REVIEW
Cell and molecular mechanisms that control cortical layer
formation in the brain
Ken-ichiro Kubo1,2 and Kazunori Nakajima1,2,3
1Department of Anatomy, Keio University School of Medicine, Tokyo, 2Department of Molecular Neurobiology
Institute of DNA Medicine, Jikei University School of Medicine, Tokyo, 3Precursory Research for Embryonic
Science and Technology (PRESTO), Japan Science and Technology Corporation (JST), Saitama, Japan(Received for publication on October 22, 2002)

Abstract. Various classes of neurons in the mammalian central nervous system (CNS) migrate fromtheir sites of origin to their ﬁnal positions, where they are arranged in elaborate laminar structures.
These precise patterns of neuronal alignment are disrupted in several human diseases and mousemutants. Among them, reeler, an autosomal recessive mouse mutant discovered half a century ago, hasbeen studied as a valuable material for investigations of neuronal layer formations. Recent identiﬁcation of a gene mutated in reeler (reelin), and subsequent characterizations of other genes underlying
mouse and human brain malformations have rapidly expanded our knowledge of the molecular programs underlying the normal brain layer formation. In this review, we summarize the cellular andmolecular mechanisms that establish highly ordered structures in the brain, in particular in the cerebral
cortex. (Keio J Med 52 (1): 8–20, March 2003)
Key words: Reelin, Dab1, VLDLR/ApoER2, Cdk5/p35, lissencephaly

tical layer formation. In this review, we focus on radialmigration, because most of the known mutants withcortical layering defects are due to the disruption ofradial migration.

Introduction
The mammalian cerebral cortex is a highly orderedstructure. The different classes of neurons reside in an
organized radial array of six cellular layers ranging
from the pial surface to the white matter. During development of laminated structures, neurons should migrate in a coordinated series of patterns from their sitesof origin to their ﬁnal positions, as neurons are usually
generated in sites distant from their ﬁnal locations.
In the developing cerebral cortex, neurons aremainly generated in the cerebral ventricular zone andthen move to the developing cortical plate via ‘‘radialmigration’’.1–4 In contrast, most GABAergic interneurons originate in the ganglionic eminences and enter
the developing cortical plate via ‘‘tangential migration’’in mice.5–8
The failure of migration leads to defects of corticallayer formation in several human diseases and mousemutants.9–11 However, the mutations of genes that are
the base cause of severe cortical dysplasias in humans
and mice have provided an entry point into the molecular pathway that controls neuronal migration and cor-

Development of the cerebral cortex
The mammalian cerebral cortex is constructed in two
distinct steps3,4,12 (Fig. 1). The ﬁrst step of neocorticaldevelopment is the formation of the preplate or primordial plexiform layer above the ventricular zone.
The preplate is composed of afferent and efferent ﬁbers
and the earliest generated neurons, including the CajalRetzius cells and the subplate neurons.12 In the second
step, cortical plate neurons are generated in the ventricular zone along the ventricle, and these earlier-bornneurons invade the preplate. Migrating neurons move
past the subplate, splitting this layer away from the
Cajal-Retzius cells. Therefore, the preplate is split intoa superﬁcial marginal zone, in which the Cajal-Retzius
cells remain adjacent to the pial surface, and a deep
subplate. The late-born neurons are generated in theventricular zone and migrate radially, passing throughReprint requests to: Dr. Kazunori Nakajima, Department of Anatomy, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku,
Tokyo 160-8582, Japan, e-mail: kazunori@sc.itc.keio.ac.jp8Keio J Med 2003; 52 (1): 8–20

9Fig. 1 Development of the neocortex. The ﬁrst step of neocortical development is the formation of the preplate (PP) above the ventricularzone (VZ). In the second step, cortical plate neurons are generated in the ventricular zone, and these earlier-born neurons invade the preplate.
Continued migration splits the preplate into a superﬁcial marginal zone (MZ), in which the Cajal-Retzius cells remain adjacent to the pial surface (PS), and a deep subplate (SP). The late-born neurons are generated in the ventricular zone and migrate radially, passing through theintermediate zone (IZ) which consists of pioneer axons, then move past the subplate and earlier generated cortical plate neurons (CP). The
systematic migration of late-generated neurons past predecessors results in the ‘‘inside-out’’ fashion of cortical layering, in which the later
generated cortical plate neurons are located at more superﬁcial positions than the earlier-born neurons. In adulthood, a six-layered neocortex isformed above the axonal tracts termed white matter (WM).

