e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y 1 5 ( 2 0 1 1 ) 3 6 1e3 6 7

Official Journal of the European Paediatric Neurology Society

Original article

ApoER2 and VLDLR in the developing human telencephalon

Lin Cheng a,1, Zhiliang Tian b,1, Ruizhen Sun a, Zhendong Wang a, Jingling Shen a,Zhiyan Shan a, Lianhong Jin a,**, Lei Lei a,*aDepartment of Histology and Embryology, Harbin Medical University, Harbin 150081, Heilongjiang Province, ChinabDepartment of Pediatrics, The Second Affiliated Hospital of Harbin Medical University, Harbin 150081, Heilongjiang Province, China

a r t i c l e i n f o

Article history:

Received 7 January 2011

Received in revised form

27 March 2011

Accepted 28 March 2011

Keywords:

Reelin-Dab1 signaling pathway

ApoER2

VLDLR

Telencephalon

Neuroblast migration

* Corresponding author. Tel.: þ86 1331461662** Corresponding author. Tel.: þ86 1390360807

E-mail addresses: wstjlh@126.com (L. Jin1 These authors contributed equally to thi

1090-3798/$ e see front matter ª 2011 Europdoi:10.1016/j.ejpn.2011.03.011

a b s t r a c t

The Reelin-Dab1 signaling pathway plays a crucial role in regulating the migration and

position of cortical neurons during the development of the cerebral cortex. Mutation in

Reelin may result in severe developmental disorders such as autosomal recessive lissen-

cephaly. Apolipoprotein E receptor type-2 (ApoER2) and very low-density lipoprotein

receptor (VLDLR) are canonical receptors of Reelin, through which extracellular Reelin

activates the intracellular adapter, Disabled1(Dab1), and subsequently interacts with other

molecules. Although it is widely accepted that ApoER2 and VLDLR are indispensable

components of the Reelin signaling pathway, little is known of their expression pattern in

the laminated developing human brain. Here, we collected 18 cases of human fetal brains

of 6e18 gestational weeks (GW) old and examined the expression of ApoER2 and VLDLR in

the their telencephalon using immunocytochemical staining. We found that both receptors

were absent in the preplate (PP) and the earliest stage of the cortical plate (CP). In later

stages of CP development, ApoER2 was expressed earlier than VLDLR in the migrating

neurons. Thus, the Reelin-Dab1 signaling pathway may not be involved in the formation of

the preplate and deep layers of the CP. Instead, the pathway may act on neurons that are

destined to form the more superficial layers of the CP. In addition, the pathway required

ApoER2 only rather than both ApoER2 and VLDLR at the initiation of activity.

ª 2011 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights

reserved.

1. Introduction the complex six-layer laminated human neocortex, which

Neuroblast migration is a key event in the development of the

human brain, and most neuroblast migration occurs between

gestational week (GW)7 and GW16 in humans.1 During this

period, millions of postmitotic neurons that are generated

from the proliferative zones migrate to their destination in

specific layers of the cerebral cortex, and eventually establish

1.9.

), leil086@yahoo.com.cn (Ls work.ean Paediatric Neurology

shows distinct cell types and functions in the different layers.

The mammalian neocortex is formed by both columnar and

laminar organizations, which are composed of ontogenic

radial clones and horizontal arrays respectively. Radial glial

cells are the major neuronal progenitors during the develop-

ment of neocortex. A radial clone is composed of one radial

glial mother cell and neurons generate sequentially from

. Lei).

Society. Published by Elsevier Ltd. All rights reserved.

e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y 1 5 ( 2 0 1 1 ) 3 6 1e3 6 7362

asymmetrical dividing of this progenitor cell which moving

out toward the pial surface. The radial glial cell’s soma locates

in the proliferative zone and axon extends throughout the

whole cerebral wall as a scaffold while the migrating neurons

along the radial glial fiber are of varying stages of matura-

tion.2,3 Neurons in a radial clone specifically form unidirec-

tional synaptic connection.4 A horizontal clone is composed of

cells generated simultaneously from multiple, related

progenitors which migrate into a common layer. Cells within

the same layer have similar morphology, connection and

biochemical features. Multiple radial and horizontal units

eventually form the radial functional columns of the primate

neocortex.2 Abnormal in these neuroblast migration may

result in several severe developmental disorders such as lis-

sencephaly, heterotopia, focal cortical dysgenesis, poly-

microgyria and schizencephaly.5 For example, during the

normal neocortex development, the numbers of ontogenetic

radial columns become decreasing and neurons in a certain

column are fewer as development progressed into postnatal

stages. Persistent and abundant microcolumnar organization

with more than eight neurons aligned in a vertical direction is

associated with focal cortical dysplasia (FCD).6

The first cohort of postmitotic neurons migrate out of

the proliferative zones, including the ventricular zone (VZ)

