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© 2016. Published by The Company of Biologists Ltd.
Reelin and cofilin cooperate during the migration of
cortical neurons: A quantitative morphological analysis
Xuejun Chai1†, Shanting Zhao1,3†, Li Fan2, Wei Zhang3, Xi Lu3, Hong Shao2, Shaobo Wang1, Lingzhen Song1, Antonio Virgilio Failla4, Bernd Zobiak4, Hans G. Mannherz5, Michael Frotscher1
1Institute for Structural Neurobiology, Center for Molecular Neurobiology Hamburg (ZMNH), University Medical Center Hamburg-Eppendorf, Hamburg, Germany, 2Institute of Zoology, School of Life Science, Lanzhou University, Lanzhou, PR China, 3College of Veterinary Medicine, Northwest A&F University, Yangling, PR China, 4UKE Microscopy Imaging Facility (UMIF), University Medical Center Hamburg-Eppendorf, Hamburg, Germany, 5Institute of Anatomy and Molecular
Embryology, Ruhr University Bochum, Bochum, Germany †equal contribution Keywords: neuronal migration, cofilin phosphorylation, Reelin signaling, actin cytoskeleton, in utero electroporation Address correspondence to: Michael Frotscher Institute for Structural Neurobiology Center for Molecular Neurobiology Hamburg (ZMNH) University Medical Center Hamburg-Eppendorf Falkenried 94 20251 Hamburg Germany Phone: ++49-40-741055028 Fax: ++49-40-741040213 E-Mail: [email protected] Summary statement: Dynamics of the actin cytoskeleton during neuronal migration is controlled by the cooperation of Reelin and cofilin.
http://dev.biologists.org/lookup/doi/10.1242/dev.134163Access the most recent version at First posted online on 18 February 2016 as 10.1242/dev.134163
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Abstract
In reeler mutant mice deficient in Reelin the lamination of the cerebral cortex is
disrupted. Reelin signaling induces phosphorylation of LIM kinase 1, which
phosphorylates the actin-depolymerizing protein cofilin in migrating neurons.
Conditional cofilin mutants show neuronal migration defects. Thus, both Reelin and
cofilin are indispensable during cortical development. To analyze the effects of cofilin
phosphorylation on neuronal migration we used in utero electroporation to transfect
E14.5 wild-type cortical neurons with pCAG-EGFP plasmids encoding either for a
nonphosphorylatable form of cofilin (cofilinS3A), a pseudophosphorylated form
(cofilinS3E) or wild-type cofilin (cofilinwt). Wild-type controls and reeler neurons were
transfected with pCAG-EGFP. Real-time microscopy and histological analyses
revealed that overexpression of each, cofilinwt, cofilinS3A, and cofilinS3E, induced
migration defects and morphological abnormalities of cortical neurons. Of note, reeler
neurons, cofilinS3A- and cofilinS3E-transfected neurons showed aberrant backward
migration towards the ventricular zone. Overexpression of cofilinS3E, the
pseudophosphorylated form, partially rescued the migration defect of reeler neurons
as did overexpression of LIM kinase1. Collectively, the results indicate that Reelin
and cofilin cooperate in controlling cytoskeletal dynamics during neuronal migration.
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Introduction
Neuronal migration is a fundamental process in brain development resulting in the
formation of neuronal layers. In the mammalian cerebral cortex, pyramidal neurons
born in the ventricular zone (VZ) migrate radially towards the marginal zone (MZ)
along the processes of radial glial cells and form the six-layered cortex (Rakic, 1971,
1972; Nadarajah et al., 2001, 2003; Cooper, 2008). In the dentate gyrus of the
hippocampus, the granule cells born in the hilus also migrate towards the MZ and
form a densely packed granular layer (Förster et al., 2002, 2006a, b; Frotscher et al.,
2003; Weiss et al., 2003; Zhao et al., 2004, 2006). In the cerebellum, Purkinje cells
migrate along Bergmann glial fibers and form the Purkinje plate beneath the transient
external granule cell layer (Yuasa et al., 1991). In all these different brain regions,
radial neuronal migration is controlled by Reelin, an extracellular matrix protein
synthesized by cells located in the target direction of the migrating neurons,
suggesting a role for Reelin in directed migration. Cajal-Retzius cells in the MZ are a
major source of Reelin in the cortex and hippocampus (D’Arcangelo et al., 1995;
Frotscher, 1997, 1998; Alcántara et al., 1998; Tissir and Goffinet, 2003); external
granule cells synthesize Reelin in the cerebellum (Schiffmann et al., 1997). In reeler
mutant mice deficient in Reelin, cortical lamination is disrupted (Falconer, 1951; Kubo
and Nakajima 2003; Tissir and Goffinet 2003; Hack et al., 2007), granule cells in the
hippocampus are loosely distributed throughout the dentate gyrus (Stanfield and
Cowan, 1979; Förster et al., 2002, 2006a, b; Zhao et al., 2004, 2006), and Purkinje
cells in the cerebellum are ectopically positioned (Yuasa et al., 1993).
Reelin binds to two lipoprotein receptors, Apolipoprotein-E receptor 2
(ApoER2) and very low-density lipoprotein receptor (VLDLR; Trommsdorff et al.,
1999; D’Arcangelo et al., 1999; Hiesberger et al., 1999) and induces the
phosphorylation of the adaptor protein Disabled 1 (Dab1; Sheldon et al., 1997;
Howell et al., 1999; Benhayon et al., 2003). ApoER2 and VLDLR double knockout
mice and Dab1 knockout mice show similar neuronal migration defects as seen in
reeler mutants (Rakic and Caviness 1995; Trommsdorff et al., 1999; Walsh and
Goffinet 2000; Drakew et al., 2002).