the intermediate zone, then move past the subplate andearlier generated cortical plate neurons. The systematic
migration of late-generated neurons past predecessors
results in the ‘‘inside-out’’ fashion of cortical layering,in which the later generated cortical plate neurons arelocated at more superﬁcial positions than the earlierborn neurons.1,13
Modes of radial migration
Two distinct modes of radial migration, locomotion
and somal translocation, have been reported (Fig. 2).
The locomotion cell migrates along a radial ﬁber of aradial glial cell;2,14 which traverse the entire thicknessof the developing cerebral wall. Neurons migrating inthis mode have a characteristic bipolar cell-morphology, with a thick leading process and a thin trailingprocess, and the entire cell moves along the radialﬁbers. In somal translocation, the cell has a long radially directed leading process that attaches to the pial
surface. The cell soma moves toward the leading edge,
which results in the shortening of the leading process, while its terminal remains attached to the pial
surface.15–19 A recent time-lapse study of radially mi-

grating neocortical neurons revealed that each modeof migration had distinct dynamics and occurred
preferentially at different developmental stages.18 At
embryonic day E12–13 in mice, neurons use somaltranslocation to split the preplate, while at E15–16,
locomotion predominates to transverse the expanding
cortical plate. Although somal translocation occurs as acontinuous movement and does not need the radial glial
ﬁbers, locomotion is interspersed by pauses and is dependent on the radial glial ﬁbers.
In contrast to the bipolar or monopolar morphology
of locomotion and somal translocation cells, histological analyses of ﬁxed sections of developing neocortex
using electron microscopy have demonstrated the existence of multipolar cells in the intermediate zone,20
which could not be explained simply by the locomotion
or somal translocation. Recently, Tabata and Nakajima established an in utero gene transfer system thatallows speciﬁc labeling of migrating cells in the developing cortex with GFP or red ﬂuorescent protein(DsRed).21,22 Using this system, we performed timelapse observations, and found that neurons dynamicallychange their morphology and migration modes during
their journeys toward the pial surface (Tabata et al.,

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Kubo K and Nakajima K: The mechanisms of cortical layer formation

Fig. 2 Modes of radial migration. (1) The locomotion cell migrates
along a radial ﬁber of a radial glial cell; which traverse the entire
thickness of the developing cerebral wall. Neurons migrating in thismode have a characteristic bipolar cell-morphology, with a thickleading process and a thin trailing process. (2) In somal translocation,
the cell has a long radially directed leading process that attaches tothe pial surface. The cell soma moves toward the leading edge, while
its terminal remains attached to the pial surface. CP, cortical plate; IZ,
intermediate zone; VZ, ventricular zone.

cell clustering or in laminar organizations have beenreported in several subcortical structures, including theamygdala, olfactory tubercle, olfactory bulb, inferiorolivary complex, dorsal cochlear nucleus, and facial
nucleus.27–31 Yip et al. showed that migration of sympathetic preganglionic neurons is affected in the spinal
cord as well, although it had been generally thoughtthat neuronal migration was not disrupted in the spinal
cord of the reeler mutant.32 The reeler mutation has its
effect without impairing the survival or vitality of neocortical neurons,33,34 and therefore has provided avaluable model for investigations of neuronal layer
formations.
In the neocortex of the reeler mutant, the ﬁrst cell
position defects arise at the splitting of the preplate.
The initial formation of the preplate proceeds normally.35,36 However, the early-born cortical neurons failto invade the preplate, and the subplate remains adjacent to the marginal zone in a structure known as the‘‘superplate’’.37 Subsequent cohorts of late-generated
neurons are unable to pass their predecessors, resulting
in an ‘‘outside-in’’ pattern of development.24,37 Consequently, a disorganized cortical plate is formed underneath the superplate, with an indistinct and almost
inverted layering.
CR-50 and the reeler genein submission). These results imply that each neuron
migrates via a combination of several modes of migration, depending on the migration phase and the developmental stage. The existence of various migration
modes is important, as the responsible genes in defects
of neocortical-layer formation might be involved in
distinct modes of migration.
Defects of Cortical Layer Formation in Mice
The analysis of malformations in mouse brains has
led to dramatic progress in the identiﬁcations of genes
important for cortical layering. Among them, the moststudied and characterized is the reeler malformation inmice.
The reeler malformation
Over 50 years, reeler (Reln rl ), the spontaneous
mouse mutant, has been studied as an important experimental model. The reeler mouse exhibits an autosomal recessive mutation located on chromosome 5.23
The reeler mutation causes ataxic and reeling gait inaffected mice. Analysis of the central nervous system ofmutant mice revealed severe architectonic abnormities
in the cortical structures of the cerebrum, hippocampus,
and cerebellum.24–26 In addition to these anomalies
in the cortical structures, more subtle abnormalities in

