Abstract

Newborn neurons migrate along the processes of radial glial cells (RGCs) to reach their final positions in the cortex. Here, we visualized individual migrating neurons and RGCs using in utero electroporation. We show that branching of migrating neurons and RGCs is closely correlated spatiotemporally with the distribution of Reelin. Time-lapse imaging revealed that the leading processes of migrating neurons gave rise to increasingly more branches once their growth cones contacted the Reelin-containing marginal zone. This was accompanied by translocation of the nucleus and gradual shortening of the leading process. Absence of Reelin in reeler mice altered these processes resulting in misorientation, loss of bipolarity, and aberrant migration of cortical neurons. Moreover, in reeler, the branching of the basal processes of RGCs in the marginal zone was severely disrupted. Consistent with previous reports, we show that in dissociated reeler cortical cultures, exposure to recombinant Reelin enhanced dendritic complexity and glial branching. Our results suggest that Reelin induces branching of the leading processes of migrating neurons and that of basal processes of RGCs when they arrive at the Reelin-containing marginal zone. Branching of these processes may be crucial for the termination of nuclear translocation during the migratory process and for correct neuronal positioning.

Introduction

The mammalian cerebral cortex consists of 6 well-organized layers extending from the pial surface to the white matter. The formation of this highly ordered structure requires the proper migration of postmitotic neurons from their sites of origin to their final destinations (Rakic 1988; Walsh and Goffinet 2000; Nadarajah and Parnavelas 2002). Disruption of neuronal migration leads to malformation of the cerebral architecture and to functional deficits (Gleeson and Walsh 2000; Ayala et al. 2007). Previous studies demonstrated that neuronal migration is precisely orchestrated by various intrinsic and extrinsic factors. Reelin is one of the crucial molecules involved in neuronal migration and cortical lamination.

Reelin is an extracellular matrix protein secreted by Cajal–Retzius (CR) cells located in the marginal zone (MZ) of the developing forebrain (D'Arcangelo et al. 1995, 1997). Absence of Reelin results in migration defects of many, but not all, neurons and in altered layer formation in laminated brain structures, such as the cerebral cortex, hippocampus, and cerebellum (Caviness and Rakic 1978; Del Rio et al. 1997; Curran and D'Arcangelo 1998; Frotscher 1998). In the Reelin-deficient mouse mutant reeler, many neurons show abnormal polarity orientation and dendritic abnormalities as well as malformation of the radial glial scaffold (Caviness and Sidman 1973; Goffinet and Lyon 1979; Frotscher et al. 2003; Hartfuss et al. 2003; Tissir and Goffinet 2003; Jossin 2004; Herz and Chen 2006; Frotscher 2010). Reelin exerts its function by binding to two lipoprotein receptors, very low-density lipoprotein receptor (VLDLR) and apolipoprotein-E receptor type 2 (ApoER2) (D'Arcangelo et al. 1999; Hiesberger et al. 1999; Trommsdorff et al. 1999). Binding of Reelin to these two receptors induces phosphorylation of the intracellular adaptor protein Disabled1 (Dab1) (Howell et al. 1997, 1999; Sheldon et al. 1997; Ware et al. 1997; Trommsdorff et al. 1999) by Src-family kinases (SFKs), particularly Src and Fyn (Arnaud et al. 2003; Bock and Herz 2003). Various signaling pathways downstream of Dab1 connect Reelin to the dynamics of the actin- and microtubule cytoskeleton (D'Arcangelo 2006; Chai et al. 2009; Förster et al. 2010; Frotscher 2010; Zhao and Frotscher 2010). Mutant mice deficient in Dab1 or in both VLDLR and ApoER2 show a reeler-like phenotype (Pinto Lord and Caviness 1979; Howell et al. 1997; Sheldon et al. 1997; Trommsdorff et al. 1999). Recent in vitro studies reported Reelin-induced branching of radial glial cell (RGC) processes and that of neuronal dendrites (Niu et al. 2004, 2008; Jossin and Goffinet 2007; Matsuki et al. 2008). Although these studies have demonstrated that Reelin is closely associated with neuronal migration and dendritic development, little is known about the underlying molecular and cellular mechanisms in space and time.

Using timed in utero electroporation in combination with immunocytochemistry for Reelin and time-lapse imaging, we demonstrate that intensive branching of migrating neurons and RGCs was closely correlated spatiotemporally with the distribution of endogenous Reelin. Our data also show that dendritic arbor complexity is inversely correlated with the length of the leading process of migrating neurons in their “terminal translocation” stage. In reeler mice, cortical neurons were unable to reach their defined destination and ectopically stopped the migratory process in the deep layers of the cortical plate (CP). Many of them lost their characteristic bipolarity and were misoriented. Moreover, branching of RGCs was strikingly impaired in reeler mutant mice leading to a dramatic reduction in the number of basal branches and end-feet. We hypothesize that Reelin promotes the branching of basal RGC processes, resulting in the stabilization of the RGC cytoskeleton, thus providing an appropriate template for migrating neurons. By inducing the branching of the leading processes of neurons, Reelin may contribute to anchoring these processes to the MZ matrix, which maintains neuronal bipolarity and orientation. Branching of the leading processes, in turn, may terminate the final phase of neuronal migration by nuclear translocation.

Materials and Methods

Animals

Reeler mice were purchased from the Jackson Laboratory (Bar Harbour). POMC-EGFP transgenic mice were generously provided by Dr. G. L. Westbrook (Vollum Institute). The animals were bred in the Experimental Animal Center of Lanzhou University and in the Experimental Animal Center of ZMNH. The animals were reared on a normal 12 h light/dark schedule. Noon of the date on which the vaginal plug was detected in the morning was designated as embryonic day 0.5 (E0.5). The first neonatal day was considered to be postnatal day 0 (P0). All experiments were carried out in accordance with the principles and guidelines of the Ethics Committees of Lanzhou University and Hamburg University. The genotype of the mutants was confirmed by PCR analysis of genomic DNA, as described (Deller et al. 1999; Overstreet et al. 2004).

Plasmids

The plasmid used in the present study, pCAG-GFP (Matsuda and Cepko 2004), was purchased from Addgene (Plasmid 11150, Addgene), which contains CAG (cytomegalovirus immediate-early enhancer/chicken β-actin hybrid) promoter (Niwa et al. 1991). The plasmid was purified using a Qiagen plasmid maxi kit.

In Utero Electroporation

In utero electroporation was performed according to the procedure described elsewhere previously (Tabata and Nakajima 2001), with slight modifications. Timed-pregnant females were deeply anesthetized by intraperitoneal injection of chloral hydrate (4.3 mg per 10 g of body weight). Then, the uterine horns of the pregnant mice were carefully exposed via a midline abdominal incision by pinching gaps between embryos with ring-forceps. 1–1.5 μL of plasmid DNA solution in normal saline was injected into the lateral ventricle of mouse embryos using a mouth-controlled pipette system and a pulled-glass micropipette. Fast Green solution (0.1%) was added to the plasmid solution in a ratio of 1 : 10 to monitor the injection. The heads of injected embryos in the uterus were placed between the tweezers-type electrodes. Electronic pulses (30–50 V; 50 ms) were charged 5 times at intervals of 900 ms with an electroporator (Gene Pulser Xcell Electroporation System). The uterine horns were placed back into the abdominal cavity to allow the embryos to continue normal development until the desired time of observation.

Slice Co-Culture and Live Imaging

Slice co-cultures were prepared as described previously (Zhao et al. 2004). To visualize individual granule cells in the reeler dentate gyrus (DG) for time-lapse imaging, POMC-EGFP transgenic mice, in which newborn granule cells in DG express GFP under the POMC promoter (Overstreet et al. 2004), were crossbred with reeler mice. For the preparation of co-cultures, newborn POMC-EGFP transgenic reeler mouse pups (P0) and young postnatal wild-type mice (P7) were used. Brains were removed following decapitation under hypothermic anesthesia. The hippocampi were dissected and sliced (300 μm) perpendicular to their longitudinal axis with a McIlwain tissue chopper. Hippocampal slices of newborn POMC-EGFP transgenic reeler mice were positioned in close vicinity to the outer molecular layer of the DG of slices from P7 wild-type mice so that the DG of the reeler slice came in direct contact with the Reelin-containing MZ of the wild-type slice. Slices were placed onto Millipore membranes and transferred to a 6-well plate with 1 mL/well nutrition medium (25% heat-inactivated horse serum, 25% Hank's balanced salt solution, 50% minimal essential medium, 2 mm glutamine, pH 7.2). Slices were first put into an incubator containing 5% CO2 at 37°C for 6 to 8 h to allow for recovery of the slices from injury during preparation. Then, the slices were transferred to a small incubator for live imaging using a confocal microscope (Improvision LiveCell Spinning Disk, PerkinElmer). The incubator was supplied with 5% CO2 and heated to 37°C. Time-lapse images were collected every 7 min for more than 12 h. Following imaging, the cultures were immediately fixed with 4% paraformaldehyde (PFA) at 4°C overnight. After rinsing in 0.1 m PB, immunostaining for Reelin and GFP was performed in the fixed cultures. Then, the cultures were counterstained with DAPI and mounted in Moviol. Images were acquired using a Zeiss LSM 510 confocal microscope.