Despite some insight into the Reelin signaling cascade it is still poorly
understood how Reelin regulates neuronal migration. Since the neurons always
migrate towards the Reelin-containing zones, Reelin has been proposed to act as a
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chemoattractive factor for radially migrating neurons (Caffrey et al., 2014).
Conversely, Reelin has been suggested to be a stop- or detachment signal since the
MZ enriched in Reelin is almost cell-free in wild-type mice but is invaded by
numerous neurons in the reeler mutant, (Frotscher, 1998; Hack et al., 2007; Chai et
al., 2009; Zhao and Frotscher 2010; Hirota et al., 2014). By binding to VLDLR or
integrin receptors on the leading processes Reelin in the MZ seems to arrest
migrating neurons (Anton et al., 1999; Dulabon et al., 2000; Sanada et al., 2004;
Schmid et al., 2005; Hack et al., 2007; Chai et al., 2009; Sekine et al., 2012; Hirota et
al., 2014).
Neuronal migration is a coordinated movement comprising extension of the
leading process, translocation of the nucleus and retraction of the trailing process.
These different processes are all associated with rearrangements of the cytoskeleton
(Bamburg 1999; Pollard and Borisy 2003; Jovceva et al. 2007). Thus, in order to
understand migration, we need to know how Reelin signaling regulates cytoskeletal
dynamics. We have shown previously that Reelin signaling enhances the activity of
LIM kinase 1 (LIMK1; Chai et al., 2009), which phosphorylates the actin-binding
protein cofilin (non-muscle cofilin, n-cofilin1; Arber et al., 1998; Yang et al., 1998).
Actin-depolymerizing factor (ADF) and cofilin sever actin filaments (F-actin) and
thereby generate new filament barbed ends (Lappalainen and Drubin 1997;
Ichetovkin et al., 2000; Andrianantoandro and Pollard, 2006), available for new
rounds of elongation, and monomeric actin, which can be reused to build new
filamentous actin structures. By these activities cofilin participates in the dynamic
reorganization of the actin cytoskeleton and defines the direction of cell migration
(Ghosh et al., 2004). Phosphorylation of cofilin at serine3 inhibits all cofilin–actin
interactions, blocks actin dynamics and subsequent process extension (Nagaoka et
al., 1996; Zebda et al., 2000; Huang et al., 2006; Bravo-Cordero et al., 2013). Reelin-
induced cofilin phosphorylation in the leading processes of migrating neurons
stabilizes their cytoskeleton, anchors them to the marginal zone and promotes somal
translocation (Chai et al., 2009; Frotscher, 2010; Förster et al., 2010; Franco et al.,
2011; Jossin and Cooper 2011). Thus, Reelin-induced phosphorylation of cofilin
appears to play a pivotal role in the proper positioning of migrating neurons. Both
Reelin and cofilin are required since conditional cofilin knockout mice show severe
migration defects of late generated cortical neurons destined to superficial layers of
the cerebral cortex (Bellenchi et al., 2007).
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In the present study, we aimed at better understanding the interplay between
Reelin and cofilin during the migration of cortical neurons by interfering with Reelin-
induced cofilin phosphorylation at serine3. We generated different cofilin constructs,
a nonphosphorylatable form of cofilin and a pseudophosphorylated form, cloned in
frame into the vector pCAG-GFP. We used timed in utero electroporation (IUE) and
real-time microscopy to monitor the migratory behavior of the transfected cells.
Results
We hypothesized that IUE with a nonphosphorylatable form of cofilin (cofilinS3A)
would result in a phenotype similar to that of reeler since cofilin phosphorylation is
significantly reduced in the reeler mutant (Chai et al., 2009). We expected a
contrasting phenotype when transfecting embryonic cortical neurons with a pseudo-
phosphorylated form of cofilin (cofilinS3E). Control neurons transfected with pCAG-
GFP alone were compared to neurons transfected with cofilinS3A, cofilinS3E, to
neurons transfected with wild-type cofilin (cofilinWT), and to neurons in reeler embryos
transfected with pCAG-GFP.
Minor migration defects two days following IUE at E14.5
On E16.5, only minor differences in the migratory behavior were observed between
neurons transfected with the different constructs two days before (Fig. 1). Thus, the
majority of all transfected cells were still found in the intermediate zone (IZ),
subventricular zone (SVZ) and ventricular zone (VZ), respectively. At this stage,
many cells in the inner portion of the IZ appeared multipolar (Hatanaka and
Yamauchi, 2013), whereas those neurons that had already reached the outer IZ or
the cortical plate (CP), such as many pCAG-GFP transfected control cells (Fig. 1A),
showed a long leading process oriented towards the marginal zone (MZ). We found
that approximately 15% of the control cells had already reached the cortical plate
(Fig. 2A, B) compared to 25% in embryos transfected with cofilinWT (Figs. 1B, 2A, B).
In contrast, in cofilinS3A-transfected slices, in slices transfected with cofilinS3E and in
reeler slices most cells were clustered in the IZ, SVZ and VZ, and very few cells were
found in the CP (Figs. 1C-E, 2A-D).
When studying the transfected neurons in the upper IZ at high-power
magnification, we observed some morphological differences between the mutant
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cells. When compared to control cells (Fig. 3A), cofilinWT-transfected neurons gave
rise to very long and thin leading processes. Occasionally several processes
originated from the cell body or proximal leading process (Fig. 3B). CofilinS3A cells
were characterized by many short processes originating from the cell body (Fig. 3C),
whereas cofilinS3E-transfected neurons had long, varicose leading processes and
several short processes, which gave rise to filopodia-like protrusions (Fig. 3D).
