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Role of radial glial cells in cerebral cortex folding§

Vı́ctor Borrell1 and Magdalena Götz2,3

Radial glial cells play key roles during cerebral cortex and even monotremes (echidna). Such widespread dis-
development, as primary stem and progenitor cells giving play of gyrencephaly has been proposed to reflect a
rise — directly or indirectly — to neurons and glia, but also common evolutionary origin across mammals [2,3], with
acting as scaffold for the cerebral cortex architecture and lissencephaly (‘smooth brain’) emerging secondarily
migrating neurons. Recent work led to the discovery of novel [2,4]. Recent evidence supports the placental mammal
types of radial glial cells with key roles in gyrification, the folding ancestor to have been gyrencephalic [5]. Despite its
of the mammalian cerebral cortex in phylogeny and ontogeny. importance, the cellular and molecular mechanisms of
Here we summarize the cellular and molecular basis of this gyrencephaly have just begun to emerge.
fascinating process allowing the expansion of the mammalian
cerebral cortex with all its functional consequences. The Radial Unit hypothesis proposed that evolutionary
Addresses increases in cortical size require increases in the number
1
Instituto de Neurociencias, Consejo Superior de Investigaciones of cortical radial units, hence on the number of founder
Cientı́ficas & Universidad Miguel Hernández, 03550 Sant Joan d’Alacant, progenitor cells populating the Ventricular Zone (VZ) at
Spain the onset of neurogenesis [6]. Although this hypothesis is
2
Institute of Stem Cell Research, German Research Center for
Environmental Health, Helmholtz Center Munich, 85764 Neuherberg,
consistent with the dramatic differences in number of
Germany founder progenitor cells in the cortical primordium of
3
Physiological Genomics, Ludwig Maximilians University, 80336 human versus mouse embryos, experimental testing
Munich, Germany demonstrated that their forced expansion in transgenic
mice eventually caused folding of the VZ, which never
Corresponding authors: Borrell, Vı́ctor ([email protected]) and Götz,
Magdalena ([email protected])
occurs in naturally folded brains [7–11]. Hence there was
more to gyrencephaly than overproliferation of radial glia
cells (RGCs) in the VZ (hereon referred to as apical
Current Opinio‘n in Neurobiology 2014, 27:39–46 RGCs). In addition to VZ, a secondary germinal layer
This review comes from a themed issue on Development and forms during cortical development basally from the VZ,
regeneration 2014 termed Subventricular Zone (SVZ). Time-lapse video
Edited by Oscar O Marin and Frank F Bradke microscopy analyses demonstrated that the SVZ is popu-
lated during neurogenesis by neurogenic Intermediate
Progenitor Cells (IPCs), also referred to as basal progeni-
tors [12], and subsequent analyses showed that these are
0959-4388/$ – see front matter, # 2014 The Authors. Published by in fact a major source of cortical neurons [13,14]. IPCs
Elsevier Ltd. All rights reserved. were found to be remarkably more abundant in large
http://dx.doi.org/10.1016/j.conb.2014.02.007 primates than in rodents [15,16], so the Intermediate
Progenitor hypothesis proposed that the abundance of
IPCs determines cortical neuron number (thus cortical
Introduction size and folding), and the heterogeneous abundance of
Brain size changes dramatically across mammals, and this IPCs across the embryonic cortex establishes the location
is mostly due to a disproportionate increase in size of the of prospective folds and fissures [17]. Yet again, functional
cerebral cortex [1]. Because the cerebral cortex is a testing of this hypothesis demonstrated that significant
laminar sheet of tissue, increased cortical size comes increases in IPCs in mice would increase cortical neuron
together with the formation of folds and fissures, which numbers, and even cortical surface area, but without
allow fitting a very large cortical sheet in a relatively small causing cortical folds [18,19]. A further refinement of
cranial volume. Although cortical folding is commonly this idea emerged as the Epithelial-Progenitor hypoth-
assumed to be a distinctive primate trait, gyrencephaly esis, proposing that the expansion of the cerebral cortex in
(‘folded brain’) occurs in all major families of mammals primates depends on the maintenance of epithelial fea-
across phylogeny, including rodents (i.e. capybara), car- tures in neural progenitors, critically including non-apical
nivores, ungulates, cetaceans, marsupials (gray kangaroo) progenitors, which in mouse are typically multipolar

