The mammalian neocortex has undergone pronounced expansion
in size and complexity during evolution and is responsible for high
cognitive function, sensory perception and consciousness
(Northcutt and Kaas, 1995). Newborn neurons migrate from the
ventricular zone (VZ) to the pial surface and settle in the cortical
plate (CP) in a sequential manner with deeper layers (DLs) formed
first, followed by upper layers (ULs) (Rakic, 1978; Super
and Uylings, 2001). The latter are developmentally and
phylogenetically the last structure to appear in the mammalian
cortex (Aboitiz et al., 2003; Molnar et al., 2006). Novel specialized
neuronal subpopulations with a high degree of complexity of
dendritic and axonal arborization have evolved in the uppermost
layers. Callosal projection neurons (CPNs), for example, represent
a heterogeneous population of UL pyramidal neurons prevalently
located in layers II and III of the neocortex (Molyneaux et al.,
2009; Ramos et al., 2008) and projecting either ipsilaterally or to
homotypic regions of the contralateral hemisphere, giving rise to
the corpus callosum (CC) (Mitchell and Macklis, 2005; Richards
et al., 2004).
It has been hypothesized that the inside-out pattern of
corticogenesis leading to the birth of the six-layered neocortex was
made possible by the appearance of glia-guided neuronal migration
(Aboitiz et al., 2003; Molnar et al., 2006). Early-born neurons give
rise to layers VI and V (phylogenetically older structures),
essentially by somal translocation (Miyata et al., 2004; Nadarajah
et al., 2002), whereas late-born neurons follow a complex pattern
of migration. During early phases, late-born neurons adopt a
multipolar morphology and migrate through the subventricular
zone (SVZ) independently of the radial glia scaffold (LoTurco and
Bai, 2006; Tabata and Nakajima, 2003). In the upper intermediate
zone (IZ), migrating neurons assume a bipolar shape, attach to
radial glia and move to the CP by glia-guided locomotion (LoTurco
and Bai, 2006; Tabata and Nakajima, 2003). Finally, during late
phases of the migratory process, bipolar neurons contact the pial
surface detaching from the glia scaffold and reach their target by
somal translocation, thus allowing glia-guided upcoming neurons
to reach upper positions (Nadarajah et al., 2001).
In the last 20 years, many molecules have been reported to be
required for different steps of this process. For example, Lis1
(Pafah1b1 – Mouse Genome Informatics), Dcx, FlnA (LoTurco and
Bai, 2006), Cdk5 (Ohshima et al., 2007) and RhoA (Ge et al., 2006;
Hand et al., 2005) control multipolar-shaped cell (MSC) migration
and the transition from MSCs to bipolar-shaped cells (BSCs) in the
IZ. Other molecules, like Mdga1 (Takeuchi and O’Leary, 2006), N-
cofilin (Bellenchi et al., 2007), 31 and 51 integrins (Dulabon
et al., 2000; Marchetti et al., 2010) and Dcx are implicated in the
maintenance of bipolar cell polarity and interaction of migrating
neurons with the radial glia. Finally, the secreted molecule Reelin
and its downstream effector Dab1 guide the detachment of
migrating neurons from the radial glia, allowing the final somal
translocation step beneath the pial surface (Dulabon et al., 2000;
Olson et al., 2006; Sanada et al., 2004). Remarkably, disruption of
many of these molecules not only leads to impairment in radial
migration and lamination, but also causes a striking alteration in
dendritic and axonal morphology of UL neurons. However, the
correlation between cell shape transition from MSC to BSC and the
final morphology and connectivity of UL neurons is still elusive.
Development 138, 4685-4697 (2011) doi:10.1242/dev.068031
© 2011. Published by The Company of Biologists Ltd
1Telethon Institute of Genetics and Medicine (TIGEM), Developmental Disorders
Program, 80131 Naples, Italy. 2INSERM, U636, Nice, F-06108, France. 3University of
Nice Sophia-Antipolis, U636, F-06108, France. 4Department of Molecular
Neurobiology, National Institute of Medical Research, London NW7 1AA, UK.
*Present address: The Australian Regenerative Medicine Institute, c/o Monash
University, Clayton VIC 3800, Australia
†Author for correspondence (email@example.com)
Accepted 16 August 2011
During corticogenesis, late-born callosal projection neurons (CPNs) acquire their laminar position through glia-guided radial
migration and then undergo final differentiation. However, the mechanisms controlling radial migration and final morphology of
CPNs are poorly defined. Here, we show that in COUP-TFI mutant mice CPNs are correctly specified, but are delayed in reaching
the cortical plate and have morphological defects during migration. Interestingly, we observed that the rate of neuronal
migration to the cortical plate normally follows a low-rostral to high-caudal gradient, similar to that described for COUP-TFI. This
gradient is strongly impaired in COUP-TFI–/–brains. Moreover, the expression of the Rho-GTPase Rnd2, a modulator of radial
migration, is complementary to both these gradients and strongly increases in the absence of COUP-TFI function. We show that
COUP-TFI directly represses Rnd2 expression at the post-mitotic level along the rostrocaudal axis of the neocortex. Restoring
correct Rnd2 levels in COUP-TFI–/–brains cell-autonomously rescues neuron radial migration and morphological transitions. We
also observed impairments in axonal elongation and dendritic arborization of COUP-TFI-deficient CPNs, which were rescued by
lowering Rnd2 expression levels. Thus, our data demonstrate that COUP-TFI modulates late-born neuron migration and favours
proper differentiation of CPNs by finely regulating Rnd2 expression levels.
KEY WORDS: Radial migration, Cerebral cortex, Callosal projection neurons, Dendrite morphology, COUP-TFI, Rnd2, Mouse
COUP-TFI promotes radial migration and proper morphology
of callosal projection neurons by repressing Rnd2 expression
Christian Alfano1,2,3, Luigi Viola1, Julian Ik-Tsen Heng4,*, Marinella Pirozzi1, Michael Clarkson2,3,
Gemma Flore1, Antonia De Maio1, Andreas Schedl2,3, François Guillemot4and Michèle Studer1,2,3,†
Although the number of molecules found to be involved in the
regulation of glia-guided radial migration has increased remarkably
in recent years, very little is known about their transcriptional
regulation. The proneural gene Ngn2 (Neurog2 – Mouse Genome
Informatics) was the first factor discovered to activate directly the
expression of one of these factors, the small Rho-GTPase Rnd2,
which is involved in the regulation of MSC-to-BSC transition and
BSC migration during mid-late stages of corticogenesis (Heng et al.,
2008; Nakamura et al., 2006). Although Rnd2 transcription is
activated by Ngn2 primarily in MSCs along the SVZ and the lower
IZ (Heng et al., 2008; Nakamura et al., 2006), its expression was also
shown to be enhanced by NeuroD2, a transcription factor that is
strongly expressed in the IZ and CP during mid-late stages of
corticogenesis (Heng et al., 2008). However, Rnd2 expression is
sharply downregulated in the upper IZ, where BSCs attach to the glia
and migrate to the CP, suggesting that an independent pathway might
negatively regulate Rnd2 expression in this compartment and that
such a step might be relevant for correct BSC migration to the CP.
In this study, we found that in the absence of COUP-TFI (Nr2f1
– Mouse Genome Informatics), a transcription factor crucial for
corticogenesis and arealization (Armentano et al., 2006; Armentano
et al., 2007; Tomassy et al., 2010), Rnd2 expression is abnormally
high in the SVZ and IZ during glia-guided radial migration. As a
consequence, a greater number of MSCs are stalled in the IZ and
BSC migration to the CP is impaired. Although normally specified,
CPNs show defects in their postnatal dendritic morphology and in
the elongation and arborization of their axons. Unexpectedly,
lowering Rnd2 levels in COUP-TFI-deficient neurons rescues
migratory defects of COUP-TFI–/–CPNs, which remarkably
recover their morphology in a cell-autonomous manner. In
conclusion, we demonstrate that Rnd2 expression levels are
precisely fine-tuned by COUP-TFI and are essential for CPN radial
migration and maturation.
MATERIALS AND METHODS
COUP-TFI null (COUP-TFI–/–) and COUP-TFI fl/fl mice were generated
and genotyped as previously shown (Armentano et al., 2006; Armentano
et al., 2007). COUP-TFI fl/fl mice were either crossed to Emx1-Cre for
inactivating COUP-TFI in progenitors (COUP-TFI CKO-Emx1)
(Armentano et al., 2007) or to Nex-Cre for COUP-TFI inactivation in post-
mitotic cells (Goebbels et al., 2006). COUP-TFI–/–were crossed to the
Ngn2KIGFPmouse line (Seibt et al., 2003) to generate double mutants,
COUP-TFI/Ngn2 dKO. Midday of the day of the vaginal plug was
embryonic day (E) 0.5. All experiments were conducted following
guidelines of the Institutional Animal Care and Use Committee, Cardarelli
Hospital, Naples, Italy.
Timed pregnant females received a single intraperitoneal injection of 5-
bromo-2?-deoxyuridine (BrdU; 50 mg/kg) at 14.5 days post-coitum (dpc)
and three wild-type (wt) and three COUP-TFI–/–embryos were collected
at E16.5. Five regions (three sections per region) were arbitrarily chosen
along the rostrocaudal axis and BrdU+ cells were counted with the NIH
ImageJ software in a 250 m-wide portion of the IZ and the CP
compartments of the lateral cortex. The number of CP BrdU+ cells was
expressed as a percentage of total CP/IZ BrdU+ cells.