Surprisingly, reeler neurons displayed a normal bipolar morphology indistinguishable
from control cells (Fig. 3E). While the lengths of the leading processes were not
significantly different between the various mutant cells (except for cofilinS3E-
transfected neurons, Fig. 4A), the number of supernumerary processes originating
from the cell body was found increased in neurons transfected with the different
cofilin constructs (Fig. 4B).
Next, we performed live imaging and measured the migratory speed and
directionality of migrating neurons using Imaris software. Statistical analysis of
migratory speed indicated no significant differences between GFP-transfected control
cells and cells transfected with the different cofilin constructs (n=50 cells for each
experimental condition; Fig. 4C). Reeler neurons also showed a normal migratory
speed when compared to control cells. However, the direction of migration varied to
some extent. While cofilinWT and cofilinS3A cells did not show obvious abnormalities,
some cofilinS3E and reeler neurons were observed that migrated back towards the VZ
(Fig. 4D), as was described before in a study of neuronal trajectories in reeler
mutants (Britto et al., 2011). Taken together, there were some morphological
abnormalities and minor migration defects as early as 2 days after IUE on E14.5.
Migration defects three days after IUE at E14.5
Three days after IUE, the majority of GFP-positive cells in controls were scattered
over the CP and IZ, and only very few cells were still seen in the SVZ and VZ,
respectively (Fig. 5A). The cells that had reached the CP had not yet formed a
distinct cell layer (Tabata and Nakajima, 2008). When dividing the CP into an upper,
middle and lower portion, almost equal numbers of neurons were found in these
subzones of CP, together amounting to more than 60% of all GFP-labeled neurons in
controls (Fig. 6A-D). Also in cofilinWT-transfected embryos numerous cells had
invaded the CP (approximately 45%), but more cells than in controls were found in
the SVZ/VZ (Figs. 5B, 6A-F). In cofilinS3A-transfected sections, the majority of labeled
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neurons were clustered in the IZ and SVZ, and only very few cells had entered the
CP (Figs. 5C, 6A-F). In cofilinS3E-transfected slices, many labeled neurons were still
seen in the IZ and SVZ, but about 30% of them had migrated into the CP (Fig. 5D,
6A-F). In contrast, in sections from reeler mutants virtually all GFP-labeled neurons
were seen in the deep layers of the cortex, many of them still present in the VZ/SVZ
(Figs. 5E, 6A-F). We hypothesized that the migration defect in reeler is at least
partially due to insufficient phosphorylation of cofilin. In fact, when we transfected
reeler embryos with the pseudophosphorylated form of cofilin (cofilinS3E) on E14.5
and studied the brains on E17.5, we observed a partial rescue of the reeler
phenotype (Figs. 5F, 6A-F). A similar partial rescue was observed when reeler
embryos were transfected with LIM kinase 1 (LIMK1) known to phosphorylate cofilin
(Figs. 5G, 6A-F).
Next, we again studied the structural characteristics and migratory behavior of
neurons transfected with the different constructs (Fig. 7; Movies 1-5). Control cells
revealed their characteristic bipolar shape and moved smoothly by nuclear
translocation (approximately 85 µm in 200 minutes; Fig. 7A; Movie 1). In contrast,
many neurons transfected with cofilinWT showed very little forward movement during
this time period (Fig. 7B; Movie 2). Moreover, cofilinWT-transfected cells were
observed to form supernumerary processes (Fig. 7B, red arrowhead) and cofilin-actin
rods (Ono et al., 1996; Bernstein and Bamburg, 2003; Bernstein et al., 2006).
Occasionally we noticed that portions of the soma were squeezed in the leading
process before the rest of the soma hooked up during the process of nuclear
translocation (Fig. 7B). However, this was similarly observed in all other types of
transfected neurons. Individual neurons of embryos transfected with cofilinS3A and
cofilinS3E were regularly found to change their shape over time but without clear
forward movement of the soma (Fig. 7C, D; Movies 3, 4). The leading processes of
cofilinS3A neurons often gave rise to branches (Fig. 7C). CofilinS3E neurons and reeler
cells, but also cofilinS3A neurons, were often oriented towards the ventricular zone
(Fig. 7D, E; Movies 4, 5). Many reeler neurons transfected with cofilinS3E or LIMK1
showed a normal migratory behavior (Fig. 7F, G; Movie 6).
These observations were confirmed by quantitative analyses (Fig. 8). We
reported recently that Reelin-induced branching of the leading processes anchors
them to the marginal zone (Chai et al., 2015). Therefore, we again measured the
lengths of leading processes in the different mutant cells and quantified the
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percentage of them that had reached the marginal zone. The data revealed that the
leading processes of cofilinWT-transfected neurons were particularly long and many of
them had reached the MZ like those of control cells when compared to the leading
processes of all other mutant cells (Fig. 8A, B).
When compared to control cells, the average migratory speed of neurons
transfected with the different cofilin constructs as well as of pCAG-GFP-transfected
reeler neurons was significantly reduced (Fig. 8C). Moreover, the percentage of
neurons migrating towards VZ was significantly increased in reeler neurons (Britto et
al., 2011) and cofilinS3A- and cofilinS3E-transfected cells (Fig. 8D), consistent with the
reduced number of leading processes of these cells reaching the MZ (Fig. 8B). Of
note, both migratory speed and migration direction towards the MZ were partially
rescued by transfecting reeler neurons with cofilinS3E or LIMK1 (Fig. 8C, D).
In sum, three days after IUE the majority of migrating neurons transfected with
nonphosphorylatable or pseudophosphorylated cofilin mutants were unable to
perceive and convey instructive positional signals from the marginal zone, which
together with structural abnormalities resulted in reduced migratory speed and
altered directionality of the migratory process. This migration defect resembled that of
reeler mutants in which the Reelin signal from the MZ is absent. A remarkable rescue
of the reeler phenotype was observed when reeler neurons were transfected with
LIMK1 or pseudophosphorylated cofilin (cofilinS3E).
