Download Layer-Specific Markers as Probes for Neuron Type Identity in

J Neuropathol Exp Neurol
Copyright Ó 2007 by the American Association of Neuropathologists, Inc.
Vol. 66, No. 2
February 2007
pp. 101Y109
REVIEW ARTICLE
Layer-Specific Markers as Probes for Neuron Type Identity in
Human Neocortex and Malformations of Cortical Development
Robert F. Hevner, MD, PhD
Abstract
Malformations of cortical development (MCDs) are heterogeneous disorders caused by abnormalities of cell proliferation,
apoptosis, cell migration, cortical organization, and axon pathfinding. In severe MCDs, the cerebral cortex can appear completely
disorganized, or may be replaced by aberrant laminar patterns, as in
B4-layered[ types of lissencephaly and polymicrogyria. Little is
known about the abnormal layers in MCDs and whether they bear
any relation to normal cortical layers or how MCDs affect specific
neuron types. Normally, each layer contains a defined mixture of
different types of pyramidal and nonpyramidal neurons. The neuron
types are distinguished by molecular expression as well as morphologic, neurochemical, and electrophysiologic criteria. Patterns of
layer-specific mRNA and protein expression reflect the segregation
of different neuron types into different layers (e.g. corticospinal
projection neurons in layer V). Numerous layer-specific markers
have been described in rodent cortex, and increasing numbers are
being documented in human and monkey cortex. Applied to MCDs,
layer-specific markers have the potential to reveal new insights on
pathogenesis, treatment possibilities, and genotypeYphenotype correlations. However, much work remains before layer-specific
markers become practical tools in diagnostic neuropathology. Additional markers, more extensive documentation of normal expression,
and better antibodies compatible with paraffin-embedded tissues will
be necessary.
Key Words: Laminar fate, Lissencephaly, Miller-Dieker syndrome,
Neurogenesis, Periventricular heterotopia, Polymicrogyria.
INTRODUCTION
Malformations of cortical development (MCDs) are
morphologic disturbances of the cerebral cortex that arise
from perturbations of fundamental processes in brain
development (1, 2). The morphologic phenotypes of MCDs
are diverse. The principal phenotypes include lissencephaly,
polymicrogyria, heterotopia, dysplasia, micrencephaly, and
megalencephaly. The severity of the brain defects in MCDs
From the Department of Pathology, University of Washington, Seattle,
Washington.
Send correspondence and reprint requests to: Robert Hevner, MD, PhD
HMC Pathology Box 359791, 325 Ninth Avenue, Seattle, Washington
98104; E-mail: [email protected]
This work was supported by the National Institutes of Health (NS045018
and NS050248), the Mallinckrodt Foundation, and the Christopher
Reeve Paralysis Foundation.
ranges from subtle microdysgenesis (ectopic neurons) to
extreme hypoplasia and cortical disorganization (e.g. microlissencephaly). MCDs usually result from genetic mutations
that affect cell proliferation, apoptosis, neuron migration,
and cortical organization, although nongenetic etiologies
(e.g. hypoxiaYischemia and viral infection) can play a role as
well. Clinically, MCDs are significant causes of seizures,
cerebral palsy, mental retardation, and neuropsychiatric
disorders (1Y3).
Current MCD classifications rely on integration of
brain morphology and genetic analysis (1, 2). Morphologic
evaluations assign MCDs to descriptive phenotypic categories, whereas genetic studies provide precise molecular
diagnoses. This approach is necessary because most MCD
phenotypes are not tied to a single gene, chromosomal
abnormality, or nongenetic etiology, and different types of
mutations in the same gene can cause different phenotypes.
Classic (type I) lissencephaly, for example, can be caused by
chromosome 17p subtelomeric deletion (Miller-Dieker syndrome) and by at least four different single gene mutations
(LIS1, DCX, RELN, and ARX) (4). Micrencephaly is linked
to at least five genetic loci, in addition to several
chromosomal syndromes (5). Likewise, periventricular heterotopia is linked to several single genes and chromosomal
abnormalities (6, 7). Polymicrogyria is even more heterogeneous, with associations to not only genetic mutations
(8Y11) but also to fetal hypoxiaYischemia (12Y15) and
intrauterine infections (8). Nevertheless, it seems likely that
morphologic evaluation will become increasingly important
as additional genes are discovered, additional subtypes of
MCDs are distinguished, and genotypeYphenotype correlations are sought. Indeed, phenotypic differences between
lissencephaly caused by LIS1 and DCX mutations have
already been found. Interestingly, LIS1 lissencephaly affects
posterior cortex worse than anterior cortex, whereas DCX
lissencephaly shows the reverse gradient (16, 17). Histologic
patterns of cortical lamination also differ between LIS1,
DCX, and other forms of lissencephaly (18, 19).
To some extent, current problems of morphologic
diagnosis reflect underlying deficiencies in our knowledge of
abnormal cortical histology in MCDs. Descriptive neuropathology has done little to elucidate how histologic
phenotypes such as 2-, 3-, and 4-layered lissencephaly (19)
and 4-layered and unlayered polymicrogyria (8), relate to
normal 6-layered cortex. In this regard, it is important to
remember that layers in normal cortex are not merely patterns of cell distribution, but are specialized compartments
J Neuropathol Exp Neurol Volume 66, Number 2, February 2007
101
Copyright @ 2007 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.
Hevner
J Neuropathol Exp Neurol Volume 66, Number 2, February 2007
containing neurons with unique properties and roles in
neural circuitry. For example, neurons that project to the
brainstem and spinal cord are located in layer V, whereas
corticothalamic neurons are located in layer VI (20).