Production of Recombinant Reelin

Preparation of Reelin-containing supernatants and control supernatants was performed as described previously (Förster et al. 2002; Zhao et al. 2004). In brief, stably transfected HEK 293-cells expressing either full-length Reelin or control cells transfected with a control vector (MOCK) were grown in Dulbecco's modified eagle medium (DMEM, low glucose, Invitrogen) with 10% fetal bovine serum, low glucose (Invitrogen), 100 U/mL penicillin, and 100 μg/mL streptomycin (Invitrogen), and 0.9 g/L G418 (Invitrogen) for 2 days to reach full confluence. The medium was then replaced by serum-free DMEM, and the cells were incubated for 2 days at 37°C in 5% CO2. Conditioned medium was collected and centrifuged. The supernatant was concentrated 10-fold using 100-kDa cut-off centrifugal filters (Millipore), sterile filtered, and stored at −80°C until used.

Neuronal Culture and Treatment with Recombinant Reelin

Dissociated cultures of neocortex were prepared as described previously (Chai et al. 2009). For primary cell cultures, 6 brains of E17.5 reeler mutants were used. The neocortex was dissected and put into ice-cold Hanks' Balanced Salt Solution (HBSS, Invitrogen). After removal of the meninges, the neocortex were collected and maintained in 15 mL Falcon tubes (BD Biosciences) containing 3 mL of 0.5% Trypsin and 0.53 mM EDTAx4Na (Invitrogen) and incubated at 37°C for 10 min. The neocortex was then washed twice in ice-cold HBSS and manually triturated with a polished glass Pasteur pipet for several times. Then, the cells were centrifuged at low speed (800g) for 3 min to discard dead cells. The sediment was collected in fresh Falcon tubes and centrifuged again. The supernatant was discarded, and cell pellets were resuspended in 1 mL of Neurobasal-A medium (Invitrogen), supplemented with 2% B27 (Invitrogen), 1 mM Glutamax (Invitrogen), 100 U/mL penicillin, and 100 μg/mL streptomycin (Invitrogen). Cortical cells were counted using a hemocytometer and suspended at a density of 5 × 105 cells in 0.5 mL aliquots on glass coverslips inserted in 24-well plates. The coverslips had been coated overnight with a solution of 20 μg/mL poly-l-ornithine (Sigma) prepared in borate buffer, pH 8.1. After 2 h of incubation in a humidified incubator at 37°C and 5% CO2 to allow for cell attachment, the medium was changed to remove dead cells. Then, 500 μL of Reelin-containing supernatant or control supernatant was added to medium. After another 24 h of incubation, cultures were fixed with 4% PFA in 0.1 m PBS, pH 7.4, for 15 min at room temperature (RT) and rinsed in 0.1 m PBS for 1 h.

Immunocytochemistry

After permeabilization and preincubation with blocking solution (5% normal goat serum and 1% bovine serum albumin in 0.1 m PB containing 0.2% Triton X-100) at RT for 1 h, the cultures were immunostained with primary antibodies against Microtubule-associated protein 2 (MAP2) and brain lipid-binding protein (BLBP) at 4°C overnight and subsequently with Alexa Fluor-488-conjugated secondary antibodies at RT for 2 h. After rinsing in 0.1 m PB for 2 h, cells were mounted in Moviol on glass slides.

The transfected brains were harvested and fixed by immersion in 4% PFA at different time points after electroporation. The fixed brains were coronally cut for 50 μm with a vibratome (Leica, VT1000S). Free-floating sections were blocked with 5% normal goat serum in 0.1 m PB for 30 min. The sections were then incubated with the solutions containing primary antibodies: rabbit anti-green fluorescent protein (GFP; 1 : 1000; Invitrogen), mouse anti-Reelin G10 (1 : 1000; Chemicon), rabbit anti-BLBP (1 : 500; Chemicon), mouse anti-Nestin (1 : 1000; Chemicon), and goat anti-doublecortin (DCX; 1 : 500; Santa Cruz Biotechnology) in 0.1 m PB overnight at 4°C. After washing in 0.1 m PB, the sections were incubated with the secondary antibodies: goat anti-rabbit Alexa Fluor 488 or 568, goat anti-mouse Alexa Fluor 568, and donkey anti-goat Alexa Fluor 568 (1 : 300, Molecular Probes) overnight at 4°C. After rinsing in 0.1 m PB for 2 h, the nuclei in some sections were labeled with propidium iodide (PI; 2.5 mg/mL; Invitrogen) or 4′, 6-diamidino-2-phenylindole (DAPI; 1 : 10 000; Roche Molecular Biochemicals). The sections were mounted in Moviol. Images were acquired using a Zeiss LSM 510 confocal microscope.

Morphometric Analysis

To analyze the morphological features of GFP-positive cells, optical z-stacks were acquired at 1-μm intervals in sections using a Zeiss LSM 510 confocal microscope with a 63×, 1.4 numerical aperture objective. These z-series were projected into a single plane (flattened) using maximum pixel intensity algorithm by the Zeiss ZEN 2010 software (Carl Zeiss). GFP-positive neurons and RGCs were traced using Neuromantic V1.6.3 software (http://www.rdg.ac.uk/neuromantic). The length of the leading processes and branches of neurons and RGCs were measured using ImageJ software (National Institutes of Health). For dendritic morphology analysis, 2 parameters were analyzed: total length of branches and number of bifurcations. A primary branch was defined as the length of any GFP-labeled structure originating from the leading process that is longer than 2.5 μm. Thus, small unstable filopodia shorter than 2.5 μm protruding from the leading processes of migrating neurons were excluded. The length of the residual leading process is measured from its origin at the soma to the first branching point.

For analysis of the cell culture experiments, the processes of MAP2-positive neurons and BLBP-positive glial cells were quantified with SIS software using images acquired on an epifluorescence microscope with a 60× objective lens (BZ-9000). A total of 200 neurons and 100 glial cells of each condition were measured. Student's t-test was used for statistical analysis. Results were presented as mean ± SD; P < 0.05 was considered to be statistically significant.

Results

Defined Cohorts of Neurons by GFP Labeling using Timed In Utero Electroporation

In the brains of wild-type mice transfected at E14.5 and fixed at E17.5, numerous GFP-positive cells were observed throughout the different layers of the developing cortex (Fig. 1A). The GFP-positive cells in the CP showed the typical bipolar morphology of migrating neurons with a thick leading process pointing toward the pial surface and a thin trailing process directed to the ventricle (Fig. 1A inset). Some GFP-positive cells already reached the upper portion of the CP, and their leading processes approached or invaded the MZ (Fig. 1A). At 5 days after electroporation, the somata of the majority of GFP-positive cells accumulated immediately beneath the MZ by forming a dense layer. They gave rise to primitive stem dendrites invading the MZ (Fig. 1B). At P5, 10 days after transfection, most GFP-positive cells showed the typical morphology of differentiated neurons with an apical dendritic tuft in the MZ (Fig. 1C).

Figure 1.

GFP-labeled neurons in the cerebral cortex transfected using in utero electroporation. (A) Numerous GFP-positive cells (green), transfected with pCAG-GFP at E14.5 and fixed at E17.5, are observed in the different cortical layers. Sections were counterstained with propidium iodide (PI, red). The inset shows a representative labeled cell with bipolar morphology, oval soma (black asterisk), a long, thick leading process (white arrow) oriented to the pial surface, and a short, thin trailing process (black arrow) extending toward the ventricle. (B) Cerebral cortex following electroporation with GFP at E14.5 and fixation at P0. The bulk of the GFP-labeled cells (green) have traversed the CP and accumulated underneath the MZ. (C) Cerebral cortex transfected with pCAG-GFP at E14.5 and fixed at P5. The GFP-labeled cells form a tight band and give rise to abundant branches. (D) Transfected cortices were double-immunostained with antibodies against GFP (green) and doublecortin (DCX, red) and counterstained with DAPI (blue). The cytoplasm (arrows) and leading process (arrowheads) of the GFP-labeled cell are DCX-positive, whereas the cell nucleus is DCX-negative (white asterisks). MZ, marginal zone; CP, cortical plate; IZ, intermediate zone; VZ, ventricular zone. Scale bars: 200 μm (A), 100 μm (B, C), and 15 μm (D).

Figure 1.

