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REVIEWS Development of the human cerebral cortex: Boulder Committee revisited Irina Bystron*||, Colin Blakemore* and Pasko Rakic§ Abstract | In 1970 the Boulder Committee described the basic principles of the development of the CNS, derived from observations on the human embryonic cerebrum. Since then, numerous studies have significantly advanced our knowledge of the timing, sequence and complexity of developmental events, and revealed important inter-species differences. We review current data on the development of the human cerebral cortex and update the classical model of how the structure that makes us human is formed. Neocortex The evolutionarily newest portion of the cerebral cortex. It is particularly enlarged in primates and underpins higher mental functions for humans. Neuroepithelium A layer of proliferating neuroepithelial cells that makes up the neural plate and neural tube. *Department of Physiology, Anatomy & Genetics, University of Oxford, Oxford, OX1 3PT, UK. ||Department of Morphology, Institute of Experimental Medicine, St Petersburg, 197376, Russia. § Department of Neurobiology and Kavli Institute of Neuroscience, Yale University Medical School, New Haven, Connecticut 208001, USA. Correspondence to P.R. & I.B. e‑mails: [email protected]; irina. [email protected] doi:10.1038/nrn2252 The mammalian cerebral cortex is a complex laminated structure that contains a bewildering diversity of neurons and has rich local and extrinsic connectivity. Regional variations in cytoarchitecture are superimposed on a common plan of layers, cell types and connections1. The explosion in the size of the cerebral hemispheres during mammalian evolution is correlated with increasing behavioural and cognitive capacity. The enormous human cortex underpins our perception, memories, thoughts and language. In all mammals, the neocortex forms at the outer surface of the embryonic cerebral vesicle, at the rostral end of the neural tube, through the migration of neurons from proliferative regions near the cerebral ventricle2,3. The arrival of migrating neurons establishes laminar compartments, some of which change or even disappear during development. The sequence of events that take place during cortical development, as it was then understood, was described more than 35 years ago by the Boulder Committee4. This committee, meeting in Boulder, Colorado, was convened by the American Association of Anatomists, the main forum for the growing fields of neuroanatomy and embryology. The committee’s remit was to standardize the heterogeneous and confusing nomenclature for the developing vertebrate CNS that had emerged from the blossoming of knowledge. Richard Sidman presented diagrams of embryonic cortical development (FIG. 1A), based on the observations of P.R., then an assistant professor in his department. The committee’s report stated: The layers and cells of the early developing central nervous system lack direct counterparts in the adult and must be designated by a special terminology. The inconsistent and inaccurate language now in use leads to misunderstanding and a revision is proposed in which the four 110 | february 2008 | volume 9 fundamental zones are termed the ventricular, subventricular, intermediate, and marginal zones. Each is defined according to the form, behaviour, and fate of its constituent cells. All neurons and macroglia of the central nervous system can be derived from these developmental zones. Thus, the committee recommended names for each of the transient embryonic cellular compartments (or ‘zones’) and gave their interpretation of the major developmental events. This model has been widely adopted as a generic description of the development of the entire vertebrate CNS. In the past three decades, experimental studies on normal and genetically altered rodents have driven the analysis of cortical development. This has given us new insights into the regulation of cell division and programmed cell death, which determine neuron number5,6, and the mechanisms of neuronal migration3,7,8. Patterns of genetic expression are being elucidated, including the expression of transcription factors that are thought to influence regional differentiation and regulate broad aspects of mitotic activity, fate determination and differentiation9–12. Recent studies have revealed new types of transient neurons and proliferative cells outside the classical neuroepithelium, new routes of cellular migration and additional cellular compartments13–18. Furthermore, we know much more about intrinsic connectivity and the formation of axonal projections into and out of the cortex19–21. Thus, our understanding of the timing and sequence of developmental events in the cerebral wall has changed radically since the work of the Boulder Committee. Despite the march of knowledge, the Boulder Committee’s nomenclature continues to be widely used. Their summary diagram (FIG. 1A) has been reproduced, www.nature.com/reviews/neuro © 2008 Nature Publishing Group REVIEWS M CP M CP I M I I M V V Aa V Ab S S V V Ac Ad Ae (SG) MZ CP MZ SP CP IZ IZ/SP PP SVZ VZ Ba PP SVZ SVZ VZ VZ VZ Bb Bc VZ Bd Be Figure 1 | The Boulder Committee’s 1970 schematic model of human neocortical development, and a proposed Neuroscience revision. A | The Boulder Committee’s original summary diagram of neocortical development.Nature B | OurReviews revised| version. Comparison of these two illustrations summarizes our redefinition of the sequence of events and the formation of transient compartments, including the preplate (PP) and the intermediate and subplate zones (IZ and SP). The panels in part B correspond to the following approximate ages (for the lateral part of the dorsal telencephalon): a: embryonic day (E) 30; b: E31–E32; c: E45; d: E55; e: gestational week 14. CP, cortical plate; I & IZ, intermediate zone; M & MZ, marginal zone; S & SVZ, subventricular zone; (SG), subpial granular layer (part of the MZ); V & VZ, ventricular zone. Part A reproduced, with permission, from REF. 4 (1970) Wiley. in its original form or modified, in virtually every monograph on neuroembryology, as well as in textbooks and research papers on the development of the CNS in a wide variety of mammals22–26. Attempts have been made to integrate more recent observations into the Boulder Commitee’s scheme24,27–29, but we believe that it is time for their description and nomenclature to be updated to reflect new developments in the field. Here we summarize the major advances in our understanding of the development of the cerebral cortex and incorporate new cell types and cellular zones into a revised version of the Boulder model. We recommend a nomenclature that preserves the essential features of the Boulder scheme but which would, if generally nature reviews | neuroscience adopted, avoid some of the difficulties that have emerged as researchers have tried to set recent discoveries into the traditional Boulder framework. Recent research has revealed potentially important differences between rodent and human development that have exacerbated the confusion and diversity in the use of Boulder terminology. Unless the species is specifically mentioned, the updated model of corticogenesis that we present here is based on observations of material derived from humans, whose