Neuroscience Research 86 (2014) 6676

Contents lists available at ScienceDirect

Neuroscience Research
journal homepage: www.elsevier.com/locate/neures

Review article

Development and evolution of cortical elds

Yoko Arai , Alessandra Pierani
Institut Jacques Monod, CNRS UMR 7592, Universit Paris Diderot, Sorbonne Paris Cit, 75205 Paris Cedex, France

a r t i c l e

i n f o

Article history:
Received 1 February 2014
Received in revised form 5 June 2014
Accepted 10 June 2014
Available online 28 June 2014
Keywords:
Neurogenesis
Cortical patterning
CajalRetzius neurons
Thalamo-cortical afferents
Evolution
Cortical areas

a b s t r a c t
The neocortex is the brain structure that has been subjected to a major size expansion, in its relative
size, during mammalian evolution. It arises from the cortical primordium through coordinated growth
of neural progenitor cells along both the tangential and radial axes and their patterning providing spatial
coordinates. Functional neocortical areas are ultimately consolidated by environmental inuences such
as peripheral sensory inputs. Throughout neocortical evolution, cortical areas have become more sophisticated and numerous. This increase in number is possibly involved in the complexication of neocortical
function in primates. Whereas extensive divergence of functional cortical elds is observed during evolution, the fundamental mechanisms supporting the allocation of cortical areas and their wiring are
conserved, suggesting the presence of core genetic mechanisms operating in different species. We will
discuss some of the basic molecular mechanisms including morphogen-dependent ones involved in the
precise orchestration of neurogenesis in different cortical areas, elucidated from studies in rodents. Attention will be paid to the role of CajalRetzius neurons, which were recently proposed to be migrating
signaling units also involved in arealization, will be addressed. We will further review recent works
on molecular mechanisms of cortical patterning resulting from comparative analyses between different
species during evolution.
2014 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

Contents
1.
2.

3.

4.

5.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Radial organization of the cerebral cortex: neurogenesis during evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.
Changes in cortical proliferative regions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.
Proliferative capacities and cell-cycle kinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3.
Proliferative capacities and environmental inuences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Tangential organization of the cerebral cortex: cortical patterning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.
Morphogens and transcription factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.
Extrinsic inuences: CajalRetzius neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.
Extrinsic inuences: thalamo-cortical afferents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Evolution of cortical elds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.
Comparative anatomy of cortical areas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2.
Genomic and transcriptomic changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3.
CR neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Conclusions and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abbreviations: NE, neuroepithelial cells; RG, radial glial cells; V1, primary visual area; A1, primary auditory area; S1, primary somatosensory area; M1, primary motor
area; AP, anteroposterior; DV, dorsoventral; VP, ventral pallium; PSB, pallial sub-pallial boundary; TCA, thalamo-cortical afferents; VHO , higher-order visual area; SGL, subpial
granular layer cells.
Corresponding author. Tel.: +33 1 57 27 81 26; fax: +33 1 57 27 80 87.
E-mail address: arai@ijm.univ-paris-diderot.fr (Y. Arai).
http://dx.doi.org/10.1016/j.neures.2014.06.005
0168-0102/ 2014 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

Y. Arai, A. Pierani / Neuroscience Research 86 (2014) 6676

1. Introduction
The mammalian neocortex, which is the control center of our
cognitive functions, responsible for behavior and social activities,
is the brain structure that shows major expansion during evolution. The neocortex arises from the dorsal telencephalon and is
composed by different types of neurons that are generated after
the exponential expansion of neural stem cells known as neuroepithelial cells (NE) and which later differentiate into radial glial
cells (RG). Among the features, which are unique to the neocortex
as opposed to other brain regions, is the radial neuronal organization in six major layers, composed of earlier and later born
neurons positioned according to an inside-out sequence. Each layer
contains multiple distinct neuronal populations and functionally
distinct connectivity. The neocortex shows a spatial organization
(in the tangential dimension) called arealization, which represents
the subdivision of the neocortex into functionally distinct cortical areas. The basic plan of a mammalian neocortex is constituted
by four primary areas: visual (V1), auditory (A1), somatosensory
(S1) and motor (M1) cortices. Primary areas relay input information from the periphery (visual, auditory and somatosensory)
and control motor output. These are functionally interconnected
to higher-order areas that act as specialized processing or integrating centers (OLeary and Sahara, 2008; Krubitzer and Dooley,
2013); the latter being largely added during neocortex evolution.
Area identity starts to be established early during development
but its ultimate determination depends also on environmental
cues brought notably by peripheral axons branching in cortical
areas (OLeary, 1989; OLeary et al., 1994). During evolution, different neocortical territories expanded unequally. Species-specic
neocortical areas were formed and coincidentally region-specic
expression of genes was also reported (Abrahams et al., 2007;
Johnson et al., 2009; Kang et al., 2011; Chen et al., 2011), suggesting
a convergent evolution between brain structure and gene regulation. Causal or as a consequence of anatomical changes, increasing
neuronal complexity and plasticity is also pronounced during evolution. For instance, the morphology of human pyramidal neurons
and their plasticity in response to environmental cues show extensive changes with area-specic differences (Elston et al., 2001; Van
Pelt and Uylings, 2002; Elston, 2003). Thus, the area-specic degree
of neuronal maturation is likely involved in functional specication
of the human brain. To understand the involvement of genetic and
environmental factors in controlling the size and unequal expansion of cortical areas of the mammalian neocortex, in this review,
we will rst discuss some fundamental mechanisms involved in
the establishment of early cortical patterning during development
and differences that may have arisen during the course of cortical
evolution.