§
This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use,
distribution and reproduction in any medium, provided the original author and source are credited.

www.sciencedirect.com Current Opinion in Neurobiology 2014, 27:39–46

40 Development and regeneration 2014

Figure 1

(a) (b)

Migrating
neuron

OSVZ

SVZ ISVZ

VZ VZ aRG bRG
Current Opinion in Neurobiology

Divergence of radial fibers and cerebral cortex expansion. (a) Lissencephalic species have few bRGCs (green), and the radial migration of neurons
(blue) mostly follows the parallel trajectories of radial fibers from aRG (red). (b) Gyrencephalic species have many bRGCs, which increases the number
of radial fibers starting at basal positions, hence causing the divergence of the entire scaffold. This creates new routes for the radial migration of
neurons, favoring their lateral dispersion (dashed pink lines) and the expansion of the cortical surface area.

non-epithelial IPCs [20]. Recent investigations provide neurogenesis. Live imaging in slice cultures shows that
partial support for this hypothesis. mouse bRGCs divide only once to generate directly two
neurons, whereas bRGCs in human brain slices either
Cellular mechanisms self-amplify generating two bRGCs or, mostly, generate
In the developing cerebral cortex of macaque monkeys, self-amplifying intermediate progenitors (Transit Ampli-
Smart and colleagues first identified the remarkable size fying Progenitors) which eventually generate neurons
of the SVZ in primates, which specializes into Inner [22,24,25,30]. Unfortunately, human tissue is very scarce
(ISVZ) and Outer (OSVZ) zones [15]. This expanded and post-mortem clinical material has room for interpret-
SVZ is present beyond primates in developing gyrence- ation. To overcome these limitations, Betizeau and col-
phalic cerebral cortices and contains a recently identified leagues have recently performed an unprecedented
type of RG cell with a long radial process contacting the extensive time-lapse analysis of progenitor cell dynamics
basal (pial) but not the apical (ventricular) surface: basal in brain slices of experimental macaque monkey
radial glial cell (bRGC; also known as oRG) [3,21,22]. The embryos, imaging over 1000 cells undergoing nearly
Tangential Divergence hypothesis proposed that the 500 divisions [29]. This comprehensive analysis demon-
abundant proliferation of bRGCs in the OSVZ of gyr- strates that bRG dynamics is much more complex and
encephalic brains adds a significant number of radial plastic than previously characterized. bRGCs in OSVZ
fibers to the preexisting scaffold in a basally expanding come in four different flavors (depending on the presence
fan-like manner (Figure 1), thus imposing divergence to of basal or apical processes, or both), they all have both
the radial migration of cortical neurons thereby generat- self-renewing and neurogenic potential, and exhibit a
ing the tangential expansion of the cortical surface and remarkable inter-class plasticity (Figure 2). The same
folding [23]. A prediction of this hypothesis is that the diversity of bRG morphologies had already been
relative abundance of bRGCs determines the degree of described previously in ferret and sheep, demonstrating
cortical folding. Accordingly, the number of bRGCs is that these specialized variations of bRGCs are common to
residual in lissencephalic cerebral cortices, as in mice and gyrencephalic species, and not primate-unique [2,28].
small primates, while species with highly folded cortices Importantly, the abundance of transit-amplifying inter-
have very large numbers of bRGCs [3,4,23–27,28,29]. mediate progenitors and their contribution to primate
cortical neurogenesis appear to be very minor in com-
In addition to modifying the radial fiber scaffold, a see- parison to bRG, contradicting previous interpretations
mingly major role for bRGCs is to contribute to cortical and predictions [21,22,31,32]. The largest neuronal

