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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: michael.frotscher@zmnh.uni-hamburg.de 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.