Immunofluorescence, immunohistochemistry and in situ
Vibratome and cryostat sections were processed for immunocytochemistry
and/or in situ hybridization as previously described (Armentano et al.,
2007). The following primary antibodies were used: rabbit anti-GFP
antibody (1:1000, Chemicon); goat anti-Cux1 (1:50, Santa Cruz); mouse
anti-BrdU (1:300, Sigma); rabbit anti-activated caspase 3 (1:100, Cell
Signaling Technology); mouse anti-human COUP-TFI (1:100, Abcam);
rabbit anti-COUP-TFI (1:500) (Tripodi et al., 2004); rat anti-CTIP2 (1:500,
Abcam); mouse anti-Nestin (1:100, Chemicon); rabbit anti-Pax6 (1:100,
Chemicon); mouse anti-Satb2 (1:20, Abcam); rabbit anti-Tbr1 (1:1000, gift
from R. Hevner, University of Washington, Seattle, USA) and rabbit anti-
Cre (1:100, Covance). The following secondary antibodies were used:
Alexa Fluor 488-conjugated anti-rabbit; Alexa Fluor 594-conjugated anti-
rabbit; Alexa Fluor 594-conjugated anti-mouse; Alexa Fluor 488-
conjugated anti-mouse (1:400, Molecular Probes). Antisense RNA Probes
were labelled using a DIG-RNA Labelling Kit (Roche).
In utero and ex vivo electroporation and organotypic slice cultures
The pCIG2-IRES-GFP, pCIG2-Cre-IRES-GFP, Rnd2-specific (SH1, SH2)
and scrambled (SCR) shRNA-IRES-GFP (Heng et al., 2008) plasmids were
injected into E14.5 embryonic vesicles with a PV820 Pneumatic PicoPump
(WPI). The electroporations were performed on whole heads using a
Tweezertrode electrode (diameter 7 mm; BTX) connected to a CUY 21
EDIT electroporator (NEPA GENE) with the following parameters: five 50
V pulses, P(on) 50 mseconds, P(off) 1 second. Brains were then embedded
in 3% agarose and cut using a vibratome (Leica VT1000S) at 300 m
thickness. Slices were cultured for 4 days in Neurobasal Medium (GIBCO)
plus N12 and B27 supplements (GIBCO), as previously described (Tripodi
et al., 2004). In utero electroporations were performed as previously
described (Nguyen et al., 2006) with the following parameters: four 40 V
pulses, P(on) 50 mseconds, P(off) 1 second. Analysis of GFP+ cell
distribution was performed on the lateral cortex along the rostrocaudal axis.
The cortex was subdivided into VZ/SVZ, IZ and CP compartments, and
the IZ and CP were further subdivided in lower, medial and upper
subcompartments (Heng et al., 2008; Nguyen et al., 2006). Cell counts in
the different subcompartments were performed on at least three 10 m
thick planes scanned at different depths of each single slide. The number
of GFP+ cells for each compartment was expressed as a percentage of the
total number of GFP+ cells. The mean value of GFP+ cells counted in each
compartment of the three planes was used to calculate the average value
among the same neocortical compartments of rostrocaudal slices from at
least three littermates for each genotype.
Real-time PCR analysis
The neocortex of three wt and three COUP-TFI–/–brains was dissected and
immediately transferred to 500 l of Trizol (Invitrogen) and processed for
total RNA isolation according to the manufacturer’s protocol. RNA (1 g)
was reverse transcribed using a QuantiTect Reverse Transcription Kit
(Qiagen). Real-time PCR (QPCR) analysis was performed for all the
littermates in triplicate on a 7900HT Fast Real Time PCR System (Applied
Biosystems). The PCR reaction was performed using Power SYBR Green
PCR Master Mix (Applied Biosystems) according to the manufacturer’s
protocol. All the assays were normalized with respect to Gapdh values.
Fold-change variations in the levels of mRNA of interest were expressed
as a percentage and normalized against wt levels (set as 100%). For
primers see Table S2 in the supplementary material.
Chromatin immunoprecipitation (ChIP)
For each experiment (n3), neocortices were dissected from 24 mice at E14.5
and diced in ice cold Hanks Buffered Saline Solution (GIBCO). Nuclei were
processed and ChIP was performed as described in Kuo and Allis, 1999 (Kuo
and Allis, 1999). Antisera directed against COUP-TFI (15 l) or 3 g Dicer
(Santa Cruz sc30226 as Rabbit IgG control) were used for
immunoprecipitation. Putative COUP-TFI binding sites were tested by
QPCR analysis of ChIP samples using the LightCycler 480 Real-Time PCR
System (Roche). Reactions were performed in triplicate on three
independently prepared ChIP samples. Amplification was expressed as fold
enrichment compared with non-specific IgG control and relative quantities
of immunoprecipitated DNA were calculated as previously described (Heng
et al., 2008). For primers see Table S2 in the supplementary material.
All the data were statistically analyzed and graphically represented using
Microsoft Office Excel software. The error bars represent s.e.m. Two-tailed
Student’s t-test was used for the analysis of statistical significance (*P≤0.05,
Development 138 (21)
**P≤0.01, ***P≤0.005). One-way analysis of variance (ANOVA) was used
to compare the means among three or more groups of samples and post-hoc
analysis was performed by two-tailed Student’s t-test.
Images were acquired with an Upright microscope Leica DM5000B
equipped with Leica IM image management software (Leica
Microsystems, Wetzlar, Germany). A Zeiss LSM 710 Laser Scanning
Microscope (Carl Zeiss MicroImaging, Jena, Germany) and a
Fluorescence Inverted Confocal Microscope LEICA SP2 AOBS (Leica
Microsystems, Heidelberg, Germany) were used for image acquisition
of ex vivo and in utero electroporated brain slices. A total of 23 z-line
scans with a step distance of 0.45 m were collected at different depths
along the z-axis of each lateral neocortex (one to four different regions
along the z-axis were scanned depending on the thickness of the
electroporated area) and maximum intensity projections were generated
either using Zeiss ZEN Confocal Software (Carl Zeiss MicroImaging)
or using Leica Confocal Software (Leica Microsystems, Wetzlar,
Cell transfection, protein extraction and western blotting
The mouse embryocarcinoma cell line P19 was transfected at 60%
confluence with pCIG2-[Flag-Rnd2]-IRES-GFP and shRNA-IRES-GFP
vectors (Heng et al., 2008) using Lipofectamine 2000 Transfection
Reagent (Invitrogen) according to the manufacturer’s protocol. Total
protein extraction and western blotting were performed as previously
described (Armentano et al., 2006). The following primary antibodies
were used: mouse monoclonal anti-Flag (1:1000, Sigma), mouse anti-3
tubulin (1:1000, Sigma); and appropriate secondary antibodies: goat
anti-mouse IgG (H+L) horseradish peroxidase conjugate (1:5000,
Bio-Rad). The signal was detected with ECL reagent (Amersham
Morphological analysis of the corpus callosum
Brains of three wt and five COUP-TFI CKO-Emx1 P60 mice were
vibratome-sectioned coronally at a thickness of 50 m and processed for
Nissl staining, as previously described (Tomassy et al., 2010). Pictures
were taken on a LEICA MZ 16 FA Stereomicroscope and converted from
RGB to BW by Adobe Photoshop software. Section images were identified
with serial numbers and analyzed in both hemispheres, spanning the entire
tract. Using a graphic pen, all the different callosal components were
labelled with different colours, and images were aligned and integrated by
Amira 4.1.2-1 software to obtain 3D pictures.
Newborn UL neurons are properly specified but
abnormally positioned in COUP-TFI–/–mice
To investigate the mechanisms underlying alteration of ULs in the
absence of COUP-TFI function (Armentano et al., 2007; Faedo et
al., 2008; Tomassy et al., 2010), we first assessed whether late-born
neurons were normally specified in COUP-TFI–/–mice. The
expression of Satb2, required for late specification of CPNs (Alcamo
et al., 2008; Britanova et al., 2008), as well as the expression of Cux1
and Cux2, involved in early and late differentiation of late-born
neurons (Cubelos et al., 2010; Nieto et al., 2004; Zimmer et al.,
2004), were affected in mutant ULs at P0 (Fig. 1A). The dense
COUP-TFI favours radial migration
Fig. 1. Abnormal distribution of differentiation
markers in COUP-TFI–/–mouse neocortices.
(A)Immunostaining for Satb2 and Cux1 of P0 coronal
sections reveals decreased thickness (dashed lines and
brackets) of upper layers (UL) in COUP-TFI mutant
(COUP-TFI–/–) brains. Cux2 expression is strongly
impaired in mutant ULs whereas it remains high in the
white matter (WM; arrowheads). Many neurons fail to
express Satb2 in upper regions of the cortex
(arrowheads in square panels), whereas scattered Cux1-
and Cux2-positive cells are abnormally positioned in
deep layers (DL) of mutant brains (lower panels and
arrowheads). Lower panels are magnifications of the
boxed areas in the respective upper panels. (B)Satb2
expression is increased in the SVZ/IZ of COUP-TFI–/–
E16.5 medial coronal sections (arrow), whereas its
expression is decreased in the mutant CP (asterisk).