and some extra-cortical sites of origin such as the medial

ganglionic eminence (MGE),7 and move radially or tangen-

tially to invade deep to the pial surface. These heteroge-

neous cell populations form a cell-sparse layer between the

VZ and the pial surface called the preplate (PP).8,9 Among

these heterogeneous cells, the most important cell pop-

ulation are the CajaleRetzius (CR) cells, which are the main

source of Reelin in the mammalian embryonic cortex and

later restrict in the molecular zone. CR cells are required for

the normal radial migration and laminar position of pyra-

midal neurons of the cortical plate, and their axonal

collaterals form synapses with early migrating neuroblasts,

establishing early synaptic circuitry in the neocortex. In

human, the CR cells are more numerous and more highly

developed than other species which extend most notably to

their axonal plexus that forms a dense, compact fiber layer,

separating the cortical plate from the MZ before the first

wave of radially migrating neuroblasts from the prolifera-

tive zone.10

At GW7, the PP splits into the abovemarginal zone (MZ, the

layer I in the mature cerebral cortex) and the below subplate

(SP) by waves of newly arriving postmitotic neurons from the

VZ, as well as subventricular zone (SVZ), another proliferative

zone that emerges at the border of the VZ at embryonic day

40-41.9,11 The compartment produced by these neurons is

known as the cortical plate (CP), which becomes the IIeVI

layers of the mature neocortex. From GW8 on, sequential

waves of neurons migrate from the VZ and SVZ, and move to

the uppermost region of the cortical plate, just beneath the

pial surface. During this process, later born neurons migrate

past the early ones that have settled at their final destination,

and occupy the superficial layers. Subsequently, they undergo

differentiation by extending processes and establishing

synaptic connections. Thus, except for layer I, which is made

of the earliest generated neurons, the six-layer neocortex

forms by an “inside-out” pattern.12,13

During this “inside-out” migration, the Reelin-Dab1

molecular pathway has been shown to play indispensable

roles.14e19 Reelin is an extracellular matrix protein secreted

mainly by CajaleRetzius(CR) cells in the MZ.15e20 The binding

of Reelin to its receptors, ApoER2 and VLDLR, onmembrane of

migrating neurons phosphorylates the intracellular adapter,

disabled1 (Dab1), in the presence of SFKs or CDK5,21e25 which

then interacts with multiple molecules to regulate the

migration and position of neurons.26e28 Reelin also plays an

important role in the development and lamination mainte-

nance of hippocampus and the entorhinal-hippocampal

network. Reelin deficiency causes dentate granule cell

dispersion that is the most common altered pattern in the

patients with medial temporal lobe epilepsy (MTLE).29 Also,

the downregulation of reelin expression has been proposed to

lead to an inhibition of maturation and plasticity of dendritic

spines and reductions in reelin expression in entorhinal

cortical related to cognitive function in aged ratswithmemory

impairment.30,31

In animal experiments, a deficiency of any component in

this pathway results in a disturbance of the brain structure to

different extents. Mutations of Reelin, Dab1 and double

mutations of ApoER2 and VLDLR all produce reeler-like

phenotypes.16 In these phenotypes, the PP forms normally,

but does not split, appearing as a “superplate” that includes

both MZ cells and subplate cells.9,32 In addition, the CP lami-

nation is inverted, with later born CP neurons settling below

early ones rather thanmigrating past them. A singlemutation

of ApoER2 or VLDLR leads to less severe phenotypes.23,33

Although remarkable progress has been made in under-

standing the role of the Reelin signal in cerebral cortex

development, those achievements were obtainedmainly from

non-human species, especially rodents. The precise role of the

Reelin pathway in human beings is poorly elucidated due to

the ethics issues. However, there might be great differences

between rodents and humans.With the extreme expansion of

the human cortex, the complexity of the morphology and

function of human cortical structure progressively increased.