GFP-labeled neurons in the cerebral cortex transfected using in utero electroporation. (A) Numerous GFP-positive cells (green), transfected with pCAG-GFP at E14.5 and fixed at E17.5, are observed in the different cortical layers. Sections were counterstained with propidium iodide (PI, red). The inset shows a representative labeled cell with bipolar morphology, oval soma (black asterisk), a long, thick leading process (white arrow) oriented to the pial surface, and a short, thin trailing process (black arrow) extending toward the ventricle. (B) Cerebral cortex following electroporation with GFP at E14.5 and fixation at P0. The bulk of the GFP-labeled cells (green) have traversed the CP and accumulated underneath the MZ. (C) Cerebral cortex transfected with pCAG-GFP at E14.5 and fixed at P5. The GFP-labeled cells form a tight band and give rise to abundant branches. (D) Transfected cortices were double-immunostained with antibodies against GFP (green) and doublecortin (DCX, red) and counterstained with DAPI (blue). The cytoplasm (arrows) and leading process (arrowheads) of the GFP-labeled cell are DCX-positive, whereas the cell nucleus is DCX-negative (white asterisks). MZ, marginal zone; CP, cortical plate; IZ, intermediate zone; VZ, ventricular zone. Scale bars: 200 μm (A), 100 μm (B, C), and 15 μm (D).

The morphology of these GFP-labeled cells indicated that they were neurons. To prove this possibility, immunostaining for doublecortin (DCX), a microtubule-associated protein being generally recognized as a marker for postmitotic migrating neurons (Francis et al. 1999; Gleeson et al. 1999), was performed in the transfected cortex. Our results showed that the majority of GFP-labeled cells in the CP, fixed at 3 days after electroporation, were DCX-positive (Fig. 1D), indicating that cells in brain sections transfected with pCAG-GFP at E14.5 and fixed 3 days or later were indeed neurons.

RGCs Were GFP-Labeled using In Utero Electroporation

In the brains of wild-type mice transfected at E17.5 and fixed at E18.5, numerous GFP-positive cells with long processes were observed in the cerebral cortex (Fig. 2A). They showed a typical bipolar radial glial morphology, characterized by a long ascending process spanning the whole developing cortical wall up to the pial surface and a short apical process contacting the ventricle and terminating by an end-foot. The oval somata of these GFP-positive cells were located in the VZ (Fig. 2B). Within the IZ and CP, the thin ascending processes of the GFP-labeled cells showed a straight course toward the MZ without branching. In contrast, upon reaching the MZ, the ascending processes of the GFP-labeled cells branched extensively and formed a distinct end-foot at the tip of each branch (Fig. 2BD).

Figure 2.

The majority of GFP-labeled cells transfected at E17.5 are RGCs. Embryonic brains were electroporated at E17.5 with GFP-plasmid and fixed at E18.5. (A) Sections stained with an antibody against GFP (green) and counterstained with PI (red) to reveal cortical organization. Numerous cells located in the VZ are strongly labeled for GFP; some of them extending parallel-oriented radial fibers (filled arrows) traversing the entire CP. (B) Higher magnifications reveal that the GFP-labeled cells exhibit a bipolar shape with an oval soma located in the VZ (black asterisk). Their short apical process (B, open arrow) contacts the ventricular surface and terminates by an end-foot (open arrowhead). Their basal processes are very thin (AD, white filled arrows) and extend from the VZ to the MZ, in which they branch extensively and form a distinct end-foot at the tip of each branch (BD, white filled arrowheads). (C, D) Double immunostaining shows that the orderly arrayed processes visualized with GFP (green) contain the radial glial markers (red) BLBP (C) and Nestin (D). Scale bars: 100 μm (A) and 20 μm (B–D).

Figure 2.

The majority of GFP-labeled cells transfected at E17.5 are RGCs. Embryonic brains were electroporated at E17.5 with GFP-plasmid and fixed at E18.5. (A) Sections stained with an antibody against GFP (green) and counterstained with PI (red) to reveal cortical organization. Numerous cells located in the VZ are strongly labeled for GFP; some of them extending parallel-oriented radial fibers (filled arrows) traversing the entire CP. (B) Higher magnifications reveal that the GFP-labeled cells exhibit a bipolar shape with an oval soma located in the VZ (black asterisk). Their short apical process (B, open arrow) contacts the ventricular surface and terminates by an end-foot (open arrowhead). Their basal processes are very thin (AD, white filled arrows) and extend from the VZ to the MZ, in which they branch extensively and form a distinct end-foot at the tip of each branch (BD, white filled arrowheads). (C, D) Double immunostaining shows that the orderly arrayed processes visualized with GFP (green) contain the radial glial markers (red) BLBP (C) and Nestin (D). Scale bars: 100 μm (A) and 20 μm (B–D).

To confirm the radial glial nature of these GFP-labeled cells, we performed immunocytochemistry to detect the expression of 2 markers for RGCs: BLBP (Feng et al. 1994) and Nestin (Lendahl et al. 1990). Our results showed that the majority of GFP-labeled cells in brains transfected at E17.5 were BLBP-positive (Fig. 2C) and Nestin-positive (Fig. 2D), indicating that they were indeed RGCs.

Reelin Promotes Branching of Neurons and Dendritic Development In Vivo

It has been demonstrated previously that migrating neurons change their migratory behavior from glia-guided locomotion to somal translocation upon contact of the leading process with the CP–MZ boundary. In this way, cortical neurons complete their migration (Nadarajah et al. 2001). To examine Reelin's function in this process, we classified the transfected neurons in wild-type cortex by their positions with respect to the CP and MZ: 1) migrating neurons with both somata and leading processes within the CP (Fig. 3A), 2) migrating neurons with their somata in the upper CP but their leading processes in contact with the Reelin-enriched MZ (Fig. 3B), and 3) neurons with their somata near the CP–MZ boundary (Fig. 3C). Drawings of representative neurons from each group are shown in Figure 3D.

Figure 3.

Spatial relationship between the Reelin-containing MZ and the locus of neuronal branching. Brains were transfected with pCAG-GFP at E14.5 and fixed at E17.5. The sections were double stained with antibodies against GFP (green) and Reelin (red) and counterstained with DAPI (blue). (A) Neurons still crawling in the CP uniformly adopted a typical bipolar morphology with a thick and long leading process directed toward the MZ, and a thin, short trailing process directed to the ventricular surface. (B) Neurons with leading processes approaching the Reelin-containing MZ develop V-shaped nascent branches (arrowheads). (C) Neurons with their somata contacting the CP–MZ boundary lose their leading processes while forming abundant ramifications that are surrounded by diffusely distributed Reelin in the MZ. (D) Drawings of the leading processes and branches of representative cortical neurons transfected with pCAG-GFP via in utero electroporation. (E) Inverse correlation of branch lengths and lengths of residual leading processes (n = 42 cells in this group, r = −0.700, P < 0.001). (F) There was a significant inverse correlation of the number of bifurcations and the lengths of residual leading processes (n = 39 cells in this group, r = −0.701, P < 0.001). Scale bars: 20 μm (AD).

Figure 3.

Spatial relationship between the Reelin-containing MZ and the locus of neuronal branching. Brains were transfected with pCAG-GFP at E14.5 and fixed at E17.5. The sections were double stained with antibodies against GFP (green) and Reelin (red) and counterstained with DAPI (blue). (A) Neurons still crawling in the CP uniformly adopted a typical bipolar morphology with a thick and long leading process directed toward the MZ, and a thin, short trailing process directed to the ventricular surface. (B) Neurons with leading processes approaching the Reelin-containing MZ develop V-shaped nascent branches (arrowheads). (C) Neurons with their somata contacting the CP–MZ boundary lose their leading processes while forming abundant ramifications that are surrounded by diffusely distributed Reelin in the MZ. (D) Drawings of the leading processes and branches of representative cortical neurons transfected with pCAG-GFP via in utero electroporation. (E) Inverse correlation of branch lengths and lengths of residual leading processes (n = 42 cells in this group, r = −0.700, P < 0.001). (F) There was a significant inverse correlation of the number of bifurcations and the lengths of residual leading processes (n = 39 cells in this group, r = −0.701, P < 0.001). Scale bars: 20 μm (AD).

We found significant differences in the branching pattern between neurons whose leading processes were in contact with the Reelin-rich MZ and those with their leading processes restricted to the CP. The leading processes of most neurons crawling through the CP displayed a straight orientation toward the MZ without bifurcation (Fig. 3A), whereas the leading processes of migrating neurons developed V-shaped bifurcations as soon as they contacted the Reelin-rich MZ (Fig. 3B). Dendritic arbors that maintained contact with Reelin became increasingly branched as their parent soma approached the MZ and the residual leading process became shorter. The somata of migrating neurons stopped at the top of the CP without entering the Reelin-rich MZ (Fig. 3C).