The ﬁrst material used to investigate the moleculardeﬁcit in the reeler mouse was obtained in a uniquestrategy. On the hypothesis that particular molecules
encoded by the reeler gene may mediate the presumptive interactions between the preplate and the forming
cortical plate neurons in normal mice, Ogawa et al.
generated a monoclonal antibody (CR-50) by immunizing reeler mice with brain extracts from normal
mice.38 CR-50 recognized a distinct allelic antigen
present in normal but not in reeler mice, expressedin the regions of normal mice where morphologicalabnormalities occur in reeler mice. In the neocortex, theCR-50 antigen was associated superﬁcially with theCajal-Retzius cells. Importantly, several experimental
approaches revealed that CR-50 functions as a blocking
antibody in culture studies.38–40 In addition, Nakajima
et al. reported that intraventricular injection of CR50 at
the embryonic stage disrupted the normal developmentof the hippocampus and converted it to a reeler patternin vivo.41 These lines of studies strongly suggested thatthe mutation of the CR-50 antigen causes the reeler
phenotype.
On the other hand, a transgene insertion produced
another reeler allele (Reln rl-tg ), providing a probe forthe reeler locus,42 which led to the isolation of thecandidate reeler gene.43 Positional cloning identiﬁed
the same candidate reeler gene at the same period.44,45

Keio J Med 2003; 52 (1): 8–20

11Fig. 3 Schematic representation of the Reelin structure. Reelin is a secreted extracellular matrix protein composed of 3,461 aa with a relative
molecular mass of 388 kDa. The N-terminus of Reelin has 25% identity with that of F-spondin. These ﬁrst 500 aa of Reelin are succeeded byeight Reelin repeats. Each Reelin repeat is composed of 350–390 aa and contains two related subrepeats A and B, separated by an EGF-like
motif of 30 aa. CR-50 antibody is recognized upstream of the Reelin repeats, although this antibody clearly blocks several Reelin-mediatedphenomena both in vitro and in vivo.

This gene was named reelin. No reelin mRNA wasdetected in the two reeler alleles (Reln rl and Reln rl-tg ).43
The reelin open-reading frame is 10,383 bases long,encoding a protein composed of 3461 amino acids (aa)which has a relative molecular mass of 388 K (Fig. 3).
An epitope of Reelin, deﬁned near the N-terminus, wasshown to be recognized by CR-50.46 Considering thefunctions of CR-50 as a blocking antibody, the deﬁciency of Reelin was in fact indicated to be responsible
for the reeler phenotype.
The region more N-terminal to the CR-50 epitopecontains a cleavable signal peptide, followed by a region which has 25% identity with that of F-spondin.47
These ﬁrst 500 aa of Reelin are succeeded by eight‘‘Reelin repeats’’. Each Reelin repeat is composed of
350–390 aa and contains two related subrepeats A andB, separated by an EGF-like motif of 30 aa.43 In thesesequences, Reelin resembles extracellular matrix proteins.
Reelin was secreted when constructed full-length
reelin cDNA was transfected to COS cells, and deletion
analysis demonstrated that a short region of positively
charged amino acids near the C-terminus is required
for secretion of Reelin.46 In the reeler Orleans mutant(Reln rl-Orl ), Reelin is made but not secreted due tothe insertion of an L1 transposonable element thatalters the amino acid sequence of the C-terminus.48,49
Recently, it was shown that secreted Reelin molecules
tend to form a homophilic dimer.50 As Reelin-Reelininteraction was inhibited by CR-50, CR-50 might neutralize the Reelin function at least partly by inhibiting
the homophilic assembly of Reelin proteins.51
In the developing brain of normal mice, Reelin issecreted from a subset of neurons, for example, by the
Cajal-Retzius cells in the cerebral cortex38 and hippocampus,41 and by granule cells in the cerebellum.39,52
Importantly, the Reelin-producing neurons are associated with regions where ﬁrst phenotypic defects ofneuronal migration are detected in reeler mice. The
spatial and temporal expression pattern suggested that
Reelin is important as the extracellular signal for
migrating neurons. This suggestion was conﬁrmed bysubsequent reports of mutations in genes encoding
molecules that transmit Reelin signal.
Yotari, scrambler, and Dab1 (disabled 1)
A number of mutations that result in a brain phenotype similar to that of reeler have expanded our knowledge of the Reelin signal cascade (Fig. 4). They include
yotari, an autosomal recessive mutant mouse whichappeared during the generation of null mice for thegene encoding the receptor for inositol-1,4,5-triphosphate.53 Despite the reeler-like phenotypes in yotari,alteration in Reelin expression was not observed.
Detailed investigations revealed that a long interspersed nuclear element (L1) insertion caused the mutation in the disabled 1 (Dab1) gene.54 This allele isreferred as Dab1 yot , and another spontaneous mutant
mice scrambler (Dab1 scm )55 with a mutation in the
Dab1 gene,56,57 or mice with a target disruption of
Dab1 (Dab1 tm1Cpr ) were also found to exhibit phenotypes indistinguishable from those of reeler.58 Dab1
protein is drastically reduced in Dab1 scm (@5% of thewild type level of Dab1 protein), and not detected in
Dab1 yot and Dab1 tm1Cpr .56,58

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Kubo K and Nakajima K: The mechanisms of cortical layer formation