67

Cortical neurons arise from NE, multipotent neural progenitor

cells characterized by their (i) self-renewing capacity and (ii) their
potential to give rise to three major neural cell types: neurons,
astrocytes and oligodendrocytes (Bertrand et al., 2002; Jandial et al.,
2008). NE are highly polarized cells arranged in a single layer that
forms the ventricular zone (VZ) (Bystron et al., 2008). The VZ is colonized by blood vessels. On one side it faces the ventricles lled
with lipoprotein- and membrane particle-rich cerebrospinal uid,
and on the other the basal lamina, a rich source of extracellular
molecules (Vaccarino et al., 1999; Raballo et al., 2000; Gtz and
Huttner, 2005). This highly dynamic and rich micro-environment
provides stem cell nichelike features to the NE during development (Lehtinen and Walsh, 2011), crucial for the regulation
of neurogenesis and neuronal diversity. Following the onset of
cortical neurogenesis, a secondary proliferative region, the subventricular zone (SVZ), is formed from NE cells. SVZ progenitor cells
continue to proliferate for approximately one-two rounds of divisions in mice before undergoing self-consuming divisions that give
rise to neurons (Noctor et al., 2004; Miyata et al., 2004; Haubensak
et al., 2004; Shitamukai et al., 2011; Wang et al., 2011). The SVZ
is further divided into an inner (ISVZ) and outer SVZ (OSVZ) in
primates and carnivoras, which corresponds to an anatomical separation by the inner ber tract (Reillo et al., 2011; Smart et al., 2002).
OSVZ progenitor cells undergo multiple rounds of self-proliferative
division followed by the direct generation of neurons (Hansen et al.,
2010; Fietz et al., 2010; LaMonica et al., 2013; Betizeau et al., 2013).
The anatomical appearance of the OSVZ is not unique to primates
but is rather common across mammalian species which have a
gyrencephalic neocortex (Smart et al., 2002; Hansen et al., 2010;
Fietz et al., 2010; Reillo et al., 2011; Shitamukai and Matsuzaki,
2012).
During mammalian cortical evolution, the number of cortical
plate neurons has massively increased, in particular the upper
(supragranular) layer neurons (layers 23), which comprise a larger
proportion of the cortex in humans compared to rodents (Hill and
Walsh, 2005). Several reports have correlated this increase with the
massive enhancement of specic types of progenitor cells found in
the OSVZ (Smart et al., 2002; Hansen et al., 2010; Fietz et al., 2010);
therefore, this acquisition and expansion of OSVZ progenitor cells
is often considered as an evolutionary adaptive change. The size
of the OSVZ is correlated with the increase in neocortical size. Is
it a consequence of prolonged neurogenesis mediated by different
environmental inuences or is it caused rst by intrinsic changes in
cell-cycle kinetics? To answer these questions, several studies analyzed the dynamics of the cell-cycle in distinct progenitor cells in
different species (Lukaszewicz et al., 2005; Arai et al., 2011; Reillo
and Borrell, 2012; Betizeau et al., 2013).
2.2. Proliferative capacities and cell-cycle kinetics

2. Radial organization of the cerebral cortex: neurogenesis

during evolution
2.1. Changes in cortical proliferative regions
To build up cytoarchitectonically and functionally different
brains as observed during evolution, various genetic and cell biological processes are involved. Changes in the number of neurons
generated may rely on changes in the proliferative capacities of
the progenitor zone, which can occur through changes of intrinsic
cell-cycle kinetics, and/or modifying the access of progenitor cells
to environmental factors. Indeed, the mammalian neocortex has
complexied its proliferative domains in the course of evolution to
give rise to different sets of progenitor cells, likely having increased
proliferative capacities, which may have resulted in the emergence
of area-specic differences in neurogenesis.

In the mouse (a lissencephalic rodentia) at embryonic day (E)

14.5, progenitor cells in the VZ have a shorter total cell-cycle length
compared to SVZ progenitor cells, due to a specic lengthening of
the S phase and a shortening of the G1 phase (Pilaz et al., 2009;
Arai et al., 2011). VZ and SVZ progenitor cells can both be further
subdivided into proliferative and neurogenic populations (Iacopetti
et al., 1999). In both VZ and SVZ, neurogenic progenitor cells have
a shorter total cell-cycle length compared to that of proliferative
progenitor cells, mainly due to a shorter S phase (Arai et al., 2011)
(Table 1), indicating that neurogenic division is linked to a total
cell-cycle shortening. Therefore, proliferative SVZ progenitor cells
have the longest cell-cycle (Arai et al., 2011) and the duration of
the total cell-cycle in VZ and SVZ progenitor cells is progressively
increased during development in rodents (Caviness et al., 1995;
Takahashi et al., 1995). In the ferret (a gyrencephalic carnivora) progenitor cells in the VZ showed no obvious differences in their total

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Y. Arai, A. Pierani / Neuroscience Research 86 (2014) 6676