On the other hand, the period of human cerebral cortex

development is much longer than that of rodents, which may

exhibit some specific events that cannot be observed in mice,

and is of great evolution consideration. For example, subpial

granular layer (SGL) is an important compartment involving in

the neuroblastmigrationwhich ismore prominent in humans

than other species. SGL is a transient cell dense layer in the

uppermost area of the MZ just beneath the pial surface. SGL

cells represent a precursor pool for reelin-producing neurons

and express the gene doublecortin-DCX.34e36 Abnormal of SGL

has been seen in association with many cortical maforma-

tions. In some cases of lissencephay, absent or thin or

discontinuous SGL can be found.37 While persistent SGL result

in marginal glioneuronal heterotopia, suggesting the SGLmay

serve a barrier function to migration space.38

Among the components of the Reelin signaling pathway,

the expression of Reelin during the development of human

brain has been well examined while the localization of the

reelin receptors ApoER2 and VLDLR were only detected in the

developing human cortex at GW22,39 the expression patterns

of both receptors at earlier stages are still unknown. In this

study, we examined the expression of ApoER2 and VLDLR in

e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y 1 5 ( 2 0 1 1 ) 3 6 1e3 6 7 363

fetal human cortex from the preplate stage at GW6 to the

middle of gestation, about GW18, which almost covers the

entire process of neuroblast migration, to gain new insight

into the first step of human corticogenesis and the role of the

Reelin signaling pathway in the development of the human

cerebral cortex.

2. Materials and methods

Human embryonic or fetal brain material (18 cases, 6e18

gestational weeks (GW) old) was collected from spontaneous

or medically induced abortions, following the national

guidelines in China and supervised by the Ethical Committee

of the Harbin Medical University.

Brains were fixed in 4% paraformaldehyde, embedded in

paraffin and cut sagittally into 5-mm-thick serial sections.

Immunohistochemistry was carried out using anti-VLDLR

(1:200, Santa Cruz Biotechnology) and anti-ApoER2 anti-

bodies (1:200, Santa Cruz Biotechnology).

Sections were deparaffinized, rehydrated and washed in

0.02 M PBS, then incubated for 30 min in blocking solution (5%

BSA) at room temperature. Subsequently, sections were

incubated with the primary antibody overnight in a humid

chamber at 4 �C. The control group received PBS. After

Fig. 1 e Compartments of the cortical wall of the human telence

eosin. A. At the PP stage, GW6, the cortical wall consists of the VZ

in the PP (arrow). B. The initial CP stage, GW7. The CP began to

Layering of the cortical wall at GW9 (C), GW11 (D) and GW18 (E, p

could not been seen in a single visual field). During these stage

CP, SP and IZ was growing thicker. PP, preplate; MZ, marginal z

SVZ, subventricular zone; VZ, ventricular zone.

washing three times in 1�PBS at room temperature, they were

incubatedwith the biotinylated secondary antibody for 30min

at room temperature, then washed and visualized using the

ABC standard kit following the manufacturer’s suggestions.

Conterstained with hematoxylin to indicate the layers.

3. Results

3.1. Development of the human telencephalon fromGW6 to GW18

At GW6 (Fig. 1A), the VZ occupied the major portion of the

cortical wall. The narrow, cell-sparse PP was superior to the

VZ, and the earliest predecessor cells, CajaleRetzius cells (CR

cells), could be clearly discriminated by their horizontal

orientation (arrow). The initial cortical plate (the prospective

neocortex) emerged at early GW7 (Fig. 1B), when a very thin

layer of neurons accumulated in the PP, splitting the PP into

the upper MZ and the lower SP. At this time, the boundary of

the SP and intermediate zone (IZ) was not clear. CR cells were

restricted to the MZ. In successive stages, huge numbers of

neurons generated from the VZ and SVZmigrated into the CP,

with successive neuronsmoving to positions superficial to the

previous neurons. The number of neurons in the CP increased

phalon from GW6 to GW18 stained with hematoxylin and

and a thin PP. CajaleRetzius cells are clearly discriminated

appear in the PP, splitting the PP into MZ and SP. CeE.

ieced together by two images of the complete structure that

s, the proliferative zones were becoming thinner while the

one; CP, cortical plate; SP, subplate; IZ, intermediate zone;

e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y 1 5 ( 2 0 1 1 ) 3 6 1e3 6 7364

progressively and the CP, SP and IZ expanded in width.

Meanwhile, the proliferative zones decreased in size gradually

(Fig. 1CeE). At GW18, approximately midgestation, the

proliferative zones, especially the VZ, became very thin,

indicating that most of the CP neurons had exited their cell

cycle and migrated to their destination. At the same time, the

SP and IZ became very thick, suggesting substantial nerve

fiber formation.

3.2. Expressions of ApoER2 and VLDLR in the cerebralwall before the CP formation

At GW6, when the PP had appeared between the proliferative

zone, the VZ, and the pial surface, both ApoER2- and VLDLR-

immunoreactivities were not detected in the cerebral wall

(Fig. 2A,B).