Dab1 encodes an intracellular adaptor or docking
protein originally identiﬁed as a Src-binding protein.59
The Dab1 p80 is predominantly expressed in neurons
in the developing CNS from one of several alternatively spliced mRNAs, and it is tyrosine phosphorylated
during brain development. Near the N-terminus, Dab1
contains a region of approximately 150 aa known as aprotein interaction/phosphotyrosine binding (PI/PTB)
domain. The Dab1 PI/PTB domain binds to phospholipid bilayers containing phosphatidylinositol 4P(PtdIns4P) or PtdIns4,5P2 and to the unphosphorylatedsequence F/YXNPXY (in the single-letter amino-acidcode, where X is any amino acid), found in the cytoplasmic tails of ﬁve members of the low density
lipoprotein receptor (LDLR) family and of threemembers of the amyloid precursor protein (APP) family, and the cytoplasmic signaling protein Ship.60–62
Unlike other PTB domains, the Dab1 PTB does
not bind to tyrosine-phosphorylated peptide ligands(NPXpY), and binds phospholipids in a manner that
does not interfere with protein binding.60 A cluster oftyrosine residues downstream of the Dab1 PI/PTB
domain serves as docking sites for SH2 domaincontaining proteins such as the non-receptor type protein tyrosine kinases (PTKs), Src, Fyn, and Abl. Src
can phosphorylate Dab1 in vitro, and tyrosine phosphorylation of Dab1 promotes the interaction withthese PTKs.59 These structures of Dab1 imply thatDab1 mediates protein-protein or protein-membrane
docking functions in kinase signaling cascades during
brain development.
The identical mutant phenotype of Reelin and Dab1
suggests that they are in a common signaling pathway that regulates layer formation. In reeler, the major
populations of displaced neurons contain elevatedlevels of Dab1 protein, although they express normal
levels of Dab1 mRNA.63 This suggests that Dab1
accumulates in the absence of a Reelin-evoked signal
and functions downstream of Reelin. In addition, Dab1
is tyrosine-phosphorylated in a Reelin-dependent manner in primary cultured neurons,64 indicating that
Reelin regulates neuronal positions by stimulating
Dab1 tyrosine phosphorylation. Genetically, thedouble-null mice for Reelin and Dab1 show no additional defect, supporting the concept that Reelin andDab1 are acting on the same signaling cascade.64
Certain tyrosines on Dab1 are responsible for theReelin signaling in vivo, as indicated by knock-in
mutation of all these tyrosines in mice, which caused abrain phenotype that is identical to that of the reeler
mice.65 The major sites of Src-catalyzed phosphorylation in vitro are also preferentially phosphorylated
by Reelin in primary cultures.65,66 These ﬁndings
suggest that Src family PTKs play important roles in
the Reelin-induced tyrosine phosphorylation of Dab1.

However, the mechanism by which Reelin induces
Dab1 phosphorylation is not yet known.67,68
Reelin receptors
After the discovery of Dab1 as a downstream molecule of Reelin, the identiﬁcation of receptors thattransmit the Reelin signal to intracellular Dab1 became
the major interest of the ﬁeld. Genetics contributed
serendipitously again. The double-knockout mice lacking both the very low density lipoprotein receptor(VLDLR) gene69,70 and the apolipoprotein E receptor
2 (ApoER2) gene71–73 exhibit behavioral and neuroanatomical defects that are indistinguishable from thoseof reeler mice.74 Moreover, Dab1 protein is increased in
the double knockout mouse, indicating an occurrenceof a failure in transmitting the Reelin signal. Biochemically, Reelin was demonstrated to bind to the ligand
binding domains of VLDLR and ApoER2.75,76 The
estimated Kd for the interaction was 0.5 nM,75 or 0.7
nM,76 suggesting a high-afﬁnity interaction. The intracellular domains of VLDLR and ApoER2 bind to Dab1
at least in vitro, implying the pivotal role of VLDLR
and ApoER2 in transmitting the Reelin signal to intracellular Dab1.
VLDLR and ApoER2 are likely to function in concert, but the presence of distinct functions is also plausible. Mice lacking only VLDLR or ApoER2 exhibit
subtle but different defects, i.e., the deﬁciency in
VLDLR predominantly affects the cerebellum, whereasa phenotype of the ApoER2 defect is obvious in the
cerebrum and hippocampus.74 Biochemically, the cytoplasmic domain of LDLR family members interacts
with various proteins implicated in the regulation of
mitogen-activating protein kinases, cell adhesion, vesicle trafﬁcking, or neurotransmission.77 ApoER2, but
not VLDLR, contains a unique insertion sequence of 59
amino acids in its cytoplasmic domain71 and binds tothe family of JNK-interacting protein (JIPs), which
act as molecular scaffolds for the JNK-signaling pathway, and to rhoGEF, an exchange factor for the small
GTPase rhoA.78 This suggests that ApoER2 is able toassemble to a multi-protein complex, which might participate in Reelin signaling. Very recently, it has beenreported that JNK is important for neuronal migrationin the neocortex.79 These additional interactions might
explain the distinct phenotypes of mice lacking either
the VLDLR or ApoER2 alone.
Although the characterization of VLDLR andApoER2 as Reelin receptors has shed light on the
Reelin-Dab1 signal, the mechanism of Reelin-induced
Dab1 phosphorylation remains unclear. VLDLR andApoER2 do not possess intrinsic tyrosine kinase activity. Neither have they been shown to associate withtyrosine kinase or phosphatases. One hypothesis is that