Table 1
Distinct cell-cycle kinetics between different species.
Species

Area

Stage

Neurons

TC
VZ

TS
(O)SVZ

TC TS

TG1

VZ

(O)SVZ

VZ

(O)SVZ

12

21

14

23

ND
ND

ND
ND

20
16

24
21

47
35

47
35

51
37

50
38

Mouse

S1

E14.5

Layer 4

19

27

Ferret

V1
V1

P0
P6

Layer 2/3
Layer 2/3

42
32

43
34

22
17

18
13

Macaque

V1
V1

E65
E78

Layer 5/6
Layer 2/3

63
46

70
50

12
9

20
12

VZ

(O)SVZ

An overview of cell-cycle kinetics in mouse (a lissencephalic rodentia, data from Arai et al., 2011), ferret (a gyrencephalic carnivora, data from Reillo and Borrell, 2012) and
macaque monkey (a gyrencephalic primate, data from Betizeau et al., 2013) with respect to the prospective cortical area and stage analyzed. Mouse data were obtained
in presumptive S1 at E14.5, a peak of layer 4 neuron generation; ferret data were obtained in presumptive V1 at P0 and P6, peaks of layer 2/3 neuron generation (see
McConnell 1988); macaque data were obtained in presumptive V1 at E65 and E78, peaks of layer 5/6 and layer 2/3 generation, respectively. The thickness of OSVZ in ferret
P6 is approximately 2.4 times that of P0 (see Reillo and Borrell, 2012) and in macaque around E78 is at least twice that of E65 (see Smart et al., 2002). Indicated cell-cycle
parameters were a representative of all progenitor cells observed in the VZ and SVZ (mouse) or OSVZ (ferret and monkey). In mouse, the total cell-cycle is lengthened during
development but it is not the case in ferret and monkey. Note: Values indicate hours. TC , total length of cell-cycle; TS , length of S phase; TG1 , length of G1 phase; TC TS ,
length of total cell-cycle minus length of S phase shows the minimum time to reach the plateau of EdU or BrdU (thymidine analogs) labeling index. ND, not measured; an
approximated duration of cell-cycle parameters from Figure 2 in Betizeau et al. (2013). E, embryonic day; P, postnatal day; S1, primary somatosensory cortex; V1, primary
visual cortex.

cell-cycle length compared to that of OSVZ progenitor cells at

the same postnatal stage (Reillo and Borrell, 2012). Furthermore,
VZ progenitor cells showed a longer S phase compared to OSVZ
progenitor cells, suggesting that VZ progenitor cells might have
either a shorter G1 or G2 + M phases or both. However, the total
cell-cycle length of both VZ and OSVZ progenitor cells decreased
during development with further shortening of the S phase (Reillo
and Borrell, 2012), which was not the case in mice (Takahashi
et al., 1995) (Table 1). In addition, a recent study using the embryonic macaque monkey (a gyrencephalic primate) has reported the
presence of ve distinct OSVZ progenitor cell types with differing
cell-cycle dynamics (Betizeau et al., 2013). Progenitor cells in the VZ
had a shorter total cell-cycle length compared to OSVZ progenitor
cells at the same developmental stage seemingly due to the shortening of their S phase and showed no obvious difference of their G1
length (Betizeau et al., 2013). However, the total cell-cycle length of
both VZ and OSVZ progenitor cells was shortened during development with shortening of both S and G1 phases, a similar tendency to
what was observed in the ferret (Reillo and Borrell, 2012) (Table 1).
Cell-cycle analyses from three different species therefore raise several points to discuss. First, the tendency of having a longer total
cell-cycle length of the SVZ/OSVZ compared with VZ progenitor
cells is observed in rodents and primates but it is not obvious in
carnivora. According to the mammalian phylogeny, an ancestor of
carnivora was separated from a common ancestor of rodents and
primates (Nishihara et al., 2006), suggesting that cell-cycle lengthening of SVZ/OSVZ with respect to VZ progenitor cells might depend
on a mechanism acquired or specically conserved in the lineage
of rodents and primates. Second, the shortening of the total cellcycle with the progression of development, which is observed in
gyrencephalic species, may reect the expansion of the OSVZ progenitor pool and consequently may have contributed to the massive
generation of neurons observed. This may explain why a similar
tendency is not found in lissencephalic rodentia. Third, it seems
there are some distinct contributions of cell-cycle phases in the VZ
and (O)SVZ progenitor cells between rodentia, carnivora and primates. For instance, in rodentia and carnivora, S phase length (Ts) is
shorter in (O)SVZ with respect to VZ progenitor cells but it is longer
in primates (Table 1). It has been suggested in mice that the lengthening of the S phase in proliferating VZ and SVZ cells reected the
DNA quality check during S phase (Arai et al., 2011). Extrapolating
this to primate OSVZ progenitor cells, it suggests that they require
a more robust DNA delity check system than that of carnivora and
rodentia SVZ progenitor cells due to their extended proliferative
periods. However, caution should be taken in these interspecies
dataset comparisons which might indicate differences in part due

