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tively. Interestingly, these divergent functions
directly result from the distinct subcellular distribu-
Dlx1 and Dlx2 were found to promote the migration of cortical
interneurons by preventing their premature differentiationbrain development result in devastating conditions, including
mental retardation, autism, and epilepsy (McManus and Golden,
2005; Wegiel et al., 2010). Active research into the molecular
mechanisms involved are unknown.
During development of the cerebral cortex, excitatory projec-
tion neurons generated in the ventricular zone (VZ) and subven-
mechanisms controlling neuronal migration has led to the dis-
covery of extrinsic cues, receptors, and intracellular pathways
tricular zone (SVZ) of the dorsal telencephalon migrate radially
through the intermediate zone (IZ) to reach the superficial layerstions of the two Rnd proteins. Because Rnd proteins
also regulate progenitor divisions and neurite
outgrowth, we propose that proneural factors,
through spatiotemporal regulation of Rnd proteins,
integrate the process of neuronal migration with
other events in the neurogenic program.
INTRODUCTION
Neurons in the embryonic mammalian brain are generated in
progenitor zones that line the ventricles. Soon after their birth,
they undergo active cell migration to reach distant locations,
where they eventually form neuronal circuits. Migration is a
fundamental behavior of neurons, and migration defects during
through transcriptional repression of several regulators of
the neuronal cytoskeleton, including microtubule-associated
protein 2 (MAP2), growth-associated protein 43 (GAP43), and
p-21-activated serine/threonine kinase 3 (PAK3) (Cobos et al.,
2007). Recent studies have shown that Neurogenin2 (also known
asNeurog2), aproneural factorwithaprominent role in neurogen-
esis in theembryonic cortex (Nieto et al., 2001;Schuurmanset al.,
2004), promotes migration in the cortex through direct induction
of the expression of the small GTPase Rnd2 and possibly other
genes involved in regulating the cytoskeleton, including RhoA,
doublecortin, and p35 (Ge et al., 2006; Hand et al., 2005; Heng
et al., 2008). Another proneural factor present in the embryonic
cortex, Ascl1 (Britz et al., 2006) has also been shown to promote
neuronal migration when overexpressed in cortical progenitors
(Geet al., 2006), although it is unclearwhether this activity reflects
a genuine role in cortical neuron migration and the downstreamzone and locomotion in the cortical plate, respec- properties of neurons have yet been reported. In one example,Article
Proneural Transcription Fa
Steps of Cortical Neuron M
Rnd-Mediated Inhibition of
Emilie Pacary,1 Julian Heng,1,4 Roberta Azzarelli,1 Philippe R
Donald M. Bell,3 Anne J. Ridley,2 Maddy Parsons,2 and Fra
1Division of Molecular Neurobiology, MRC National Institute for Medi
2Randall Division of Cell and Molecular Biophysics, King’s College Lo
3Confocal and Image Analysis Laboratory, National Institute for Medi
4Present address: Australian Regenerative Medicine Institute, Monas
5Present address: CRICM UPMC/INSERM, UMR-S 975/CNRS UMR
Paris 750013, France
*Correspondence: fguille@nimr.mrc.ac.uk
DOI 10.1016/j.neuron.2011.02.018
SUMMARY
Little is known of the intracellular machinery that
controls the motility of newborn neurons. We have
previously shown that the proneural protein Neurog2
promotes the migration of nascent cortical neurons
by inducing the expression of the atypical Rho
GTPase Rnd2. Here, we show that another proneural
factor, Ascl1, promotes neuronal migration in the
cortex through direct regulation of a second Rnd
family member, Rnd3. Both Rnd2 and Rnd3 promote
neuronal migration by inhibiting RhoA signaling, but
they control distinct steps of the migratory process,
multipolar to bipolar transition in the intermediatetors Regulate Different
gration through
hoA Signaling
u,2 Diogo Castro,1 Me´lanie Lebel-Potter,1 Carlos Parras,1,5
¸ ois Guillemot1,*
l Research, Mill Hill, London NW7 1AA, UK
don, London SE1 1UL, UK
l Research, Mill Hill, London NW7 1AA, UK
University, Clayton, Victoria 3800, Australia
225, Hoˆpital de la Pitie´-Salpeˆtrie`re, 47 Boulevard de l’Hoˆpital,
that together guide neurons to their destination (Marı´n and
Rubenstein, 2003; Sobeih and Corfas, 2002). However, much
less is known of the intracellular machinery that confers a motile
behavior to newly generated neurons and how this machinery is
activated when neurons are born.