protracted development allows more precise sequencing of cellular events. However, we have attempted to provide basic nomenclature and definitions that are widely applicable to other species. volume 9 | february 2008 | 111 © 2008 Nature Publishing Group REVIEWS a Bis/H3 E30 b Bis/TU20 E32 c Bis/TU20 < PP < < < VZ < VZ < d Bis/H3 E40–E41 < < PP 25 µm e Bis/H3 E50–E51 < VZ Mes Mes 25 µm E33 Mes 25 µm f Bis/H3 E56 < SVZ MZ SVZ PP IZ/SP MZ < > Mes VZ CP VZ VZ < IZ/SP CP * 50 µm < * 100 µm > > < < 100 µm < > > < > < > < < Figure 2 | Early formation of layers in the embryonic human ventrolateral cerebral wall. The ventricular zone (VZ) is seen in all sections. In a–d arrowheads demarcate the pial surface, with the mesenchyme (Mes) outside the |surface. Nature Reviews Neuroscience Bisbenzimide (Bis) staining of cell nuclei is shown in blue. Phosphohistone‑H3 staining of mitotic cells is shown in pink in a and d–f. TU20 immunoreactivity, which labels postmitotic neurons, is shown in yellow in b and c. a | Initially the cerebral wall consists entirely of neuroepithelial cells. b | Predecessor neurons, migrating into the cortical primordium from the subpallium, initiate formation of the preplate (PP) at embryonic day (E) 31–E32. c | From E33, postmitotic neurons, generated in the VZ, migrate radially to join the predecessor neurons. d | As shown in ref. 14, and illustrated in another example here, at E40–E41 mitotic cells (indicated by the arrows) start to accumulate at the basal border of the VZ, initiating formation of the subventricular zone (SVZ) before the cortical plate (CP) begins to form. e | As shown in ref. 64, at E50–E51 radially migrating neurons initiate formation of the CP. The emerging CP splits the preplate into the marginal zone (MZ) and a compartment that contains heterogeneous cell populations. At this stage the cell density in the compartment between the SVZ and the CP is nearly homogeneous, and the boundary between the intermediate zone and the incipient subplate is unclear. Thus, this compartment is called the intermediate/subplate zone (IZ/SP). f | By E56, at the end of the human embryonic period, the lower part of the IZ/SP is seen as a cell-sparse area (demarcated with arrowheads). Extrinsic axons invade this area in the most differentiated part of the ventrolateral cortex (see FIG. 4e–g). Asterisks in e and f mark the striatocortical boundary. Parts a–c reproduced, with permission, from REF. 13 (2006) Macmillan Publishers Ltd. Parts e and f reproduced from REF. 108. In humans there is a clear gradient of maturation across the hemisphere that is more prominent and prolonged than in species with smaller forebrains. Estimates in this article of the timing of events are based primarily on observations of the ventrolateral part of the human dorsal telencephalon. Neural plate The neuroectodermal epithelium before the neural groove and neural tube form. Interkinetic nuclear movement The apical-basal-apical migration of the neuroepithelial cell nucleus during the cell cycle. The proliferative zones: factories of the cortex In humans the neural tube closes at embryonic day (E) 30 (ref. 30). Initially the telencephalic primordium is composed entirely of dividing neuroepithelial cells (often called neural stem cells), the direct descendants of cells of the neural plate (FIG. 2a). These proliferative cells form the ‘matrix’, ‘germinal epithelium’ or ‘primitive ependyma’ described by classical embryologists, which was termed the ventricular zone (VZ) by the Boulder Committee4. 112 | february 2008 | volume 9 Before the onset of neurogenesis, the proliferative cells of the VZ constitute a homogeneous pseudo-stratified epithelium. They have radial processes31 and divide symmetrically. The nucleus moves through the apical process towards the ventricular surface shortly before metaphase and cytokinesis, and migrates towards the pial surface during DNA synthesis (a process termed interkinetic nuclear movement)32,33.These cells have highly polarized features, most notably a localized concentration of various transmembrane proteins (including prominin‑1; also known as CD133), tight junctions and adherens junctions at the apical pole (BOX 1), and receptors for constituents of the basal lamina (including integrin-α6) at the basal pole34,35. Early proliferation increases the surface area and thickness of the VZ. This is particularly dramatic in the human www.nature.com/reviews/neuro © 2008 Nature Publishing Group REVIEWS Box 1 | What is up and what is down? In naming the subventricular zone (SVZ) the Boulder Committee followed the convention of classical embryology: the ventricular surface was considered the top of the proliferative zone and the layers were described downwards from the ventricular surface. Similarly, the term ‘apical’ traditionally signifies orientation towards, or proximity to, the ventricular surface, whereas ‘basal’ refers to features further from the ventricle. To the contemporary observer, focused on the eventual functions of the cerebral cortex, it is logical to view the pial surface as the top and therefore to think of structures or layers that are closer to the pia as being superficial, outer or apical. This is the convention for numbering the mature layers of the cortex. Layers closer to the pia than layer 4 — the granular layer — are called supragranular; deeper layers are infragranular. Equally, the major dendrites of pyramidal neurons, which are directed towards the pia, are called ‘apical’ dendrites, whereas those at the lower pole of the cell are ‘basal’ dendrites — this nomenclature is directly opposite in orientation to that of the apical and basal poles of early neuroepithelial cells. It might be logical to take the pial surface consistently as the reference for all terminology. In this case the SVZ would be the ‘supraventricular zone’. However, the term SVZ has been universally accepted and will undoubtedly be retained. (Consistency, though, would demand that when the VZ disappears in the adult cerebrum and is replaced by the ependyma, the SVZ ought to be called the ‘subependymal zone’, as it was originally in the neuropathological literature!) We can see no way in which the inconsistency in defining what is up and what is down can be eliminated, but it should be acknowledged and embraced. Current terminology is fairly uniform in describing the orientation of features in the proliferative layers in relation to the ventricular surface, whereas features in the developing layers of postmigratory cells (preplate, plate and subplate) are defined with respect to the pial surface. We suggest that this convention be explicitly adopted. embryo, in which the forebrain primordium is much larger and the VZ much thicker than in rodents13. At a certain point, neuroepithelial stem cells begin to switch to an asymmetrical mode of cell division36–38, marking the onset of neurogenesis — this occurs at approximately E33 in the lateral part of the cortical wall in humans13 and at approximately E10 in mice39. One daughter remains a progenitor, the other is a postmitotic cell that is destined to become a neuron or a glial cell40–42. At the start of neurogenesis neuroepithelial stem cells downregulate their epithelial characteristics, including the presence of tight junctions and the apical polarity of some of their plasma membrane