Profound neuronal migration defects five days after IUE at E14.5
Next, we studied the transfected animals after birth. At this time point, most neurons
have terminated their migration and reached their destinations in the CP. Control
cells formed a compact cell layer subjacent to the MZ (Fig. 9A). More than 80% of all
labeled cells in controls were found in the upper cortical plate (layer II/III of the cortex;
Fig. 10A-D). The IZ and SVZ/VZ, contained only very few still migrating neurons. In
cofilinWT-transfected slices there were mainly gradual differences to controls; these
neurons were also capable of forming a cell layer near the marginal zone. However,
significantly fewer cells than in controls (about 50%) were found in the upper portion
of the CP (Figs. 9B, 10A-D). The vast majority of GFP-positive neurons in animals
transfected with cofilinS3A (Figs. 9C, 10A-D) or cofilinS3E (Figs. 9D, 10A-D) were
observed in the deep layers of the cortex. In reeler slices GFP-positive cells were
scattered all over the cortex with many cells showing an inverted orientation towards
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the VZ. Very few cells had invaded the upper layers of the CP subjacent to the MZ
(Figs. 9E, 10A-D).
Since at that stage most neurons transfected at E14.5 have terminated their
migration – having arrived at normal or abnormal locations – we refrained from
measuring migratory speed and documenting migratory behavior but analyzed their
morphological characteristics. For this purpose we focused on still migrating neurons
in each experimental group that were behind the main cohort of cells and were
located at the border between the upper IZ and the CP.
Such late neurons in control slices still showed the characteristic asymmetric
shape of migrating cells (Fig. S1A, F). In cofilinWT-transfected cells many small
branches originated from the cell bodies and leading processes (Fig. S1B, F).
CofilinS3A cells displayed an asymmetric bipolar shape with short, branched leading
processes. Some neurons exhibited two or more processes directed towards the MZ
(Fig. S1C, F). CofilinS3E neurons had lost the characteristic asymmetric polarity of
migrating neurons. The two processes originating from the opposite poles of the cell
body appeared equally short and thick, and no typical leading or trailing processes
could be identified (Fig. S1D, F). GFP-labeled reeler neurons displayed a variety of
different shapes with leading processes oriented in all directions, often towards the
white matter (Fig. S1E, F).
Taken together, five days after IUE at E14.5 the migration defects observed
after shorter survival times have become more obvious since GFP-labeled neurons
have now terminated their migration and in controls formed a cell layer in the upper
portion of the cortical plate. While cofilinWT-transfected neurons largely hooked up
with controls, the majority of cofilinS3A and cofilinS3E cells were unable to migrate to
the upper cortical plate, pointing to a central role of phosphorylatable cofilin in the
migratory process. Numerous reeler neurons had invaded the cortical plate but only
very few of them had reached its upper portion.
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Discussion
Directed neuronal migration is a process that requires the coordinated reorganization
of the actin cytoskeleton. Changes in cell shape during migration are associated with
cytoskeletal remodeling; however, stability of the leading process is needed for
nuclear translocation to take place (Nadarajah et al., 2001; Nadarajah and
Parnavelas, 2002; Miyata and Ogawa, 2007; Cooper, 2013). Hence, neuronal
migration is characterized by well-coordinated, consecutive periods of cytoskeletal
stability and reorganization of the leading process. Actin-depolymerizing molecules
such as ADF and cofilin play important roles since they control the turnover and
stability, respectively, of actin filaments. Forced expression of cofilinWT, but in
particular transfection with a nonphosphorylatable form of cofilin (cofilinS3A) or a
pseudo-phosphorylated form (cofilinS3E) resulted in alterations of this fine-tuned
balance, in structural abnormalities of the migrating neurons and in migration defects.
To some extent the observations in these cofilin mutant cells resembled those in
reeler neurons, likely because Reelin is crucially involved in cofilin phosphorylation
(Chai et al., 2009). As an example, cofilinS3A-transfected cells, cofilinS3E cells as well
as reeler neurons showed backward migration towards the VZ (Britto et al., 2011;
present study), suggesting that a Reelin gradient from the MZ is pivotal for the
stabilization of the leading process to the MZ by cofilin phosphorylation and thus for
directed migration to the CP.
Both cofilin and Reelin are involved in cell proliferation (Bellenchi et al., 2007;
Zhao et al., 2007). In particular, cofilin plays an important role in cell cycle exit
(Bellenchi et al., 2007). Thus, the results presented in the present study have to be
interpreted with the caveat that IUE with cofilin mutants might also have affected
progenitor cell proliferation.
Migration of neurons overexpressing wild-type cofilin
Our present results have shown that overexpression of cofilinWT leads to the
formation of supernumerary processes resulting in a loss of the normal asymmetric
bipolarity of migrating cells entering the CP (Miyata et al., 2001; Noctor et al., 2004,
Kriegstein and Noctor 2004; Tsai and Gleeson 2005; Ayala et al., 2007; Noctor et al.,
2007). These structural abnormalities most likely contribute to the migration defects
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seen after 3 and 5 days following IUE. Overexpression of ADF and cofilin increases
cytoskeletal remodeling and neurite outgrowth (Meberg and Bamburg, 2000; Endo et
al., 2003; Flynn et al., 2012), including the formation of rods within the cells (Ono et
al., 1996; Bernstein and Bamburg, 2003; Bernstein et al., 2006). We found that many
neurons overexpressing cofilinWT formed a relatively long leading process and cofilin-
actin rods; they largely reached their appropriate destinations in the CP, but layer
formation was delayed, consistent with a reduction in migratory speed. These results
are in line with studies reporting that overexpression of ADF/cofilin inhibits motility
and invasiveness of different types of cancer cells (Lee et al., 2005; Yap et al., 2005).