Keio J Med 2003; 52 (1): 8–20

Fig. 4 The Reelin signaling pathway. Binding of Reelin to cell surface receptors results in tyrosine phosphorylation of Dab1. Src family
protein kinases (Src, Fyn, Abl) are potential candidates that phosphorylate Dab1. The Reelin signal is transmitted to Dab1 through avery low density lipoprotein receptor (VLDLR) or ApoE receptor 2(ApoER2). Cadherin-related neuronal receptors (CNRs) and a3b1
integrin are the candidates for co-receptors. Dab1 binds to the cytoplasmic tails of VLDLR and ApoER2 and to those of members of theamyloid precursor protein (APP) family. Dab1 can bind to phospholipids at the same time, including PtdIns(4,5)P2 which is a cofactor insignaling to the actin cytoskeleton.

Dab1 is tyrosine-phosphorylated by a coreceptor. It wasreported that Reelin binds to cadherin-related neuronal
receptors (CNRs),80 and a3b1 integrin81 in biochemical studies. As CNRs bind Fyn in their cytoplasmic
tails82 and Src associates with integrin-dependent
cytoskeletal complexes, both receptors could contribute to Reelin-induced Dab1 tyrosine phosphorylation.
Further studies are required to elucidate the complex
interactions between Reelin and its various receptors.
In particular, the functional importance should beshown genetically in vivo.
How does Reelin regulate neocortical layering?
Despite the identiﬁcation of molecules in the Reelinsignaling pathway, the response of cortical neurons to
the Reelin signal remains unclear.
Several lines of studies indicate that Reelin inﬂuences neuronal migration by altering the adhesive properties of neurons. In reeler mutants, early-born neurons
maintain abnormally extensive contacts with the radialﬁbers and obstruct the movement of late-born neurons.83 Neurons from reeler mice exhibit increased adhesive properties in cell aggregation assays.84 Earlierborn neurons from normal and reeler brains form
aggregates with different properties, probably reﬂecting
altered adhesion in reeler mice.38,85 In addition, a recent study demonstrated that Reelin acts as a detach-13

ment signal in chain-migrating interneuron precursors
in the olfactory bulb.86 Reelin might alter the adhesive
properties of neurons to release neurons from radialﬁbers and/or to place them in distinct layers. The future
identiﬁcation of molecular cues, for instance, the adhesion molecules that interact with neurons within eachlayer and their altered properties in reeler mice, will be
important to test this model.
Reelin was also suggested to detach neurons fromradial ﬁbers by binding to a3b1 integrin, which regulates neuron-glia interactions.81 The Reelin-binding
to these integrins has been thought to switch migratingneurons from a gliophilic adhesive preference to aneurophilic adhesive preference. However, the phenotypes of mice lacking the b1 gene are quite different
from those of reeler, and a major role of b1 integrin in
the brain seems to be in establishing the integrity of thepial basement membrane.87 To what extent the inhibitory effects of Reelin binding to a3b1 contribute toneuronal migration remains unknown.
The phenotypes of mutant mice that lack the Cterminal region of Dab1 are interpreted in terms ofthe ‘‘stop signal’’ model,88 but also consistent with the‘‘detachment model’’. Unlike full length Dab1, a single
copy of the truncated gene does not support normal
migration of the CA1 pyramidal cell in the hippocampus and the late-born cortical plate neurons. In the
neocortex, the late-born neurons mistakenly enter layer
1, probably because they fail to receive the Reelin signal and over-migrate. It will be extremely interesting
to test the conditional ablation of Reelin or Dab1 during late cortical development to verify the function of
Reelin on late-born cortical neurons.
With the aim of elucidating the Reelin function atransgenic mouse was produced, in which an ectopic
expression system was used to target Reelin to theventricular zone under the control of the nestin
promoter/enhancer.89 In the presence of Reelin protein, ectopic Reelin did not alter cell migration. However, in the reeler background mice that express ectopic
Reelin, some, but not all, cortical neurons migrate past
the subplate in the presence of ectopic Reelin in theventricular zone. Consequently, subplate neurons weresplit away from the Cajal-Retzius cells by the insertion of a subpopulation of cortical neurons. From theseresults, Reelin does not appear to function simply as astop signal or an attractant. Rather, ectopic Reelin was
thought to promote cell-cell interactions that are sufﬁcient for the preplate splitting. However, studies areneeded to elucidate where ectopic Reelin is secreted, asa small amount of Reelin might be transported to themarginal zone though the ﬁbers and might inﬂuence thesplitting of the preplate.
The role of Reelin in promoting splitting of the preplate may be distinct from its effect on lamination, as