to the analysis of different cortical regions or stages rather than

existing ones among species. Indeed, area specic changes in cellcycle kinetics are reported in primates. For instance, total cell-cycle
length of progenitor cells in the thick OSVZ of area 17 is shorter
rather than that of the thin OSVZ of area 18 in primates (macaque)
(Lukaszewicz et al., 2005). The difference in the thickness of the
OSVZ along the rostralcaudal axis is also reported in both ferrets
and macaques (Smart et al., 2002; Reillo and Borrell, 2012); therefore, cell-cycle parameters could be variable in different cortical
areas along the rostralcaudal axis. Moreover, cell-cycle analyses
in the mouse were performed in the presumptive S1 at E14.5 (the
peak of layer 4 generation) (Arai et al., 2011; Kwan et al., 2012)
and both ferret and macaque analyses were done in presumptive
area 17 (V1) in the occipital cortex coinciding with the appearance of upper layer neurons (McConnell, 1988; Reillo and Borrell,
2012; Betizeau et al., 2013). Therefore, the difference of cell-cycle
kinetics that have been reported in the above three species may
reect the difference in cortical areas analyzed. Taken together,
cell-cycle regulation differs in distinct subtypes of cortical progenitor cells and it is likely to be involved in regulating the number of
neurons produced. Nevertheless, since cell-cycle parameters vary
between different cortical regions and developmental stages, this
should be considered when comparative analyses between species
are performed.
2.3. Proliferative capacities and environmental inuences
The cell-cycle and morphological differences observed across
distinct species may also be correlated with environmental
changes. The morphological characteristics of OSVZ progenitor cells
allow them to be exposed to more signaling molecules than the
majority of mouse SVZ progenitor cells as OSVZ cells maintain a
long process that spans the length of the cortical wall from the ventricle to the pia matter. For instance, Notch (Hansen et al., 2010) and
Integrin signaling (Fietz et al., 2010) from the basal extracellular
matrix, and Shh, FGF2 and Igf2 from the apical cerebrospinal uid
(Huang et al., 2010; Lehtinen and Walsh, 2011) have been shown to
inuence their proliferation. This rich environment is further complexied with the arrival of embryonic thalamo-cortical axons that
have a mitotic effect on progenitor cells through the function of
basic FGF (Dehay et al., 2001). These afferent bers arrive around
E14.5 in the intermediate zone in mice and are in close proximity to the SVZ, when the production of SVZ progenitor cells is still
high. Then the distance between afferent bers and proliferative VZ
and SVZ zones further increases during development (Dehay et al.,
2001; Dehay and Kennedy, 2007). In monkeys, thalamic axons are

Y. Arai, A. Pierani / Neuroscience Research 86 (2014) 6676

located in the outer ber layer above the OSVZ (Dehay and Kennedy,
2007) and the appearance of the thalamic bers occurs much earlier in development compared to that in the mouse (Smart et al.,
2002; Dehay and Kennedy, 2007). With the progression of development, bers remain close to the OSVZ in area 17 (Smart et al., 2002;
Betizeau et al., 2013). It is therefore tempting to link the two events
and thus that thalamic axons might inuence cell-cycle progression
of OSVZ progenitor cells. However, much remains to be elucidated
about the molecular aspects of the environmental proliferative cues
which may act on different types of progenitor cells, and which
cell-cycle parameters these inuence, as well as how these may
vary between different species and may have contributed to cortical
evolution.
3. Tangential organization of the cerebral cortex: cortical
patterning
Among the key changes observed during evolution is the different size of specic cortical territories dedicated to distinct
neocortical functions. The prefrontal neocortical territories, dedicated to integrative function, are preferentially expanded in the
primate lineage including humans, together with associative areas,
which are devoted to higher-order cortical processing (Krubitzer,
2007). However, the precise molecular mechanisms responsible for
the increase of associative areas have not yet been fully investigated.
3.1. Morphogens and transcription factors
The specication of cortical territories begins at early stages of
development, starting from organizing centers which express different morphogens, such as Fgfs, Wnts, Bmps and Shh, and are
crucial for the establishment of future cortical territories (Fig. 1a
and b) (Shimamura et al., 1995; Grove et al., 1998; Assimacopoulos
et al., 2003; Shimogori et al., 2004; OLeary et al., 2007). Ectopic Fgf8
expression in the caudal cortex at an early stage of development
has been shown to cause a functional duplication of cortical areas
at postnatal stages (Assimacopoulos et al., 2012). Thus, the correct
establishment of morphogen gradients is required for setting up
cortical area identity.
Cortical progenitor cells are exposed to different concentrations of morphogens (Viti et al., 2003; Lillien and Gulacsi, 2006;
Toyoda et al., 2010) that function to set up the graded expression
of transcription factors, such as Pax6, Emx2, COUP-TF1 and SP8
(Bishop et al., 2000; Mallamaci et al., 2000; Zembrzycki et al., 2007;
Armentano et al., 2007; OLeary et al., 2007). As a consequence,
the regionalization of the dorsal telencephalon is therefore established along the anteroposterior (AP) and dorsoventral (DV) axes by
E12.5 (Fig. 1c). In rodents, extensive work has been carried out to
demonstrate the importance of these transcription factors in precisely controlling the positioning and size of primary cortical areas
(OLeary and Sahara, 2008). For instance, loss of function of the Emx2
and COUP-TF1 genes resulted in the reduction of caudal cortical
areas (Bishop et al., 2000; Mallamaci et al., 2000; Armentano et al.,
2007) and the expansion of the anterior motor cortex, whereas
gain of function of Emx2 resulted in the expansion of caudal cortical areas (Hamasaki et al., 2004) (Fig. 1d). On the contrary, loss
of Pax6 and Sp8 led to the expansion of the caudal V1 area and
in the reduction of anterior most territories (Bishop et al., 2000,
2002; Zembrzycki et al., 2007; OLeary et al., 2007) (Fig. 1d). The
specication of neocortical areas is therefore controlled by intrinsic information in the progenitor domain in agreement with the
protomap hypothesis, which postulates that progenitor cells are
programmed to generate area-specic cohorts of cortical plate neurons (Rakic, 1988, 2009). Nevertheless, this expression is controlled