Whether the overexpressed cofilin induced elongation of processes or initiated new
branches is much dependent on its relative concentration to F-actin
(Andrianantoandro and Pollard, 2006) and its cooperation with the Arp2/3 protein
complex. The Arp2/3 complex has been shown to bind to the site of a pre-existing
"mother" filament, which then induces the lateral outgrowth of a new filament, leading
to a branched F-actin network (Volkmann et al., 2001; DesMarais et al., 2004; Egile
et al., 2005; Rouiller et al., 2008). Cofilin supports Arp2/3 complex activity by
severing existing capped, i.e., elongation-blocked actin filaments, thus generating
free fast-growing barbed ends that will then elongate and form new binding sites for
active Arp2/3 complex (DesMarais et al., 2004), supporting the notion that the
synergistic interaction between cofilin and the Arp2/3 complex might have been
responsible for the branching and process extension observed in cofilinWT-transfected
neurons.
There are other proteins involved in the organization of the cytoskeleton.
Pacary et al. (2011) have previously shown that co-transfection of a non-
phosphorylatable form of cofilin (cofilinS3A) together with shRNA for the proneural
transcription factors Rnd2 and Rnd3 rescued the migration defect of Rnd2-silenced
neurons, but not that of Rnd3-silenced cells, pointing to a significant role of Rnd
proteins in the reorganization of the actin cytoskeleton.
Cofilin phosphorylation in the leading process is required for directed neuronal
migration
Although essentially two modes of neuronal migration have been described, i.e.,
somal translocation and locomotion along guiding radial glial fibers (Rakic, 1971,
1972; Nadarajah et al., 2001; Nadarajah and Parnavelas, 2002; Kriegstein and
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Noctor 2004; Cooper, 2008, 2013), translocation of the nucleus into the leading
process is common to both forms. Soma translocation requires that the leading
process is under tension (Miyata and Ogawa, 2007; Cooper, 2013) and that the soma
is pulled forward by a myosin II-dependent flow of actin filaments towards the tip of
the leading process (He et al., 2010). Movement of the nucleus involves SUN-domain
proteins connecting microtubule-based motor proteins with the nuclear membrane
(Zhang et al., 2009). We have shown here that the majority of neurons expressing a
form of cofilin that cannot be phosphorylated do not populate the CP. Since soma
translocation and directed migration necessitates a leading process with a stabilized
actin cytoskeleton (Chai et al., 2009), overexpression of cofilinS3A might result in the
depolymerization of the actin filaments necessary for the functionality of the leading
process, loss of directed migration and eventually even backward migration.
CofilinS3A is likely to produce many free barbed F-actin ends, which might
initiate the branching activity of the Arp2/3 complex (Svitkina, et al., 1999; Pantaloni
et al., 2000; Volkmann et al., 2001; Amann and Pollard, 2001; Ichetovkin et al.,
2002). Ongoing reorganization of the actin cytoskeleton in S3A neurons might not
only lead to the formation of supernumerary processes but might also result in
remodeling of microtubules as well as altered localization of the centrosome (Solecki
et al., 2009; Sakakibara et al., 2014), which determines neuronal polarity (de Anda et
al., 2005). As a consequence, the cells will change migration direction. We
hypothesize that exuberant process growth and insufficient stabilization of the actin
cytoskeleton are crucially involved in the inability of cofilinS3A-transfected cells to
migrate properly towards the cortical surface.
The migration defect of cofilinS3A-transfected neurons is reminiscent of that in
reeler mutants. Reelin, concentrated in the MZ of the developing cortex, was
previously found to phosphorylate cofilin by activating LIM kinase 1 (Chai et al.,
2009). Thus, the leading processes of migrating neurons are stabilized as they
approach the MZ, which supports directed migration towards the surface of the
cortex. Reeler neurons and cofilinS3A-transfected neurons have in common that
deficient stabilization of the leading processes prevents them from becoming
anchored to the MZ, resulting in aberrant (non-directed) migration including backward
migration towards the VZ (Britto et al., 2011). The inverted course of many pyramidal
cell apical dendrites, the former leading processes, in the adult reeler cortex reflects
this mal-orientation of cortical neurons during the developmental period (Terashima
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et al., 1985, 1992; Frotscher et al., 2009). Mal-orientation of migrating neurons in
reeler eventually results in the malformation of cortical layers as visualized by using
layer-specific markers (Hack et al., 2007; Dekimoto et al., 2010).
We regard it as an important result exemplifying the cooperation of Reelin and
cofilin in the migratrion of cortical neurons that transfection of neurons in reeler slices
with LIMK1 or pseudophosphorylated cofilin (cofilinS3E) partially rescued the reeler
phenotype. Future studies will aim at downregulating Cyclin-dependent kinase 5
(Cdk5), because suppression of Cdk5 was found to increase cofilin phosphorylation
in cortical neurons (Kawauchi et al., 2006).
During development Reelin is also detectable in the germinal zone and in layer
V neurons of the cortical plate (Alcantara et al. 1998; Schiffmann et al., 1997; Jossin
et al., 2007; Chai et al., 2009). It has been suggested that Reelin activates Rap1,
which in turn upregulates N-cadherin through Rab-GTPase-dependent endocytic
pathways (Kawauchi et al. 2010; Jossin and Cooper, 2011; Franco et al., 2011). N-
cadherin is needed for the orientation of migrating multipolar neurons in the
intermediate zone and their transformation to a bipolar shape (Kadowaki et al., 2007;
Shikanai et al., 2011; Jossin and Cooper 2011). We hypothesize that Reelin in the
germinal zone and in layer V enables late-generated neurons to bypass their
predecessors and to migrate to their destination in superficial cortical layers (Pinto
Lord et al., 1982; Nadarajah et al., 2001, 2003).