Abnormal layers in MCDs arise by redistribution of neurons,
transforming the normal 6 layers into an abnormal pattern,
but the effects on layer-specific neuron types generally
cannot be discerned on routine histologic staining. In
principle, it should be possible to map the pathways of
redistribution and thus link abnormal laminar patterns to
their origins from normal cortical layers, using layer-specific
molecular markers.
So far, attempts to relate abnormal patterns in MCDs
to normal cortical layers have generated reasonable hypotheses but no definitive answers. In polymicrogyria, different
explanations for 4-layered and unlayered patterns have been
proposed. Caviness and colleagues (12), using cresyl violet
histology, observed continuity of certain layers between
normal and 4-layered polymicrogyric cortex and proposed
that the 4-layered pattern was produced by laminar destruction of the middle cortical layers. Ferrer and Catalá (13, 14)
studied unlayered polymicrogyria, using Golgi impregnations to identify neuronal morphologies characteristic
of upper and lower cortical layers. They found that the relative positions of neurons along the radial (deep-superficial)
axis were generally preserved, although the apical dendrites
of many pyramidal neurons were maloriented. They suggested that the cortical disorganization in unlayered polymicrogyria resulted from irregular scarring due to vascular
compromise. Interestingly, the same studies suggested that
periventricular heterotopia (present in association with polymicrogyria) contained mainly upper-layer type neurons (14).
Studies of laminar relations in lissencephaly have
reached divergent conclusions. Using routine histologic
stains (hematoxylin and eosin, Luxol fast blue, and cresyl
violet), Encha-Razavi and colleagues (18) suggested that the
cortical layers were overall inverted in LIS1 lissencephaly
but were relatively preserved in DCX lissencephaly. Golden
and colleagues (19), relying on neurofilament protein
immunohistochemistry in addition to routine stains, reported
a Bnear inversion[ of cortical layers in both LIS1 and DCX
lissencephaly, although subtle differences between them
were also found. In contrast, Sheen et al (21) reached a
completely different conclusion. Using layer-specific neuronal markers to study a fetal case of LIS1 lissencephaly in
Miller-Dieker syndrome, they found that the positions of
cortical layers were relatively preserved and not inverted as
suggested by other studies (18, 19). In particular, neurons
immunoreactive for transcription factor FOXP1 were located
in deep layers of lissencephalic as well as normal cortex.
Additional work will be required to resolve the discrepancies
among these studies.
As the recent article by Sheen et al (21) illustrates,
layer-specific molecular markers are beginning to be
introduced in neuropathologic studies of MCDs, and moreover, this trend is likely to continue. Scores of layer-specific
markers have been identified in developing mouse cerebral
cortex (20, 22Y33), and increasing numbers are being documented in monkeys and humans. Layer-specific markers
102
have the potential to enhance both diagnosis and research in
developmental neuropathology by revealing how neuronal
positions map from layers in normal cortex to abnormal
patterns in MCDs. In this article, I review the neurobiology
and applications of layer-specific molecular markers, with an
emphasis on practical considerations for neuropathologists,
including technical aspects, limitations, and caveats. Whereas layer-specific markers are already well-established probes
for mouse neurobiology, expanding applications to human
neuropathology will require much additional groundwork.
Diversity of Neuron Types and Subtypes Within
Cortical Layers
It has been known since the work of Cajal that the
cerebral cortex contains 2 main classes of neurons, pyramidal and nonpyramidal neurons, which both encompass
multiple distinct types. Pyramidal neurons are the more
abundant class, accounting for 75% to 85% of cortical neurons, whereas nonpyramidal neurons account for 15% to
25% (34, 35). Neurons of the same type frequently occupy
the same cortical layer or layers, such as Betz cells
(corticospinal neurons) in layer 5 of the motor cortex. In
turn, the characteristic anatomical and functional properties
of each neuron type depend on its unique molecular
expression profile. This fundamental linkage between cortical neuron type, laminar position, and molecular profile is
the conceptual foundation for understanding layer-specific
gene expression (20). Thus, layer-specific markers may be
understood more accurately as neuron type-specific markers.
The principle that layer-specific patterns represent the radial
distribution of distinct neuron types is essential for understanding how these patterns arise and for avoiding pitfalls in
the interpretation of layer-specific markers.
The diversity of neuron types within layers has been
studied most extensively in layer V of rodent cerebral cortex. Layer V contains several distinct types of pyramidal
neurons (29, 36, 37) and interneurons (38) (Fig. 1A). The
two main types of pyramidal neurons in layer V are known
as tufted (type I) and nontufted (type II), according to the
degree of dendrite branching in layer I (37). Tufted pyramidal neurons make corticobulbar or corticospinal axon
projections, contain high levels of neurofilament proteins,
and fire bursts of action potentials (29, 37). In contrast,
nontufted pyramidal neurons make corticocortical or corticostriatal axon projections, do not contain neurofilament proteins in the cell body, and fire nonbursting action potentials.
Among interneuron types in layer V, the most abundant are
large basket cells, although chandelier, Martinotti, bipolar,
double bouquet, and bitufted interneurons are also found in
layer V (14, 38). In general, most interneuron types are
broadly distributed among layers (38), although a few types
do show laminar specificity, such as calretinin+ interneurons
in layers II to III (39).
Neuronal diversity within layers is apparent even in
the developing cortex (Fig. 1B). Indeed, many layer-specific
molecular expression patterns and differences between neuron types in the same layer are most prominent during
Ó 2007 American Association of Neuropathologists, Inc.
Copyright @ 2007 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.