Right-hand panels are magnifications of the boxed
areas in the respective left-hand panels. (C)Tbr1
expression increases in the IZ of COUP-TFI–/–E16.5
coronal sections (arrowheads). (D)Cux1 and Cux2
expression is undetectable in the CP of COUP-TFI–/–
E16.5 coronal sections (asterisks). Scale bars: 200m
for A-D except C, square panel (100m). uSVZ, upper
Satb2- and Cux1-positive upper cell layer was reduced in thickness
and scattered Cux1- and Cux2-positive cells were observed along the
radial extent of the mutant cortex. At E16.5, when late-born neurons
are predominantly migrating, increased expression of Satb2 and
Tbr1, a marker of post-mitotic projection neurons (Hevner et al.,
2001) was observed in the upper SVZ and IZ of mutant brains (Fig.
1B,C). No double positive cells for Satb2 and Tbr2 (Eomes – Mouse
Genome Informatics), an intermediate progenitor marker (Englund
et al., 2005; Kowalczyk et al., 2009; Sessa et al., 2008) were found
in COUP-TFI–/–brains, similarly to control brains (see Fig. S1A in
the supplementary material), excluding precocious expression of
Satb2 in early post-mitotic neurons. In addition, Satb2 expression
was decreased in the CP and Cux1 and Cux2 showed weak or absent
expression in the CP (asterisks in Fig. 1D), suggesting overall
abnormal cell migration rather than abnormal specification of late-
born neurons in the absence of COUP-TFI function.
Loss of COUP-TFI affects neuronal radial migration
in presumptive somatosensory cortex
To investigate further whether absence of COUP-TFI has an effect
on radial cell migration during mid-late corticogenesis, we injected
BrdU into pregnant females at 14.5 dpc and evaluated the
percentage of BrdU+ cells in the CP of E16.5 wt and COUP-TFI–/–
brains along their rostrocaudal axis (Fig. 2A,B; see Table S1 in the
supplementary material). The number of BrdU+ cells in the CP was
expressed as a percentage of the BrdU+ cells in the IZ/CP regions
(Fig. 2B) to avoid any interference derived from increased
proliferation of mutant progenitor cells in the VZ/SVZ (Faedo et
al., 2008). Interestingly, we observed a significantly lower
percentage of cells reaching the CP in mediolateral regions of
mutant neocortices (presumptive somatosensory area), whereas
rostral- and caudal-most regions were unaffected (Fig. 2B; see
Table S1 in the supplementary material).
Next, we assessed directly the behaviour of abnormally
migrating cells by ex vivo electroporation (Hand et al., 2005) of a
green fluorescent protein (GFP)-expressing vector in the VZ of wt
and COUP-TFI–/–E14.5 brains (Fig. 2C). Slices from rostral to
caudal regions of electroporated brains were analyzed after 4 days
of culture in vitro (DIV). We observed a striking reduction of
neurons reaching the CP principally from rostro- to caudomedial
regions of mutant cortices (Fig. 2C-E; see Table S1 in the
supplementary material). This defective migration was neither due
to impairment of the glial scaffolding nor to abnormal specification
of migrating neurons, because almost all GFP-positive cells failing
to leave the IZ were Satb2-positive (UL) and Ctip2 (Bcl11b –
Mouse Genome Informatics)-negative (deep layers) (see Fig. S2A-
D in the supplementary material).
Notably, cell counting revealed a gradual increase in the percentage
of GFP+ cells reaching the CP from rostral to caudal regions of wt
cortices, with a maximum rate of migration in the caudomedial cortex
(Fig. 2F). This migration gradient was lost in COUP-TFI–/–brains,
although no differences were found between rostral- and caudal-most
neocortical regions of wt and COUP-TFI–/–brains (Fig. 2F; see Table
S1 in the supplementary material). To exclude any interference from
the neurogenic gradient, we limited our statistical analysis of GFP+
cell distribution to the IZ and CP as well as to the lower, middle and
upper regions within the CP (Fig. 2E; see Fig. S3A,B in the
supplementary material). This analysis confirmed defective cell
transition from the IZ to the CP in rostro- to caudomedial regions and
showed a significant decrease in cells reaching the upper CP also in
caudal-most slices. Analysis of cell migration within the CP also
revealed impaired transition from lower to upper CP in rostromedial
to caudal regions of COUP-TFI–/–cortices (see Fig. S3B in the
supplementary material). Together, these data indicate that COUP-
TFI regulates cell migration from IZ to CP in presumptive
somatosensory and occipital cortex during mid-late corticogenesis.
Development 138 (21)
Fig. 2. Radial cell migration is
impaired in COUP-TFI–/–mouse
brains. (A)Immunostaining for BrdU of
E16.5 medial coronal sections indicates
decreased BrdU-positive cells in the CP of
showing the percentage of BrdU-positive
cells migrating from the IZ to the CP
from rostral to caudal regions of wt and
mutant cortices (n3). (C)Ex vivo
electroporation of a GFP-expressing
vector in E14.5 wt and COUP-TFI–/–
neocortical VZ and organotypic slices
from three distinct regions along the
rostrocaudal axis after 4 days in culture
(DIV). In diagrams to the left, black areas
indicate the regions shown.
(D)Histograms describing GFP-positive
cells radial distribution in wt and COUP-
TFI–/–brains (n3). (E)Quantification of
IZ/CP GFP-positive cells reaching the CP.
(F)Line graph showing the percentage of
GFP+ cells in the CP along the
rostrocaudal axis of wt and COUP-TFI–/–
brains (n3). *P<0.05, **P<0.01,
***P<0.005. Scale bars: A (top left
panel), 400m; A (right panel) and C,
Rnd2 rostrocaudal gradient is strongly altered in
We found that the small Rho-GTPase Rnd2, which we reported to
be altered in COUP-TFI–/–brains (Armentano et al., 2006), is
expressed in a high-rostral to low-caudal gradient, complementary
to that of COUP-TFI (Fig. 3A) and to the migratory gradient (Fig.
2). Because Rnd2 normally modulates cell migration (Heng et al.,
2008), we hypothesized that COUP-TFI represses Rnd2 expression
along the rostrocaudal axis. Indeed, we found that at E13.5, COUP-
TFI inactivation abolished the Rnd2 gradient and increased Rnd2
expression in caudomedial cortical regions (Fig. 3B). Double
labelling of E13.5 medial coronal sections with Tbr2 and Rnd2
confirmed higher levels of Rnd2 expression in the IZ, and more
specifically in the CP, despite a normal distribution of basal
progenitors (Fig. 3C).
At E15.5, when the majority of UL neurons are generated and start
their migration to the CP, high levels of Rnd2 expression were
maintained in COUP-TFI–/–SVZ and IZ (Fig. 3D,E). Abnormal
expression levels of Svet1, a marker of SVZ progenitors and
multipolar-shaped cells (MSCs) (Sasaki et al., 2008; Tarabykin et al.,
2001), and accumulation of Satb2+ cells were also observed in the
SVZ and IZ of mutant brain adjacent sections, confirming that UL
neurons, and more particularly CPNs, were properly specified but
stalled as MSCs in the upper SVZ and IZ (Fig. 3E). Finally, Rnd2
expression levels were still slightly higher in the deep cortex of
COUP-TFI–/–brains at P0 (Fig. 3F). Taken together, our data indicate
that the Rnd2 gradient is abolished in COUP-TFI–/–brains and that
Rnd2 expression is increased in abnormally migrating cells.
COUP-TFI negatively regulates Rnd2 expression
through an Ngn2-independent pathway
In light of the role for Ngn2 in promoting Rnd2 expression (Heng
et al., 2008), we hypothesized that COUP-TFI and Ngn2 either
regulate each other or act through independent pathways to fine-
tune Rnd2 expression. To distinguish between these two
possibilities, we assessed Rnd2 transcript levels relative to Ngn2
and other factors shown to promote Rnd2 expression (Heng et al.,
2008). Despite the remarkable increase in Rnd2 expression in
E15.5 COUP-TFI–/–neocortices (57.9±7.8%, n3, P0.002), we
found no significant changes in Ngn2, NeuroD2 and Tbr2 levels,
and a mild decrease in NeuroD1 expression (12.1±3.1%, n3,
P0.02) (Fig. 4A; see Table S1 in the supplementary material).
This suggests independent control of Rnd2 by COUP-TFI. To
confirm this hypothesis further, we generated double COUP-TFI
Ngn2 compound mutants (dKO). Interestingly, we found that in
dKO brains Rnd2 levels were increased in comparison with Ngn2–/–
brains (Fig. 4B), indicating that repression of Rnd2 expression by
COUP-TFI occurs independently of Ngn2. This is confirmed
further by the abnormally high levels of Rnd2 in COUP-
TFIfl/flNex-Crebrains (Fig. 2C), in which COUP-TFI is specifically
inactivated in early post-mitotic cells (see Fig. S4 in the
Finally, by using MatInspector (Quandt et al., 1995) (http://
ECR browser (Loots and Ovcharenko, 2007) (http://
ecrbase.dcode.org) and ChromAnalyzer (Montemayor et al., 2010),
we identified five putative COUP-TFI binding sites at the Rnd2
locus with variable evolutionary conservation in mammals (Fig.