Asymmetric bipolarity is absent in neurons expressing a pseudophos-
phorylated form of cofilin
A thick leading process pointing towards the marginal zone and a thin trailing
process, the future axon, originating from the opposite pole of the cell body, are
characteristic features of a migrating neuron. Controlled actin turnover is likely to be
involved in this asymmetric cell differentiation, but is altered in neurons transfected
with cofilinS3E. In these cells, the leading and the trailing processes are equally thick
and poorly differentiated. CofilinS3E mimics phosphorylated cofilin (Moriyama et al.,
1996) and thus inhibits the supply of G-actin, which is essential for actin
polymerization. In conditional cofilin knockout mice layer II and III of the cerebral
cortex are missing, implying migration defects of the neurons destined to these layers
(Bellenchi et al., 2007). We conclude that the severing activity of cofilin and
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remodeling of the actin cytoskeleton, respectively, are required for the differentiation
of asymmetric cell polarity, process extension and motility of migrating neurons.
The present results point to an important role of cofilin and its coordinated
regulation by Reelin-dependent phosphorylation during the directed migration of
cortical neurons. Further studies will be necessary to specify the involvement of the
Reelin receptors ApoER2 and VLDLR, which were found previously to have specific
functional roles (Zhao et al., 2006; Hack et al., 2007; Zhao and Frotscher 2010) and
distinct spatiotemporal expression patterns (Hirota et al., 2014).
Materials and Methods
Animals
Pregnant wild-type mice (C57/BI/6J) (n=50) and heterozygous reeler mutants
(C57/BI/6J-reln) (n=12) were purchased from the Jackson Laboratory (Bar Harbour,
ME, USA). Animals were bred in the Experimental Animal Center of the University
Medical Center Hamburg-Eppendorf. All animals were maintained in accordance with
the institutional guidelines of the University of Hamburg (License No.: 48/13). Care
was taken to minimize suffering of the animals during surgical procedures. The day
on which the vaginal plug was detected was designated as embryonic day 0.5 (E0.5).
The first neonatal day was considered to be postnatal day 0 (P0). Reeler genotypes
were confirmed by PCR analysis of genomic DNA and immunostaining for Reelin
(Deller et al. 1999; Chai et al., 2009).
Plasmid construction
CofilinWT was generated by PCR from a mouse cofilin cDNA library (Genbank
accession No. NM_007687) using the forward primer: 5'-CCG GAA TTC GCC ACC
ATG GCC TCT GGT GTG GCT GTC-3' and reverse primer: 5'-GCG GGA TCC CCC
AAA GGC TTG CCC TCC AGG-3. Two cofilin mutants were generated that mimic
either the dephosphorylated (constitutively active) or phosphorylated (dominant
negative) form by changing Ser3 to alanine (cofilinS3A) or Ser3 to aspartate
(cofilinS3E), respectively. Wild-type LIMK1 was generated by PCR from a mouse
LIMK1 cDNA library (Genbank accession No. NM-010717) using the forward primer:
5'- GTC GAC GCC ACC ATG AGG TTG ACG CTA CTT TGT TGC A -3' and reverse
primer: 5'- AGA TCT GTC AGG GAC CTC GGG G -3’. The resulting fragments were
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cloned in frame into the vector pCAG-GFP, a mammalian expression vector driven by
the chicken actin promoter (Plasmid 11150, Addgene, Cambridge, UK; Niwa et al.,
1991). The constructs were further tested by sequence analysis and enzyme
restriction. The resulting plasmids were then purified with Endo-free maxi prep kit
from QIAGEN (Hilden, Germany).
In utero electroporation
In utero electroporation (IUE) was performed as described previously (Saito and
Nakatsuji, 2001; Tabata and Nakajima, 2001). Briefly, timed pregnant mice were
deeply anesthetized with isoflurane (Abbott, Wiesbaden, Germany) at embryonic day
(E) 14.5 (wild-type mice n=50; reeler mice n=12) and then firmly fixed on a heating
matte. After unhairing and disinfecting the abdomen with iodine tincture, a 3 cm
midline laparotomy was performed, and the uterus was exposed. Plasmids were
dissolved in phosphate-buffered saline (PBS; pH 7.4) at a concentration of 1 µg/µl.
Fast Green solution (0.1%) was added to the plasmid solution in a ratio of 1:10 to
monitor the injection. Approximately 1 µl of the plasmid solution was injected into one
of the lateral ventricles with a glass micropipette made from a microcapillary tube
(GB100TF-10; Science Products, Hofheim, Germany), which was connected to an
aspirator tube (Sigma-Aldrich, Taufkirchen, Germany). The heads of embryos in the
uterus were placed between the 7-mm platinum tweezers electrodes (Model 520;
Harvard Apparatus, AHN Biotechnologie, Nordhausen, Germany). Electronic pulses
(30 V, 50 ms) were given five times at intervals of 950 ms with an ECM830 BTX
square wave electroporator (ECM 830 BTX; Harvard Apparatus). The uterine horns
were put back into the original location. The abdominal wall and skin were sewed up
with surgical sutures, and the embryos were allowed to continue development.
Plasmid pCAG-GFP was injected into the lateral ventricles of wildtype embryos as a
control. Alternatively, the different n-cofilin constructs (cofilinWT, cofilinS3A, and
cofilinS3E) were injected. Reeler mutants were injected either with pCAG-GFP alone,
with cofilinS3E or LIMK1. After electroporation, the uterine horns were put back to their
original location. Dams were sacrificed and the brains of the embryos used for the
preparation of slice cultures (see below). Alternatively, 2, 3 or 5 days after IUE brains
were fixed in 4% paraformaldehyde (PFA) and sectioned transversally into 50 μm-
thick sections.
