical progenitors, and TCAs that operatewithin this framework, become translated into the pre-cisely patterned anatomical and functionally specializedareas that characterize the adult cortex.

Acknowledgments

Work in the authors’ lab on the topic of this article is funded by NIHgrants R01 NS31558 and R01 MH086147, the Vincent J. Coates Chairof Molecular Neurobiology (D.D.M.O’Leary), and a fellowship toA.S. Stacker from T 32 EY020503. The authors thank current and formermembers of the O’Leary lab, particularly Zoila Babot, Shen-ju Chou,and Setsuko Sahara, for thoughtful discussions.

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