proteins34. The morphological and molecular differentiation of the subsequent proliferative cells is currently a subject of intensive investigation, but one type — radial glial cells — is particularly prominent and distinctive (BOX 2). Neuroepithelial cells are induced to generate radial glia by a set of genes that includes forkhead box G1 (FOXG1), LIM homeobox 2 (LHX2), paired box 6 (PAX6) and empty spiracles homologue 2 (EMX2)10. Radial glia share some molecular characteristics with earlier neuroepithelial cells, including the expression of nestin and antigens that are recognized by the RC1 and RC2 antibodies43. Radial glia differ in aspects of their gene expression from region to region in the brain, a factor that might influence regional differences in their postmitotic progeny. Cortical radial glia express PAX6, which is required for their normal development44. Until recently there was thought to be much greater precursor diversity in primates than in lower vertebrates45–47. Even shortly after the start of neurogenesis not nature reviews | neuroscience all mitotic cells in the primate VZ have extensive radial processes, and distinct neuronal and glial progenitors have been described in both the monkey and human VZ48,49. Recent evidence has shown that the VZ of rodents also contains a variety of progenitors, which become progressively restricted in their differentiation potential as development progresses6,10,50. The use of specific markers and electron microscopy has revealed a distinct population of short neural precursors. Like radial glia, these cells have radial processes. However, whereas the apical process contacts the ventricular surface, the basal process is of variable length and is retracted during mitotic division47. Short neural precursors are distinguished by their expression of the tubulin-α1 promoter. At some point after the onset of neurogenesis, dividing cells start to appear at the basal border of the VZ4,51. Accumulation of these intermediate or ‘basal’ progenitors creates a distinct new compartment above the VZ, which the Boulder Commitee named the subventricular zone (SVZ)4 (BOX 1). These cells are not attached to the ventricular surface and do not undergo interkinetic nuclear movement41. Studies in rodents have shown that intermediate progenitors are generated by the asymmetrical division of radial glia, and they subsequently migrate into the SVZ52,53. Originally the SVZ was thought to generate mainly glia41,54, but it is now clear that early SVZ progenitors are largely neurogenic: many cortical neurons originate from the SVZ in mice, monkeys and humans16,55–57. Most early SVZ progenitors are thought to undergo terminal symmetrical division to create pairs of neurons 58. Unlike radial glia, intermediate precursors express the transcription factors TBR2 , neurogenin 2 (NGN2), cut-like homeobox 1 (CUX1) and CUX2, but not GLAST or PAX6 (Ref. 35). The progressive attenuation of PAX6 expression and the initiation of TBR2 expression characterize the transition to intermediate progenitors59. Recently, rodent radial glia and early intermediate precursors have been shown to differ in their downstream signalling from the Notch receptor60. Signalling through the classical C‑promoter-binding factor 1 (CBF1) pathway, which inhibits neurogenesis, is attenuated in early intermediate precursors. Hence, radial glia are more multipotent than intermediate progenitors: transplantation experiments suggest that radial glia produce similar proportions of neurons, astrocytes and oligodendrocytes, whereas early intermediate precursors produce mainly neurons60. The degree of SVZ-progenitor diversity and the precise relationship of these cells to cells of the VZ are still largely unknown. Unlike in rodents, cells isolated from the human VZ and SVZ, even at early stages of cortico genesis, generate separate neuron-restricted and gliarestricted precursors in vitro61. Furthermore, cell-lineage analysis in human embryonic slices has demonstrated that VZ and SVZ progenitors undergo multiple divisions before they begin radial migration to the neocortex56. Subdivision of the neuroepithelium into the VZ and the SVZ is particularly distinctive in humans62 (FIG. 2e,f). The Boulder Committee (FIG. 1Ad) and many volume 9 | february 2008 | 113 © 2008 Nature Publishing Group REVIEWS Box 2 | What are radial glial cells? Early silver-impregnation methods revealed a population of elongated, non-neuronal cells in the fetal human brain; these cells were initially called epithelial cells or fetal glia57. However, the Boulder Committee did not refer to such cells specifically in their model. Later electron-microscopic and immunohistochemical studies in humans and non-human primates revealed the presence of glial fibrillary acidic protein (GFAP) in these cells, confirming their glial nature141,142. The basal end feet of these bipolar cells form the pial surface of the fetal cerebrum57,142. P.R. proposed that these ‘radial glial cells’, as he called them143, might act as a guidance scaffold for neuronal migration. However, it has since become clear that radial glia are also true precursors that can divide to produce postmitotic cells and other progenitors144. A subset of GFAP-positive radial glial cells stop dividing in mid-gestation of the primate, presumably to serve as a migratory scaffold, but they subsequently resume mitosis and generate astrocytes57. Although GFAP is not expressed in rodent VZ cells until the completion of corticoneurogenesis57, it is widely accepted that there are comparable cells in rodents. Indeed, it has been suggested that the neocortical VZ is composed primarily of multipotential radial glia, and that these are the sole type of VZ precursor144,145. However, it is generally agreed that the VZ in murine dorsal telencephalon contains multiple types of neuronal precursor, as is the case in humans and nonhuman primates47. Radial glial cells in rodents, as in primates, are derived from neuroepithelial cells and do not appear until after the start of neurogenesis. Although GFAP expression is delayed, the radial glia can be identified by their astroglial characteristics, such as the presence of glycogen granules and their expression of vimentin, brain-lipid-binding protein and the astrocyte-specific glutamate transporter, GLAST34,57. Initially it was assumed that proliferation of radial glial cells produces only additional radial glia and glioblasts48. However, it is now recognized that radial glial cells can be neurogenic and produce neuronal precursors as well as postmitotic neurons40,46,146. Some authors refer to radial glial cells as neural stem cells60, whereas others use this term for true neuroepithelial cells147. Until a consensus emerges regarding the definition of neural stem cells, it is important to retain the accepted names of distinct cell types. Predecessor cell A type of neuron that arrives beneath the pial surface of the human embryonic forebrain before the neural tube has even finished closing. Predecessor cells appear in the developing cortex before any other post-mitotic cells. subsequent authors2,4,16,63 assumed that the SVZ appears only after the cortical plate (CP; see below) has begun to form. However, recent reports indicate that it arises earlier14,29,64,65. Our data, which were obtained using antiphosphohistone‑H3 