69

by morphogens secreted at organizing centers which are extrinsic

to neocortical progenitors.
3.2. Extrinsic inuences: CajalRetzius neurons
In addition to the classical role of patterning centers, recent
studies point to the importance of the postmitotic compartment in
inuencing cortical patterning (Pierani and Wassef, 2009; Borello
and Pierani, 2010). Among the rst-born neurons are CajalRetzius
(CR) neurons, which are mainly generated from different organizing centers at the borders of the developing pallium: the pallial
septum, the ventral pallium (VP)/pallial sub-pallial boundary (PSB)
and the cortical hem (see Fig. 1b) (Takiguchi-Hayashi et al., 2004;
Bielle et al., 2005; Yoshida et al., 2006; Tissir et al., 2009). Analyses
of Fgf8 and Tgf signaling showed the importance of organizing
centers in inducing the generation of septum and cortical hem
CR neurons, respectively (Siegenthaler and Miller, 2008; Zimmer
et al., 2010). Depending on their origin, CR neurons express distinct
molecular markers and preferentially populate specic regions of
the developing cerebral cortex (Bielle et al., 2005; Yoshida et al.,
2006). CR neurons, together with other pioneer neurons, form the
preplate, a neuronal dense layer, which is split into two layers: the
supercial marginal zone (layer 1) and the deep subplate by incoming radially migrating cortical plate neurons which will form layers
26 (Kwan et al., 2012). The CR neurons secrete reelin, an extracellular matrix glycoprotein and the mutation of reelin resulted in
a disorganization of cortical laminar formation (DArcangelo et al.,
1995; Ogawa et al., 1995). During early stages of development, CR
neurons migrate tangentially underneath the pial surface to cover
the entire cortical surface where they are also in close proximity
with cortical progenitor cells. Specic ablation of a subpopulation
of CR neurons derived from Dbx1-expressing cells at the pallial
septum (Dbx1-derived septum CR neurons in short) results in the
redistribution of CR subtypes in the rostromedial pallium between
E10.5 and E11.5 and leads to arealization defects in the postnatal
brain (Fig. 2a). Redistribution of CR subtypes leads to changes in the
expression of Pax6 and Sp8 transcription factors within the cortical
progenitor cells and in the proliferation properties of medial and
dorsal cortical progenitor cells at E11.5. This early regionalization
defects correlate with shifts in the positioning of cortical areas at
postnatal stages. Notably, transcriptomic analysis of Dbx1-derived
neurons showed that they express distinct morphogens (Griveau
et al., 2010), suggesting that morphogen secretion by migrating CR
cells could inuence areal patterning. Thus, by signaling to cortical progenitors in the mitotic compartment, these neurons serve
as organizers during development, therefore acting as mobile
signaling units. This work points toward a novel general strategy
for long-range patterning in large structures, in addition to passive
diffusion of morphogens, via migration of signaling cells, a mechanism which could be of use in the expansion of the cortical surface
in primates.
3.3. Extrinsic inuences: thalamo-cortical afferents
The relative size of cortical elds and their functional connectivity are also inuenced by the environment. Cortical neurons receive
information from the periphery through the thalamus via thalamocortical projections. Developmental studies of the thalamo-cortical
afferents (TCA) have shown that they have a negligible effect on
the size, position and molecular identity of cortical areas during
mouse embryonic stages (Nakagawa et al., 1999; Garel et al., 2002).
Recent studies using conditional transgenic mice have since conrmed that while no early regionalization defects occurred upon
modulation of specic TCA projections, area changes in the postnatal brain were observed (Vue et al., 2013; Chou et al., 2013). In these
studies, loss and gain of specic TCA projections showed that within

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Fig. 1. Patterning centers in the developing mouse telencephalon, sources of CR neurons and transcription factors mediating cortical arealization. (a) Schematic representation
of the developing mouse telencephalon from E10.0 to E12.5. Morphogens are secreted from signaling centers. Fgfs (Fgf8, 15 and 17) are secreted from the pallial septum
(green), Wnts (Wnt2b, 3a and 5a) and Bmps (Bmp4 and 7) are secreted from the cortical hem (blue), Tgf, Sfrp2 and Fgf7 are expressed at the pallial-subpallial boundary
(PSB or anti-hem in red). (b) Coronal views of the developing mouse forebrain showing the position of signaling centers that are highlighted in colors. These territories also
represent the sites of CR subtype generation. (c) Graded expression of transcription factors (TFs) along the anterior-posterior and medial-lateral axes. While Emx2 and COUPTF1 showed rostral low/caudal high expression patterns, Pax6 and Sp8 show rostral high/caudal low expression. (d) Role of TFs in arealization by gain- and loss-of-function
studies. Primary motor cortex (M1) is in green, primary somatosensory cortex (S1) in red, primary auditory cortex (A1) in orange and primary visual cortex (V1) in blue.
Overexpression (OE) of Emx2 (NestinEmx2 ) leads to the expansion of V1 and an anterior shift of caudal areas. An opposite defect is observed in Emx2 knock out (KO) mice.
Conditional KO of COUP-TF1 shows a massive expansion of motor cortex (M1). Small eye mutant mice (Pax6 null mice) and conditional KO of SP8 shows a reduction of rostral
M1 and an anterior shift of caudal areas.