J Neuropathol Exp Neurol Volume 66, Number 2, February 2007
Layer-Specific Markers in Cerebral Cortex
FIGURE 1. Diversity of neuron types in layer V of rodent cerebral cortex. (A) Adult cortex. Layer V contains glutamatergic
pyramidal projection neurons (multiple types) and GABAergic nonpyramidal interneurons (multiple types). Some wellcharacterized examples include tufted and nontufted pyramidal neurons and large basket and double bouquet interneurons.
Axons are shown in blue (projection neurons) or brown (interneurons). CB, calbindin; PV, parvalbumin; VIP, vasoactive intestinal
polypeptide. SMI-32, FNP-7, and N200 are neurofilament epitopes. (B) Developing cortex. Even while neurogenesis is still
ongoing, different types of projection neurons and interneurons can be distinguished. Labeling with DiI, a fluorescent cell stain,
reveals a Martinotti type interneuron (IN) and a radially oriented immature projection neuron (PN) in layer V. The axon (a) of
each neuron is indicated. Layer V projection neurons express different combinations of transcription factors, as revealed by twocolor immunofluorescence to detect Otx1 (green) in combination with SCIP (red) or ER81 (red). Transcription factor ER81 is
expressed by corticospinal (C-Sp) projection neurons, labeled retrogradely with a fluorescent tracer (green) and by other layer V
projection neurons. Red and green arrows indicate single-labeled cells; yellow arrows indicate double-labeled cells. Scale bars =
100 Km (morphology and molecular expression); 40 Km (axons).
development, when axon pathways, cell positions, and dendritic morphology are being established. Thus, fewer molecular markers show layer-specific patterns in the mature
cortex than during development. Neuron type and laminar
Ó 2007 American Association of Neuropathologists, Inc.
fate are specified early in development during cortical neurogenesis and subsequently remain set throughout life. The
developmental mechanisms that specify neuron type are
discussed in greater detail below.
103
Copyright @ 2007 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.
Hevner
J Neuropathol Exp Neurol Volume 66, Number 2, February 2007
Layer-Specific Molecular Markers
and Neuron Types
FIGURE 2. Layer-specific markers in developing and adult
human cerebral cortex. (A) In fetal cortex (32 gestational
weeks), MAP1B was expressed in a subset of layer V projection
neurons. Formalin-fixed, paraffin-embedded tissue. (B) In
adult cortex, nonphosphorylated neurofilament heavy chain
(NF-H) was expressed in a subset of layer V projection
neurons. Formalin-fixed, paraffin-embedded tissue. (C) Calretinin (green) and ROR-A (red) were expressed mainly in layers
II and IV of adult cortex, respectively. Frozen section. (D)
Ctip2 (green) and TLE4 (red) were expressed mainly in layers
V and VI of adult cortex, respectively, with a few double
labeled cells (yellow) in layer V. Frozen section. Scale bars =
(A) 200 Km; (B) 500 Km; (C, D) 100 Km.
104
Many molecules with layer-specific expression patterns have been identified in mice (20, 22Y33). Using
appropriate panels of markers, it is now possible to map
cortical layers and areas with remarkable precision in mice
(20, 40Y42). Eventually, it seems likely that molecular
markers will be used to define all cortical neuron types,
although that capability is still a distant goal. Many different
families of molecules exhibit layer-specific expression
patterns, including axon guidance molecules, secreted
morphogens, calcium-binding proteins, cytoskeletal proteins,
transcription factors, and others. Depending on the type of
molecule and its subcellular distribution, layer-specific
patterns may be evident from expression of mRNA, protein,
or both. Discrepancies between mRNA and protein expression patterns can be seen, for example, with genes in the Eph
and ephrin families. These axon guidance molecules show
layer-specific mRNA expression, but the proteins are
redistributed across layers by axonal transport (43). In
contrast, mRNA and protein laminar patterns are typically
identical for molecules with protein localization to the
nucleus or perikaryon, such as transcription factors.
Fewer layer-specific markers have been documented in
human and monkey than in mouse cerebral cortex, but this is
likely to change as additional studies are completed. Not
surprisingly, many layer-specific markers in primates belong
to the same molecular families as those identified in mice,
including Eph and ephrin genes (44), transcription factors
(21, 30, 31, 45, 46), secreted molecules (47Y49), cytoskeletal
proteins (50Y54), and calcium-binding proteins (39, 46, 48,
53, 55, 56). Cytoskeletal proteins and calcium-binding proteins are especially useful for studies of human cortex,
because the antigens are relatively resistant to autolysis,
formalin fixation, and paraffin embedding and are thus
amenable to immunohistochemical analysis using routinely
processed brain tissue. For example, cytoskeletal proteins
microtubule-associated protein (MAP1B) and neurofilamentH, which label subsets of layer V neurons, are consistently
detectable in formalin-fixed, paraffin-embedded autopsy
material (Fig. 2A, B). Many other antigens are detectable
only in cortex that has been specially preserved, for example, by snap freezing. Examples of such antigens include
RORA, Ctip2, and TLE4, which exhibit layer-specific
expression patterns in frozen sections (Fig. 2C, D) but not
in paraffin sections. Finally, probes for mRNA expression
could potentially reveal additional layer-specific patterns in
humans as in monkeys (30, 31, 44), but mRNA is usually
too degraded for sufficient detection in most human brain
specimens.
In humans as in all mammals, Blayer-specific[ markers
label a limited set of one or a few cortical neuron types with
restricted laminar distribution, not the entire complement of
neurons in one layer. Accordingly, many layer-specific
markers are actually expressed in multiple layers, and no
single marker labels all the neurons in a particular layer. For
example, nonphosphorylated neurofilament-H is only
expressed in a subset of layer V pyramidal neurons, leaving
Ó 2007 American Association of Neuropathologists, Inc.