To quantify the morphological differences between neurons within the CP and those contacting the MZ, we measured the lengths of leading processes and their apical branches and counted the number of bifurcation points. The length of leading processes restricted to the CP was 65.73 ± 10.88 μm. The length of the residual leading process correlated negatively with the total length of all branches (n = 42 cells for this group, r = −0.700, P < 0.001; Fig. 3E) and the number of bifurcations (n = 39 cells for this group, r = −0.701, P < 0.001; Fig. 3F). In summary, the close spatiotemporal relationship of Reelin in the MZ and the initiation of branching suggest that it is Reelin that induces branching of the leading processes, thus contributing to the early development of the apical dendritic arbor.

Reelin Induces Branching of Leading Processes of Migrating Neurons in Slice Culture

As a next step, we wanted to visualize the Reelin-induced branching dynamics using real-time microscopy. For this purpose, slice co-cultures were performed, an experimental model in which the migration defect of granule cells in the reeler DG was rescued by Reelin from a co-cultured wild-type hippocampus (Zhao et al. 2004). To visualize the granule cells in the reeler DG in the living slice, reeler mice were crossbred with POMC-EGFP transgenic mice, in which EGFP is specifically expressed in dentate granule cells under the POMC promoter. The EGFP-positive reeler hippocampus (P0) was then co-cultured with P7 wild-type hippocampus, which provided a Reelin-rich MZ. Time-lapse imaging was performed for more than 12 h. With this approach, we were able to monitor the migration process and branching of the migrating neurons. Neurons that did not yet contact the Reelin-containing zone showed their typical bipolar morphology, and their leading processes did not give rise to branches (Fig. 4A). However, the leading process started to branch as soon as its growth cone came in contact with the Reelin-containing zone. The more the soma moved toward the Reelin-containing zone, the more branches were formed in that zone, accompanied by shortening of the leading process (Supplementary Video 1, Fig. 4A). The migration was terminated as the soma reached the border of the Reelin-containing zone and the leading process disappeared. As a result, only its branches remained in the Reelin-containing zone (Fig. 4AC). These results indicate that Reelin induces the dynamic branching of the leading process during somal translocation, the last step of the migratory process. We have shown previously that the effects were mediated via canonical Reelin signaling involving ApoER2, VLDLR, and the adapter molecule Dab1 (Zhao et al. 2006).

Figure 4.

Time-lapse imaging illustrating branching of the leading process upon contact with the Reelin-containing zone, followed by nuclear translocation. The hippocampus of a P0 POMC-EGFP transgenic reeler mouse was co-cultured with the hippocampus of a P7 wild-type mouse. Granule cells in reeler DG were GFP-positive. (A) Time-lapse imaging demonstrating the morphological changes and migratory behavior of a neuron (highlighted with green color) in the reeler DG at different time points after its leading process approached and contacted the Reelin-rich MZ of the wild-type DG (highlighted with red color). The leading process gradually gave rise to more and more branches in the Reelin-containing zone, followed by upward movement of the cell body. Nuclear translocation was terminated upon reaching the Reelin-containing zone (see Supplementary Video 1). (B) Immunostaining for Reelin (red) and GFP (green) of the co-culture shown in (A). Counterstaining with DAPI (blue). Arrow points to the granule cell highlighted in (A). Dashed line indicates the border between wild-type (wt) DG and reeler DG. (C) High magnification of the boxed area in (B). Asterisk labels the cell body of the neuron in (A). Solid arrowheads indicate dendritic branches that had emerged from the former leading process. Hollow arrowhead points to the trailing process that will become the axon. Scale bars: 20 μm (A), 30 μm (B), and 10 μm (C).

Figure 4.

Time-lapse imaging illustrating branching of the leading process upon contact with the Reelin-containing zone, followed by nuclear translocation. The hippocampus of a P0 POMC-EGFP transgenic reeler mouse was co-cultured with the hippocampus of a P7 wild-type mouse. Granule cells in reeler DG were GFP-positive. (A) Time-lapse imaging demonstrating the morphological changes and migratory behavior of a neuron (highlighted with green color) in the reeler DG at different time points after its leading process approached and contacted the Reelin-rich MZ of the wild-type DG (highlighted with red color). The leading process gradually gave rise to more and more branches in the Reelin-containing zone, followed by upward movement of the cell body. Nuclear translocation was terminated upon reaching the Reelin-containing zone (see Supplementary Video 1). (B) Immunostaining for Reelin (red) and GFP (green) of the co-culture shown in (A). Counterstaining with DAPI (blue). Arrow points to the granule cell highlighted in (A). Dashed line indicates the border between wild-type (wt) DG and reeler DG. (C) High magnification of the boxed area in (B). Asterisk labels the cell body of the neuron in (A). Solid arrowheads indicate dendritic branches that had emerged from the former leading process. Hollow arrowhead points to the trailing process that will become the axon. Scale bars: 20 μm (A), 30 μm (B), and 10 μm (C).

Absence of Reelin Results in Severe Migration Defects and Morphological Abnormalities of Neurons

It is well known that in reeler mice deficient in Reelin neuronal migration is severely altered and cortical layers are nearly inverted. In addition, dendrites and axons of the neurons are misoriented. However, the time point at which these abnormalities occur has remained unclear. To address this issue, we compared the positioning and morphology of GFP-labeled neurons in reeler mice with those in wild-type littermates at 2 critical time points, i.e., 3 and 5 days after electroporation at E14.5, respectively. In the wild-type cortex fixed at E17.5, GFP-labeled neurons were found in all layers of the cerebral cortex (Fig. 1A), most of them migrating through the different layers of CP. Some neurons had already reached their final destination, future layer 2/3 of the cerebral cortex directly underneath the MZ. In the reeler cortex fixed at E17.5, we similarly observed numerous GFP-labeled neurons traversing the CP (Fig. 5A), displaying the typical bipolar morphology as known from migrating neurons in the wild-type cortex (Fig. 5A,B). However, the vast majority of GFP-labeled neurons were found in the deep portion of the CP. No GFP-labeled neurons were observed that reached the border between CP and MZ (Fig. 5A). These observations suggest that the transformation of migrating neurons from the multipolar to the bipolar stage in the intermediate zone (IZ) and the migration from IZ to CP were not affected in reeler. However, the migration within CP appeared disrupted. One possibility might be that neurons in the reeler CP migrated slower than their counterparts in the wild-type cortex. Alternatively, neurons in reeler cortex were unable to migrate further toward superficial layers. To examine these possibilities, we fixed brains at P0, i.e., 5 days after electroporation. At this time point, most of the GFP-labeled neurons in the wild-type cortex had reached their final destination, forming a densely packed cell layer underneath the MZ (Fig. 1B). In contrast, in the reeler cortex, most GFP-labeled neurons were loosely scattered throughout the deep portion of the CP, far from their definitive destination, the upper cortical layers. At this time point, we also observed severe morphological abnormalities of the neurons in the reeler cortex transfected at E14.5 when compared with wild-type mice. GFP-labeled neurons in the wild-type cortex were characterized by their bipolarity and preferential polarity orientation. Thus, they give rise to a short, thick apical dendrite, the former leading process that forms an apical tuft in layer 1, the former MZ containing Reelin (cf. Figs 1B,C and 5E). The thin axon (the former trailing process) emerges from the opposite side of the cell body (Figs 1C and 5E). No basal dendrites were observed at this stage. In the reeler cortex, many neurons have lost their bipolarity and preferential orientation, and many apical dendrites are oriented toward the white matter (Fig. 5C,D,F). Moreover, the axons of many neurons in the reeler cortex do not originate from the soma, but from one of the dendrites. The above-mentioned results suggest that Reelin does not only play a crucial role in the migratory process but is involved in neuronal polarization, polarity orientation, and axon formation.

Figure 5.

Migration defects and severe morphological abnormalities of neurons in reeler cortex. Reeler brains were transfected at E14.5 with plasmids expressing GFP and then fixed at E17.5 (A, B) and P0 (C, D), respectively. Sections were immunostained with an antibody against GFP (green) and counterstained with PI (red). (A) Overview of the reeler cortex fixed at 17.5. Most GFP-labeled neurons migrated into CP. However, they were found only in the deep layers of CP. No GFP-labeled neuron reached the border between CP and MZ. (B) High magnification at E17.5. The GFP-labeled cells showed the same typical bipolar morphology of migrating neurons as in wild-type mice. No obvious morphological abnormalities were observed at this time point. (C) Overview of reeler cortex fixed at P0. The GFP-labeled neurons show a severe migration defect and morphological abnormalities (cf. Fig. 1B showing the same stage in a wild-type animal). The reeler neurons were unable to reach their destination, the upper layers of the cortex. Moreover, most neurons displayed an inverted orientation (arrows). Their “apical” dendrites pointed toward the white matter. (D) High magnification to illustrate loss of bipolarity and altered orientation of neurons. Asterisks label neurons with several dendrites directly derived from soma and pointing to random directions. Arrows label neurons with aberrant course of their main dendrites. They reversed their apical dendrites to VZ. Note: the cell-free rifts in A and C, a characteristic morphological feature in reeler cortex. (E,F) Drawings of representative neurons in wild-type (E) and reeler cortex (F) at P0. (E) Wild-type neurons that have reached their definitive positions near MZ. Dendrites are shown in green, the axon in red. (F) Neurons in the reeler cortex that had stopped their migration showed altered orientation and paucity of dendritic branches. Note: origin of the axon from a dendritic process in some of the cells. Scale bars: 80 μm (A,C), 20 μm (B,D), and 15 μm (E,F).