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Preparation of embryonic cortical slice cultures
Two or three days after electroporation, pregnant mice at E16.5 (wild-type mice n =
16; heterozygous reeler mice n = 2) and E17.5 (wild-type mice n = 24; heterozygous
reeler mice n = 4), respectively, were decapitated under hypothermic anesthesia. The
embryos at E16.5 (for each cofilin construct and control plasmid: n = 24; for reeler n =
5) and at E17.5 (for each cofilin construct and control plasmid n = 36; for reeler mice
n = 14) were collected and rapidly placed in ice-cold Hank’s balanced salt solution
(HBSS, Invitrogen, Darmstadt, Germany). The brains were dissected from the skull
and checked under a fluorescence microscope.
GFP-positive cerebral cortices were then dissected and sliced (300 μm)
perpendicularly to the longitudinal axis of the cerebral cortex using a McIlwain tissue
chopper. The slices were placed onto culture inserts (Millipore, Schwalbach,
Germany) and transferred to 6-well plates with 1 mL/well nutrition medium containing
25% heat-inactivated horse serum, 25% Hank’s balanced salt solution, 50% minimal
essential medium, and 2 mM glutamine (pH 7.2; Invitrogen), and incubated in 5%
CO2 at 37°C at least for 3 hours. After recovery the culture inserts were put into Petri
dishes (30 mm diameter) with a glass bottom containing fresh medium and then
transferred for live- imaging.
Live imaging of slice cultures
For live imaging of slice cultures, an Improvision confocal spinning-disc microscope
(Zeiss, Jena, Germany) and 20x air immersion objective were used to acquire z-
series of cortical slices at 5 µm intervals through a tissue depth of about 20 µm. The
z–series were then visualized as single optical scans with concurrent orthogonal
views using Volocity6 software (Perkin Elmer, Waltham, Mass., USA). The time
interval was 10 minutes. Duration of imaging was up to 15 hours. This microscope
was equipped with a chamber for the control of temperature, humidity and CO2.
Results of time-lapse imaging of the slice cultures were analyzed by using Volocity6
software, and the average speed of neuronal cell migration was measured by using
Imaris software. A total of 50 GFP-expressing cells were collected from 3 videos of
each different plasmid transfection and stage (two and three days after IUE). Only
neurons were included whose migration could be tracked for a substantial distance.
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In these cells the average speed of movement and the direction of migration (towards
the marginal zone or ventricular zone) were measured in the x- and y-axis.
Dissection of brains for histology
Two, three and five days after IUE brains were prepared for histological analysis. Of
the embryos transfected with the different cofilin constructs at least 3 per different
construct and stage and 3 GFP-transfected reeler brains were used. Three reeler
mice each were used for the rescue experiments with cofilinS3E and LIMK1,
respectively, three days after IUE on E14.5. Two and three days after IUE pregnant
wild-type mice and heterozygous reeler mutants were anesthetized with isoflurane
under hypothermic anesthesia and the embryos dissected in ice-cold HBSS. Five
days after IUE, corresponding to the day of birth, newborn mice were sacrificed by
cervical dislocation under hypothermia and the brains dissected and rapidly placed in
HBSS. Brains were viewed under a fluorescence microscope to verify transfection.
GFP-positive hemispheres were fixed in 4% PFA overnight at 4°C. After washing in
0.1M PBS at room temperature (RT) for several hours, brains were embedded in 5%
agar and cut transversally into 50 µm-thick slices on a LeicaVT 1000S vibratome
(Leica Microsystems, Frankfurt, Germany) and the sections were placed in a 24-well
plate containing 0.1 M PBS, counterstained with propidium iodide (PI, Sigma-Aldrich),
and mounted in Moviol on glass slides. Sections were photographed using a confocal
laser scanning microscope (Leica TCS SP5) and 20x air or 63x oil immersion
objectives. Z-series of brain sections at 0.5 μm intervals through a tissue depth of 9
μm were acquired and visualized as single optical scans with concurrent orthogonal
views using Leica LAS software (Leica).
Quantitative assessments
In fixed slices, all GFP-positive neurons in the different zones of the cortex were
counted. Two days after IUE, cells were counted in CP, IZ, and SVZ/VZ (n = 3
sections from 3 different mice). Three days after IUE, when the cortex had increased
in thickness, the cortical plate was subdivided into three portions, an upper CP
(UCP), middle CP (MCP), and lower CP (LCP) (n = 3 sections from 3 different mice).
Five days after IUE, cells were counted in the UCP, IZ and SVZ/VZ (n = 3 slices from
3 different mice). Cell counts were performed using ImageJ software.
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In 50-100 neurons from each transfected group of cells the lengths of the
leading processes two days and three days after IUE were measured using iTEM
software (Zeiss). In addition, two days after IUE the percentage of migrating neurons
giving rise to more than two leading processes, and three days after IUE the
percentage of GFP-positive neurons with leading processes reaching the marginal
zone was determined.
Results were documented using excel software and presented as mean ±
s.e.m. Differences between groups were tested for statistical significance (1-way
ANOVA with Tukey's multiple comparison test, *p < 0.05; **p < 0.01; *** p < 0.001).
Acknowledgments
The authors thank Dr. Froylan Calderon de Anda for supplying the ECM830 BTX
electroporator.
Competing interests
The authors declare that they have no conflict of interest.
Author Contributions
X.C., S.Z. and M.F. developed the concept of the study, X.C., L.F., W.Z., X.L., L.S.
and H.S. performed the experiments, S.W., A.V.F. and B.Z. helped with live imaging,
H.G.M. provided plasmids, and X.C. and M.F. wrote the manuscript.