Copyright @ 2007 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.
J Neuropathol Exp Neurol Volume 66, Number 2, February 2007
many layer V pyramidal and nonpyramidal neurons unlabeled (Fig. 2B). Conversely, nonphosphorylated neurofilament-H is also expressed by some neurons outside layer V,
mainly in layers III and VI (51, 53, 54). Further complications arise from changes in the laminar specificity of markers
across cortical areas (30, 31, 40, 42) and developmental
stages (22, 26, 33). Finally, some layer-specific markers can
be actively regulated in response to neural activity (51). All
of these factors must be carefully controlled when molecular
markers are used as probes for laminar identity.
In human neocortex, layer-specific markers have been
used to identify neurons in layer 1 (marginal zone), layers 2
to 3, layer 4, layer 5, layer 6, and the subplate (Fig. 3).
Nevertheless, because of the diversity of neuron types in
each layer, the available markers do not cover all neuron
types with restricted laminar distribution. For example, no
marker specific for layer II/III projection neurons has been
documented in human neocortex. Thus, the markers listed in
Figure 3 represent a starting point toward the ultimate goal
of developing a comprehensive panel of markers to analyze
all neuron types and their distribution in the human
neocortex. Such a panel would be able to detect abnormalities in the position and number of each neuron type and
Layer-Specific Markers in Cerebral Cortex
would be tremendously valuable for evaluating not only
malformations of cortical development but also diseases
involving selective loss of specific neuron types.
Cortical Neurogenesis, Fate Specification,
and Cell Migration
Cortical neurons are produced by neurogenesis in
progenitor zones near the lateral ventricles, followed by
extensive postmitotic cell migration along radial and nonradial axes (Fig. 4A). Interestingly, the two main classes of
cortical neurons are produced in separate regions of the
developing forebrain. Projection neurons are produced in the
cortical neuroepithelium (pallium), whereas many or most
interneurons are produced in the basal telencephalon
(ganglionic eminences) (57Y60). Accordingly, the subsequent migrations of projection neurons and interneurons are
also quite different. Projection neurons migrate primarily
along the radial axis from progenitor zone to cortical plate,
whereas interneurons must migrate both nonradially (to enter
cortex from basal forebrain) and radially (to enter the
cortical plate). Despite these differences, both classes of
neurons demonstrate similar Binside-out[ relations between
cell birthday and laminar fate, i.e. early-born neurons
FIGURE 3. Layer-specific markers in human neocortex. Expression of each antigen in specific layers of fetal and adult cerebral
cortex is indicated by shading. Darker shading indicates stronger expression. The expression of MAP1B, NSE, and FoxP1 in adult
cortex has not been reported. This figure is only a rough guide because layer-specific patterns differ among cortical areas and
change throughout development. The neuron class and type, optimal fixation conditions, and literature references are also
indicated for each marker. ER81 has been described in monkey cortex (30, 31) and is presumably expressed in human layer V,
although this has not been verified.
Ó 2007 American Association of Neuropathologists, Inc.
105
Copyright @ 2007 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.
Hevner
J Neuropathol Exp Neurol Volume 66, Number 2, February 2007
FIGURE 4. Development of neuron types. (A) Distinct origins and migrations of projection neurons and interneurons in
the embryonic brain viewed in coronal section. Pyramidal
projection neurons originate from the cortical (Ctx) neuroepithelium (green), whereas interneurons originate mainly
from the medial ganglionic eminence (MGE) (red) and lateral
ganglionic eminence (LGE) (orange). New projection neurons
migrate radially (green arrows), whereas new interneurons
migrate both nonradially and radially (red and orange
arrows). (B) Sequential generation of different projection
neuron types. Within the cortical neuroepithelium (corresponding to the boxed region in A), progenitor cells (brown
nuclei) in the ventricular zone (VZ) and subventricular zone
(SVZ) divide and produce new projection neurons that
migrate through the intermediate zone (IZ) to the cortical
plate (CP). The neuron type (colored nuclei) and laminar fate
are specified during neurogenesis, before the new neuron
migrates out of the progenitor compartments. Different
neuron types are produced sequentially and migrate to
increasingly superficial layers of cortex.
migrate to deep/lower cortical layers and late-born neurons
migrate to superficial/upper layers (61Y66).
Much evidence indicates that neuron identity, type,
and laminar fate are specified during neurogenesis, before a
new neuron begins migrating (Fig. 4B) (67, 68). Neurogenesis and fate specification appear to be controlled by an
intrinsic genetic program that can also be modulated by
factors or interactions in the progenitor zone (69, 70). Diversity of neuron types is achieved by the sequential production
of different types of neurons from patterned progenitor
compartments, according to well-defined temporal and
spatial coordinates. Once specified, the neuron type identity
and laminar fate are apparently impervious to change, even
if cell migration is abnormal and the neuron migrates to an
inappropriate location. The best example of this situation
occurs in reeler mutant mice, in which deficiency of a
secreted glycoprotein (reelin) causes abnormal cell migrations, leading to inversion of the laminar organization
(Boutside-in[ cortex) (71). Remarkably, the misplaced
neurons in reeler express appropriate properties for the
laminar fate they should occupy in normal cortex, despite
the abnormal laminar organization. For example, subplate
(layer VIb) neurons migrate to abnormally superficial
positions in reeler cortex but still function to guide pioneer
thalamocortical axons into the cortex, along abnormal
subpial pathways (72). Corticospinal axon connections and
cortical barrels likewise develop in reeler despite the cortical
disorganization (73).