Figure 5.

Migration defects and severe morphological abnormalities of neurons in reeler cortex. Reeler brains were transfected at E14.5 with plasmids expressing GFP and then fixed at E17.5 (A, B) and P0 (C, D), respectively. Sections were immunostained with an antibody against GFP (green) and counterstained with PI (red). (A) Overview of the reeler cortex fixed at 17.5. Most GFP-labeled neurons migrated into CP. However, they were found only in the deep layers of CP. No GFP-labeled neuron reached the border between CP and MZ. (B) High magnification at E17.5. The GFP-labeled cells showed the same typical bipolar morphology of migrating neurons as in wild-type mice. No obvious morphological abnormalities were observed at this time point. (C) Overview of reeler cortex fixed at P0. The GFP-labeled neurons show a severe migration defect and morphological abnormalities (cf. Fig. 1B showing the same stage in a wild-type animal). The reeler neurons were unable to reach their destination, the upper layers of the cortex. Moreover, most neurons displayed an inverted orientation (arrows). Their “apical” dendrites pointed toward the white matter. (D) High magnification to illustrate loss of bipolarity and altered orientation of neurons. Asterisks label neurons with several dendrites directly derived from soma and pointing to random directions. Arrows label neurons with aberrant course of their main dendrites. They reversed their apical dendrites to VZ. Note: the cell-free rifts in A and C, a characteristic morphological feature in reeler cortex. (E,F) Drawings of representative neurons in wild-type (E) and reeler cortex (F) at P0. (E) Wild-type neurons that have reached their definitive positions near MZ. Dendrites are shown in green, the axon in red. (F) Neurons in the reeler cortex that had stopped their migration showed altered orientation and paucity of dendritic branches. Note: origin of the axon from a dendritic process in some of the cells. Scale bars: 80 μm (A,C), 20 μm (B,D), and 15 μm (E,F).

Reelin Induces Branching of RGC Processes In Vivo

To examine whether Reelin also induces branching of RGCs, we performed in utero electroporation with pCAG-GFP in embryonic wild-type mice and reeler mutants at E17.5 and fixed the tissue at E18.5. The sections were double stained with antibodies against Reelin and GFP. We found that in wild-type mice, RGCs projected long and thin processes to the pial surface, spanning the entire developing CP. The segments of radial glial fibers that were located in the IZ and CP were straight with characteristic varicosities, whereas those contacting the Reelin-rich MZ gave rise to extensive branches (Figs 2 and 6A,C). Each branch formed an end-foot that attached these processes to the pial surface (Figs 2BD and 6A). In reeler mice, radial glial fibers also extended into the MZ but rarely exhibited branches (Fig. 6B). Quantitative analysis showed that each wild-type radial glial fiber gave rise to various branches in the MZ ranging from 6 to 12. In contrast, the number decreased significantly to 1.85 ± 0.96 in reeler mice (Fig. 6E). Total branch length, total number of branches and average branch length of glial fibers were dramatically reduced in reeler mutants compared with wild-type mice (Fig. 6DF). Furthermore, branches of radial glial fibers in the MZ mainly ran perpendicular to the pial surface, whereas their course was more irregular in reeler. Notably, the first branch points in wild-type mice were close to the CP–MZ boundary, whereas the few branch points in reeler were often found within the MZ (Fig. 6G).

Figure 6.

Branching of RGCs is decreased in reeler. Embryonic brains were transfected with pCAG-GFP at E17.5 and fixed 24 h later. The sections were double stained with antibodies against GFP (green) and Reelin (red) and counterstained with DAPI (blue). (A) In wild-type mice, RGCs extend regular-oriented ascending processes to the MZ-CP boundary that branch extensively in the MZ. Six to 12 branches emanate from each single glial fiber shaft, terminating with end-feet (arrowheads). Most branches extend perpendicularly to the pial surface. (B) In reeler mice, RGCs traverse the whole developing CP, but the number of bifurcations emanating from each single glial fiber is strongly reduced. (C) Drawings of fiber shafts and branches of representative RGCs in wild-type mice and reeler mutants. (DG) Total branch length, branch number, average branch length, and distance from primary branch point to the MZ-CP boundary of radial glial fibers in wild type and reeler. Compared with wild-type mice, all parameters are significantly decreased in reeler. *** P < 0.001. Data represent mean ± SEM. Scale bars: 20 μm (AC).

Figure 6.

Branching of RGCs is decreased in reeler. Embryonic brains were transfected with pCAG-GFP at E17.5 and fixed 24 h later. The sections were double stained with antibodies against GFP (green) and Reelin (red) and counterstained with DAPI (blue). (A) In wild-type mice, RGCs extend regular-oriented ascending processes to the MZ-CP boundary that branch extensively in the MZ. Six to 12 branches emanate from each single glial fiber shaft, terminating with end-feet (arrowheads). Most branches extend perpendicularly to the pial surface. (B) In reeler mice, RGCs traverse the whole developing CP, but the number of bifurcations emanating from each single glial fiber is strongly reduced. (C) Drawings of fiber shafts and branches of representative RGCs in wild-type mice and reeler mutants. (DG) Total branch length, branch number, average branch length, and distance from primary branch point to the MZ-CP boundary of radial glial fibers in wild type and reeler. Compared with wild-type mice, all parameters are significantly decreased in reeler. *** P < 0.001. Data represent mean ± SEM. Scale bars: 20 μm (AC).

The observed disruption of the radial glial scaffold in Reelin-deficient reeler mice indicates that Reelin is indispensable for the branching of basal processes of RGCs. The total length of branches of a wild-type RGC was 402.69 ± 131.07 μm, i.e., approximately 6 times the width of the MZ.

Reelin Promotes Dendritic Arborization and Glial Process Outgrowth In Vitro

To determine whether Reelin directly promotes dendritic development independent of the neuron's integration into its normal environment and developing neural circuitry, we established primary neural cultures of neocortex from E17.5 reeler mice and treated them with recombinant Reelin for 24 h. To analyze dendritic development and glial process outgrowth separately, antibodies against MAP2, a marker for neurons, and BLBP, a specific marker for glial cells in the embryonic neocortex, were used in this study.

Treatment of recombinant Reelin significantly induced process formation, elongation, and branching of both neurons and glial cells (Fig. 7AD). After treatment, the number of terminal tips of neuronal and glial processes, an index of process formation and branching, increased from 2.1 ± 0.6 to 3.4 ± 1.0 (P < 0.001) and from 1.8 ± 0.4 to 2.1 ± 0.6 (P < 0.05), respectively (Fig. 7E). Average length of processes increased from 7.4 ± 2.5 to 13.7 ± 2.8 μm in neurons (P < 0.001) and from 14.8 ± 4.2 to 18.7 ± 6.0 μm in glial cells (P < 0.001; Fig. 7F). The total process length per neuron increased from 20.3 ± 6.5 to 45.0 ± 14.1 μm (P < 0.001), whereas the total process length per glial cell increased from 27.5 ± 8.2 to 38.2 ± 16.1 μm (P < 0.05; Fig. 7G). These results indicate that Reelin promotes both dendritic complexity and glial process outgrowth.

Figure 7.

Reelin treatment promoted neuronal dendritic arborization and glial process outgrowth. Primary cultures were prepared from the cerebral cortex of E17.5 reeler mutants and treated with recombinant Reelin for 24 h. Immunostaining for MAP2 (A, B) and BLBP (C, D) in cultures treated with control medium (A, C) and recombinant Reelin (B, D). Both the number and length of neuronal and glial processes increased after Reelin treatment. Quantitative analysis of the number of terminal tips (E), average length of process (F), and total length of processes (G) of each cell in the control and Reelin-treated groups. *P < 0.05, ***P < 0.001. Data represent mean ± SD. Scale bar: 35 μm.

Figure 7.

Reelin treatment promoted neuronal dendritic arborization and glial process outgrowth. Primary cultures were prepared from the cerebral cortex of E17.5 reeler mutants and treated with recombinant Reelin for 24 h. Immunostaining for MAP2 (A, B) and BLBP (C, D) in cultures treated with control medium (A, C) and recombinant Reelin (B, D). Both the number and length of neuronal and glial processes increased after Reelin treatment. Quantitative analysis of the number of terminal tips (E), average length of process (F), and total length of processes (G) of each cell in the control and Reelin-treated groups. *P < 0.05, ***P < 0.001. Data represent mean ± SD. Scale bar: 35 μm.