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71

Fig. 2. Postnatal arealization defects caused by extrinsic signals. (a) Arealization defects by conditional ablation of Dbx1-derived septum CajalRetzius (CR) neurons. Both
M1 and S1 shift laterally (dorsal view). (b) Arealization defects by conditional loss- or gain-of-function of thalamo-cortical axonal (TCA) projections derived from the dorsal
lateral geniculate (dLG) nucleus in the thalamus. Loss of TCA projections causes lack of differentiation of V1. Conversely, V1 is expanded in the case of gain-of-function.

the visual eld the differentiation between V1 and a surrounding

higher-order visual area (VHO ) depends on TCA at postnatal day
(P) 7 (Chou et al., 2013) (Fig. 2b). All together these studies show
that TCA inputs are involved in rening cortical areas identity at
postnatal stages (see also Pouchelon et al., 2014). However, since
arrival of TCA in the developing cortex occurs at mid-gestation in
mice, it leaves open the possibility that they may also function during embryogenesis and, thus, will have a double role. Several open
questions still remain, such as which type of cells are rst targeted
by TCA to cause arealization defect later at postnatal brain either
the proliferation of progenitor cells or specication of post-mitotic
neurons or both, and which molecules are involved in changes in
cortical patterning imprinted by TCA projections and the environment (Rakic et al., 1991; OLeary et al., 1994; Dehay et al., 1996;
Kahn and Krubitzer, 2002; Karlen et al., 2006), and if it is emphasized during evolution.
In nature also there are examples which suggest that cortical
arealization depends on the environmental niche and, thus, it is
inuenced by the use of sensory systems (Campi and Krubitzer,
2010). A comparative analysis between adult wild-caught Norway
rat and Norway rat bred in the laboratory reported variations of
the size of cortical elds (Campi and Krubitzer, 2010) exemplifying
the impact of environmental sensory input on cortical plasticity.
Despite both being the same species, wild-caught Norway rat was
clearly exposed to a richer living environment than that of the
laboratory. The relative size of S1 and A1 in laboratory rats was
signicantly bigger than that of wild-caught. In addition, although
no obvious differences in V1 size were observed, a greater density
of neurons was observed in wild-caught rats than in laboratory rats

(Campi et al., 2011) in this area. These results strongly suggested

that the use of visual sensory system inputs change not only the size
of cortical elds but also the cellular composition of the V1 cortex. It
would be very interesting to know which genetic mechanisms are
responsible for such changes in cortical elds and whether these
alterations are partially reversible or epigenetically xed to propagate it into the progenies, which is an important aspect from the
evolutionary point of view.
Altogether, many ndings revealed that cortical arealization is
a multi-step process initiated from early stages of development at
the progenitor level, through morphogen release from signaling
center and migrating CR regulating the expression of cell intrinsic transcription factors, and ne-tuned at postnatal stages by
TCA projections. Peripheral stimuli and niches are involved in the
proper formation of the neuronal circuitry and cortical elds which
aspects might be emphasized during evolution. This might provide
important cues to elucidate the interplay between niches and brain
evolution.

4. Evolution of cortical elds

4.1. Comparative anatomy of cortical areas
The human neocortex is different compared to that of other
mammalian neocortices not only its relative size but also its structure and organization. The most pronounced total neocortical
expansion is observed in anthropoid primates including humans
whereas an unequal expansion of cortical areas is already detected

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Y. Arai, A. Pierani / Neuroscience Research 86 (2014) 6676

Fig. 3. A schematic representation of cortical area evolution. The neocortex from different mammalian species, like mouse and human, maintains basic organization of primary
M1 (green), S1 (red), A1 (orange) and V1 (blue) areas (see more details in Krubitzer and Seelke, 2012), however, their allocation and relative size are variable. In humans,
the size and number of anatomically distinct but functionally associated higher-order cortices are increased (indicated in white). Areas under strong genetic inuences are
marked by purple dots in human neocortex (see details in Chen et al., 2011). Changes in neocortical organization are initiated at the cellular level during development and,
end up in sophisticated adult behavior. There are multiple steps possibly involved in the acquisition of functionally distinct areas in humans. Corresponding Brodmanns
areas are indicated in brackets.

in hominids. To better understand these changes, a categorical

analysis of anatomical neocortical structures that are conserved
or which diverged in mammalians, primates and hominids is
necessary, as well as an understanding of their potentially divergent activities. Comparative anatomical studies reported a degree
of similarity in the organization of primary cortical areas in all
mammalian species examined (Krubitzer and Dooley, 2013). Furthermore, in the human fetal cerebral cortex, the expression of
transcription factors involved in cortical patterning showed similar
gradients compared to that in mice, strongly suggesting that fundamental anatomical features together with the basic molecular cues
involved in cortical patterning are conserved across mammalian
species (Bayatti et al., 2008). The main differences among mammals appear to reside in the relative size and location of the primary
cortical areas and in the number of association areas (Krubitzer
and Seelke, 2012) (Fig. 3). Moreover, the relative size of some cortical areas was found to be variable within the same species, and

even within the same individual, across its lifetime (Larsen and
Krubitzer, 2008). Thus, the origin of this anatomical variability is
multiple and relies on both the genetic background and the environment (Larsen and Krubitzer, 2008). In support of this notion, a
recent study using magnetic resonance imaging (MRI) of human
twins provided an estimation of the importance of genetic versus
environmental inuences on cortical patterning (Chen et al., 2011).
The strongest genetic inuence was observed around the frontal
(anterior end of the frontal lobe) and temporal poles (anterior end of
the temporal lobe), S1 and V1, suggesting that the remaining areas
developed under environmental inuences (Fig. 3). Particularly,
it was emphasized that language-related Brocas and perisylvian
areas showed the highest divergence between twins (Chen et al.,
2011). Therefore, intraspecies studies strongly suggest that some
cortical areas are indeed inuenced by the environment. This areal
plasticity might also reect the evolutionary-acquired plasticity of
these specic territories and it is interesting to speculate whether