14laminar formation was not rescued despite the successful prelate splitting in reeler mice that express ectopic
Reelin.89 Similarly, in the brains of chimeras consistingof Dab1 mutant (Dab1=) and wild type (Dab1þ=þ)cells, the preplate was split by Dab1þ=þ neurons, but
the Dab1= neurons could not pass the formed subplate, indicating failure in splitting the preplate is not
the sole defect in Dab1= mutant (Dab1 scm ).90 Thus
Reelin might exert different inﬂuences on early- andlate-born neurons. Moreover, Reelin does not function
exclusively in layer formation. Reelin is thought to act
as a branching signal for the entorhinohippocampal
projections40 and for the glial processes in the hippocampus91. In addition, abnormalities in retinal synaptic
layering are also observed in reeler.92 Different subsequent intracellular signaling events evoked by ReelinDab1 signal might underlie the speciﬁc responses.
Identiﬁcations of effector molecules are hoped to elucidate the roles of Reelin-Dab1 signal in each phase.
Cdk5/p35 and cross talk
Genetics has provided another group of mutant
mice that have defects of neocortical layer formation.
They include mice with a targeted disruption of cyclindependent kinase 5 (Cdk5)93 or its regulatory subunit
p35.94 Cdk5 is a serine/threonine kinase, and unlike
other members of the Cdk family, its activity is detected
mainly in postmitotic neurons95 Association of Cdk5
with a neuron-speciﬁc regulatory subunit, either p35 or
its isoform p39, is critical for its kinase activity.96–98
Null mice for Cdk5 exhibit perinatal death associatedwith cortical laminar defects,93 whereas mice lacking
p35 display a milder phenotype because of the redundancy of Ckd5 regulatory subunits.94,99,100 Moreover,
p35 and p39 double mutant mice show a phenotype
identical to that of Cdk5 null mice, conﬁrming that p35
and p39 are essential for Cdk5 function during brain
development.101
In mice that are null for either Cdk5 or p35, theﬁrst cohort of migrating cortical neurons successfully
invades and splits the preplate.93,94,99,102 However, thelate-generated neurons are unable to migrate past theirpredecessors and pile up in an inverted layer under
the subplate. Consequently, neurons are packed in an‘‘outside-in’’ pattern, in an indistinct laminated structure. Despite similarities between the phenotypes of
the reeler mice and those of Cdk5 and p35 mutants,
differences are also apparent. For instance, the marginal zone (layer 1) is not present in the reeler mice andpresent in Cdk5 and p35 mutants, albeit very thin in
Cdk5 mutants. The corpus callosum is largely intact inreeler but diminished in Cdk5 and p35 mutants. Unlike
reeler, Cdk5 mutants die during the perinatal period,
while p35 mutants suffer from seizure activity. The

Kubo K and Nakajima K: The mechanisms of cortical layer formation

Fig. 5 Cyclin dependent protein kinase 5(Cdk5)/p35 signaling. Cdk5/
p35 may regulate the dynamics of microtubules though phosphorylating microtuble-associating proteins Tau, MAP2, and Nudel.
Associated with Nudel, mNudE, and dynein, lissencephaly 1 (Lis1)
interacts with the microtubules, probably in concert with Doublecortin (Dcx). Cdk5/p35 also causes Pak1 hyperphosphorylation in a
RacGTP-dependent manner and modulates dynamics of the actin
cytoskeleton. Dab1 is serine-phosphorylated by Cdk5/p35. Active
Fyn and Abl can tyrosine-phosphorylate Cdk5, which is enhanced by
Cables.

large neurons of Cdk5 mutants in the brainstem andin the spinal cord show degenerative chromatolyticchanges, partly explaining the perinatal mortality.93
These differences imply that Reelin-Dab1 and Cdk5/
p35 have different roles in neuronal migration. Furthermore, the double-null mice for p35 and Reelin/
Dab1 demonstrate more severe migration defects than
the single mutants, indicating that Cdk5/p35 andReelin/Dab1 act on the different signaling pathways.100
This is in contrast with the phenotypes in mutant micelacking both Reelin and Dab1 that resemble those of
the individual mutants of these genes.64
However, it is also obvious that Cdk5/p35 andReelin/Dab1contribute synergistically to neuronal migration, and biochemical studies suggest cross talkbetween these pathways (Fig. 5). For instance, as
above-mentioned, Dab1 is phosphorylated by Src
family PTKs in vitro, and active c-Abl, a member of Src
family PTKs, leads to Cdk5 phosphorylation on tyrosine 15 (Y15), which stimulates p35/Cdk5 kinase activity. This phosphorylation is enhanced by Cables, a protein which exhibits limited homology to cyclin A,
suggesting that Cables may mediate the interaction
between Cdk5 and Dab1 by binding to both Cdk5 andc-Abl.103 Fyn, another member of Src family PTKs,
also associates with Cdk5 and facilitates the activationof Cdk5 through Y15 phosphorylation, coordinately
mediating neuronal guidance regulated by Semaphorin3A.104 Hyperphosphorylation of Tau, a microtuble-