Discussion

During development, postmitotic neurons migrate along radial glial fibers from the VZ toward the MZ, forming the well-organized 6-layered structure of the mammalian cerebral cortex in an “inside-out” manner (Rakic 1972; Nadarajah and Parnavelas 2002). Reelin, synthesized and secreted by CR cells, is located in the MZ of the developing cerebral cortex as revealed by in situ hybridization and immunocytochemistry for Reelin (D'Arcangelo et al. 1995, 1997; Ogawa et al. 1995; de Bergeyck et al. 1997; Schiffmann et al. 1997; Drakew et al. 1998). Previous studies demonstrated that Reelin is required for correct positioning of neurons in the developing neocortex (Rakic and Caviness 1995). However, the precise mechanism of Reelin's action on neuronal migration and layer formation has remained unclear. By combining in utero electroporation with immunocytochemistry against Reelin and by studying migrating neurons using real-time microscopy, we provide evidence that Reelin promotes the branching of neurons and RGCs in vivo, which might contribute to the completion of the migratory process and correct positioning of radially migrating cortical neurons.

In Utero Electroporation Labels Individual Neural Cells at Defined Points in Time

In utero electroporation is a simple and powerful method, which has proven to efficiently introduce genes into cells of the embryonic cortex (Saito and Nakatsuji 2001; Tabata and Nakajima 2001). During the past decade, it has been successfully applied to study the proliferation, migration, and differentiation of neurons (Tabata and Nakajima 2002; Olson et al. 2006; Chen et al. 2008; Kubo et al. 2010; Jossin and Cooper 2011). Thus, this technique enables the investigator to visualize the morphology of individual neurons and RGCs in different developmental stages. In previous studies, DiI labeling was used to trace RGCs (Miyata et al. 2001; Noctor et al. 2002; Hartfuss et al. 2003). However, passive lateral diffusion of the tracer did not result in the complete staining of cell types with branches, and staining with this lipophilic dye is incompatible with immunocytochemical techniques employing Triton X-100 treatment (Matsubayashi et al. 2008; Staffend and Meisel 2011). Various antibodies against specific markers expressed in different developmental phases were also used to label diverse cohorts of neurons or to identify distinct neural cell types. However, nearly all the cells of the selected population are immunolabeled, which makes it difficult to trace individual cells with all their processes. Therefore, in utero electroporation appears to be an appropriate method to trace individual neurons and RGCs.

Different Neural Cells Were Labeled at Different Embryonic Stages

In the present study, we transfected embryonic brains with pCAG-GFP at distinct embryonic days using in utero electroporation. Different experimental results were obtained when the transfected brains were harvested after different survival times. When the electroporation was performed at E14.5 and the transfected brains were fixed at E17.5, numerous migrating bipolar neurons with a long, thick leading process and a short, thin trailing process were labeled by GFP throughout the entire CP, whereas in brains fixed 5 days after transfection, the majority of the somata of GFP-labeled cells accumulated immediately beneath the MZ. This indicates that newly born neurons require 4–5 days to complete their migration. When we fixed E14.5-transfected brains at P5, the majority of the neurons were differentiated with abundant branches. When electroporation was performed at embryonic stage E17.5 and the transfected brains were fixed 24 h later, the majority of GFP-positive cells were identified as RGCs, many of them exhibiting the typical spindle-shaped soma, a short apical process contacting the ventricular surface, and a long ascending basal process spanning the whole developing cortical wall. Thus, in utero electroporation allowed us to study defined types of cells as well as different stages of their migration and differentiation during the development of the cerebral cortex.

Reelin Induces Branching of Migrating Neurons and RGCs

Previous studies on the role of Reelin during embryonic and early postnatal development have focused on radial neuronal migration and the formation of the laminated cortical structure (Zhao et al. 2004; Hack et al. 2007; Frotscher 2010; Zhao and Frotscher 2010; Jossin and Cooper 2011). Here, we focused on Reelin's role in process differentiation of late-generated cortical neurons and RGCs. In agreement with previous reports, we found that addition of recombinant Reelin to the culture medium promoted dendritic extension and branching of reeler cortical neurons and glial cells in vitro (see Förster et al. 2002; Niu et al. 2004; Niu et al. 2008).

The thin radial glial fibers follow a straight course traversing the IZ and CP and then branch extensively as soon as they reach the Reelin-rich MZ. They terminate with bulbous swellings, the characteristic radial glial end-feet. A similar branching pattern was found for the leading processes of neurons. Our results raise the possibility that the Reelin-induced bifurcation of the leading process, which results in two branches thinner than the main leading process, forms a mechanical obstacle, preventing further migration by nuclear translocation. Along this line, branching of the radial fiber may similarly be involved in ceasing the migration process, probably facilitating detachment of the neuron from the radial glial fiber. At least, this hypothesis would be compatible with the results in reeler mice where the branching of leading processes and radial glial fibers is severely altered and many neurons “overmigrate” and populate the MZ, a cell-poor layer in wild-type animals. This interpretation is also concordant with previous studies in which Reelin was found to induce the detachment of migrating neurons from their radial glial substrate (Dulabon et al. 2000; Hack et al. 2002; Sanada et al. 2004).

Our real-time microscopy studies have shown that the assumed sequence of events, i.e., branching of the leading process upon contact with the MZ followed by nuclear translocation associated with the shortening of the leading process, does take place in one and the same migrating neuron as it approaches its definitive destination. In future real-time microscopy studies, migrating neurons and radial glial fibers might be labeled by different fluorescent proteins in order to get more insight into the interactions of both cell types in the terminal phase of the migratory process. The paucity of branches of leading processes and radial glial fibers and their misorientation in reeler mutants strongly suggests that Reelin not only induces branching—as was shown before (Niu et al. 2004; Olson et al. 2006) and confirmed by the present culture experiments—but is also important for anchoring the leading processes to the MZ by these branches. Attachment to the MZ, in turn, might create the tension of the leading process that is required for nuclear translocation to occur. Finally, it may not only be ramification of the leading process in the MZ that attaches it to the cortical surface. Another likely presumption is the direct involvement of Reelin in morphological alterations of the migrating neurons themselves. Chai et al. (2009) have previously shown that Reelin phosphorylates the actin-depolymerizing protein cofilin. Phosphorylation of cofilin renders it unable to depolymerize actin, thereby stabilizing the actin cytoskeleton (Arber et al. 1998; Yang et al. 1998). Consisting with this function of Reelin, p-cofilin immunostaining was strongest in the leading processes approaching the Reelin-rich MZ (Chai et al. 2009). Immunostaining for p-cofilin was dramatically reduced in reeler tissue, likely resulting in an altered cytoskeletal stability of the leading process. This in turn would be compatible with the altered orientation of many apical dendrites, the former leading processes, in reeler. In the wild-type cortex, apical dendrites are regularly directed toward the pial surface, resulting from the Reelin-induced branching of the leading process and its stabilization via cofilin phosphorylation.

In conclusion then, our data provide evidence for a role of Reelin in inducing the branching of migrating neurons and RGCs. Thus, Reelin may act as a “stop signal” in two ways, on the one hand by inducing branching, on the other by phosphorylating cofilin, which stabilizes the actin cytoskeleton thus interfering with the flow of actin filaments required for the translocation of the nucleus during migration (Chai et al. 2009, 2014; He et al. 2010). Finally, we would like to point out that Reelin's effect on the branching of leading processes of neurons and the processes of RGCs should not be generalized. Other guiding molecules and mechanisms are involved in the formation of axonal projections, axonal collateralization, and targeting, as for instance in the development of thalamocortical projections (Molnár et al. 1998; Higashi et al. 2005).

Supplementary Material

Supplementary material can be found at: http://www.cercor.oxfordjournals.org/

Funding

This work was supported by grants from the Deutsche Forschungsgemeinschaft (FR 620/12-1 to M.F.) and the National Natural Science Foundation of China (No. 31071873 to S.Z.).

Notes

Michael Frotscher is Senior Research Professor of the Hertie Foundation. The authors are grateful to Ms. Ludmila Butenko for her excellent technical assistance and Ms. Pingjing Guo for genotyping the mutants. The authors thank the members of the Core Facility Imaging (UMIF) for their help with real-time microscopy. Conflict of Interest: None declared.