In addition to cell migration disorders, abnormal
specification or differentiation of neuron type and laminar
106
fate can also contribute to cortical disorganization and
MCDs. In mice with targeted mutations of Fezl or Ctip2,
the specification and differentiation of corticospinal neurons
are defective (27, 74). Similarly, Tbr1 inactivation perturbs
the differentiation of early-born neuron types, leading to
defects of layer-specific molecular expression, axon pathfinding, and cell migration (26). In malformations involving
defects of neuron type specification or differentiation, layerspecific markers can be particularly useful to demonstrate
reduction or absence of certain neuron types, as well as
altered laminar distribution.
Layer-Specific Markers in Cortical
Malformations
Layer-specific markers have been used extensively in
mice as probes for neuron types in cortical malformations as
well as normal development. The reeler mutant cortex is
particularly well studied as it has been the focus of intense
research for many years. The overall inversion of the reeler
neocortex was first demonstrated by cell birth-dating
methods. Those early studies revealed that the reeler cortex
developed by migration of early-born neurons to superficial
positions and late-born neurons to deep positions, that is,
opposite to the usual inside-out sequence (71). More recent
studies with numerous layer-specific markers have shown
that patterns of molecular expression shift to abnormal
laminar positions in reeler, as predicted from the overall
cortical inversion (20, 22, 28). Moreover, double labeling for
cell birthday and molecular expression has verified that neurons in reeler express markers that would be appropriate for
the same birthday in normal mice, despite their abnormal
positions (20). Thus, the correlations between cell birthday
and neuron type are generally preserved, although some
slight alterations in the neurogenesis of certain neuron types
have been observed and attributed to effects of reelin deficiency on progenitor cells (73). Finally, layer-specific markers
have also revealed that neuron types do not simply conform
to a strict inverted phenotype in reeler but also scatter more
widely than normal across radial positions (20, 22, 28).
Examples of layer-specific markers (Cux1 and Ctip2)
in mutant mouse cortical malformations are shown in
Figure 5, along with corresponding cresyl violetYstained
sections. Normally, transcription factors Cux1 and Ctip2 are
expressed in upper and lower cortical layers of the neonatal
mouse cortex, respectively (Fig. 5A, B) (27, 28, 74). In the
roughly inverted cortex of reeler, Cux1+ neurons shift to
deep positions and Ctip2+ neurons to superficial positions,
and laminar boundaries are blurred (Fig. 5C, D). Tbr1
mutant cortex shows a strikingly different phenotype, with
alternating patches of deep (Ctip2+) and superficial (Cux1+)
type neurons, suggesting an abnormality of nonradial as well
as radial cortical organization (Fig. 5E, F) (26). These
examples illustrate how molecular markers can reveal
aspects of cortical organization that are not apparent with
routine stains such as Nissl. The layer-specific markers act as
molecular tags that not only identify individual cells but also
precisely report the number and positions of different types
of neurons. This type of analysis is now a mainstay of
Ó 2007 American Association of Neuropathologists, Inc.
Copyright @ 2007 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.
J Neuropathol Exp Neurol Volume 66, Number 2, February 2007
Layer-Specific Markers in Cerebral Cortex
cortex, despite the cortical thickening and laminar distortion
due to impairment of cell migration. Their analysis contrasted sharply with conclusions reached by other groups
using routine stains and neurofilament immunohistochemistry (18, 19), and further studies will be necessary to resolve
the discrepancies. These limited examples provide exciting
glimpses of the tremendous potential of layer-specific
markers to open new insights into the neuropathology of
MCDs.
Present Challenges and Future Directions
Current trends suggest that layer-specific markers will
be increasingly used to study all kinds of MCDs. It is hoped
FIGURE 5. Layer-specific markers in developing mouse neocortex (postnatal day 1) and genetically induced cortical
malformations. (A, B) Normal cortex. Cux1 (red) was
expressed in superficial layers (IIYIV). Ctip2 (green) was
expressed in deep layers, strongly in layer V but weaker in
layer VI and subplate (SP). (C, D) Reeler (rl/rl) mutant cortex.
Cortical laminar disorganization was obvious on Nissl staining
(cresyl violet). Layer-specific markers revealed a shift of superficial layer type neurons (Cux1+) to deep positions, with
scattering of layer V neurons (strongly Ctip2+) and blurring of
laminar boundaries. (E, F) Tbr1 null mutant cortex. Cortical
laminar disorganization was likewise obvious with Nissl stain.
Layer-specific markers showed patchy, alternating aggregates
of superficial layer type neurons (Cux1+) and layer V neurons
(strongly Ctip2+). Scale bar = 100 Km.
research on mouse cerebral cortex development but has only
recently been introduced as an approach to studying human
development and neuropathology.
Very few studies have used layer-specific markers to
analyze normal human cortex, MCDs, or epileptic foci (21,
39, 45Y56). My laboratory has recently focused on transcription factor Tbr1, a specific marker for early-born
neurons in mice (26) and normal humans (45). In preliminary studies of a case of type II lissencephaly (WalkerWarburg syndrome), we found that the number of Tbr1+
neurons was overall reduced and that the remaining Tbr1+
neurons were widely scattered and did not form a distinct
layer (Fig. 6). Recently, Sheen et al (21) used Tbr1, as well
as FoxP1 and other layer-specific markers, to study type I
lissencephaly (Miller-Dieker syndrome). They found that the
cortical organization was unexpectedly similar to normal
Ó 2007 American Association of Neuropathologists, Inc.
FIGURE 6. Tbr1 protein expression in normal human fetal
cortex (22 gestational weeks) and type II lissencephaly due to
Walker-Warburg syndrome (WWS). (A, B) Normal cortex.