3.3. Expression of ApoER2 and VLDLR during the CPformation

3.3.1. Expression of ApoER2 and VLDLR at the onset of the CPformationAt GW7, neurons generated in the VZ began to migrate and

invaded the PP, which initiated formation of the CP. These

earlier born neurons were destined to be deeper layers of the

future six-layer cerebral cortex. Later generated neurons

migrated past these early ones and formed the upper layers.

During this time, ApoER2-immunoreactivity was apparent in

the proliferative zone, while VLDLR-immunoreactivity was

not detected. Another interesting observation was that, in this

period, neither ApoER2- nor VLDLR-immunoreactivity was

detectable in the newly formed CP (Fig. 3A,B).

3.3.2. Expressions of ApoER2 and VLDLR from GW8 to GW18From GW8 to GW18, the expression of ApoER2 and VLDLR

were similar, and both were detected throughout the cerebral

wall, with their immunoreactivity stronger in the top of the CP

than in other compartments. As to specific layers, it was

difficult to distinguishwhether these two receptors differed in

expression. Most of the CR cells expressed both receptors

(Fig. 4AeH).

Fig. 2 e The expression of ApoER2 and VLDLR at GW6.

Immunostaining for ApoER2 (A) and VLDLR (B) sagittal

brain sections at PP stage. Both receptors were absent

throughout the cerebral wall. PP, preplate; VZ, ventricular

zone.

4. Discussion

During the past three decades, the Reelin signaling pathway

has been examined extensively due to its crucial role in brain

development. However, the precise mechanisms of its action

and exact roles in every compartment of this pathway

remains unclear.40Because of ethics issues, few studies have

examined the role of this pathway in human brain develop-

ment, and animal experiments may not exhibit specific

evolutionary changes. In the present study, we examined the

expression of Reelin receptors in the developing human

telencephalon during neuroblast migration. Our results indi-

cate that Reelin receptors are not expressed in early stages of

neuron migration, and ApoER2 and VLDLR show different

expression patterns at the onset of Reelin-dependent

migration.

4.1. Cortical stratification during the development ofhuman brain

Initially, the cerebral wall is consisted of proliferative cells

only, in which the proliferative cells undergo symmetric

division to enlarge the size of the proliferative area. As early as

embryonic day (E) 33,8 postmitotic cells are observed in the

cortical wall and then exit the VZ and move to the area

between the pia and the VZ, forming the PP together with

predecessor neurons from other proliferative zones.8,9 Our

results on the development of the cerebral cortex were

consistent with previous studies. Before GW7, the cerebral

wall consisted of a cell dense proliferative zone, the VZ, and

a more superficial cell-sparse PP, consisting of CR cells and

other postmitotic cells of different originations. From GW7,

the CP neurons inserted into the PP. Previous studies indicated

that before formation of the CP, the PP shows sub-

compartmentalization, resulting in CR cells located in the

Fig. 3 e Expression of ApoER2 and VLDLR at GW7. At the

initial CP stage, immunostaining for ApoER2 and VLDLR on

sagittalbrainsectionsshowedthatApoER2 (A)wasexpressed

mainly in the VZ, while ApoER2-immunoreactivity was

negative in the CP andMZ. By contrast, VLDLR-immunore-

activity (B) was not observed in any compartment of the

cerebral wall. MZ, marginal zone; CP, cortical plate; SP,

subplate; IZ, intermediatezone;SVZ, subventricular zone;VZ,

ventricular zone.

Fig. 4 e The expression of ApoER2 and VLDLR in GW8 (A,B), GW10 (C,D), GW11 (E,F) and GW18 (G,H, both were pieced

together by three images of the complete structure that could not been seen in a single visual field). During the main period

for neuroblast migration, immunostaining for ApoER2 and VLDLR on sagittal brain sections showed that both receptors

distribute in every layer of the cerebral wall, with heavier staining in the superficial CP than in other layers. MZ, marginal

zone; CP, cortical plate; SP, subplate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone.

e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y 1 5 ( 2 0 1 1 ) 3 6 1e3 6 7 365

superficial region of the PP and future SP cells in the deep

portion of the PP.8,41 Our findings are consistent with these

observations, with the horizontal CR cells restricted to theMZ.

From GW8 to GW18, the cerebral cortex underwent peak

migration and extreme enlargement in size.

