* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Layer-Specific Markers as Probes for Neuron Type Identity in
Adult neurogenesis wikipedia , lookup
Holonomic brain theory wikipedia , lookup
Synaptogenesis wikipedia , lookup
Biochemistry of Alzheimer's disease wikipedia , lookup
Types of artificial neural networks wikipedia , lookup
Activity-dependent plasticity wikipedia , lookup
Axon guidance wikipedia , lookup
Neurogenomics wikipedia , lookup
Neurotransmitter wikipedia , lookup
Subventricular zone wikipedia , lookup
Cognitive neuroscience of music wikipedia , lookup
Nonsynaptic plasticity wikipedia , lookup
Caridoid escape reaction wikipedia , lookup
Neural oscillation wikipedia , lookup
Central pattern generator wikipedia , lookup
Multielectrode array wikipedia , lookup
Human brain wikipedia , lookup
Molecular neuroscience wikipedia , lookup
Environmental enrichment wikipedia , lookup
Stimulus (physiology) wikipedia , lookup
Cortical cooling wikipedia , lookup
Metastability in the brain wikipedia , lookup
Convolutional neural network wikipedia , lookup
Eyeblink conditioning wikipedia , lookup
Single-unit recording wikipedia , lookup
Neural coding wikipedia , lookup
Biological neuron model wikipedia , lookup
Apical dendrite wikipedia , lookup
Mirror neuron wikipedia , lookup
Neuroeconomics wikipedia , lookup
Spike-and-wave wikipedia , lookup
Aging brain wikipedia , lookup
Clinical neurochemistry wikipedia , lookup
Pre-Bötzinger complex wikipedia , lookup
Neuroplasticity wikipedia , lookup
Development of the nervous system wikipedia , lookup
Anatomy of the cerebellum wikipedia , lookup
Neuroanatomy wikipedia , lookup
Neural correlates of consciousness wikipedia , lookup
Optogenetics wikipedia , lookup
Premovement neuronal activity wikipedia , lookup
Neuropsychopharmacology wikipedia , lookup
Nervous system network models wikipedia , lookup
Channelrhodopsin wikipedia , lookup
Feature detection (nervous system) wikipedia , lookup
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.