Hematoxylin and eosin showed developing cortical layers.
Tbr1 protein (yellow) was strongly expressed in deep layers,
especially layer VI and subplate (SP). Note scattering of
subplate neurons throughout the intermediate zone (IZ). (C,
D) Type II lissencephaly. Tbr1+ neurons were reduced in
number compared with normal, and were widely scattered in
the disorganized cortex. Formalin-fixed, paraffin-embedded
tissues with antigen retrieval. Scale bar = 500 Km.
107
Copyright @ 2007 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.
J Neuropathol Exp Neurol Volume 66, Number 2, February 2007
Hevner
that such markers will reveal important details of cortical
organization and neuronal composition that influence not
only interpretations of pathogenesis but also diagnostic
classification. Eventually, layer-specific markers may even
become standard immunohistochemical probes in the neuropathologic diagnosis of MCDs. The biggest immediate barriers to wider use of layer-specific markers in human
neuropathology are: 1) the paucity of layer-specific antibodies compatible with formaldehyde-fixed, paraffinembedded brain specimens and 2) the daunting task of
documenting expression in normal human cortex at various
developmental stages and in different cortical areas. Progress on the first problem will require the production of
better antibodies and improvements to antigen retrieval in
immunohistochemistry. Progress on the second problem will
be driven by the need to compare MCDs with appropriate
normal controls and by interest in basic mechanisms and
principles of cerebral cortex development and organization
in humans and nonhuman primates.
More broadly, layer-specific markers represent an expanding universe of cell type-specific markers expressed in
all areas of the CNS. Just as layer-specific markers will
influence neuropathologic studies of MCDs, it is very likely
that many other new markers will have a similar impact on
studies of malformations in the spinal cord, hindbrain,
cerebellum, midbrain, thalamus, and basal ganglia. As our
knowledge of the genetic underpinnings of CNS malformations advances, molecular markers of neuron type will play
an important role in establishing genotype-phenotype correlations and determining the range of phenotypic heterogeneity for particular mutations. Molecular markers of cell
identity may also be valuable for identifying affected neuron
types in neurodegeneration, hypoxiaYischemia, and other
causes of neuron loss and for evaluating the efficacy of
neural tissue regeneration using exogenous stem cells or
endogenous progenitors (75).
ACKNOWLEDGMENTS
I thank Ray Daza and Randy Small for expert
technical assistance, Dr. Tom Jessell for anti-ER81, Dr.
Stefano Stifani for anti-TLE4, Dr. Greg Lemke for antiSCIP, and Dr. Henk Stunnenberg for anti-RORA. All other
antibodies were purchased from commercial suppliers.
REFERENCES
1. Sarnat HB, Flores-Sarnat L. Integrative classification of morphology
and molecular genetics in central nervous system malformations. Am J
Med Genet A 2004;126:386Y92
2. Barkovich AJ, Kuzniecky RI, Jackson GD, et al. A developmental and
genetic classification for malformations of cortical development.
Neurology 2005;65:1873Y87
3. Guerrini R, Marini C. Genetic malformations of cortical development.
Exp Brain Res 2006;173:322Y33
4. Golden JA. Lissencephaly, type I. In: Golden JA, Harding BN, eds.
Pathology & Genetics: Developmental Neuropathology. Basel, Switzerland: ISN Neuropath Press, 2004:34Y41
5. Ferrer I, Armstrong J. Microcephaly. In: Golden JA, Harding BN, eds.
Pathology & Genetics: Developmental Neuropathology. Basel, Switzerland: ISN Neuropath Press, 2004:26Y31
6. Parrini E, Ramazzotti A, Dobyns WB, et al. Periventricular heterotopia:
Phenotypic heterogeneity and correlation with filamin A mutations.
Brain 2006;129:1892Y1906
108
7. Neal J, Apse K, Sahin M, et al. Deletion of chromosome 1p36 is
associated with periventricular nodular heterotopia. Am J Med Genet A
2006;140:1692Y995
8. Harding BN, Pilz DT. Polymicrogyria. In: Golden JA, Harding BN,
eds. Pathology & Genetics: Developmental Neuropathology. Basel,
Switzerland: ISN Neuropath Press, 2004:49Y51
9. Piao X, Hill RS, Bodell A, et al. G protein-coupled receptor-dependent
development of human frontal cortex. Science 2004;303:2033Y36
10. Powers JM. Peroxisomal disorders. In: Golden JA, Harding BN, eds.
Pathology & Genetics: Developmental Neuropathology. Basel, Switzerland: ISN Neuropath Press, 2004:287Y95
11. Piao X, Chang BS, Bodell A, et al. Genotype-phenotype analysis of
human frontoparietal polymicrogyria syndromes. Ann Neurol 2005;58:
680Y87
12. Richman DP, Stewart RM, Caviness VS Jr. Cerebral microgyria in a
27-week fetus: An architectonic and topographic analysis. J Neuropathol Exp Neurol 1974;33:374Y84
13. Ferrer I. A Golgi analysis of unlayered polymicrogyria. Acta Neuropathol (Berlin) 1984;65:69Y76
14. Ferrer I, Catalá I. Unlayered polymicrogyria: Structural and developmental aspects. Anat Embryol 1991;184:517Y28
15. Hevner RF, Horoupian DS. Pena-Shokeir phenotype associated with
bilateral opercular polymicrogyria. Pediatr Neurol 1996;15:348Y51
16. Pilz D, Matsumoto N, Minnerath S, et al. LIS1 and XLIS (DCX)
mutations cause most classical lissencephaly, but different patterns of
malformation. Hum Mol Genet 1998;7:2029Y37
17. Dobyns WB, Truwit CL, Ross ME, et al. Differences in the gyral
pattern distinguish chromosome 17-linked and X-linked lissencephaly.