4D). Two binding sites were very close and mapped in the Rnd2
last intron (sites A, A1), whereas the other three mapped in the
downstream genomic region of the Rnd2 locus (sites B, C and D).
Strikingly, site B mapped between the two previously identified
E-boxes enhancing Rnd2 expression in the developing cortex
(Heng et al., 2008). To assess whether COUP-TFI binds to these
sites in vivo, we performed chromatin immunoprecipitation (ChIP)
on E14.5 telencephalic tissue with a COUP-TFI antibody (Tripodi
et al., 2004). QPCR analysis of immunoprecipitated material
showed enrichment of all five sites, although to different extents
COUP-TFI favours radial migration
Fig. 3. Increased Rnd2 expression levels in
COUP-TFI–/–mouse brains. (A)Dorsal and
ventral views of E13.5 brains stained for COUP-TFI
(black arrows) and Rnd2 (white arrows). The
dashed line demarcates COUP-TFI-positive
territories. (B)In COUP-TFI–/–brains, expression of
Rnd2 in caudal-most regions is increased (arrows
in lateral views and arrowheads in ventral views).
(C)Double labelling for Tbr2 protein and Rnd2 of
E13.5 medial coronal sections indicates increased
Rnd2 expression in mutant SVZ and IZ, and
ectopic expression in the CP (black arrows) and
lateroventral cortex (open arrowheads).
(D)Coronal sections shows increased Rnd2
expression in dorsal and ventral E15.5 COUP-TFI–/–
neocortex (black arrows). (E)Rnd2 and Svet1
transcript enhancement and Satb2+ cell
accumulation in the mutant IZ of adjacent E15.5
coronal sections (asterisks and high magnification
views). (F)Rnd2 expression in wt and mutant P0
coronal sections. UL, upper layers; DL, deep
layers. Scale bars: A,B, 1 mm; C (top left panel),
200m; C (bottom left panel), 100m; D,
800m; E,F, 200m.
(Fig. 4D; see Table S1 in the supplementary material), suggesting
that COUP-TFI binds to these sequences with different affinities.
Together, our data strongly suggest a direct repression of Rnd2 by
COUP-TFI in post-mitotic late-born cortical neurons.
Loss of COUP-TFI function alters morphology of
Next, we carefully assessed the morphology of migrating GFP+ cells
in wt and COUP-TFI–/–medial brain slices at the sites where Rnd2
expression is highly increased (Fig. 5A) and observed a significant
increase of MSCs in mutant IZ (Fig. 5B; wt: 39.4±2.1%, n3; KO:
59.1±1.8%, n4; P<0.005), although their radial distribution along
the neocortex was unchanged compared with wt brains (Fig. 5C; see
Table S1 in the supplementary material). Accordingly, the BSC
percentage decreased from 59.3±1.7% in wt to 38.8±2.1% in COUP-
TFI–/–brains (P<0.005) (Fig. 5B), whereas no alterations were
observed in the percentage of round-shaped cells (RSCs, another
morphology adopted by migrating neurons) (see Table S1 in the
supplementary material). We observed an abnormal BCS distribution
along the radial extent of the cortex. Normally, the highest
percentage of BSCs is found in the upper IZ and the CP; however,
mutant BSCs were mainly located in the IZ (Fig. 5C), indicating that
although they adopt a bipolar morphology, COUP-TFI–/–neurons fail
to reach the CP. Thus, our data indicate that COUP-TFI is required
for both the morphological transition from multipolar to bipolar
shape and the intrinsic migratory property of BSCs within the IZ and
CP during UL neuron radial migration.
Rnd2 downregulation rescues the distribution and
morphological defects of COUP-TFI-deficient
To test whether abnormally high levels of Rnd2 expression
causes the migratory and morphological defects observed in
COUP-TFI–/–brains, we reduced Rnd2 expression levels by
Development 138 (21)
Fig. 4. COUP-TFI represses Rnd2 expression post-mitotically in an Ngn2-independent manner. (A)Histogram showing no significant
changes in Ngn2, Tbr2, NeuroD1 (ND1) and NeuroD2 (ND2) expression in COUP-TFI–/–mouse cortices despite the strong increase in Rnd2
expression. (B)Rnd2 expression decreases in Ngn2–/–neocortex and archicortex (open arrowheads) and is rescued to normal levels in COUP-
TFI–/–;Ngn2–/–(dKO) neocortex. Ectopic expression of Rnd2 in lateroventral neocortex of COUP-TFI and double KO brains (black arrows). Right-hand
panels show high magnification views of boxed areas in the left-hand panels. (C)In COUP-TFIfl/flNexCreE15.5 coronal sections, in which COUP-TFI is
inactivated post-mitotically, Rnd2 expression is strongly enhanced (bracket in lower panels) and ectopically detected in the CP and ventral cortex
(arrows). Lower panels are magnifications of boxed areas in upper panels. (D)Snapshot from ECR browser showing the conservation of COUP-TFI
binding sites (A, A1, B, C, D) along the Rnd2 locus. Immunoprecipitation of chromatin (ChIP) from E14.5 wt embryos indicates that COUP-TFI binds
these sites with different affinities (n3). E1 and E2 are previously identified Rnd2 enhancers. mFabp7 and mCycA sites are positive and negative
controls, respectively. All data are expressed as ‘relative’ fold enrichment and normalized against ChIP performed on the same samples with an anti-
Dicer antibody. *P<0.05, **P<0.01. Scale bars: 400m.
electroporation of two Rnd2-specific shRNA–IRES-GFP
expressing vectors (noted SH1 and SH2 below) (Fig. 6; see Fig.
S5A in the supplementary material) (Heng et al., 2008). As a
control we used a scrambled shRNA (SCR). In P19 cells, a strong
silencing effect on Rnd2 protein expression was observed with
both sh-RNAs, although they exhibited different efficacies (see
Fig. S5A in the supplementary material). Electroporation of SH2
in mutant cortices resulted in a remarkable rescue of the number
of GFP+ mutant cells reaching the CP (Fig. 6A,B; NULL-SCR:
4.1±0.2%, n3; NULL-SH2: 19.8±1.6%; n4; P<0.001) and,
within the CP, in the upper cortical region (NULL-SCR:
2.3±1.2%; NULL-SH2: 14.0±0.9%; P0.003) (Fig. 6A,B). SH1
electroporation instead resulted in a mild decrease in the number
of mutant GFP+ cells in the VZ/SVZ compared with mutant
SCR-expressing cells. However, mutant SH1+ cells accumulated
in the IZ and failed to reach the CP (Fig. 6A,B; see Table S1 in
the supplementary material). SH2 was, thus, more effective in
rescuing mutant cell transition from VZ/SVZ to CP (Fig. 6B; see
Table S1 in the supplementary material), possibly because Rnd2
levels were approximately restored to normal. Conversely, SH1
reduced Rnd2 expression, both in wt and mutant cells, below a
critical threshold leading to poor migratory rescue (see Fig. S5A-
C and Table S1 in the supplementary material).
Next, we assessed whether rescued GFP+ cells re-acquired their
correct shape during migration. The number of MSCs significantly
decreased in SH2-electroporated COUP-TFI–/–cortices, whereas
there was a significant and concomitant increase of BSCs (Fig. 6C;
NULL-SCR, MSC: 66.5±1.8%, BSC: 31.4±1.2%, n3; NULL-SH2,
MSC: 54.3±1.7%, BSC: 43.8±1.7%, n4; P<0.05). Furthermore,
mutant BSC radial distribution was, remarkably, rescued by SH2
electroporation, whereas no changes were observed in the
localization of SH2+ mutant MSCs (Fig. 6D; see Fig. S5D and
Table S1 in the supplementary material). Our findings strongly
support a role for COUP-TFI, via the regulation of Rnd2, in
promoting transition from MSC to BSC and in controlling correct
distribution of BSCs in the IZ and CP during migration of late-born
Callosal axonal extension and dendritic
morphology of CPNs are dependent on proper
Rnd2 expression levels
Next, to assess whether migratory impairment would affect CPN
maturation, we inactivated COUP-TFI in single migrating neurons
via in utero electroporation of a Cre-IRES-GFP-expressing vector
(referred to as CRE) in E14.5 COUP-TFIflox/flox(floxed) mice. To
rescue any eventual morphological defect in mutant neurons we co-
electroporated SCR- or SH2 together with CRE. Analysis of Cre
expression in electroporated brains at E18.5 confirmed that COUP-
TFI was inactivated in single Cre-positive cells of floxed mice and
that CRE electroporation did not cause cell death (see Fig. S6A-C
in the supplementary material). Thus, GFP-expressing cells could
be followed as individual migrating cells extending their axons in
a wild-type environment (Fig. 7).