Transcription factors play leading roles in developmental
programs that direct the differentiation of progenitor cells into
mature neurons. Over the past few years, transcription factors
have been shown to contribute significantly to the control of
neuronal migration, with proteins such as Hoxa2 and Hoxb2 in
the hindbrain and Nkx2.1 in the ventral forebrain regulating the
expression of cell adhesion molecules and receptors for
guidance molecules in migrating neurons (Che´dotal and Rijli,
2009; No´brega-Pereira and Marı´n, 2009). However, few exam-
ples of transcription factors regulating the intrinsic migratoryNeuron 69, 1069–1084, March 24, 2011 ª2011 Elsevier Inc. 1069

Neuronof the cortical plate (CP). Distinct phases of neuronal migration
and correlated morphologies of migrating neurons can be distin-
guished (LoTurco and Bai, 2006). Neurons initiate migration in
the VZ with a bipolar morphology, they become transiently multi-
polar in the SVZ and IZ, and they convert back to a bipolar
morphology to enter the CP. Bipolar neurons migrate along
radial glial fibers by using a mode of migration termed locomo-
tion, which involves a reiterative succession of steps affecting
different cellular domains. Neurons extend their leading process
along radial glia fibers and translocate their nucleus and perinu-
clear region into the proximal leading process, a process known
as nucleokinesis, which is followed by retraction of the trailing
process, resulting in overall movement of the neuron (Marı´n
et al., 2006). The different steps of neuronal migration involve
extensive reorganization of the cytoskeleton and, not surpris-
ingly, RhoGTPases, which control many aspects of cytoskeleton
dynamics (Heasman and Ridley, 2008), have been implicated in
migration of different types of neurons (Govek et al., 2005; Heas-
man andRidley, 2008;Marı´n et al., 2006). Rac1 is required for the
formation of the leading process in cortical neurons (Kawauchi
et al., 2003; Konno et al., 2005), while Cdc42 is important for
nuclear movements in postmitotic cerebellar granule neurons
(Kholmanskikh et al., 2006), and RhoA activity is required for
nucleokinesis and organization of the cytoskeleton at the rear
end of migrating precerebellar neurons (Causeret et al., 2004).
Although many pathways are known to control the activity of
Rho, Rac, and Cdc42 in nonneuronal cells, much less is known
of how the activity of these small GTPases is controlled in
migrating neurons. The atypical Rho protein Rnd3/Rho8/RhoE
is an important regulator of migration of fibroblasts and tumor
cells (Chardin, 2006; Guasch et al., 1998; Klein and Aplin,
2009; Nobes et al., 1998) that acts by inhibiting RhoA through
stimulation of the Rho GTPase-activating protein p190RhoGAP
(Wennerberg et al., 2003), and/or inhibition of the activity of
ROCKI, one of the main effectors of RhoA (Riento et al., 2003).