Funding
This work was supported by grants from the Deutsche Forschungsgemeinschaft (FR
620/12-2 and FR 620/14-1 to M.F.) and the National Natural Science Foundation of
China (No. 31071873 to S.Z.). Michael Frotscher is Senior Research Professor of the
Hertie Foundation.
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Figures
Figure 1. Migration of cortical neurons two days following IUE) on E.14.5. (A-E)
In control sections, sections transfected with cofilinWT, cofilinS3A or cofilinS3E and in
sections from reeler mutants most neurons are still located in the IZ and SVZ/VZ.
PI, propidium iodide (red) to label cell nuclei. Scale bar (A-E): 100 µm.
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Figure 2. Quantitative analysis of neuron numbers in CP, IZ and SVZ/VZ two
days after IUE. (A) Percentage of GFP-labeled neurons in CP, IZ, and SVZ/VZ in
controls, sections transfected with the different cofilin constructs, and in sections from
reeler mice. (B-D) Statistical analysis of the percentage of neurons in CP (B), IZ (C),
and SVZ/VZ (D). Mean ± s.e.m.; two-tailed Student’s t-test; ns, not significant; *p <
0.05.
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Figure 3. Morphological characteristics of transfected neurons in the IZ two
days after IUE. (A) In control slices, most transfected neurons show a thick leading
process (white arrows) oriented towards the MZ. (B) Neurons in cofilinWT-transfected
slices have very thin and long leading processes (white arrows). (C) CofilinS3A-
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transfected cells give rise to supernumerary, branching processes (white arrows). (D)
CofilinS3E-transfected cells show varicose short processes with filopodial extensions
(white arrows). (E) Reeler neurons display thick, vertically oriented leading processes
similar to control cells (white arrows). (A-E) Sections counterstained with PI. Scale
bar: 50 µm.
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Figure 4. Quantitative assessment of structural characteristics and migratory
behavior of transfected neurons two days after IUE. (A) Lengths of leading
processes. (B) Percentage of neurons with more than two leading processes. (C)
Migratory speed of migrating neurons. (D) Migratory directions of neurons in the
different groups. Mean ± s.e.m; n = 50 cells for each experimental condition; two-
tailed Student’s t-test; ns, not significant. *p < 0.05; ***p < 0.001.
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Figure 5. Migration of cortical neurons three days after IUE. (A, B) In control
sections and cofilinWT-transfected sections, many neurons have entered the CP but
do not yet form a compact cell layer. (C, D) In cofilinS3A-transfected slices and in
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slices transfected with cofilinS3E, the majority of neurons are still observed in the IZ.
(E) In reeler slices very few labeled neurons have entered the CP. (F, G) In contrast,
in reeler slices transfected with cofilinS3E (F) or LIMK1 (G) significantly more neurons
were found in the CP. (A-G) Sections counterstained with PI. Scale bar: 100 µm.
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Figure 6. Quantitative analysis of neuron numbers in CP, IZ and SVZ/VZ three
days after IUE. (A) Percentage of GFP-labeled neurons in the upper, middle, and
lower portion of the CP (UCP, MCP, LCP), IZ, and SVZ/VZ in controls, sections
transfected with the different cofilin constructs, in sections from reeler mice, and in
reeler sections transfected with cofilinS3E or LIMK1. (B-D) Statistical analysis of the
percentage of neurons in UCP (B), MCP (C), LCP (D), IZ (E), and SVZ/VZ (F). Mean
± s.e.m.; two-tailed Student’s t-test; ns, not significant; *p < 0.05; **p < 0.01; ***p <
0.001.
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Figure 7. Migratory behavior of neurons transfected with the different
constructs three days after IUE. (A-G) Individual neurons monitored over a period
of 200 minutes (selected neurons from Movies 1-6). Red arrowheads label cell
bodies, white arrows leading processes. MZ is at the top of the figures, VZ at the
bottom. Scale bar: 15 µm.
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Figure 8. Quantitative assessment of structural characteristics and migratory
behavior of transfected neurons three days after IUE. (A) Lengths of leading
processes. (B) Percentage of neurons with leading processes reaching MZ. (C)
Migratory speed of migrating neurons. (D) Migratory directions of neurons in the
different groups. Mean ± s.e.m; n = 50 cells for each experimental condition; two-
tailed Student’s t-test; ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001.
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Figure 9. Migration of cortical neurons five days after IUE. (A) In control slices,
most neurons have arrived at their destination and form a compact cell layer (white
arrows). (B) In cofilinWT-transfected slices, labeled neurons were capable of forming a
cell layer near the MZ (white arrows), but there were still many cells in the deeper
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layers. (C) In cofilinS3A-transfected slices, almost all GFP-positive neurons were
found in the deep layers. (D) In cofilinS3E-transfected slices, most neurons were
observed in deep cortical layers but a few cells have migrated into the CP. (E) In
reeler slices, the neurons are scattered over the CP, often oriented towards the VZ.
Very few cells are seen in upper cortical layers. Sections counterstained with PI.
Scale bar (A-E): 100 µm.
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Figure 10. Quantitative analysis of neuron numbers in UCP, IZ and SVZ/VZ five
days after IUE. (A) Percentage of GFP-labeled neurons in UCP, IZ, and SVZ/VZ in
controls, sections transfected with the different cofilin constructs, and in sections from
reeler mice. (B-D) Statistical analysis of the percentage of neurons in the UCP (B), IZ
(C), and SVZ/VZ (D). Mean ± s.e.m.; two-tailed Student’s t-test; ns, not significant;
**p < 0.01; ***p < 0.001.