Acute inactivation of COUP-TFI resulted in a significant
decrease of GFP+ neurons reaching the upper CP at E18.5 in floxed
brains (Fig. 7A,B; wtCRE/SCR: 54.8±2.3%; fl/flCRE/SCR: 35.4±0.8%;
n4; P<0.005). The migratory defect of mutant cells was partially
rescued by co-electroporation of SH2 and CRE in floxed brains
(fl/flCRE/SCR: 35.4±0.8%; fl/flCRE/SH2: 43.7% ± 2.4%; n4, P<0.05)
(Fig. 7A,B; see Table S1 in the supplementary material), indicating
that COUP-TFI acts cell-autonomously through Rnd2 to control
migratory properties. Moreover, CRE/SCR-electroporated floxed
brains showed a strong impairment in the elongation of callosal
axons, which failed to reach the midline as a compact fibre tract
(Fig. 7C; n4). Remarkably, co-electroporation of CRE and SH2 in
floxed brains rescued the midline crossing by GFP+ callosal axons
(Fig. 7C), strongly indicating an important role for Rnd2 in axonal
To assess whether mutant callosal axon elongation was
permanently arrested or just delayed, we repeated the above
experiment and collected brains at P8. We observed that both wt
and mutant GFP+ callosal projections were able to reach the
contralateral cortex (Fig. 7D). As it is not possible to quantify the
percentage of crossing fibres in electroporated brains owing to the
variability in the number of cells incorporating GFP, we analyzed
the extension and volume of the callosal commissure in COUP-
TFIflox/floxEmx1Creadult brains (Armentano et al., 2007).
Interestingly, we found that the rostrocaudal extension and total
volume of the callosal commissure were reduced to 70.7±3.8% and
63.0±7.1%, respectively, in COUP-TFI conditional adult brains
(see Fig. S7 in the supplementary material), suggesting that most
callosal axons are just delayed in crossing the midline after
COUP-TFI favours radial migration
Fig. 5. Abnormal morphology and distribution of migrating cells
in COUP-TFI–/–mouse brains. (A)Ex vivo electroporation of a GFP-
expressing vector in the VZ of E14.5 neocortices. After 4 days of
organotypic culture (4 DIV) multipolar-shaped cells (MSCs, yellow
arrowheads) accumulate in the mutant IZ. (Ai-iv) High magnification
views of boxed areas in A. Dashed line indicates boundary of CP and
IZ. (B)The ratio of MSCs is increased at the expense of bipolar shaped
cells (BSCs, open arrowheads in A) in COUP-TFI–/–neocortices (n3).
No changes were found in the percentage of round shaped cells
(RSCs). (C)Distribution of MSCs and BSCs along the radial extent of
the neocortex indicates that the majority of mutant BSCs are stalled in
the IZ and lower CP (lCP), whereas mutant MSCs accumulate
principally in the IZ (n3). uCP, upper CP; mCP, middle CP; uIZ, upper
IZ; mIZ, middle IZ; lIZ, lower IZ. *P<0.05, **P<0.01, ***P<0.005. Scale
inactivation of COUP-TFI. Whereas callosal axons normally
arborize in layers II/III of contralateral somatosensory regions (S1
and S2) at P8 (Wang et al., 2007) (Fig. 7Di,ii,vii), mutant callosal
axons showed poor arborization in these regions (Fig. 7Diii,iv,viii).
Strikingly, in SH2-electroporated floxed brains, GFP+ axon
innervation of contralateral parietal areas was partially rescued,
particularly in S2 (Fig. 7Dv,vi,ix).
In wt control brains, Satb2-positive callosal neurons had a
branched apical dendrite and complex apical tufts in layer I at P8
(Fig. 8A). Although the cell-autonomous inactivation of COUP-
TFI strongly affected the morphology of these tufts, which barely
reached the marginal zone (MZ) (Fig. 8A,B; see Fig. S8A,B and
Movies 1 and 2 in the supplementary material), the floxed
CRE/SH2-electroporated neurons restored proper contact with the
pial surface and apical tuft complexity in layer I (Fig. 8A,B; see
Fig. S8A,B and Movie 3 in the supplementary material). However,
either mutant or SH2-rescued mutant neurons abnormally
aggregated in small clusters in deep positions, which were never
observed for wt neurons (Fig. 8A; see Fig. S8B,C in the
supplementary material). Finally, dendrite branching, strongly
altered in SCR-expressing mutant neurons, was also rescued in
SH2-electroporated mutant neurons (Fig. 8B; see Fig. S8A in the
supplementary material), although the dendrite length was
increased in COUP-TFI-deficient neurons (wtCRE/SCR: 193.9±12.1
m; fl/flCRE/SCR: 287.3±31.2 m; n3; P<0.05) but not restored in
CRE/SH2-electroporated brains (wtCRE/SCR: 193.9±12.1 m;
fl/flCRE/SH2: 285.9±5.4 m; n3; P<0.005) (see Fig. S8D in the
supplementary material). Taken together, our analyses demonstrate
a cell-autonomous role for COUP-TFI in migration, targeting, and
axonal and dendritic arborization of CPNs and indicate that fine-
tuning of Rnd2 levels by COUP-TFI during radial migration
favours CPN maturation.
COUP-TFI and Ngn2 independently promote radial
migration by fine-tuning Rnd2 levels
Our data contribute to the delineation of a genetic circuitry
underlying Rnd2 expression during glia-guided radial migration
and confirms that the fine control of its expression during
different phases of this process is crucial for correct radial
distribution of UL newborn neurons. We demonstrate that the
sharp decrease of Rnd2 in the upper IZ favours glia-guided BSC
migration to the CP. We demonstrate that COUP-TFI represses
Rnd2 expression levels in the IZ independently of its main
activator (Ngn2) in post-mitotic migrating neurons, indicating a
direct role of COUP-TFI during early phases of glia-guided radial
migration. Moreover, we show that COUP-TFI binds directly to
various Rnd2 downstream sequences, probably recruiting
chromatin remodelling factors or other co-repressors
(Montemayor et al., 2010; Nishihara et al., 2004; Zhang et al.,
2009). Strikingly, one of the COUP-TFI binding sites is
positioned between two Ngn2-bound sequences in a
transcriptional enhancer previously identified in the downstream
region of the Rnd2 locus (Heng et al., 2008). COUP-TFI might
thus prevent or destabilize the binding of Ngn2 and other basic
helix-loop-helix (bHLH) factors to the Rnd2 enhancer in the IZ.
Development 138 (21)
Fig. 6. Lowering Rnd2 expression levels in COUP-TFI–/–mice
rescues impaired neuronal radial migration. (A)Wt and
COUP-TFI–/–brains were electroporated with a control (SCR; n3)
and two different Rnd2-specific shRNA-IRES-GFP expressing
vectors (SH1 and SH2; n4). SH1 does not appreciably recover
neuronal migration with respect to mutant brains electroporated
with SCR (asterisks in CP). SH2 rescues radial migration.
(B)Histogram of the experiments described in A. (C)Lowering of
Rnd2 levels partially rescues the defective multipolar (MSC) to
bipolar (BSC) shape transition of mutant cells (wt, null SCR n3;
null SH2 n4). (D)Radial distribution of mutant BSCs is rescued
by SH2 (yellow) with respect to the SCR+ mutant neurons,
whereas no relevant changes are observed in the distribution of
SH2+ MSC (wt, null SCR n3; null SH2 n4). See Table S1 in the
supplementary material for statistical analysis of data. uCP, upper
CP; mCP, middle CP; lCP, lower CP; uIZ, upper IZ; mIZ, middle IZ;
lIZ, lower IZ. *P<0.05, **P<0.01, ***P<0.005. Scale bars: A,
This finding correlates with a previous study showing that Ngn2
overexpression does not alter radial distribution of migrating cells
in this compartment (Hand et al., 2005). Overall, our data
demonstrate that Ngn2 and COUP-TFI are independently
involved in the fine-tuning of Rnd2 expression and indicates that
COUP-TFI normally promotes glia-guided radial migration by
progressively repressing Rnd2 expression along medial and
caudal regions of the neocortex during mid- to late corticogenesis.
COUP-TFI favours radial migration
Fig. 7. COUP-TFI-deficient CPNs show defective axonal elongation and arborization, which is cell-autonomously rescued by lowering
Rnd2 expression. (A)Acute deletion of COUP-TFI in single migrating neurons by in utero co-electroporation of a Cre-expressing vector with either
a control or Rnd2-specific shRNA-expressing vectors (SCR and SH2, respectively) in the lateral cortex of E14.5 wt and COUP-TFIflox/flox mouse
brains. SCR+ mutant neurons show altered migration at E18.5 (arrows). Affected migration is partially rescued by CRE/SH2 co-electroporation.
(B)Quantification of the experiments described in A (n4). Dashed lines indicate boundaries between subcompartments. (C)CRE/SCR expressing
floxed CPNs, in contrast to CRE/SCR+ wt neurons, fail to project their axons towards the midline (arrows and asterisk) (n3). Callosal axons from
SH2-rescued mutant CPNs elongate properly and cross the midline (arrows) (n3). (D)P8 wt and COUP-TFIflox/flox coronal slices from brains co-
electroporated at E14.5 either with CRE/SCR or with CRE/SH2, as described in A. Arrows indicate crossing of GFP-positive callosal axons. (Di-vi)
Details of cortical regions corresponding to somatosensory areas 1 and 2 (S1, S2) of electroporated brains elaborated by Transparent plug-in (Zeiss
ZEN Confocal Software). (Di-ii) 3D images show almost complete innervation of wt contralateral cortex by callosal axons. (Diii-iv) GFP+ mutant
axons poorly innervate contralateral S1 and S2 regions. (Dv-vi) A remarkable rescue of mutant axon arborization in S2 and a partial recovery of their
innervation in S1 (limited to deeper regions) of the contralateral cortex is obtained after CRE/SH2 co-electroporation. (Dvii-ix) Quantification of GFP
intensity in lateroventral cortex (S2 region) from wt and COUP-TFI floxed brains obtained by Profile plug-in (Zeiss ZEN Confocal Software) confirms
poor innervation in S2 of CRE/SCR-electroporated floxed axons, which is recovered after CRE/SH2 co-electroporation. *P<0.05, ***P<0.005. Scale
bars: A, 200m; C, 300m; D, 900m.