Current Opinion in Neurobiology 2014, 27:39–46 www.sciencedirect.com

 Radial glia dynamics in cortex expansion Borrell and Götz 41

Figure 2

Previous model Current model

N
N N
IPC

aRG bRG IPC aRG bRG

l

r
l

th
ns
sa

ica

ola
bo
tra
ba

ap

bip

Current Opinion in Neurobiology

Models of corticogenesis. The prevailing model (left) acknowledged only the existence of aRG, bRG and multipolar IPCs, and proposed that most
cortical neurons (N) are generated by IPCs, derived by self-amplification, aRGCs or bRGCs [23,32]. The current model (right) distinguishes 5 different
classes of bRGCs depending on the nature of their radial processes [28,29]. IPCs and every type of bRGCs self-renew and can generate virtually
any other type of bRGCs or IPCs (far right). In this model, cortical neurons are generated by all basal progenitors, but most abundantly by bipolar
RGCs.

output in primate OSVZ comes from bipolar bRGCs across brain regions during evolution for the expansive
(bpRGCs) containing both apical and basal processes. generation of neurons during a brief period of time.
This novel type of bpRGC was first discovered in the
murine ganglionic eminence (GE), where it divides at Molecular mechanisms
abventricular positions and generates a particularly large The fascinating question emerging from these exciting
neuronal output supplying the entire telencephalon with new insights is which molecular mechanisms govern
interneurons [28]. In the developing cerebral cortex, the these processes, such as the generation of OSVZ
abundance of bpRGCs is largest in brains with a high RGCs.
gyrification index [28].
Delamination of aRGCs
Betizeau and colleagues also distinguish two develop- One major difference between VZ and OSVZ RGCs is
mental phases in cortical progenitor dynamics: an early the epithelial nature, including adherens junctions (AJs)
phase with slow cell cycles and little OSVZ progenitor that anchor the former but not the later to the apical,
amplification, in favor of neurogenesis, and a later phase ventricular surface. Hence, molecules regulating apical
with significant amplification of progenitors, when short anchoring and cell delamination may contribute to dis-
cell cycles lead to the rapid generation of large lineage tinguish between these progenitor cell fates. Accordingly,
trees over a brief period of time. A rapid amplification of loss of AJ proteins (like cadherins), their links to the
bRG would be responsible for expanding the primate cytoplasm (like a-catenin and b-catenin), or regulators of
OSVZ to its outstanding size, and to prepare it for an the F-actin belt linking AJs (like RhoA), all result in
eventual tsunami of upper-layer neuron production partial or complete loss of apical anchoring followed by
[16,33,52]. Interestingly speeding up the cell cycle has delamination of the progenitor cells [12,20,35]. Accord-
also been described in the murine GE [28], suggesting ingly, orienting cell division planes in an oblique or
that this strategy may have been selectively co-opted horizontal angle with respect to the ventricular surface,

www.sciencedirect.com Current Opinion in Neurobiology 2014, 27:39–46

42 Development and regeneration 2014

such that only one daughter cell remains anchored, Up-stream regulators of the decision to delaminate or not
resulted in an excess of daughter cells delaminating from are transcription factors of the snail family, which mediate
the ventricular surface [34,36]. However, this had limited epithelial–mesenchymal transition (EMT) and the dela-
effects on cell fate decisions, and failed to elicit the mination of cells from the apical surface by repressing
generation of an enlarged SVZ or cortical folding. More- cadherins [38]. Cadherin repression also comes via Robo
over, process formation is highly dynamic as described receptors, which are expressed in the VZ and positively
above for the SVZ also in the VZ and not necessarily regulate the delamination of nascent IPCs from the apical
predictive of progenitor fate [37]. surface [39]. In Robo1/2 double mutants VZ cells