Neurology 1999;53:270Y777
18. Viot G, Sonigo P, Simon I, et al. Neocortical neuronal arrangement in
LIS1 and DCX lissencephaly may be different. Am J Med Genet A
2004;126:123Y28
19. Forman MS, Squier W, Dobyns WB, et al. Genotypically defined
lissencephalies show distinct pathologies. J Neuropathol Exp Neurol
2005;64:847Y57
20. Hevner RF, Daza RAM, Rubenstein JLR, et al. Beyond laminar fate:
Toward a molecular classification of cortical projection/pyramidal
neurons. Dev Neurosci 2003;25:139Y51
21. Sheen VL, Ferland RJ, Neal J, et al. Neocortical neuronal arrangement
in Miller Dieker syndrome. Acta Neuropathol 2006;111:489Y96
22. Ferland RJ, Cherry TJ, Preware PO, et al. Characterization of Foxp2
and Foxp1 mRNA and protein in the developing and mature brain. J
Comp Neurol 2003;460:266Y79
23. Funatsu N, Inoue T, Nakamura S. Gene expression analysis of the late
embryonic mouse cerebral cortex using DNA microarray: Identification of
several region- and layer-specific genes. Cereb Cortex 2004;14:1031Y44
24. Gray PA, Fu H, Luo P, et al. Mouse brain organization revealed through
direct genome-scale TF expression analysis. Science 2004;306:2255Y57
25. Guillemot F, Molnár Z, Tarabykin V, et al. Molecular mechanisms of
cortical differentiation. Eur J Neurosci 2006;23:857Y68
26. Hevner RF, Shi L, Justice N, et al. Tbr1 regulates differentiation of the
preplate and layer 6. Neuron 2001;29:353Y66
27. Arlotta P, Molyneaux BJ, Chen J, et al. Neuronal subtype-specific genes
that control corticospinal motor neuron development in vivo. Neuron
2005;45:207Y21
28. Nieto M, Monuki ES, Tang H, et al. Expression of Cux-1 and Cux-2 in
the subventricular zone and upper layers II-IV of the cerebral cortex.
J Comp Neurol 2004;479:168Y80
29. Voelker CCJ, Garin N, Taylor JSH, et al. Selective neurofilament
(SMI-32, FNP-7 and N200) expression in subpopulations of layer V
pyramidal neurons in vivo and in vitro. Cereb Cortex 2004;14:1276Y86
30. Watakabe A, Ichinohe N, Ohsawa S, et al. Comparative analysis of
layerYspecific genes in mammalian neocortex. Cereb Cortex, in press
31. Yoneshima H, Yamasaki S, Voelker CCJ, et al. ER81 is expressed in a
subpopulation of layer 5 neurons in rodent and primate neocortices.
Neuroscience 2006;137:401Y12
32. Zhong Y, Takemoto M, Fukuda T, et al. Identification of the genes that
are expressed in the upper layers of the neocortex. Cereb Cortex 2004;
14:1144Y52
33. Zimmer C, Tiveron M, Bodmer R, et al. Dynamics of Cux2 expression
suggests that an early pool of SVZ precursors is fated to become upper
cortical layer neurons. Cereb Cortex 2004;14:1408Y20
Ó 2007 American Association of Neuropathologists, Inc.
Copyright @ 2007 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.
J Neuropathol Exp Neurol Volume 66, Number 2, February 2007
34. Hendry SHC, Schwark HD, Jones EG, et al. Numbers and proportions
of GABA-immunoreactive neurons in different areas of monkey
cerebral cortex. J Neurosci 1987;7:1503Y19
35. Meinecke DL, Peters A. GABA immunoreactive neurons in rat visual
cortex. J Comp Neurol 1987;261:388Y404
36. Tsiola A, Hamzei-Sichani F, Peterlin Z, et al. Quantitative morphologic
classification of layer 5 neurons from mouse primary visual cortex. J
Comp Neurol 2003;461:415Y28
37. Molnár Z, Cheung AFP. Towards the classification of subpopulations of
layer V pyramidal projection neurons. Neurosci Res 2006;55:105Y15
38. Markram H, Toledo-Rodriguez M, Wang Y, et al. Interneurons of the
neocortical inhibitory system. Nat Rev Neurosci 2004;5:793Y807
39. González-Albo MC, Elston GN, DeFelipe J. The human temporal
cortex: Characterization of neurons expressing nitric oxide synthase,
neuropeptides and calcium-binding proteins, and their glutamate
receptor subunit profiles. Cereb Cortex 2001;11:1170Y81
40. Rubenstein JLR, Anderson S, Shi L, et al. Genetic control of cortical
regionalization and connectivity. Cereb Cortex 1999;9:524Y32
41. Grove EA, Fukuchi-Shimogori T. Generating the cerebral cortical area
map. Annu Rev Neurosci 2003;26:355Y80
42. Rash BG, Grove EA. Area and layer patterning in the developing
cerebral cortex. Curr Opin Neurobiol 2006;16:25Y34
43. Kudo C, Ajioka I, Hirata Y, et al. Expression profiles of EphA3 at both
the RNA and protein level in the developing mammalian forebrain. J
Comp Neurol 2005;487:255Y69
44. Donoghue MJ, Rakic P. Molecular evidence for the early specification
of presumptive functional domains in the embryonic primate cerebral
cortex. J Neurosci 1999;19:5967Y79
45. Hevner RF, Zecevic N. Pioneer neurons and interneurons in the developing subplate: Molecular markers, cell birthdays, and neurotransmitters. In: Erzurumlu R, Guido W, Molnár Z, eds. Development and