labelling in the human cerebral vesicle, show mitotic cells accumulating above the VZ nearly a week before the CP begins to form14 (FIG. 2d). By the end of the embryonic period the SVZ is of similar thickness to the VZ in the ventrolateral cortex64 (FIG. 2f ). The SVZ has increased in size and complexity during evolution and its cellular organization in primates is significantly different from that in other species15,16,29. Later in development — after the twentieth gestational week (GW) in humans and after approximately E72 in monkeys — a band of tangentially orientated fibres divides the SVZ into inner and outer sublayers in some parts of the cortex. The inner part of the SVZ (the part closest to the VZ) in humans shows higher expression of PAX6, TBR2 and Ki67 (Ref. 66). PAX6 is thought to suppress cell cycle exit and the expression of postmitotic markers in progenitor cells67. Therefore PAX6 expression, which is much higher in the human SVZ than in the mouse SVZ, indicates that there is a large reservoir of progenitor cells in the human SVZ. By GW25–GW27, when the SVZ is still proliferating, the human VZ has reduced in size to a one-cell-thick ependymal layer16. Just as in the macaque monkey15, this occurs before the cessation of the major phase of neuron production, indicating that the SVZ becomes the principal source of cortical neurons in the expanded cerebrum68. The Boulder Committee model assumed that mitosis is restricted to the VZ and SVZ4. However, dividing cells have been described outside the classical proliferative zones in the rodent and human cortex 14,62,69. In our human material, occasional ‘abventricular’ mitotic cells 114 | february 2008 | volume 9 are seen directly under the pial surface, even before the formation of the preplate13, but it is unclear what types of postmitotic cells they produce. Preplate: a community of transient neurons The Boulder Committee suggested that before any postmitotic cells appear in the prospective cerebral cortex the sub-pial processes of ventricular cells constitute a cell-sparse layer, which they termed the marginal zone (MZ) (FIG. 1Ab). They assumed that all neurons arise in the local neuroepithelium and migrate radially4, and they proposed that the first such neurons migrate through the intermediate zone (IZ), which lies below the MZ (FIG. 1Ac; see below). Since then, many investigators and most textbooks have used the term MZ70 or early MZ (Refs 71,72) to refer to a cell-sparse layer under the pial surface before the CP forms. Our own analysis of the embryonic human telencephalon shows cell bodies of neuroepithelial cells extending right up to the pial surface until the first neurons (predecessor cells; see below) invade tangentially, directly below the surface, to initiate formation of the preplate13 (FIG. 2a,b). At no earlier stage is there a distinct cell-free sub-pial layer. We therefore propose that the term marginal zone should not be used before the CP forms. In the past two decades, data from various mammalian species have revealed a complex sequence of early events that take place above the proliferative neuro epithelium. Neurons with various molecular signatures, some of which migrate from extra-cortical sites of origin, occupy a thickening layer under the pial surface (BOX 3). In recognition of its cellular complexity and the fact that it precedes the formation of the CP, investigators have introduced the terms primordial plexiform layer and preplate to describe this compartment73–76. We suggest www.nature.com/reviews/neuro © 2008 Nature Publishing Group REVIEWS Box 3 | Neurons of the preplate In 1893 Retzius described unusual horizontal bipolar neurons located beneath the surface of the fetal human forebrain at approximately mid-gestation148. He acknowledged that Cajal had found similar cells in other species149, and they are thought to exist in all amniotes. The cells have radial processes that contact the pial surface and a plexus of horizontal axons that synapse with the apical dendrites of later-arriving pyramidal neurons (FIG. 5a). These Cajal–Retzius cells, which disappear during corticogenesis, produce developmentally important proteins — in particular reelin, which regulates the formation of cortical layers150,151. Cajal–Retzius cells have been reported to have diverse origins in rodents. Some cells arise in the neocortical neuroepithelium6,152; others migrate tangentially from the medial cortical hem39 and other extra-cortical sites17,153. It was widely thought that these cells are the first to occupy the cortical preplate84. However, a population of locally generated pioneer neurons that arise before the Cajal–Retzius cells has been described in rats154. In humans, we recently identified an even earlier population of neurons, called predecessor cells, which have not yet been reported in any other species13 (FIG. 2b,c). These cells arrive under the pial surface of the ventrolateral cerebral wall at approximately embryonic day (E) 31, before the neural tube completely closes and neurogenesis begins in the local VZ. They are bipolar and express the neuronal marker TU20, but they lack axons. They have long horizontal processes, through which the soma translocates dorsally from the basal telencephalon. They form an extensive network over the forebrain that might act as a guidance scaffold for subsequent cellular or axonal navigation. Predecessor neurons co-express TBR1, which was thought to be specific to the cells of the dorsal telencephalon in rodents13. In humans the first reelin-expressing cells appear in the rostral telencephalon at approximately E40: many of these are generated in the local VZ and migrate radially13,72 (FIG. 5b,c). Others migrate tangentially from diverse origins91,151. Neurons labelled with antibodies to MAP2, golli and calbindin were reported in the early preplate64,91. Cells labelled with interneuron markers, such as calretinin and GABA (γ-aminobutyric acid), appear in the dorsal telencephalon from approximately E43 (Refs 72,91). A number of preplate GABAergic cells co-express either NKX2.1 or the DLX transcription factors that are characteristic of the basal telencephalon91. A few days later glutamic acid decarboxylase (GAD)-positive interneurons migrate tangentially into the preplate from the basal telencephalon91, just before the arrival of the first neurons of the cortical plate — at E50–E51 in the rostral ventrolateral cortex30,72. Thus, in the preplate, various cell types and their processes intermingle with each other and with the processes of proliferative cells, providing an opportunity for cell–cell interactions. that ‘preplate’ be used to describe the entire compartment of heterogeneous post-migratory cells and neuropil that forms between the proliferative zone and the pial surface of the dorsal telencephalon before the appearance of the CP. The preplate is a dynamic, evolving, largely-transient structure that comprises various cell types, most of which are destined to die. Immunocytochemical analysis in humans reveals subcompartmentalization of the preplate shortly before the CP forms. Cajal–Retzius cells, which express reelin, shift into a superficial sub-pial position and delineate the future MZ13,72. Because reelin terminates the migration of cortical neurons, the bottom of this subcompartment sets the upper