Figure 3

(a) E12 Tangential growth

Fast radial expansion

Mixed growth
E14

shTrnp1

Radial growth

E16 High Trnp1

Low Trnp1

(b)
Neuron

Migrating
neuron

IPC

aRG bRG bpRG

High
High Trnp1 Low Trnp1 High Trnp1 Cdk4/CcnD1
Current Opinion in Neurobiology

Molecular regulation of cerebral cortex folding. (a) In the developing mouse cerebral cortex, early progenitors (E12) express high levels of Trnp1 (dark
red), leading to their self-renewal and tangential expansion of the neuroepithelium. At mid-stages (E14) Trnp1 expression is downregulated (pale red) in
a salt-and-pepper fashion, leading to a mixed tangential and radial expansion. Focal knock-down of Trnp1 (i.e. by shRNA) causes a rapid radial
expansion of this region (right). At late stages (E16) Trnp1 is downregulated, causing radial growth in detriment of tangential growth. (b) The
downregulation of Trnp1 in an entire region (not salt-and-pepper), either by shRNA in mouse or by the natural expression pattern in humans, increases
the delamination of aRG (red) to generate abundant bRGCs (including bpRG) and IPCs (green), which locally increase the number of neurons
generated (blue) and the divergence of radial fibers (black lines), hence expanding cortical surface area. Because this occurs only locally, a cortical fold
is formed in between regions with relatively higher Trnp1 expression and hence less radial/basal expansion. Importantly, the outer cortical surface
folds (top), but not the ventricular surface (bottom). Overexpression of Cdk4/CyclinD1 (green background) in basal progenitors causes their
overproliferation and increased cortical folding in ferrets, but not in mice.

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 Radial glia dynamics in cortex expansion Borrell and Götz 43

progress to an IPC fate but often retain apical anchoring. generate few IPCs [48]. Overexpression of Trnp1, a
However, even in cases of increased delamination, none nuclear protein acting as mitotic book-marking factor
of these models was sufficient to generate OSVZ-like tightly associated with chromatin including M-phase
RGCs. [49], led to expansion of aRGCs at the expense of IPCs,
causing a tangential growth of the cortical VZ surface
Amplification of OSVZ progenitors [49]. More strikingly its downregulation caused not only
As described above, a second key mechanism for OSVZ IPC expansion, but also massive increases in bpRG and
expansion is the continued proliferation of bRGCs and bRG abundance (otherwise residual in murine cortex),
IPCs. Indeed, overexpression of Cdk4 and CyclinD1 (both with the subsequent establishment of an OSVZ-like layer
positive regulators of cell cycle speed and re-entry) in that led to the eventual formation of fissures and folds in
OSVZ progenitors of ferret cortex caused a significant the newborn mouse cortex [49] (Figure 3). Time-lapse
increase in surface area and the formation of additional imaging of cultured slices revealed how this phenomenal
cortical folds, while preserving the normal six-layered increase in bRG number caused the fanning-out of the
organization [19] (Figure 3). Intriguingly, the same radial fiber scaffold, with the subsequent tangential dis-
manipulations in the mouse only amplified IPCs, enlar- persion of radially migrating neurons and the formation of
ging the brain but not generating any folds [19]. As gyrus-like structures, much like is seen in gyrencephalic
amplification of Tbr2+ IPCs is not sufficient a key feature species [3,49]. Unlike previous manipulations [7] fold-
of the OSVZ in gyrified brains is the extraordinary variety ing of the mouse cortex by Trnp1 knock-down did not
of RGCs, including the combinatorial expression of Pax6, involve folding of the germinal layers, which remained
Tbr2, Olig2, Ascl1 and Sox2 [2,3,21,22,29,40–43], smooth, but only of the neuronal layers, as in naturally
prompting the question of which molecular regulators folded brains [47,49] (Figure 3). These experiments
may be involved in achieving this cellular diversity. provided strong proof for the Tangential Divergence
hypothesis, including a key role for bRG abundance in
Regulation of basal progenitor heterogeneity this process.
One way to tackle this key question is the genome-wide
expression analysis of the developing cortex in humans Consistent with the mechanisms discussed above, Trnp1
[44] and non-human primates [45], and its comparison knock-down initially modified the angle of cell division
with expression in mouse SVZ [46]. This has led to the (Table 1), favoring oblique or horizontal cleavage planes.
intriguing finding that extracellular matrix (ECM) com- Concomitantly, Trnp1 knock-down elicited many
ponents are strongly enriched in OSVZ of human fetal bpRGCs to remain anchored to the apical surface while
cortex compared to its murine counterparts [46]. Impor- dividing at abventricular positions without undergoing
tantly, signaling via ECM, and also fibroblast growth interkinetic nuclear migration [28], thus allowing lin-
factors, expands and self-amplifies basal progenitor cells eage amplification, as previously predicted [20]. This
[46,47]. While these are interesting mechanisms, prolonged apical anchoring may be caused by Robo2
neither manipulations of ECM receptors nor growth and Rnd2 downregulation after Trnp1 knock-down (Table
factors has successfully generated in mice the remarkable 1) [39,50]. Also multipolar IPCs proliferate actively and
cellular heterogeneity observed in the OSVZ of species repetitively upon Trnp1 knock-down, mediated, for
with gyrated cerebral cortices. example, by reduced expression levels of NeuroD1 and
NeuroD4 (Table 1). Thus various mechanisms regulated
However, this has been achieved by manipulating Trnp1 by Trnp1 contribute to amplify the different cell types
which was identified in a screen as enriched in a sub- populating an OSVZ-like structure. Finally, live imaging
population of aRGCs that are highly self-amplifying and also revealed faster migration of neurons upon Trnp1