References

Arber
S
Barbayannis
FA
Hanser
H
Schneider
C
Stanyon
ca
Bernard
O
Caroni
P
.
1998
.
Regulation of actin dynamics through phosphorylation of cofilin by LIM-kinase
.
Nature
 .
393
:
805
809
.
Arnaud
L
Ballif
BA
Förster
E
Cooper
JA
.
2003
.
Fyn tyrosine kinase is a critical regulator of disabled-1 during brain development
.
Curr Biol
 .
13
:
9
17
.
Ayala
R
Shu
T
Tsai
LH
.
2007
.
Trekking across the brain: the journey of neuronal migration
.
Cell
 .
128
:
29
43
.
Bock
HH
Herz
J
.
2003
.
Reelin activates SRC family tyrosine kinases in neurons
.
Curr Biol
 .
13
:
18
26
.
Caviness
VS
Rakic
P
.
1978
.
Mechanisms of cortical development: a view from mutations in mice
.
Ann Rev Neurosci
 .
1
:
297
326
.
Caviness
VS
Jr
Sidman
RL
.
1973
.
Time of origin of corresponding cell classes in the cerebral cortex of normal and reeler mutant mice: an autoradiographic analysis
.
J Comp Neurol
 .
148
:
141
151
.
Chai
X
Förster
E
Zhao
S
Bock
HH
Frotscher
M
.
2009
.
Reelin stabilizes the actin cytoskeleton of neuronal processes by inducing n-cofilin phosphorylation at serine3
.
J Neurosci
 .
29
:
288
299
.
Chai
X
Münzner
G
Zhao
S
Tinnes
S
Kowalski
J
Häussler
U
Young
C
Haas
CA
Frotscher
M
.
2014
.
Epilepsy-induced motility of differentiated neurons
.
Cereb Cortex
 .
24
:
2130
2140
.
Chen
G
Sima
J
Jin
M
Wang
KY
Xue
XJ
Zheng
W
Ding
YQ
Yuan
XB
.
2008
.
Semaphorin-3A guides radial migration of cortical neurons during development
.
Nat Neurosci
 .
11
:
36
44
.
Curran
T
D'Arcangelo
G
.
1998
.
Role of reelin in the control of brain development
.
Brain Res Brain Res Rev
 .
26
:
285
294
.
D'Arcangelo
G
.
2006
.
Reelin mouse mutants as models of cortical development disorders
.
Epilepsy Behav
 .
8
:
81
90
.
D'Arcangelo
G
Homayouni
R
Keshvara
L
Rice
DS
Sheldon
M
Curran
T
.
1999
.
Reelin is a ligand for lipoprotein receptors
.
Neuron
 .
24
:
471
479
.
D'Arcangelo
G
Miao
GG
Chen
SC
Soares
HD
Morgan
JI
Curran
T
.
1995
.
A protein related to extracellular matrix proteins deleted in the mouse mutant reeler
.
Nature
 .
374
:
719
723
.
D'Arcangelo
G
Nakajima
K
Miyata
T
Ogawa
M
Mikoshiba
K
Curran
T
.
1997
.
Reelin is a secreted glycoprotein recognized by the CR-50 monoclonal antibody
.
J Neurosci
 .
17
:
23
31
.
de Bergeyck
V
Nakajima
K
Lambert de Rouvroit
C
Naerhuyzen
B
Goffinet
AM
Miyata
T
Ogawa
M
Mikoshiba
K
.
1997
.
A truncated Reelin protein is produced but not secreted in the ‘Orleans’ reeler mutation (Reln[rl-Orl])
.
Brain Res Mol Brain Res
 .
50
:
85
90
.
Deller
T
Drakew
A
Heimrich
B
Forster
E
Tielsch
A
Frotscher
M
.
1999
.
The hippocampus of the reeler mutant mouse: fiber segregation in area CA1 depends on the position of the postsynaptic target cells
.
Exp Neurol
 .
156
:
254
267
.
Del Rio
JA
Heimrich
B
Borrell
V
Förster
E
Drakew
A
Alcantara
S
Nakajima
K
Miyata
T
Ogawa
M
Mikoshiba
K
et al
1997
.
A role for Cajal-Retzius cells and reelin in the development of hippocampal connections
.
Nature
 .
385
:
70
74
.
Drakew
A
Frotscher
M
Deller
T
Ogawa
M
Heimrich
B
.
1998
.
Developmental distribution of a reeler gene-related antigen in the rat hippocampal formation visualized by CR-50 immunocytochemistry
.
Neuroscience
 .
82
:
1079
1086
.
Dulabon
L
Olson
EC
Taglienti
MG
Eisenhuth
S
McGrath
B
Walsh
CA
Kreidberg
JA
Anton
ES
.
2000
.
Reelin binds alpha3beta1 integrin and inhibits neuronal migration
.
Neuron
 .
27
:
33
44
.
Feng
L
Hatten
ME
Heintz
N
.
1994
.
Brain lipid-binding protein (BLBP): a novel signaling system in the developing mammalian CNS
.
Neuron
 .
12
:
895
908
.
Förster
E
Bock
HH
Herz
J
Chai
X
Frotscher
M
Zhao
S
.
2010
.
Emerging topics in Reelin function
.
Eur J Neurosci
 .
31
:
1511
1518
.
Förster
E
Tielsch
A
Saum
B
Weiss
KH
Johanssen
C
Graus-Porta
D
Muller
U
Frotscher
M
.
2002
.
Reelin, Disabled 1, and beta 1 integrins are required for the formation of the radial glial scaffold in the hippocampus
.
Proc Natl Acad Sci USA
 .
99
:
13178
13183
.
Francis
F
Koulakoff
A
Boucher
D
Chafey
P
Schaar
B
Vinet
MC
Friocourt
G
McDonnell
N
Reiner
O
Kahn
A
et al
1999
.
Doublecortin is a developmentally regulated, microtubule-associated protein expressed in migrating and differentiating neurons
.
Neuron
 .
23
:
247
256
.
Frotscher
M
.
1998
.
Cajal-Retzius cells, Reelin, and the formation of layers
.
Curr Opin Neurobiol
 .
8
:
570
575
.
Frotscher
M
.
2010
.
Role for Reelin in stabilizing cortical architecture
.
Trends Neurosci
 .
33
:
407
414
.
Frotscher
M
Haas
CA
Förster
E
.
2003
.
Reelin controls granule cell migration in the dentate gyrus by acting on the radial glial scaffold
.
Cereb Cortex
 .
13
:
634
640
.
Gleeson
JG
Lin
PT
Flanagan
LA
Walsh
CA
.
1999
.
Doublecortin is a microtubule-associated protein and is expressed widely by migrating neurons
.
Neuron
 .
23
:
257
271
.
Gleeson
JG
Walsh
CA
.
2000
.
Neuronal migration disorders: from genetic diseases to developmental mechanisms
.
Trends Neurosci
 .
23
:
352
359
.
Goffinet
AM
Lyon
G
.
1979
.
Early histogenesis in the mouse cerebral cortex: a Golgi study
.
Neurosci Lett
 .
14
:
61
66
.
Hack
I
Bancila
M
Loulier
K
Carroll
P
Cremer
H
.
2002
.
Reelin is a detachment signal in tangential chain-migration during postnatal neurogenesis
.
Nat Neurosci
 .
5
:
939
945
.
Hack
I
Hellwig
S
Junghans
D
Brunne
B
Bock
HH
Zhao
S
Frotscher
M
.
2007
.
Divergent roles of ApoER2 and Vldlr in the migration of cortical neurons
.
Development
 .
134
:
3883
3891
.
Hartfuss
E
Förster
E
Bock
HH
Hack
MA
Leprince
P
Luque
JM
Herz
J
Frotscher
M
Gotz
M
.
2003
.
Reelin signaling directly affects radial glia morphology and biochemical maturation
.
Development
 .
130
:
4597
4609
.
He
M
Zhang
ZH
Guan
CB
Xia
D
Yuan
XB
.
2010
.
Leading tip drives soma translocation via forward F-actin flow during neuronal migration
.
J Neurosci
 .
30
:
10885
10898
.
Herz
J
Chen
Y
.
2006
.
Reelin, lipoprotein receptors and synaptic plasticity
.
Nat Rev Neurosci
 .
7
:
850
859
.
Hiesberger
T
Trommsdorff
M
Howell
BW
Goffinet
A
Mumby
MC
Cooper
JA
Herz
J
.
1999
.
Direct binding of Reelin to VLDL receptor and ApoE receptor 2 induces tyrosine phosphorylation of disabled-1 and modulates tau phosphorylation
.
Neuron
 .
24
:
481
489
.
Higashi
S
Hioki
K
Kurotani
T
Kasim
N
Molnár
Z
.
2005
.
Functional thalamocortical synapse reorganization from subplate to layer IV during postnatal development in the reeler-like mutant rat (shaking rat Kawasaki)
.
J Neurosci
 .
25
:
1395
1406
.
Howell
BW
Hawkes
R
Soriano
P
Cooper
JA
.
1997
.
Neuronal position in the developing brain is regulated by mouse disabled-1
.
Nature
 .
389
:
733
737
.
Howell
BW
Herrick
TM
Cooper
JA
.
1999
.
Reelin-induced tyrosine phosphorylation of disabled 1 during neuronal positioning
.
Genes Dev
 .
13
:
643
648
.
Jossin
Y
.
2004
.