Keio J Med 2003; 52 (1): 8–20

stabilizing protein and a substrate of p35/Cdk5, isreported in mice that lack either Reelin or bothVLDLR and ApoER2, supporting the links between
Reelin signaling and p35/Cdk5.75 However, it is not yet
known whether Reelin signaling actually stimulates
p35/Cdk5 kinase activity in vitro or in vivo.
On the other hand, Dab1 itself is a substrate for
Cdk5, and is phosphorylated by Cdk5 on serine 491
in vitro and in vivo.105 As already mentioned above,mutant mice that lack the C-terminal region containing
serine 491 show normal development, implying that
the C-terminal region might not be essential for Reelinsignaling.88 However, unlike full length Dab1, a single
copy of the truncated gene does not support normal
migration of the CA1 pyramidal cell in the hippocampus and late-born CP neurons. These results might
imply that the Dab1 C-terminus affects the strength ofReelin signaling. Although Dab1 knock-in mice should
be made to assess the precise functional signiﬁcanceof Dab1 serine phosphorylation, Cdk5 might serve tomodulate Reelin signaling.
How do p35/Cdk5 contribute to neuronal migrationand neocortical layering? Several studies indicate that
p35/Cdk5 may regulate actin/microtubule dynamics(Fig. 5). The interactions between p35/Cdk5 and Rac/
Pak1 suggest that p35/Cdk5 may modulate dynamics ofthe actin cytoskeleton in neurons.106,107 Microtuble
associating proteins Tau, MAP2, and Nudel are substrates of p35/Cdk5.108–111 Additionally, p35/Cdk5 may
regulate cell adhesion by N-cadherin during corticaldevelopment by binding to b-catenin.112
Defects of Cortical Layer Formation in Humans
In humans, the malformation due to abnormal
neuronal migration often appears as lissencephaly9(‘‘smooth brain’’, from ‘‘lissos’’, meaning smooth, and‘‘encephalos’’, meaning brain). Concerning lissencephaly, analogous mutations in mice have been produced by gene targeting in detailed investigations.
Type 1 (classical ) lissencephaly
In patients with type 1, or classical lissencephaly(LIS), both agyric (convolution is absent) and pachygyric (convolution is broad) regions have a characteristic ‘‘four-layered’’ cortex, composed of (1) a molecularlayer, (2) an outer cellular layer (true cortex), (3) a cellsparse layer, and (4) a deep cellular layer composedof heterotopic incompletely migrated neurons.113,114
Clinical manifestations are mainly epilepsy and mental
retardation. Two genes associated with LIS have beencloned: lissencephaly 1 (Lis1, also known as MillerDieker syndrome chromosome region, MDCR, or more
properly, the b subunit of platelet activating factor

15

acetylhydrolase, PAFAH1B1)115,116 and doublecortin(Dcx, also known as XLIS).117,118 In more than 90%
of patients with Miller-Dieker syndrome (MDS) and@40% of those with isolated lissencephaly sequence(ILS), heterozygous mutations in the Lis1 gene areobserved.115,119 Dcx is responsible for X-linked ILS;
only hemizygous males develop X-linked ILS, and heterozygous females have a subcortical band of heterotoipia (SBH).117 Whereas the brain malformation due
to Lis1 mutations was more severe over the parietal
and occipital regions, Dcx mutations produced the reverse gradient, which was more severe over the frontal
cortex.119 The distinct LIS patterns suggest that Lis1
and Dcx may work in a partly overlapping, but distinct
manner, in human neuronal migration.
To further address the function of Lis1, mutant
alleles in mouse Lis1 were produced.120 Lis1 nullmice die soon after implantation, indicating that Lis1
is an essential gene. Lis1 heterozygotes show cellautonomous delayed neuronal migration. Cortical development progressed in a normal inside-out fashion,
but formed cortical layers are indistinct. Further
reduction of Lis1 activity displays more severe brain
disorganization, suggesting a dosage-sensitive role inneuronal migration. Lis1 contains a WD (tryptophanaspartic acid) repeat, and interacts with multiple proteins, including an ATP-driven microtubule motor
dynein121–124 and Nudel/mNudeE.110,111,125 These
interactions have profound effects on the microtubule
structure of a cell, and may play central roles in microtubule dynamics for nuclear movement, cellular transport, and migration. Furthermore, Nudel is a substrate
for Cdk5 and is regulated by Cdk5/p35 to modify neuronal morphology.110,111 However, a precise model for
how these interactions are regulated during brain development is still unclear and further investigations are
required.
A mouse carrying a targeted mutation in the Dcx
gene has also been created.126 Hemizygous male Dcx
mice, however, show neocortical lamination largely
indistinguishable from the wild type and show normal
patterns of neocortical neurogenesis and neuronal migration. Migrating neurons in the human brain may
have a greater dependence on Dcx function than mice.
Proteins homologous to Dcx, such as DCAMKL1,127
may sufﬁce to support migration in the Dcx mutant
mouse. As Dcx binds to microtubules and stimulates
the polymerization of puriﬁed tubulin,128,129 Dcx
seems to regulate the organization of microtubules incooperation with Lis1.
Whereas hemizygous Dcx mutations and heterozygous Lis1 mutations produce lissencephaly and profound neocortical disorganization in humans, analogous
mutations in mice cause more subtle (in the case ofLis1) or negligible (in the case of Dcx) neocortical