A graded rate of migration along the rostrocaudal
axis might regulate important processes of
In this report, we show that the expression of the atypical Rho-
GTPase Rnd2 follows a high-rostral to low-caudal gradient, which
is complementary to the one described for COUP-TFI (Armentano
et al., 2007; Liu et al., 2000; Tomassy et al., 2010; Zhou et al.,
2001). This gradient is also complementary to the UL neurons
migratory gradient detailed in this study. Thus, high expression
levels of Rnd2 in rostral cortex might restrict migrating cells from
reaching the CP more than in caudal regions. In the absence of
COUP-TFI function, the Rnd2 gradient is abolished and higher
Rnd2 expression levels are detected caudally. Accordingly, a lower
percentage of cells reach the cortical plate. Thus, our data, together
with previous reports, demonstrate that Rnd2 expression is
downregulated by increasing levels of COUP-TFI along the
rostrocaudal axis, leading to differential rates of migration from
rostral to caudal regions of the cortex.
The biological meaning of this migratory gradient is still unclear
and deserves further investigation. However, other factors involved
in cortical arealization, besides COUP-TFI, seem to modulate
neuron radial migration (O’Leary et al., 2007). Furthermore,
neurons in ULs (and particularly in layer IV) represent a source of
signals for thalamocortical innervation (Shimogori and Grove,
2005), an important process refining cortical arealization. It is thus
tempting to hypothesize that COUP-TFI might, at least in part,
control the topography of thalamocortical innervation by
modulating the timing of neuronal migration to layer IV along the
rostrocaudal axis of the neocortex.
Accurate Rnd2 expression levels promote proper
dendritic morphology of callosal pyramidal
Here, we also provide evidence that COUP-TFI is involved in
the development of correct dendritic morphology of UL neurons.
The precise morphology of these neurons could be controlled
during early phases of pyramidal neuron radial migration. A
previous report suggested that initial apical dendrite formation
occurs during early stages of glia-guided radial migration and
that RhoA activity inhibits this process by promoting MSC
morphology (Hand et al., 2005). As Rnd2 modulates RhoA
activity via pragmin interaction (Pacary et al., 2011; Tanaka et
al., 2006), higher Rnd2 expression levels observed in COUP-TFI
mutants might alter RhoA activity and thus disrupt the formation
of the initial dendrite, ultimately leading to an increased number
of MSCs. Our data suggest that altered initial dendritic formation
might also affect late dendritic arborization of pyramidal
neurons. However, although lowering Rnd2 levels in mutant
neurons restores late dendritic complexity, it only partially
rescues the morphological transition from MSC to BSC,
indicating that most rescued cells were defective BSCs stalled in
the IZ and lower CP regions. We cannot exclude that our shRNA
strategy failed to re-establish precise levels of Rnd2; however,
our results are consistent with previous data showing that
artificially enhanced Rnd2 levels only lead to a slight increase in
MSC (Heng et al., 2008).
Another possibility is that the development of an abnormal
apical dendrite with no or few apical tufts, as observed in COUP-
TFI-deficient neurons, might be due to the failure of their leading
process to reach the MZ and activate reelin-mediated dendritic
branching (Hunter-Schaedle, 1997; Niu et al., 2004; Ohshima et al.,
2007; Sanada et al., 2004). It has been shown previously that reelin
in the MZ stabilizes the leading process by cofilin phosphorylation
(Chai et al., 2009). Lack of proper stabilization of the leading
process might not only affect final phases of radial migration but
also dendritic maturation. Thus, the remarkable rescue of dendritic
complexity observed in SH2-rescued COUP-TFI-deficient CPNs
might be due to restored contact of the MZ by their leading
Development 138 (21)
Fig. 8. Impaired dendritic morphology of COUP-TFI-deficient
pyramidal neurons in mouse is rescued by lowering Rnd2 levels.
(A)The apical tuft morphology of P8 COUP-TFIflox/flox callosal neurons
co-electroporated in utero at E14.5 with Cre- and control shRNA-IRES-
GFP-expressing vectors (CRE and SCR) is strongly impaired (arrows). This
defect was remarkably rescued by co-electroporation of CRE and Rnd2-
specific shRNA-IRES-GFP-expressing vector 2 (SH2). Lower panels show
high magnified details of upper panels showing that GFP+ pyramidal
neurons correctly expressed Satb2 in all three conditions (arrows).
Numbers 1-5 indicate arbitrary division of UL into five bins (see Fig. S8C
in the supplementary material). (B)CRE/SCR-expressing wt pyramidal
neurons at P8 have a branched apical dendrite (white arrowheads in Bi)
with a complex shaped apical tuft (white arrows). COUP-TFI-deficient
neurons show a thin and almost unbranched apical dendrite with no or
simple apical tufts (Bii). Co-electroporation of SH2 with CRE in COUP-
TFI-floxed neurons restores their defective morphology (Biii). *P<0.05,
**P<0.001. Scale bars: A (top left panel), 200m; A (bottom left panel)
50m; B, 100m.
processes. Nevertheless, although rescued COUP-TFI-deficient
dendrites successfully target the MZ, both mutant and SH2-rescued
mutant cells remain beneath the upper CP with abnormally long
apical dendrites and form abnormal cell clusters in the ULs. We
suggest that these processes might be independent from Rnd2 as it
was already reported that the expression of two microtubule-
associated proteins, MAP1B (Mtap1b – Mouse Genome
Informatics) and MAP2 (Map2 – Mouse Genome Informatics),
strongly decreases in COUP-TFI-deficient dendrites (Armentano
et al., 2006), whereas cell adhesion increases in the absence of
COUP-TFI function (Adam et al., 2000). Furthermore, Rnd2
expression in the CP seems to be very low during late stages of
Thus, correct positioning of UL neurons does not seem to be a
prerequisite for the acquisition of proper dendritic morphology.
However, correct timing in reaching the CP, and more particularly
the MZ, might be crucial for late-born migrating neurons to acquire
a fully mature morphology.
COUP-TFI promotes BSC radial migration and
callosal axon elongation by repressing Rnd2
Beside dendritic abnormalities, COUP-TFI-deficient callosal
pyramidal neurons show delayed axonal elongation and impaired
contralateral innervation. Although previous studies suggested a
link between neuron radial migration and CC formation, a direct
relationship between proper cell migration and callosal neuron
differentiation has not been fully established (Donahoo and
Richards, 2009; Richards et al., 2004). Inactivation of various
molecules involved in radial migration, such as Dcx and Dclk,
leads to affected migration and defective commissural outgrowth
(Deuel et al., 2006; Koizumi et al., 2006); however, their
expression is maintained at high levels in the CP during late
events of differentiation. In this study, we show that altered
callosal neuron migratory properties in COUP-TFI null mice
correlate with a strong delay in callosal axon midline crossing at
perinatal stages as well as reduced arborization at P8. In
accordance with previous observations suggesting that axon
elongation starts during BSC migration (Hatanaka and
Murakami, 2002; Noctor et al., 2004; Norris and Kalil, 1991),
we observed altered distribution of mutant BSCs along the IZ
and CP and delayed callosal axon elongation, which were both
restored by lowering Rnd2 levels in mutant neurons.
Accordingly, a previous study demonstrated that the interaction
of Rnd2 with plexin D1 inhibits neurite outgrowth in cortical
neurons (Uesugi et al., 2009). Finally, because Rnd2 modulates
actin dynamics through interaction with the WASP proteins
(Kakimoto et al., 2004), it is highly possible that increased Rnd2
levels might alter BSC neurite outgrowth, a process that is at the
base of both glia-guided migration and CC formation.
Finally, in light of its expression gradient, we propose that Rnd2
might play an important role in CC formation by modulating the
axonal elongation rate along the rostrocaudal axis of the neocortex.
Previous studies demonstrated that all components of the CC
(rostral, medial and caudal) cross the midline at similar
developmental stages. As the midline allows callosal axon crossing
mainly in the anterior half of the medial cortex and only for a
restricted time, caudal callosal axons should cover greater distances
to reach the crossing site and their homotypic regions (Richards et
al., 2004). One can thus speculate that caudomedial and caudal
CPNs that express lower levels of Rnd2 during migration, elongate
their axons at a higher rate to reach the crossing regions at the
In conclusion, our study clearly demonstrates that COUP-TFI
repression of Rnd2 in the IZ is crucial for proper migration of late-
born BSC neurons. This suggests that progressive lowering of
Rnd2 levels along the rostrocaudal cortical axis modulates the
timing of CPN settlement as well as the rate of callosal axon
elongation in different regions of the neocortex, ultimately
favouring CC formation.
We would like to thank V. Tarabykin for the Svet1 plasmid and Satb2
polyclonal antibody; M. Nieto for the Cux1 and Cux2 plasmids; R. Hevner for
the Tbr1 antibody; G. Andolfi for genotyping; M. Giordano for animal
husbandry; D. Di Nucci and G. Esposito for surgical assistance; and S. Arbucci,
and F. A. Martín for confocal imaging. We are very thankful to R. Rispoli and
the TIGEM Bioinformatics Core; and particularly to F. A. Pereira and C.