boundary of the CP13,72 (FIG. 3a,b). The reelin-negative subcompartment, which contains a heterogeneous cell population, extends down to the SVZ. After the emergence of the CP, these former preplate cells remain below the CP and contribute to the subplate (SP; see below)30. Golgi staining A staining technique introduced by Camillo Golgi in 1873 that selectively stains neurons with silver nitrate. Cortical plate: the emerging blueprint In all mammalian species the arrival of the first neurons of the future neocortex is a critical developmental event. These post-migratory cells accumulate to form a progressively thickening layer — the CP. In humans, as in other species, there is a clear temporal gradient of maturation across the hemisphere, with the CP appearing first in the most lateral part of the rostral telencephalic wall, at E50, and then approximately 1 week later in the dorsocaudal pole30,64 (FIGS 2e,3a,b). Early [3H]thymidine autoradiographic studies in both primates and rodents established that radially nature reviews | neuroscience migrating neurons accumulate in the CP in an insideout sequence77,78. The earliest-born neurons are destined to become the future layer 6, and the last-born neurons will become layer 2. At every stage the most recently arrived, closely packed neurons form a band in the uppermost part of the CP, which is sometimes called the ‘dense cortical plate’. According to the Boulder Committee model, migrating neurons form the CP between the IZ and the MZ. Marin-Padilla proposed that CP cells accumulate within the primordial plexiform layer (the preplate), splitting it into the MZ above and the SP below79,80. Although this hypothesis was widely accepted, recent observations indicate that additional cells become incorporated into the MZ and the SP after the CP forms81,82. Differences across species in the organization of the region between the CP and the germinal layer have fuelled uncertainty about the definition of this region (see below). Golgi staining and other techniques have provided information about the differentiation of human cortical neurons83. Our study of the morphology of individual cells at GW8–GW9 confirms that early differentiated neurons of the CP have a fusiform cell body, a descending axon and an apical dendrite (BOX 1) with a dense terminal tuft in the MZ79 (FIG. 3c,d). In humans, as in rodents, the length of the apical dendrite of a cortical neuron reflects its developmental age84. Several developmental stages are recognized after the initial formation of the human CP85. Peak migratory activity is thought to occur between the third and fifth months of gestation, and migration is completed during the third trimester2,86,87. The period after GW22 is the most significant time for areal, laminar and cytological volume 9 | february 2008 | 115 © 2008 Nature Publishing Group REVIEWS a Bis b Bis/reelin E52–E55 c Bis/DiI E52–E55 GW9 CP PP PP * * SVZ ** ** MZ MZ SVZ VZ SVZ ** IZ/SP IZ/SP IZ/SP ** ** 100µm MZ ** CP 50µm CP d Bis/DiI e Bis GW9 E52–E55 SP SVZ VZ IZ/SP * IZ > MZ > < < CP LE CP 25µm 200µm f Bis g Bis/DiI VZ SVZ IZ/SP Figure 3 | Subcompartmentalization within the cortical wall. At E52–E55, the cortical plate (CP; indicated by asterisks in parts a and b) is appearing in the Nature (e). Reviews | Neuroscience dorsolateral cortex (a, b) but is more mature in the ventral cortex At this stage the boundary between the intermediate zone (IZ) and the incipient subplate (SP) is unclear30,64,120. The IZ/SP is much thinner in the dorsolateral cortex (a–c) than it is more ventrally (e–g). a | Bisbenzimide (Bis) staining of cell bodies in the cortical wall. b | The same image, merged with one showing reelin staining, reveals that reelin-expressing preplate (PP) neurons form a distinct subcompartment, which becomes the marginal zone (MZ)13,72. c | A DiI crystal placed ventrally in the cerebral wall at gestational week (GW) 9 has labelled axons running in the IZ/SP, as well as some cells of the CP and MZ, and a single radial glial cell (indicated by the arrow). d | A similar DiI crystal placement has labelled two CP cells with apical dendritic tufts in the MZ, and a SP cell with long tangential processes. e | In the ventral cortex at E52–E55, the lower part of the IZ/SP is seen as a cell-sparse area (demarcated with arrowheads). f,g | Higher power views of the striocortical boundary, indicated by the asterisk in part e. In part g, a DiI crystal placed in the thalamus reveals axons running through the lower part of the IZ/SP. All sections show Bis counterstaining. LE, lateral ganglionic eminence; SVZ, subventricular zone;VZ, ventricular zone. Parts a and b reproduced, with permission, from REF. 13 (2006) Macmillan Publishers Ltd. Parts c and d reproduced, with permission, from REF. 108. Parts e–g reproduced from REF. 120. 116 | february 2008 | volume 9 differentiation of the CP63. By the seventh month the cortex is clearly divided into six layers (including the MZ (layer 1); see below). It remains to be elucidated whether neocortical neurons are still being generated in humans during the last few months of gestation and, if so, which cortical layers such cells are destined to enter. Studies of transcription-factor expression in mice and heterochronic transplantation of cortical precursors in ferrets suggest that the VZ generates the projection neurons of the deeper layers of the neocortex whereas the SVZ produces those of the more superficial layers29,88–90. However, the situation might be more complex in humans in view of the early appearance of the SVZ14,64 (FIG. 2d–f). In rodents nearly all cortical GABAergic interneurons originate in, and migrate tangentially from, the basal telencephalon, whereas in humans a subpopulation of interneurons is born locally, in the SVZ of the dorsal telecephalon16,56,91. In all mammals a common basic pattern of cortical lamination can be recognized. In mice, precursors in the local neuroepithelium have an intrinsic timing mechanism that determines the laminar specification of successive postmitotic cells6. However, lamination is elaborated differently in different species and in different cytoarchitectonic areas of the hemisphere2,29. The upper cortical layers are disproportionately thickened in larger brains, which have a greater proportion of late-derived neurons92. Each layer has a distinct cellular appearance and connectivity that are characteristic of the specific neuronal types that comprise it. A major challenge for the future will be to identify the genetic interactions that underlie the generation of unique cellular phenotypes and the mechanisms that are involved in the specification of the laminar identity of cortical neurons. In many mammals, variation in neuronal activity, especially in the sensory areas of the cortex, has been shown to influence the distribution of incoming axons and the morphology and functional characteristics of cortical neurons, and hence to contribute to cytoarchitectonic differentiation in cortical areas93,94. The primary visual cortex provides one of the best-known examples. Thalamic axons from the right-eye and left-eye laminae of the lateral geniculate nucleus terminate in segregated, surface-parallel bands in layer 4 of the cortex, and the relative width of these bands can be modified by early postnatal deprivation of vision in