Table 1

Effects of Trnp1 knock-down relevant for forming folds at cellular and molecular level

Region/location Cell biological events after Trnp1 knock-down Gene expression changes after Trnp1 knock-down
(as possible mechanism for the cellular events on the left)
Apical surface More non-vertical cell divisions Reduced expression of Trio and Drg2
Many more abventricular dividing RG remaining Reduced expression of Robo2 and Rnd2
apically anchored (generating bRGCs)
Increased delamination Reduced expression of Cdh6, Cdh11 and Trio
Basal–SVZ Fast dividing basal progenitors Reduced expression of Unc5d, NeuroD1 and NeuroD6
Further basal — OSVZ More bRGCs and bpRGCs cells with increased Reduced expression of Unc5d, NeuroD1 and NeuroD6
proliferation rates
Neuronal layers Faster neuronal migration Reduced expression of Robo2 and Map2k6 in neurons

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44 Development and regeneration 2014

knock-down, implicating Trnp1 as a key regulator in by grants from Spanish Ministry of Economy and Competitivity (BFU2012-
33473, CONSOLIDER CSD2007-00023), European Research Council
RGCs and postmitotic neurons depending on its expres- (309633) and European Union Seventh Framework Programme FP7/2007-
sion levels in these cell types. Thus, Trnp1 acts as up- 2013 under the project DESIRE (602531); work in MG’s lab is supported by
the Collaborative Research Grant 870 and the Excellence Cluster for
stream ‘master’ regulator of a combination of mechanisms Systems Neurology (SYNERGY) of the German Research Council (DFG).
all contributing to expanding cell number and diversity,
as the basis for gyrification (Table 1). Most importantly,
these findings demonstrate for the first time that the References and recommended reading
genetic program required for expanding the SVZ and Papers of particular interest, published within the period of review,
have been highlighted as:
generating massive numbers of bRGCs is already present
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and that the temporal and spatial regulation of a single  of outstanding interest
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This study shows that OSVZ in the macaque embryo contains a diversity evidence for an outer subventricular zone in rodents. PLoS
of progenitor cell classes much richer than previously identified, namely ONE 2012, 7:e30178.

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46 Development and regeneration 2014

44. Kang HJ, Kawasawa YI, Cheng F, Zhu Y, Xu X, Li M, Sousa AM, lineage and transcriptome analysis. Mol Cell Neurosci 2008,
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