Montemayor for the ChromAnalyzer-driven bioinformatic analysis of the Rnd2
locus; to J. Hazan for critical reading; and to members of the Studer laboratory
for fruitful discussions.
This work was supported by the Italian Telethon Foundation [TMSC24TELB to
M.S.]; the ‘Compagnia di San Paolo’, Program of Neuroscience [TMSP14CSPA
to M.S.]; and the L’Agence nationale de la recherche Chaire d’Excellence
Program [R09125AA to M.S.].
Competing interests statement
The authors declare no competing financial interests.
Supplementary material for this article is available at
Aboitiz, F., Morales, D. and Montiel, J. (2003). The evolutionary origin of the
mammalian isocortex: towards an integrated developmental and functional
approach. Behav. Brain Sci. 26, 535-552.
Adam, F., Sourisseau, T., Metivier, R., Le Page, Y., Desbois, C., Michel, D. and
Salbert, G. (2000). COUP-TFI (chicken ovalbumin upstream promoter-
transcription factor I) regulates cell migration and axogenesis in differentiating
P19 embryonal carcinoma cells. Mol. Endocrinol. 14, 1918-1933.
Alcamo, E. A., Chirivella, L., Dautzenberg, M., Dobreva, G., Farinas, I.,
Grosschedl, R. and McConnell, S. K. (2008). Satb2 regulates callosal
projection neuron identity in the developing cerebral cortex. Neuron 57, 364-
Armentano, M., Filosa, A., Andolfi, G. and Studer, M. (2006). COUP-TFI is
required for the formation of commissural projections in the forebrain by
regulating axonal growth. Development 133, 4151-4162.
Armentano, M., Chou, S. J., Srubek Tomassy, G., Leingartner, A., O’Leary, D,
D. and Studer, M. (2007). COUP-TFI regulates the balance of cortical
patterning between frontal/motor and sensory areas. Nat. Neurosci. 10, 1277-
Bellenchi, G. C., Gurniak, C. B., Perlas, E., Middei, S., Ammassari-Teule, M.
and Witke, W. (2007). N-cofilin is associated with neuronal migration disorders
and cell cycle control in the cerebral cortex. Genes Dev. 21, 2347-2357.
Britanova, O., de Juan Romero, C., Cheung, A., Kwan, K. Y., Schwark, M.,
Gyorgy, A., Vogel, T., Akopov, S., Mitkovski, M., Agoston, D. et al. (2008).
Satb2 is a postmitotic determinant for upper-layer neuron specification in the
neocortex. Neuron 57, 378-392.
Chai, X., Forster, E., Zhao, S., Bock, H. H. and Frotscher, M. (2009). Reelin
stabilizes the actin cytoskeleton of neuronal processes by inducing n-cofilin
phosphorylation at serine3. J. Neurosci. 29, 288-299.
Cubelos, B., Sebastian-Serrano, A., Beccari, L., Calcagnotto, M. E., Cisneros,
E., Kim, S., Dopazo, A., Alvarez-Dolado, M., Redondo, J. M., Bovolenta,
P. et al. (2010). Cux1 and Cux2 regulate dendritic branching, spine
morphology, and synapses of the upper layer neurons of the cortex. Neuron 66,
Deuel, T. A., Liu, J. S., Corbo, J. C., Yoo, S. Y., Rorke-Adams, L. B. and Walsh,
C. A. (2006). Genetic interactions between doublecortin and doublecortin-like
kinase in neuronal migration and axon outgrowth. Neuron 49, 41-53.
Donahoo, A. L. and Richards, L. J. (2009). Understanding the mechanisms of
callosal development through the use of transgenic mouse models. Semin.
Pediatr. Neurol. 16, 127-142.
Dulabon, L., Olson, E. C., Taglienti, M. G., Eisenhuth, S., McGrath, B., Walsh,
C. A., Kreidberg, J. A. and Anton, E. S. (2000). Reelin binds alpha3beta1
integrin and inhibits neuronal migration. Neuron 27, 33-44.
COUP-TFI favours radial migration
Englund, C., Fink, A., Lau, C., Pham, D., Daza, R. A., Bulfone, A., Kowalczyk,
T. and Hevner, R. F. (2005). Pax6, Tbr2, and Tbr1 are expressed sequentially by
radial glia, intermediate progenitor cells, and postmitotic neurons in developing
neocortex. J. Neurosci. 25, 247-251.
Faedo, A., Tomassy, G. S., Ruan, Y., Teichmann, H., Krauss, S., Pleasure, S. J.,
Tsai, S. Y., Tsai, M. J., Studer, M. and Rubenstein, J. L. (2008). COUP-TFI
coordinates cortical patterning, neurogenesis, and laminar fate and modulates
MAPK/ERK, AKT, and beta-catenin signaling. Cereb. Cortex 18, 2117-2131.
Ge, W., He, F., Kim, K. J., Blanchi, B., Coskun, V., Nguyen, L., Wu, X., Zhao, J.,
Heng, J. I., Martinowich, K. et al. (2006). Coupling of cell migration with
neurogenesis by proneural bHLH factors. Proc. Natl. Acad. Sci. USA 103, 1319-
Goebbels, S., Bormuth, I., Bode, U., Hermanson, O., Schwab, M. H. and
Nave, K. A. (2006). Genetic targeting of principal neurons in neocortex and
hippocampus of NEX-Cre mice. Genesis 44, 611-621.
Hand, R., Bortone, D., Mattar, P., Nguyen, L., Heng, J. I., Guerrier, S., Boutt,
E., Peters, E., Barnes, A. P., Parras, C. et al. (2005). Phosphorylation of
Neurogenin2 specifies the migration properties and the dendritic morphology of
pyramidal neurons in the neocortex. Neuron 48, 45-62.
Hatanaka, Y. and Murakami, F. (2002). In vitro analysis of the origin, migratory
behavior, and maturation of cortical pyramidal cells. J. Comp. Neurol. 454, 1-14.
Heng, J. I., Nguyen, L., Castro, D. S., Zimmer, C., Wildner, H., Armant, O.,
Skowronska-Krawczyk, D., Bedogni, F., Matter, J. M., Hevner, R. et al.
(2008). Neurogenin 2 controls cortical neuron migration through regulation of
Rnd2. Nature 455, 114-118.
Hevner, R. F., Shi, L., Justice, N., Hsueh, Y., Sheng, M., Smiga, S., Bulfone, A.,
Goffinet, A. M., Campagnoni, A. T. and Rubenstein, J. L. (2001). Tbr1
regulates differentiation of the preplate and layer 6. Neuron 29, 353-366.
Hunter-Schaedle, K. E. (1997). Radial glial cell development and transformation
are disturbed in reeler forebrain. J. Neurobiol. 33, 459-472.
Kakimoto, T., Katoh, H. and Negishi, M. (2004). Identification of splicing
variants of Rapostlin, a novel RND2 effector that interacts with neural Wiskott-
Aldrich syndrome protein and induces neurite branching. J. Biol. Chem. 279,
Koizumi, H., Tanaka, T. and Gleeson, J. G. (2006). Doublecortin-like kinase
functions with doublecortin to mediate fiber tract decussation and neuronal
migration. Neuron 49, 55-66.
Kowalczyk, T., Pontious, A., Englund, C., Daza, R. A., Bedogni, F., Hodge, R.,
Attardo, A., Bell, C., Huttner, W. B. and Hevner, R. F. (2009). Intermediate
neuronal progenitors (basal progenitors) produce pyramidal-projection neurons
for all layers of cerebral cortex. Cereb. Cortex 19, 2439-2450.
Kuo, M. H. and Allis, C. D. (1999). In vivo cross-linking and immunoprecipitation
for studying dynamic Protein:DNA associations in a chromatin environment.
Methods 19, 425-433.
Liu, Q., Dwyer, N. D. and O’Leary, D. D. (2000). Differential expression of COUP-
TFI, CHL1, and two novel genes in developing neocortex identified by
differential display PCR. J. Neurosci. 20, 7682-7690.
Loots, G. and Ovcharenko, I. (2007). ECRbase: database of evolutionary
conserved regions, promoters, and transcription factor binding sites in vertebrate
genomes. Bioinformatics 23, 122-124.
LoTurco, J. J. and Bai, J. (2006). The multipolar stage and disruptions in neuronal
migration. Trends Neurosci. 29, 407-413.
Marchetti, G., Escuin, S., van der Flier, A., De Arcangelis, A., Hynes, R. O.
and Georges-Labouesse, E. (2010). Integrin alpha5beta1 is necessary for
regulation of radial migration of cortical neurons during mouse brain
development. Eur. J. Neurosci. 31, 399-409.
Mitchell, B. D. and Macklis, J. D. (2005). Large-scale maintenance of dual
projections by callosal and frontal cortical projection neurons in adult mice. J.
Comp. Neurol. 482, 17-32.
Miyata, T., Kawaguchi, A., Saito, K., Kawano, M., Muto, T. and Ogawa, M.
(2004). Asymmetric production of surface-dividing and non-surface-dividing
cortical progenitor cells. Development 131, 3133-3145.