16

Kubo K and Nakajima K: The mechanisms of cortical layer formation

defects. Perhaps in humans, in whom cortical neurons
transverse a much longer absolute distance than in mice
during brain development, migrations are more subject
to various impairments, resulting in more severe disorganizations.

six broad classes, LCHa-f, that are grouped according
to their distinguishing features.10,138 Among them, it
has recently been reported that an autosomal recessive
form of human lissencephaly (LCHb) was mapped tochromosome 7q22 and was associated with two independent mutations in the human gene encoding reelin(RELN ).139 This distinctive pattern possesses a moderately thickened cortex and pachygyria, markedly abnormal hippocampal formation, and severe cerebellar
hypoplasia with absent folia.139,140 Some humans withreelin mutations show a severe delay in neurologicaland cognitive development, accompanied with epilepsy.139
Additionally, some patients show abnormal neuromuscular connectivity and congenital lymphedema, andone showed accumulation of fatty ascites ﬂuid that
required peritoneal shunting.139 These unsuspected
symptoms might reﬂect Reelin functions via LDL receptor families outside the brain.

Type 2 (cobblestone) lissencephaly
Type 2 or cobblestone lissencephaly is the common
brain abnormality seen in some types of congenital
muscular dystrophies (CMDs), in which abnormal neuronal migration results in a brain with a bumpy, cobblestone appearance and loss of the normal folding
pattern.130 In cobblestone lissencephaly, the pia is disrupted and discontinuous, and through the gaps corticalneurons migrate over the outer surface of the pia,
forming piles of neurons.
Two of these for which human gene mutations havebeen found are Fukuyama congenital muscular dystrophy (FCMD) and muscle-eye-brain disease (MEB).
The FCMD gene fukutin,131 shares homology withfringe-like glycosyltransferases, and the MEB gene,
POMGnT1, seems to be a new glycosyltransferase.132
Very recently, it was reported that reduced (¼hypo)glycosilation of a-dystroglycan is involved in these two
diseases, as well as in a spontaneous mouse mutant, themyodystrophy (myd) mouse.133 In the myd mouse, amutation in the LARGE gene, which is thought to encode another glycosyltransferase, has been linked to a
muscular dystrophy phenotype. The hypoglycosylationof a-dystroglycan results in impairment in the binding
components of the basement membrane and disruptionof the pia. Moreover, brain-selective deletion of dystroglycan in mice is sufﬁcient to cause CMD-like brain
malformation, convincing proof of the role of dystroglycan in stabilizing the pial basement membrane.134
Similar cortical dysplasias are also found in mice withtargeted disruption of the basement membrane protein hepalan sulfate proteoglycan Perlecan,135 of thenidogen-binding site of Laminin g1,136 of a6 integrin,137 and of a brain-speciﬁc b1 integrin.87 In all
mutants, including human CMDs, the continuity of thepial basement membrane is disrupted, accompanied bycortical dysplasia. Perlcan, Nidogen1, and Laminin1
are distributed in the pial basement membrane, while
Laminin receptors a6 integrin and b1 integrin are
expressed in the developing neocortex, implying thatthe interaction between the pia and neocortex is indispensable for normal neuronal migration, as well as thecontinuity of the pial basement membrane.
Lissencephaly caused by mutation in the reelin gene
The nomenclature for lissencephaly with cerebellar
hypoplasia (LCH) is only now evolving, and comprises

Concluding Remarks
Recent genetic studies have provided detailed information about molecules important for neuronal migrations over a relatively short period of time. On theother hand, direct observation of migrating neurons is
expanding our knowledge of the different behavior of
migrating neurons in the formation of the cortex. In the
coming years, it is hoped that these molecular and cellular approaches will work together to rapidly elucidate
the mechanisms that regulate cortical layer formations,the basis of the elaborate networks in the brain.
Acknowledgements: This work was supported by Kowa Life Science
Foundation, Keio Health Counseling Center, Japan Science andTechnology Corporation, Ministry of Education, Culture, Sports, andScience and Technology of Japan, and Japan Society for the Promotion of Science.

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