4.2. The PP and deeper layers of the CP formindependently of the Reelin-Dab1 signaling pathway

Our study on the expression of two receptors of Reelin

showed that at GW6 in the PP stage of the human cortex,

e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y 1 5 ( 2 0 1 1 ) 3 6 1e3 6 7366

neither ApoER2- nor VLDLR were expressed in any compart-

ment of the cerebral wall. It could be reasonably explained

that neurons in the PP were primarily from sub-proliferative

zones, using tangential migration to move to the PP, while

the Reelin signaling pathway mainly regulates the radial

migration. An interesting observation was that there were no

VLDLR- or ApoER2-immunoreactive cells in the early cortical

plate, which suggests that the Reelin signal may not be

necessary for the migration of deep layer neurons in the

human cerebral cortex. A real-time imaging study suggested

that there were two modes of radial migration. One mode

occurs in early stages of cortex development when the

distance between the VZ and the pia is relatively short.

Neurons move toward the pial surface by somal trans-

location, that is, they maintained their pial attachment while

losing their ventricular attachments gradually. In contrast, at

later stages when the migrating path is longer, neurons

migrate on radially oriented glial fibers that span the cerebral

wall.42,43 From our results, we conclude that neurons both in

the PP and in deep layers of the CP experience somal trans-

location, which was unaffected by the Reelin pathway, and

the radial glial guided migration began to predominate from

approximately GW8. Evidence supporting our conclusion is

that in the ApoER2 mutant mice, layer V and part of layer IV

were formed almost normally.44

However, previous studies showed human brain Reelin

expressed as early as GW5. Thus, whether Reelin plays roles

during the early stages of CP via other receptors or Reelin is

inactive at the very beginning of neuroblast migration needs

further research.

4.3. VLDLR and ApoER2 in the developing human cortex

VLDLR and ApoER2 are two members of the low-density lipo-

protein (LDL) receptor family and had been shown to be

indispensable components of the Reelin signaling pathway.

After their ectodomains bind to Reelin, their cytoplasmicNpxY

motifs are a docking site for Dab1.22,45 In mice carrying double

mutations of these two receptors, the animals displayed the

same phenotype as reeler, while only a mutation in one of the

receptors led to divergent abnormalities. VLDLR is more

important for development of the cerebellum, while ApoER2 is

more important for cortical lamination.33 Another experiment

in mice indicated that VLDLR deficiency resulted in the inva-

sion of CP neurons into theMZ, suggesting that VLDLR acted as

a stop signal in neuroblast migration, while ApoER2 deficiency

led to a disturbance of layers IIeIV neurons, proposing its role

in themigration of later generated neurons.44 In our study, we

investigated the expression of these two receptors in the

developing human brain, and found greater ApoER2-

immunoreactivity than VLDLR-immunoreactivity at GW7, the

earlier stage of cerebral migration. This result suggests

different roles of these two receptors in humans aswell.While

inGW8 toGW18, theywere both detected in thewhole cerebral

wall especially on the topof theCP, and it ishard to tellwhether

they have different expression patterns. Our result is differed

from the observation of Perez-Garcıa,39 who found that at

GW22, the ApoER2 and VLDLR expressed in the upper CP and

the future layers III and IV but not thewhole cerebral wall. The

possible reason of this difference is that during GW8 to GW18

the neuroblast migration was still ongoing while at GW22, the

migration had already completed. Further studies on both

receptors may elucidate the exact role of the Reelin signaling

pathway in the migration of cerebral neurons.

In conclusion, our findings suggest that the formation of

the PP and deep layers of the CP may form independent of the

Reelin-Dab1 signaling pathway. Furthermore, its rolemight be

prominent from GW8, for the neurons destined to form the

more superficial layers of the CP. At the onset of its function,

Reelin acts mainly via ApoER2 rather than through both

VLDLR andApoER2. It is known that the dentate gyrus is one of

the few sites that undergo continued neurogenesis

throughout life. Granule cells migrate from the subgranular

zone to the granular layer. Since recent studies have demon-

strated ApoER2 played critical role in the reelin-induced

dendritic development46 and the fact we found that reelin

played its role initially by ApoER2 at the beginning of the

neuroblast migration, we presume that the lamination and

synaptic development of hippocampus may be more associ-

ated with ApoER2. Further work is required to fully elucidate

the function of the Reelin signaling pathway in the develop-

ment of the human telencephalon.

Acknowledgment

This work was supported by the Project of Abroad Researcher

Foundation of Heilongjiang Province, China, Grant No.

LC07C17 and the Innovative Fund of Harbin Medical Univer-

sity Graduate Student, Grant No. HCXB201004.

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