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the human specic ability for language, considered to be the prominent neocortical function which evolved very recently, may also be
the most susceptible to this plasticity.
4.2. Genomic and transcriptomic changes
A high quality genomic sequence of a ca. 50,000-year-old Neanderthal woman was completed recently and showed changes of
protein-coding DNA sequences compared to great apes, Denisovans and the present-day human, that may be related to language
and higher cognitive functions in hominids during evolution (Prfer
et al., 2014). This study permitted to estimate the time when
modern humans split from both Neanderthals and Denisovans
(approximately 600,000 years ago) and Neanderthals from Denisovans (approximately 400,000 years ago) (Prfer et al., 2014).
Comparison of modern humans to Neanderthals, Denisovans and
great apes, revealed 87 genes which showed non-synonymous
mutations (protein coding changes), of which 90% were expressed
in the developing cortex. Among these, 40% showed restricted
spatiotemporal pattern of expression. Some of these genes were
expressed in the developing cortex at mid-fetal stages, with a
frontotemporal gradient suggesting that they might be involved
in patterning of cortical areas dedicated to language and cognitive functions. Some other genes were highly enriched in the
VZ suggesting stem cell maintenance functions (Prfer et al.,
2014). Similarly, specic frontotemporal expansion related to the
orbitofrontal cortex (devoted to decision-making) was suggested in
modern humans compared with Neanderthals using morphological
analysis of skull endocasts from fossils (Pearce et al., 2013). Thus,
the comparative neuroanatomy using paleontological evidence
such as endocasts and hominid fossils together with molecular
genetics will shed light on the when and how unique human
features may have emerged and might bring about some of the
molecular clues to human evolution.
The genetic modications which occurred during cortical evolution emphasized not only the change of genomic but also of
transcriptomic information. Recently, the easy access to genomewide analysis techniques has allowed the revealing of differences in
gene expression in areas that specically expanded or were added
in the human lineage. Lists of specic candidate genes likely to
be involved in the acquisition of novel areas, for instance regions
related to language acquisition (Abrahams et al., 2007) or cognitive
function (Johnson et al., 2009; Kang et al., 2011), are now available. A comprehensive analysis of these datasets will bring many
interesting clues to cortical area evolution.
In addition to gene expression level changes, alternative isoforms can now be studied from exon-array platforms (Johnson et al.,
2009) increasing the pool of possible splicing and/or transcriptional regulations involved in cortical area evolution (Johnson et al.,
2009; Kang et al., 2011). For instance, an axon guidance molecule
expressed both in the temporal lobe and the prefrontal cortex presented with a different enrichment of its isoforms in these two
territories (Johnson et al., 2009), eventually highlighting its possible
role in region-specic axonal trajectories. More insightful to human
evolution, genes expressed in the neocortex in a region-specic
manner were twice as likely to be associated with cis-regulatory
elements that appeared to have undergone human-specic accelerated substitutions (Prabhakar et al., 2006). Such species-specic
activity of cis-regulatory elements seems to be one of the molecular mechanisms for spatiotemporally controlling gene expression
during evolution.
4.3. CR neurons
Neocortical evolution also relies on an increase in neuronal
types. As mentioned previously, a neuronal population of interest