Neuronal migration and the role of reelin during early development of the cerebral cortex
.
Mol Neurobiol
 .
30
:
225
251
.
Jossin
Y
Cooper
JA
.
2011
.
Reelin, Rap1 and N-cadherin orient the migration of multipolar neurons in the developing neocortex
.
Nat Neurosci
 .
14
:
697
703
.
Jossin
Y
Goffinet
AM
.
2007
.
Reelin signals through phosphatidylinositol 3-kinase and Akt to control cortical development and through mTor to regulate dendritic growth
.
Mol Cell Biol
 .
27
:
7113
7124
.
Kubo
K
Honda
T
Tomita
K
Sekine
K
Ishii
K
Uto
A
Kobayashi
K
Tabata
H
Nakajima
K
.
2010
.
Ectopic Reelin induces neuronal aggregation with a normal birthdate-dependent “inside-out” alignment in the developing neocortex
.
J Neurosci
 .
30
:
10953
10966
.
Lendahl
U
Zimmerman
LB
McKay
RD
.
1990
.
CNS stem cells express a new class of intermediate filament protein
.
Cell
 .
60
:
585
595
.
Matsubayashi
Y
Iwai
L
Kawasaki
H
.
2008
.
Fluorescent double-labeling with carbocyanine neuronal tracing and immunohistochemistry using a cholesterol-specific detergent digitonin
.
J Neurosci Methods
 .
174
:
71
81
.
Matsuda
T
Cepko
CL
.
2004
.
Electroporation and RNA interference in the rodent retina in vivo and in vitro
.
Proc Natl Acad Sci USA
 .
101
:
16
22
.
Matsuki
T
Pramatarova
A
Howell
BW
.
2008
.
Reduction of Crk and CrkL expression blocks reelin-induced dendritogenesis
.
J Cell Sci
 .
121
:
1869
1875
.
Miyata
T
Kawaguchi
A
Okano
H
Ogawa
M
.
2001
.
Asymmetric inheritance of radial glial fibers by cortical neurons
.
Neuron
 .
31
:
727
741
.
Molnár
Z
Adams
R
Goffinet
AM
Blakemore
C
.
1998
.
The role of the first postmitotic cortical cells in the development of thalamocortical innervation in the reeler mouse
.
J Neurosci
 .
18
:
5746
5765
.
Nadarajah
B
Brunstrom
JE
Grutzendler
J
Wong
RO
Pearlman
AL
.
2001
.
Two modes of radial migration in early development of the cerebral cortex
.
Nat Neurosci
 .
4
:
143
150
.
Nadarajah
B
Parnavelas
JG
.
2002
.
Modes of neuronal migration in the developing cerebral cortex
.
Nat Rev Neurosci
 .
3
:
423
432
.
Niu
S
Renfro
A
Quattrocchi
CC
Sheldon
M
D'Arcangelo
G
.
2004
.
Reelin promotes hippocampal dendrite development through the VLDLR/ApoER2-Dab1 pathway
.
Neuron
 .
41
:
71
84
.
Niu
S
Yabut
O
D'Arcangelo
G
.
2008
.
The Reelin signaling pathway promotes dendritic spine development in hippocampal neurons
.
J Neurosci
 .
28
:
10339
10348
.
Niwa
H
Yamamura
K
Miyazaki
J
.
1991
.
Efficient selection for high-expression transfectants with a novel eukaryotic vector
.
Gene
 .
108
:
193
199
.
Noctor
SC
Flint
AC
Weissman
TA
Wong
WS
Clinton
BK
Kriegstein
AR
.
2002
.
Dividing precursor cells of the embryonic cortical ventricular zone have morphological and molecular characteristics of radial glia
.
J Neurosci
 .
22
:
3161
3173
.
Ogawa
M
Miyata
T
Nakajima
K
Yagyu
K
Seike
M
Ikenaka
K
Yamamoto
H
Mikoshiba
K
.
1995
.
The reeler gene-associated antigen on Cajal-Retzius neurons is a crucial molecule for laminar organization of cortical neurons
.
Neuron
 .
14
:
899
912
.
Olson
EC
Kim
S
Walsh
CA
.
2006
.
Impaired neuronal positioning and dendritogenesis in the neocortex after cell-autonomous Dab1 suppression
.
J Neurosci
 .
26
:
1767
1775
.
Overstreet
LS
Hentges
ST
Bumaschny
VF
de Souza
FS
Smart
JL
Santangelo
AM
Low
MJ
Westbrook
GL
Rubinstein
M
.
2004
.
A transgenic marker for newly born granule cells in dentate gyrus
.
J Neurosci
 .
24
:
3251
3259
.
Pinto Lord
MC
Caviness
VS
Jr
.
1979
.
Determinants of cell shape and orientation: a comparative Golgi analysis of cell-axon interrelationships in the developing neocortex of normal and reeler mice
.
J Comp Neurol
 .
187
:
49
69
.
Rakic
P
.
1972
.
Mode of cell migration to the superficial layers of fetal monkey neocortex
.
J Comp Neurol
 .
146
:
335
345
.
Rakic
P
.
1988
.
Specification of cerebral cortical areas
.
Science
 .
241
:
170
176
.
Rakic
P
Caviness
VS
Jr
.
1995
.
Cortical development: view from neurological mutants two decades later
.
Neuron
 .
14
:
1101
1103
.
Saito
T
Nakatsuji
N
.
2001
.
Efficient gene transfer into the embryonic mouse brain using in vivo electroporation
.
Dev Biol
 .
240
:
237
246
.
Sanada
K
Gupta
A
Tsai
LH
.
2004
.
Disabled-1-regulated adhesion of migrating neurons to radial glial fiber contributes to neuronal positioning during early corticogenesis
.
Neuron
 .
42
:
197
211
.
Schiffmann
SN
Bernier
B
Goffinet
AM
.
1997
.
Reelin mRNA expression during mouse brain development
.
Eur J Neurosci
 .
9
:
1055
1071
.
Sheldon
M
Rice
DS
D'Arcangelo
G
Yoneshima
H
Nakajima
K
Mikoshiba
K
Howell
BW
Cooper
JA
Goldowitz
D
Curran
T
.
1997
.
Scrambler and yotari disrupt the disabled gene and produce a reeler-like phenotype in mice
.
Nature
 .
389
:
730
733
.
Staffend
NA
Meisel
RL
.
2011
.
DiOlistic labeling of neurons in tissue slices: a qualitative and quantitative analysis of methodological variations
.
Front Neuroanat
 .
5
:
14
.
Tabata
H
Nakajima
K
.
2001
.
Efficient in utero gene transfer system to the developing mouse brain using electroporation: visualization of neuronal migration in the developing cortex
.
Neuroscience
 .
103
:
865
872
.
Tabata
H
Nakajima
K
.
2002
.
Neurons tend to stop migration and differentiate along the cortical internal plexiform zones in the Reelin signal-deficient mice
.
J Neurosci Res
 .
69
:
723
730
.
Tissir
F
Goffinet
AM
.
2003
.
Reelin and brain development
.
Nat Rev Neurosci
 .
4
:
496
505
.
Trommsdorff
M
Gotthardt
M
Hiesberger
T
Shelton
J
Stockinger
W
Nimpf
J
Hammer
RE
Richardson
JA
Herz
J
.
1999
.
Reeler/Disabled-like disruption of neuronal migration in knockout mice lacking the VLDL receptor and ApoE receptor 2
.
Cell
 .
97
:
689
701
.
Walsh
CA
Goffinet
AM
.
2000
.
Potential mechanisms of mutations that affect neuronal migration in man and mouse
.
Curr Opin Genet Dev
 .
10
:
270
274
.
Ware
ML
Fox
JW
Gonzalez
JL
Davis
NM
Lambert de Rouvroit
C
Russo
CJ
Chua
SC
Jr
Goffinet
AM
Walsh
CA
.
1997
.
Aberrant splicing of a mouse disabled homolog, mdab1, in the scrambler mouse
.
Neuron
 .
19
:
239
249
.
Yang
N
Higuchi
O
Ohashi
K
Nagata
K
Wada
A
Kangawa
K
Nishida
E
Mizuno
K
.
1998
.
Cofilin phosphorylation by LIM-kinase 1 and its role in Rac-mediated actin reorganization
.
Nature
 .
393
:
809
812
.
Zhao
S
Chai
X
Bock
H-H
Brunne
B
Förster
E
Frotscher
M
.
2006
.
Rescue of the reeler phenotype in the dentate gyrus by wild-type coculture is mediated by lipoprotein receptors for Reelin and Disabled 1
.
J Comp Neurol
 .
495
:
1
9
.
Zhao
S
Chai
X
Förster
E
Frotscher
M
.
2004
.
Reelin is a positional signal for the lamination of dentate granule cells
.
Development
 .
131
:
5117
5125
.
Zhao
S
Frotscher
M
.
2010
.
Go or stop? Divergent roles of Reelin in radial neuronal migration
.
Neuroscientist
 .
16
:
421
434
.

Author notes

X.C. and L.F. contributed equally to this work.