Rnd3 has been shown to induce neurite outgrowth in pheochro-
mocytoma PC12 cells, but its role in neuronal migration has not
been examined (Talens-Visconti et al., 2010). A related protein,
Rnd2/Rho7/RhoN, has been shown to promote the radial migra-
tion of cortical neurons (Heng et al., 2008; Nakamura et al., 2006)
and to inhibit neurite growth and induce neurite branching in
PC12 cells (Fujita et al., 2002; Tanaka et al., 2006), but themech-
anisms mediating Rnd2 activity in neurons remain unclear. Rnd2
and Rnd3 belong to the small Rnd family of atypical Rho proteins
that lack intrinsic GTPase activity and are therefore constitutively
bound to GTP (Chardin, 2006). Rnd proteins are thought to be
regulated at the level of their expression, phosphorylation, and
subcellular localization (Madigan et al., 2009; Riento et al.,
2005a). We have previously shown that the proneural protein
Neurog2 promotes the migration of nascent cortical neurons
through induction of Rnd2 expression as part of an extensive
subtype-specific transcriptional program controlling cortical
neurogenesis (Heng et al., 2008). In this study, we have further
investigated how the cell behavior of radial migration of cortical
neurons is regulated in the context of a global developmental
program. We show that another proneural factor expressed in
the embryonic cortex, Ascl1, promotes neuronal migration
through regulation of Rnd3. Importantly, we demonstrate that
1070 Neuron 69, 1069–1084, March 24, 2011 ª2011 Elsevier Inc.both Rnd2 and Rnd3 inhibit RhoA signaling in cortical neurons,
but that they regulate steps of migration by interfering with
RhoA activity in different cell compartments. Together, our
results demonstrate that proneural factors, through regulation
of different Rnd proteins, integrate the process of neuronal
migration with other events in the neurogenic program.
RESULTS
Rnd3 Is a Direct Transcriptional Target of Ascl1
We began this study by asking whether the proneural transcrip-
tion factor Ascl1, which has been shown to enhance cell migra-
tion when overexpressed in cultured cortical cells (Ge et al.,
2006), is required for neuronal migration during development of
the cerebral cortex. We examined the consequence of acute
Ascl1 loss of function in the embryonic cortex by introducing
an expression construct encoding the Cre recombinase in the
cortex of embryos carrying a conditional mutant allele of Ascl1
(Ascl1flox/flox; Figures S1A–S1C and Supplemental Experimental
Procedures). In utero electroporation of the Cre construct
together with GFP to label electroporated cells in embryonic
day (E) 14.5 Ascl1flox/flox mice resulted in a significant reduction
of the radial migration of electroporated cells at E17.5 when
compared with electroporation of only GFP (Figure 1A), demon-
strating thatAscl1 is required for proper neuronal migration in the
embryonic cortex. We next asked whether Rnd2, which medi-
ates the promigratory activity of Neurog2, is also regulating
cortical neuron migration downstream of Ascl1. We found that
Rnd2 transcripts are normally present in the telencephalon of
Ascl1 mutant embryos, whereas they are clearly depleted in
Neurog2 mutants (Heng et al., 2008; Figure S1D), suggesting
that Ascl1 does not regulate Rnd2 expression. To identify alter-
native mechanisms through which Ascl1 promotes migration,
we searched for candidate target genes of Ascl1 that might be
involved in regulating cell migration (Gohlke et al., 2008; Fig-
ure S1E). By using gene expression microarrays, we found that
Rnd3/RhoE, a member of the Rnd family of small GTP-binding
proteins that also includes Rnd2 (Chardin, 2006), was signifi-
cantly downregulated in the embryonic cortex of Ascl1 null
mutant embryos and upregulated in the ventral telencephalon
of embryos electroporated with an Ascl1 expression construct
(Figure S1E). Rnd3 transcripts are found throughout embryonic
development in the VZ and the CP of the cerebral cortex (Figures
1B–1E), as well as in the VZ and SVZ of the ventral telencephalon
(Figures 1C–1E). Rnd3 transcript levels were markedly reduced
in embryos mutant for Ascl1, while they were unaffected in
Neurog2 mutant embryos (Figures 1F–1H and Figure S1D). To
determine whether Rnd3 is a direct transcriptional target of
Ascl1, we performed an in silico search for putative Ascl1-regu-
lated elements within the Rnd3 gene locus and identified 21
distinct evolutionarily conserved regions which contained
a consensus Ascl1 binding motif (CAGSTG) (Figure S1F). To
evaluate Ascl1 occupancy within these putative regulatory
Ascl1-Rnd3-RhoA Pathway in Neuronal Migrationregions, we carried out chromatin immunoprecipitation (ChIP)
with an antibody against Ascl1 and chromatin prepared from
embryonic telencephalon and found that Ascl1 was bound
in vivo to two of these conserved elements (Rnd3 E1, located
59 kb 30 of the gene and Rnd3 E5, located 110 kb 30 of Rnd3;