one eye95. A recent study reveals similar effects resulting from early loss of vision in one eye in humans96. However, little is known about the extent to which activity influences the functional maturation and differentiation in the rest of the human cortex. Intermediate zone: migration and connectivity His recognized a compartment, which he called the ‘mantle layer’, above the VZ, through which postmitotic cells migrate into what he saw as a cell-sparse ‘marginal zone’. Kershman97 first used the term IZ to describe the compartment that lies between the proliferative layers and the cortical plate at a later stage in development. The Boulder Committee tried to conflate these concepts. They defined the early IZ, which exists before the appearance www.nature.com/reviews/neuro © 2008 Nature Publishing Group REVIEWS Projection neuron A type of glutamatergic neuron, characterized by a typical pyramidal morphology, that extends its axon to distant intracortical, subcortical or subcerebral targets. Projection neurons are born in the VZ, and their lineage is different from that of the interneurons. Interneurons A heterogeneous group of nonpyramidal, mostly GABAergic neurons that project locally and appear to be mainly inhibitory. In rodents most interneurons originate in the ganglionic eminence of the ventral forebrain and then migrate tangentially to the neocortex, whereas in humans and non-human primates they originate from both the ganglionic eminence and the SVZ of the dorsal telencephalon. In the human brain there might be pronounced differences in the proportions of neurons from the two sources. Corticofugal axon An axon that originates from a cortical neuron but projects ouside the cortex to subcortical structures. Internal capsule A large bundle of axons that reciprocally connects the cortex with the subcortical structures of the brain. of the CP, as a layer containing radially-migrating postmitotic cells that directly adjoins the VZ, below what they called the MZ (FIG. 1Ac). For the later stages they applied the same term, IZ, to the entire area between the SVZ and the CP. They pointed out that long-range axons course through this compartment (FIG. 1Ad,e). Thus, the white matter of the cortex forms within, and eventually replaces, the IZ. Many subsequent authors have tried to retain various aspects of the Boulder definition of the IZ for humans24,63,98 and other species27,99. Birthdating, cellular labelling and axon-tracing studies in various mammalian species have greatly clarified the status of the cells and axons that lie directly above the germinal layer. At early stages, before the appearance of the CP, early-born cells of the preplate fill this space. Some of these cells give rise to corticofugal axons, which in rodents project towards the putative internal capsule, even before the first CP cells arrive100–103. These axons are later joined by early ascending axons. Cells born in the basal telencephalon, which eventually enter the cortex to become GABAergic inhibitory interneurons, migrate tangentially above the SVZ 102. In humans such cells are seen as early as GW6–GW7, before the appearance of the CP91. Recognition of the existence of the preplate and the absence of a distinct, cell-sparse MZ at early stages demands the modification of the Boulder Committee’s concept of the early IZ. Radially migrating ‘pioneer’ neurons do contribute to the preplate, but no separate compartment of such migratory cells is visible at an early stage72. Conversely, there is general agreement that a compartment does emerge at later stages between the proliferative layers and the sub-pial post-migratory cells. This compartment contains both radially and tangentially migrating neurons, and a thickening band of long-range axonal projections, both afferent and efferent, forms within it. However, an examination of the literature reveals no agreed definition of this IZ, and there is a wide divergence of opinion on when it first appears and what its boundaries are. Some authors state that in rodents the early IZ is the space between the VZ and the preplate, and that the lower part of the IZ becomes the SVZ104. According to this definition the IZ would contribute to cortical neurogenesis99. However, most researchers follow the Boulder Committee in defining the lower border of the IZ as the top of the entire germinal compartment. Others27, attempting to reconcile the Boulder nomenclature with more recent evidence, represent the early IZ as equivalent to the preplate, extending from the germinal layer right up to a narrow, cell-sparse MZ. The recognition of the SP as a distinct and functionally important cellular compartment (see below) has produced further confusion with regards to how to define the upper border of the IZ (BOX 1). Some authors consider the IZ to abut the lower border of the CP99, and therefore define it as containing most if not all of the SP27. However, as the SP is largely or at least partly the lower part of the original preplate (see below), and as it constitutes a distinct and functionally important nature reviews | neuroscience compartment of post-migratory cells, most authors take the SP to be separate from the IZ28,82,105. We suggest that the term IZ be used to describe the heterogeneous compartment that lies between the proliferative layers and the postmigratory neurons above. It is defined by the virtual absence of precursors or postmigratory cells. It contains radially and tangentially migrating cells and a thickening layer of extrinsic axons. Thus, the lower border of the IZ abuts the SVZ. At early stages it is difficult to be sure whether there is a separate zone containing only migrating cells between the SVZ and the preplate. In rodents, a distinct fibre layer can sometimes be discerned even before the appearance of the CP84, and some authors have called this layer the IZ84,106. Our axon-tracing studies in humans64 did not reveal a distinct layer of corticofugal fibres to be present before the formation of the CP. The arrival of the first CP cells creates the appearance of an apparently uniform, loosely filled compartment between the CP and the SVZ (FIGS 2e,3a,b), and many authors have called this entire compartment the IZ72,107 (FIG. 2e). Presumably the lower part of this compartment consists of radially and tangentially migrating cells, but undoubtedly the upper part contains former preplate cells (part of the SP). If we are to be rigorous in our functional definition of the IZ (that is, if we define it as containing migrating cells and axons), we should avoid the temptation to describe this entire compartment as the IZ and simply admit that, in the absence of markers of post-migratory cells, the boundary between the IZ and the incipient SP is unclear. At slightly later stages (approximately GW8–GW9), starting in the ventral cortex, a cell-sparse compartment filled with extrinsic axons emerges directly above the SVZ64,108 (FIGS 2f,3e–g). It is tempting to describe this fibre layer as the entire IZ but, again, we cannot be certain that its upper border coincides with the lowest postmigratory cells. During the establishment of the CP at GW8–GW9, the compartment above the SVZ and below the CP has sometimes been called the intermediate/subplate zone (IZ/SP)66; we suggest that this term be adopted. It has been shown that, at