Molnar, Z., Metin, C., Stoykova, A., Tarabykin, V., Price, D. J., Francis, F.,
Meyer, G., Dehay, C. and Kennedy, H. (2006). Comparative aspects of
cerebral cortical development. Eur. J. Neurosci. 23, 921-934.
Molyneaux, B. J., Arlotta, P., Fame, R. M., MacDonald, J. L., MacQuarrie, K.
L. and Macklis, J. D. (2009). Novel subtype-specific genes identify distinct
subpopulations of callosal projection neurons. J. Neurosci. 29, 12343-12354.
Montemayor, C., Montemayor, O. A., Ridgeway, A., Lin, F., Wheeler, D. A.,
Pletcher, S. D. and Pereira, F. A. (2010). Genome-wide analysis of binding sites
and direct target genes of the orphan nuclear receptor NR2F1/COUP-TFI. PLoS
ONE 5, e8910.
Nadarajah, B., Brunstrom, J. E., Grutzendler, J., Wong, R. O. and Pearlman,
A. L. (2001). Two modes of radial migration in early development of the cerebral
cortex. Nat. Neurosci. 4, 143-150.
Nadarajah, B., Alifragis, P., Wong, R. O. and Parnavelas, J. G. (2002).
Ventricle-directed migration in the developing cerebral cortex. Nat. Neurosci. 5,
Nakamura, K., Yamashita, Y., Tamamaki, N., Katoh, H., Kaneko, T. and
Negishi, M. (2006). In vivo function of Rnd2 in the development of neocortical
pyramidal neurons. Neurosci. Res. 54, 149-153.
Nguyen, L., Besson, A., Heng, J. I., Schuurmans, C., Teboul, L., Parras, C.,
Philpott, A., Roberts, J. M. and Guillemot, F. (2006). p27kip1 independently
promotes neuronal differentiation and migration in the cerebral cortex. Genes
Dev. 20, 1511-1524.
Nieto, M., Monuki, E. S., Tang, H., Imitola, J., Haubst, N., Khoury, S. J.,
Cunningham, J., Gotz, M. and Walsh, C. A. (2004). Expression of Cux-1 and
Cux-2 in the subventricular zone and upper layers II-IV of the cerebral cortex. J.
Comp. Neurol. 479, 168-180.
Nishihara, E., O’Malley, B. W. and Xu, J. (2004). Nuclear receptor coregulators
are new players in nervous system development and function. Mol. Neurobiol.
Niu, S., Renfro, A., Quattrocchi, C. C., Sheldon, M. and D’Arcangelo, G.
(2004). Reelin promotes hippocampal dendrite development through the
VLDLR/ApoER2-Dab1 pathway. Neuron 41, 71-84.
Noctor, S. C., Martinez-Cerdeno, V., Ivic, L. and Kriegstein, A. R. (2004).
Cortical neurons arise in symmetric and asymmetric division zones and migrate
through specific phases. Nat. Neurosci. 7, 136-144.
Norris, C. R. and Kalil, K. (1991). Guidance of callosal axons by radial glia in the
developing cerebral cortex. J. Neurosci. 11, 3481-3492.
Northcutt, R. G. and Kaas, J. H. (1995). The emergence and evolution of
mammalian neocortex. Trends Neurosci. 18, 373-379.
Ohshima, T., Hirasawa, M., Tabata, H., Mutoh, T., Adachi, T., Suzuki, H.,
Saruta, K., Iwasato, T., Itohara, S., Hashimoto, M. et al. (2007). Cdk5 is
required for multipolar-to-bipolar transition during radial neuronal migration and
proper dendrite development of pyramidal neurons in the cerebral cortex.
Development 134, 2273-2282.
O’Leary, D. D., Chou, S. J. and Sahara, S. (2007). Area patterning of the
mammalian cortex. Neuron 56, 252-269.
Olson, E. C., Kim, S. and Walsh, C. A. (2006). Impaired neuronal positioning and
dendritogenesis in the neocortex after cell-autonomous Dab1 suppression. J.
Neurosci. 26, 1767-1775.
Pacary, E., Heng, J., Azzarelli, R., Riou, P., Castro, D., Lebel-Potter, M., Parras,
C., Bell, D. M., Ridley, A. J., Parsons, M. et al. (2011). Proneural transcription
factors regulate different steps of cortical neuron migration through Rnd-
mediated inhibition of RhoA signaling. Neuron 69, 1069-1084.
Quandt, K., Frech, K., Karas, H., Wingender, E. and Werner, T. (1995). MatInd
and MatInspector: new fast and versatile tools for detection of consensus
matches in nucleotide sequence data. Nucleic Acids Res. 23, 4878-4884.
Rakic, P. (1978). Neuronal migration and contact guidance in the primate
telencephalon. Postgrad. Med. J. 54, 25-40.
Ramos, R. L., Tam, D. M. and Brumberg, J. C. (2008). Physiology and
morphology of callosal projection neurons in mouse. Neuroscience 153, 654-
Richards, L. J., Plachez, C. and Ren, T. (2004). Mechanisms regulating the
development of the corpus callosum and its agenesis in mouse and human. Clin.
Genet. 66, 276-289.
Sanada, K., Gupta, A. and Tsai, L. H. (2004). Disabled-1-regulated adhesion of
migrating neurons to radial glial fiber contributes to neuronal positioning during
early corticogenesis. Neuron 42, 197-211.
Sasaki, S., Tabata, H., Tachikawa, K. and Nakajima, K. (2008). The cortical
subventricular zone-specific molecule Svet1 is part of the nuclear RNA coded by
the putative netrin receptor gene Unc5d and is expressed in multipolar
migrating cells. Mol. Cell. Neurosci. 38, 474-483.
Seibt, J., Schuurmans, C., Gradwhol, G., Dehay, C., Vanderhaeghen, P.,
Guillemot, F. and Polleux, F. (2003). Neurogenin2 specifies the connectivity of
thalamic neurons by controlling axon responsiveness to intermediate target cues.
Neuron 39, 439-452.
Sessa, A., Mao, C. A., Hadjantonakis, A. K., Klein, W. H. and Broccoli, V.
(2008). Tbr2 directs conversion of radial glia into basal precursors and guides
neuronal amplification by indirect neurogenesis in the developing neocortex.
Neuron 60, 56-69.
Shimogori, T. and Grove, E. A. (2005). Fibroblast growth factor 8 regulates
neocortical guidance of area-specific thalamic innervation. J. Neurosci. 25, 6550-
Super, H. and Uylings, H. B. (2001). The early differentiation of the neocortex: a
hypothesis on neocortical evolution. Cereb. Cortex 11, 1101-1109.
Tabata, H. and Nakajima, K. (2003). Multipolar migration: the third mode of
radial neuronal migration in the developing cerebral cortex. J. Neurosci. 23,
Takeuchi, A. and O’Leary, D. D. (2006). Radial migration of superficial layer
cortical neurons controlled by novel Ig cell adhesion molecule MDGA1. J.
Neurosci. 26, 4460-4464.
Tanaka, H., Katoh, H. and Negishi, M. (2006). Pragmin, a novel effector of Rnd2
GTPase, stimulates RhoA activity. J. Biol. Chem. 281, 10355-10364.
Tarabykin, V., Stoykova, A., Usman, N. and Gruss, P. (2001). Cortical upper
layer neurons derive from the subventricular zone as indicated by Svet1 gene
expression. Development 128, 1983-1993.
Development 138 (21)
Tomassy, G. S., De Leonibus, E., Jabaudon, D., Lodato, S., Alfano, C., Mele,
A., Macklis, J. D. and Studer, M. (2010). Area-specific temporal control of
corticospinal motor neuron differentiation by COUP-TFI. Proc. Natl. Acad. Sci.
USA 107, 3576-3581.
Tripodi, M., Filosa, A., Armentano, M. and Studer, M. (2004). The COUP-TF
nuclear receptors regulate cell migration in the mammalian basal forebrain.
Development 131, 6119-6129.
Uesugi, K., Oinuma, I., Katoh, H. and Negishi, M. (2009). Different
requirement for Rnd GTPases of R-Ras GAP activity of Plexin-C1 and Plexin-D1. J.
Biol. Chem. 284, 6743-6751.
Wang, C. L., Zhang, L., Zhou, Y., Zhou, J., Yang, X. J., Duan, S. M., Xiong, Z.
Q. and Ding, Y. Q. (2007). Activity-dependent development of callosal
projections in the somatosensory cortex. J. Neurosci. 27, 11334-11342.
Zhang, L. J., Liu, X., Gafken, P. R., Kioussi, C. and Leid, M. (2009). A chicken
ovalbumin upstream promoter transcription factor I (COUP-TFI) complex
represses expression of the gene encoding tumor necrosis factor alpha-induced
protein 8 (TNFAIP8). J. Biol. Chem. 284, 6156-6168.
Zhou, C., Tsai, S. Y. and Tsai, M. J. (2001). COUP-TFI: an intrinsic factor for early
regionalization of the neocortex. Genes Dev. 15, 2054-2059.
Zimmer, C., Tiveron, M. C., Bodmer, R. and Cremer, H. (2004). Dynamics of
Cux2 expression suggests that an early pool of SVZ precursors is fated to
become upper cortical layer neurons. Cereb. Cortex 14, 1408-1420.
COUP-TFI favours radial migration