73

are CR neurons whose number and intensity of Reelin expression

has increased during cortical evolution (Meyer and Gofnet, 1998;
Aboitiz et al., 2005). Marginal zone/layer 1, which CR neurons populate, is expanded in size and diversity in primates including humans
(Zecevic and Rakic, 2001; Meyer, 2010), suggesting the number
of CR neurons have increased in humans. The contribution of CR
neurons to human evolution was emphasized by the characterization of a novel RNA gene (HAR1F), rapidly evolving in humans,
that happens to be expressed by CR neurons at early stages in
the future neocortex (Pollard et al., 2006). CR neurons express the
secreted protein Reelin, and two types of Reelin producing cells
have been characterized in humans. First layer1 is populated by
Reelin-positive large neurons, and later by smaller ones in the subpial granular layer cells (SGL) (Meyer and Gofnet, 1998). It has
been proposed that SGL are transiently present underneath the pial
surface in the human fetal telencephalon, and are almost absent or
scarce in other mammals (Meyer and Gofnet, 1998; Meyer et al.,
1998), thus suggesting a complexication of layer 1 formation in
primates. In addition to these morphologically distinct neurons in
layer 1, a comparative study of distinct LIM transcription factor
expression at the cortical hem in different species (Abelln et al.,
2010) suggested a potential increase of subtypes of CR neurons
in the primate layer 1 (Abellan et al., 2010) and a contribution of
LIM transcription factors family in this process. Note that layer 1
include not only CR neurons but also types of neurons including
GABergic interneurons (Zecevic and Rakic, 2001). Therefore, more
comparative gene expression analyses will be informative to distinguish different types of neurons in layer 1 including CR neurons,
and it still remains to know how many molecularly distinct CR subtypes exist in primates and whether they populate different cortical
areas.
In all species examined so far (turtles, crocodiles, lizard, rodent
and primates including humans, see a review by Molnr et al.,
2006), CR neurons express Reelin, and can also be divided into
subpopulations by the expression of p73, a family member of the
tumor suppressor p53 (Yang and McKeon, 2000), which marks septum and cortical hem-derived CR neurons (Meyer et al., 2002).
Reelin-positive and p73-negative CR-like neurons are prominent
in the lizard cortex, while the population of double positive Reelin
and p73 is increased in mammals (Cabrera-Socorro et al., 2007). In
parallel with the p73-positive subpopulation increase, an expansion of the cortical hem progenitor domain size was apparent in
humans (Meyer and Gofnet, 1998). These observations led to
the hypothesis that increased cortical hem-derived CR neurons
are required to cover the enlarged surface of the human neocortex (Meyer, 2010). Furthermore, a comparison of mouse and
chick telencephalon showed the presence of a novel site of Dbx1expression at the mouse VP/PSB progenitor domain, from which CR
neurons originate (Bielle et al., 2005). This absence of Dbx1 expression at the VP/PSB can also be correlated to the presence of fewer CR
neurons in chick than in the mouse (Bar et al., 2000; Nomura et al.,
2008). Together with the capacity of the Dbx1 gene to induce the
production of reelin positive cells when overexpressed ectopically
(Nomura et al., 2008), it supports the idea that novel specialized
progenitor domains for CR genesis may have been acquired during
evolution.
Another mechanism that may regulate the number of CR neurons is to switch the competence of progenitor cells devoted to the
production of cortical plate neurons into CR neuron producing progenitor cells. The analysis of Fgf8 gain-of-function indicates that the
ectopic Fgf8 signaling promoted the generation of CR neurons from
cortical progenitor cells (Zimmer et al., 2010) and studies using
Foxg1 mutant mice showed a massive increase of CR neurons, indicating that Foxg1 is a key gene controlling reversible progenitor cell
competence (Muzio and Mallamaci, 2005; Hanashima et al., 2007;
Kumamoto et al., 2013). In a similar line, the lack of Lhx2 expression

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Y. Arai, A. Pierani / Neuroscience Research 86 (2014) 6676

led also to an increase in CR neuron numbers by expanding CR

generation sites (Bulchand et al., 2001; Roy et al., 2014).
Although suggested by all these studies, the relevance of the
increased generation of CR neurons to neocortical evolution is still
to be addressed. The study of the molecular control of CR generation throughout evolution should shed light on whether and how
a qualitative and/or quantitative expansion of CR neurons plays a
role in the evolution of cortical areas.
5. Conclusions and perspectives
The work of many laboratories points to the importance of regulating cortical patterning during neocortical development as the
foundation to neocortical evolution. Cortical patterning is tightly
associated with the temporal and spatial regulation of neurogenesis, which support neuronal diversity. Increasing genomic and
transcriptome analyses provide comprehensive lists of genes that
reveal their susceptibility to evolutionary changes. Whether these
genes evolved rst or as a consequence of environmental inuences
affecting their expressions and in turns were xed through epigenetic mechanisms, are questions that still remain unanswered.
Multi-disciplinary approaches combined with genetic manipulations, including classical mutant analysis, behavioral analysis,
electrophysiological data, mathematical modeling, bioinformatics
and inference from paleogenetics will be powerful to answer these
evolutionary questions.
In the cortical arealization eld, there are still many questions
which remain to be answered from the developmental and specifically evolutionary point of views. The development of imaging
techniques of the neocortex allows us to better dene cortical
elds and functions of specic areas, and thus to more precisely
understand brain network activities. Together with the increasing
precision in genomic and transcriptomic information, important
genes can be pinned down and evolutionary questions can be
addressed. Whether cortical areas develop and evolve individually or in an orchestrated manner, it is still an open question.
Whether or not these mechanisms are reversible, and whether
there is some window of plasticity sustaining area formation are
examples of many questions which remain to be fully investigated
at the molecular level. The anatomical changes of cortical elds are
linked to their neuronal connectivity. This last degree of complexity
also raises relevant questions concerning species-specic behaviors and intraspecies-specic differences, particularly in humans,
in the context of the susceptibility to mental diseases, which are
often associated with evolutionary highlighted genes.
Acknowledgements
The authors apologize for not being able to cite the work of
many contributors to the eld. We thank Drs Eva-Maria Geigl,
Melissa Barber and Veronique Dubreuil for critical reading of
the manuscript. Drs Miguel Turrero Garcia, YoonJeung Chang and
Jeremy Pulvers, and Misses Betty Freret-Hodara and Iffat Sumia for
valuable discussions. Y.A was the recipient of fellowships from the
Association pour la Recherche sur le Cancer (ARC), Fondation pour
la Recherche Medicale (FRM) and Japan Society for the Promotion of
Science Invitation. A.P. is a CNRS (Centre National de la Recherche
Scientique) Investigator and was supported by grants from the
Agence Nationale de la Recherche (ANR-07-NEURO-046-01), FRM
(DEQ20130326521) and ARC (SFI20111203674).
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