GW9–GW10 in humans, the upper half of this compartment expresses markers that are characteristic of synaptic connectivity and axonal outgrowth66. Hence, the authors called this the SP and used IZ for the lower half of the compartment. At subsequent stages, the invasion of large numbers of corticocortical fibres and the beginning of myelination2 transform the original IZ into the white matter. Some cells (which are usually considered to be part of the SP) terminate their migration in the white matter to become interstitial cells of the white matter, which marks the end of the IZ as we have defined it. Subplate: enigmatic post-Boulder discovery The Boulder Committee did not recognize a distinct neuronal compartment between the CP and the IZ (FIG. 1Ad,e). However, a few years later such a lamina was described, first in the human109 and then in the monkey telencephalon110 — this region was called the SP111. The SP is present in all mammals, but its morphological volume 9 | february 2008 | 117 © 2008 Nature Publishing Group REVIEWS a Bis GW12 b TU20 GW12 c Bis/TU20 MZ MZ MZ IZ SVZ SVZ CP IZ GW12 CP IZ SVZ VZ SP VZ 100µm 50µm VZ 100µm d Bis IZ SP SP 100µm GW12 e DiI GW12 f Bis/DiI CP CP 50µm SP GW12 CP 50µm Figure 4 | Human cerebral wall at GW12. The subplate (SP) in humans is a separate 30,64,66 Nature(CP) Reviews Neuroscience layer that forms shortly after the emergence of the cortical plate .|Here we provide examples of the complexity of this zone in humans. a,c,d,f | Bisbenzimide (Bis) staining shows that the cells of the SP are sparsely distributed in radial palisades. b,c | TU20 staining, which reveals both neurons and their processes, suggests that the spaces between SP cells are filled with axons. The arrangement of the cells in palisades gives the impression that the axons are also radially orientated, but DiI staining from a crystal placed in the ventral cortex (e,f) reveals that the fibres pass obliquely between the cells. IZ, intermediate zone; MZ, marginal zone; SVZ, subventricular zone; VZ, ventricular zone. Figure reproduced, with permission, from REF. 156. characteristics and persistence in adulthood vary among species. In rodents (mice and rats) and carnivores (cats and ferrets), the SP is present as a distinct layer from the start of the formation of the CP82. Indeed, in these species [3H]thymidine birthdating has shown that nearly all SP neurons are preplate cells — that is, they are born before the first cells of the CP27,112,113. A significant minority of the SP interneurons are produced late in neurogenesis114. Precocious, mainly transient SP neurons are thought to have important roles in development, giving rise to early descending axons that form a scaffold over which thalamocortical afferent axons navigate103,115, as well as forming transient connections into the developing CP116. The SP contains heterogeneous cell populations, including glutamatergic neurons that migrate radially from the germinal zone, and interneurons, which are mainly produced from progenitors in the basal telencephalon82. Early-born glutamatergic neurons extend subcortical pioneer axons and express the transcription factors TBR1 and EMX1, the transgene golli–lacZ, the p75 neurotrophin receptor, kynurenine aminotransferase and other markers82,117. Interneurons in the SP express transcription factors such as DLX and LHX6, as well as β‑galactosidase, neuropeptides, calbindin, and other markers associated with GABAergic interneurons82,105,118. Many of these neurons appear to be migrating through the SP82. 118 | february 2008 | volume 9 Although the SP was first described in humans and monkeys, its origin and early development in primates are not yet fully understood30,107. The dynamic changes in the fibre content and cell morphology of the early SP and the IZ, as well as their complex spatial arrangement, limit their clear delineation. Although a number of molecular markers for the SP have been reported, most are also expressed by other cell populations. A recent study of the developing SP in mice and humans highlighted the inadequacy of current molecular markers for distinguishing between the multiple types of neuron in this compartment82. There is no consensus on the origin and timing of formation of the SP in humans. Many consider that a thin, cell-sparse ‘pre-SP’ appears at GW10, with the SP proper forming after GW12 (REFS 105,107). Some recognize no component of the SP even as late as GW11 (Ref. 98). Others see at least a thin SP layer as soon as the CP emerges30,64. As described above, there is no simple way of defining a boundary between IZ and SP at early stages (FIGS 2,3). However, there is recent evidence that cells of the upper half of this IZ/SP compartment express molecular markers that are characteristic of synaptic connectivity66. By GW9–GW10, partition into upper (SP) and lower (IZ) layers becomes obvious14,30,66. The balance of this evidence suggests that, as in other mammals, at least some human SP cells are born before the first cells of the CP. However, unlike in rodents, the SP of primates (including humans) thickens substantially during corticogenesis, especially under regions of association cortex. Although there are no neuronal birthdating data for humans, the general timescale of appearance of the SP in humans is similar to that in monkeys, in which the SP expands rapidly between E72 and E100 (REFs 15,107). Between GW9 and GW12 the human SP thickens considerably and its cell density decreases14,66 (FIG. 4). Accumulation of axons and neuropil107,119, causing early-born cells to disperse downwards, certainly contributes to the expansion of the SP, but the SP is so thick in some parts of the human cortex that it seems likely that many late-born cells contribute to it82. On the other hand, most human SP cells express TBR1, like all the early-born cells with pioneering axons in the mouse SP82. A minority of SP cells in humans are GABAergic interneurons that express DLX. In mice, a fraction of such cells are born after CP formation, but many appear to be migrating through the SP82. The extent to which later-generated neurons enter the primate SP remains to be elucidated. On the basis of silver staining it was concluded that thalamic afferents invade the human dorsal telencephalon at E47–E50, before the CP forms, and that they are present in the SP at E50–E56 (Ref. 30). However, our carbocyanine dye tracing data indicate that thalamo cortical axons reach the lateral ganglionic eminence by E47–E50 and invade the cortical wall only after the formation of the CP64,120. As an example, FIG. 3g shows thalamocortical axons running tangentially above the SVZ at approximately E53, but not substantially invading the upper part of the IZ/SP. www.nature.com/reviews/neuro © 2008 Nature Publishing Group REVIEWS a Cajal, 1893 b Reelin-positive cells c Reelin-positive cells E42 E52 PP PP VZ 25µm 25µm Figure 5 | Morphology and origin of Cajal–Retzius cells. a | Cajal’s original drawing155 of Golgi-stained neonatal human visual cortex shows Naturetransient Reviews neurons | Neuroscience (labelled A, B and C) that were named Cajal–Retzius cells in honour of their co-discoverers. b,c | Cajal–Retzius cells in the embryonic human cortex at early stages of development, revealed by their reelin immunoreactivity. At embryonic day (E) 42 (b), radial