Plasticity in Sensory Thalamus and Cortex. New York: Springer, 2006:
1Y18
46. Zecevic N, Chen Y, Filipovic R. Contributions of cortical subventricular
zone to the development of the human cerebral cortex. J Comp Neurol
2005;91:109Y22
47. Meyer G, Goffinet AM. Prenatal development of reelin-immunoreactive
neurons in the human neocortex. J Comp Neurol 1998;397:29Y40
48. Meyer G, Schaaps JP, Moreau L, et al. Embryonic and early fetal
development of the human neocortex. J Neurosci 2000;20:1858Y68
49. Deguchi K, Inoue K, Avila WE, et al. Reelin and Disabled-1 expression
in developing and mature human cortical neurons. J Neuropathol
Exp Neurol 2003;62:676Y84
50. Bourne JA, Rosa MGP. Hierarchical development of the primate visual
cortex, as revealed by neurofilament immunoreactivity: Early maturation of the middle temporal area (MT). Cereb Cortex 2006;16:405Y14
51. Duffy KR, Murphy KM, Frosch MP, et al. Cytochrome oxidase and
neurofilament reactivity in monocularly deprived human primary visual
cortex. Cereb Cortex, in press
52. Haynes RL, Borenstein NS, DeSilva TM, et al. Axonal development in
the cerebral white matter of the human fetus and infant. J Comp Neurol
2005;484:156Y67
53. Thom M, Martinian L, Parnavelas JG, et al. Distribution of cortical
interneurons in grey matter heterotopia in patients with epilepsy.
Epilepsia 2004;45:916Y23
Ó 2007 American Association of Neuropathologists, Inc.
Layer-Specific Markers in Cerebral Cortex
54. Thom M, Martinian L, Sen A, et al. Cortical neuronal densities and
lamination in focal cortical dysplasia. Acta Neuropathol 2005;110:
383Y92
55. Ferrer I, Tuñon T, Soriano E, et al. Calbindin immunoreactivity in
normal human temporal neocortex. Brain Res 1992;572:33Y41
56. Pancoast M, Dobyns W, Golden JA. Interneuron deficits in patients with
the Miller-Dieker syndrome. Acta Neuropathol 2005;109:400Y404
57. Anderson SA, Kaznowski CE, Horn C, et al. Distinct origins of
neocortical projection neurons and interneurons in vivo. Cereb Cortex
2002;12:702Y9
58. Marı́n O, Rubenstein JLR. Cell migration in the forebrain. Annu Rev
Neurosci 2003;26:441Y83
59. Wonders CP, Anderson SA. The origin and specification of cortical
interneurons. Nat Rev Neurosci 2006;7:687Y96
60. Letinic K, Zoncu R, Rakic P. Origin of GABAergic neurons in the
human neocortex. Nature 2002;417:645Y49
61. Angevine JB, Sidman RL. Autoradiographic study of cell migration
during histogenesis of the cerebral cortex in the mouse. Nature 1961;
192:766Y68
62. Fairén A, Cobas A, Fonseca M. Times of generation of glutamic acid
decarboxylase immunoreactive neurons in mouse somatosensory cortex.
J Comp Neurol 1986;251:67Y83
63. Peduzzi JD. Genesis of GABA-immunoreactive neurons in the ferret
visual cortex. J Neurosci 1988;8:920Y31
64. Rakic P. Specification of cerebral cortical areas. Science 1988;241:
170Y76
65. Jackson CA, Peduzzi JD, Hickey TL. Visual cortex development in the
ferret. I. Genesis and migration of visual cortical neurons. J Neurosci
1989;9:1242Y53
66. Hevner RF, Daza RAM, Englund C, et al. Postnatal shifts of interneuron
position in the neocortex of normal and reeler mice: Evidence for
inward radial migration. Neuroscience 2004;124:605Y18
67. McConnell SK. Fates of visual cortical neurons in the ferret after isochronic and heterochronic transplantation. J Neurosci 1988;8:945Y74
68. McConnell SK, Kaznowski CE. Cell cycle dependence of laminar
determination in developing neocortex. Science 1991;254:282Y85
69. Shen Q, Wang Y, Dimos JT, et al. The timing of cortical neurogenesis
is encoded within lineages of individual progenitor cells. Nat Neurosci
2006;9:743Y51
70. Desai AR, McConnell SK. Progressive restriction in fate potential by
neural progenitors during cerebral cortical development. Development
2000;127:2863Y72
71. Caviness VS Jr. Neocortical histogenesis in normal and reeler mice: a
developmental study based upon [3H]thymidine autoradiography. Brain
Res Dev Brain Res 1982;4:293Y302
72. Molnár Z, Adams R, Goffinet AM, et al. The role of the first postmitotic
cortical cells in the development of thalamocortical innervation in the
reeler mouse. J Neurosci 1998;18:5746Y65
73. Polleux F, Dehay C, Kennedy H. Neurogenesis and commitment of
corticospinal neurons in reeler. J Neurosci 1998;18:9910Y23
74. Molyneaux BJ, Arlotta P, Hirata T, et al. Fezl is required for the birth
and specification of corticospinal motor neurons. Neuron 2005;47:817Y31
75. Sohur US, Emsley JG, Mitchell BD, et al. Adult neurogenesis and
cellular brain repair with neural progenitors, precursors and stem cells.
Philos Trans R Soc Lond B Biol Sci 2006;361:1477Y97
109
Copyright @ 2007 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.