columns of reelin-positive cells extend through the ventricular zone (VZ), indicating that some Cajal–Retzius cells in humans derive from the local neuroepithelium of the dorsal telencephalon13,151. By E52 (c), reelin expression has increased and these early-born neurons are restricted to the upper part of the preplate (PP)13,72. Parts b and c reproduced, with permission, from REF. 13 (2006) Macmillan Publishers Ltd. At GW12, staining with the neuronal marker TU20 reveals palisades of neurons in the SP, creating the impression that axons might be streaming radially through the spaces between these columns (FIG. 4c). However, DiI labelling reveals that extrinsic axons run obliquely from the lower IZ into the SP. As in other mammalian species, the human SP appears to serve as a ‘waiting compartment’ for thalamic axons, although in primates the network formed by these axons is more elaborate than in other mammalian species (FIG. 4). Much later, presumably when the CP becomes growth-permissive21, these axons invade and find their ultimate targets in layer 4 (Ref. 121). The SP in humans reaches its maximum thickness roughly two-thirds of the way through gestation122,123, when it is approximately four times thicker than the CP63. Cell density then gradually decreases, leaving only scattered ‘interstitial’ cells in the white matter, which are still visible during the sixth postnatal month70,107,124. The marginal zone: from preplate to layer 1 We have proposed that the term MZ should be used to refer to the part of the former preplate that lies above the emerging CP and that later becomes layer 1 of the mature cortex. In rodents most MZ cells are generated before the CP forms125; however, the MZ is more complex in primates107. Neurons are continuously added to the monkey MZ by tangential migration during the first two-thirds of gestation, and this is also likely to be the case in humans91,126. nature reviews | neuroscience At GW11, cells originating in the basal periolfactory SVZ migrate tangentially to the surface of the ventro lateral cortex, giving rise to a sub-pial granular (SG) layer at the top of the MZ91,127–129. By GW14 this layer, which contains granular GABAergic interneurons and Cajal–Retzius cells, covers the entire cortical surface. A subset of layer 1 neurons express NKX and DLX transcription factors, which are characteristic of cells born in the basal telencephalon in rodents91. However, in the human brain the expression of these transcription factors extends dorsally into regions of the cortical VZ. The transient SG layer, which is non-existent or negligible in mice, is prominent in the fetal cortex of humans and non-human primates at later stages. [3H]thymidine analysis in the macaque monkey revealed an uncommonly thick SG in some regions of the MZ, where it might supply local circuit neurons to the underlying cortex, as it does in the area 17 (Ref. 126). The SG layer progressively disappears after GW28 and is not present in the newborn brain130. At GW18–GW28 the MZ is remarkably complex in neuronal differentiation and laminar composition, and six sublayers have been distinguished. However, no sublamination is visible in layer 1 of the newborn child’s brain131. The mature layer 1 contains few neurons and is largely filled with arborizations of apical dendrites and instrinsic tangential axons. Concluding remarks A huge corpus of data on the development of the cere bral cortex has accumulated since 1970, demanding revision of the Boulder Committee’s classical schematic model. Much of the new evidence comes from rodents, but it generally fits remarkably well with what is known of human corticogenesis, with studies on non-human primates providing a valuable bridge. Our revised model and nomenclature emphasize the timing, sequence, scale and peculiarities of events in the develop ment of the human cortex (BOX 4). However, we believe that they are broadly applicable to all mammals. Although the known differences in corticogenesis between rodents and humans are mainly small and often neglected, they might indicate important evolutionary steps that have considerable functional significance. Purely quantitative differences (for example, in the period of neurogenesis and the duration of the cell cycle) might relate simply to the expansion of the forebrain. Apparently unique human characteristics are more intriguing. We need to learn the functional implications of distinctive features of the developing human cortex, such as the early expression of GFAP in radial glia at the onset of neurogenesis45,48,132, the existence of non-dividing radial glia133, the production of interneurons in the SVZ, the particularly distinctive SG layer, and the existence of entirely new classes of neurons, such as the fusiform neurons134, precocious predecessor cells13 and late-born SP cells. Do any of these features relate to the role of the human cortex in distinctively human aspects of behaviour and cognition? Might partly heritable developmental cognitive disorders, including autism and dyslexia, involve genetic anomalies in the regulation of human-specific features? volume 9 | february 2008 | 119 © 2008 Nature Publishing Group REVIEWS Box 4 | Summary of the revisions of the Boulder model •A transient layer with a diverse population of neurons forms between the neuroepithelium and the pial surface of the dorsal telencephalon before the appearance of the cortical plate (CP). We suggest that the term preplate, which is already widely used, should be adopted for this layer. •The subventricular zone appears as a distinctive proliferative layer before the emergence of the CP, earlier than previously recognized. •There is no distinct cell-sparse layer under the pial surface before the CP forms. Thus, the term marginal zone should be used only after the appearance of the CP, to refer to the residual superficial part of the preplate, which becomes the layer 1 of the mature cortex. •The term intermediate zone (IZ) has been used in various ways. We propose that it should be reserved for the heterogeneous compartment that lies between the proliferative layers and the postmigratory cells above. The IZ contains radially and tangentially migrating cells and a thickening layer of extrinsic axons that eventually constitutes the white matter. •The subplate (SP) is a distinct and functionally important transient layer, located directly below the cortical plate, which was not recognized by the Boulder Committee. In rodents and carnivores most SP neurons are born before the first CP cells. In humans, preplate cells also contribute to the SP, but its substantial thickening at later stages probably involves the addition of later-born neurons. 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This work was supported by the Kavli Institute for Neuroscience at Yale (P.R. & I.B.), the National Institute of Neurological Disorders and Stroke of the US Public Health Service (P.R.) and the Hill Foundation (I.B.) DATABASES Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query. fcgi?db=gene CUX1 | CUX2 | EMX1 | EMX2 | FOXG1 | integrin-α6 | Ki67 | LHX2 | LHX6 | NGN2 | PAX6 | prominin‑1 | reelin | TBR1 | TBR2 | FURTHER INFORMATION Pasko Rakic’s homepage: http://rakiclab.med.yale.edu All links are active in the online pdf www.nature.com/reviews/neuro © 2008 Nature Publishing Group