Molecular Gradients and Compartments in the Embryonic Primate Cerebral Cortex

Maria J. Donoghue and Pasko Rakic

Yale University School of Medicine, Section of Neurobiology, 333 Cedar Street, New Haven, CT 06510, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The mature cerebral cortex is divided into morphologically distinct, functionally dedicated and stereotypically connected cortical areas. How might such functional domains arise during development? To investigate possible intrinsic programs within the embryonic cerebral cortex we examined patterns of gene expression early in corticogenesis. We performed these studies using the developing macaque monkey because of the size, complexity, areal make-up and the extended nature of its cortical development. Here, we present results for two types of molecules. (i) Transcription factors – gene products that bind DNA and activate transcription, directing cellular fates through cascades of gene expression. We find that the transcription factors TBr-1, Lhx-2, Emx-1 and a novel POU domain-containing gene are differentially expressed within the forming primate forebrain, and are present in gradients across the neocortex. (ii) The EphA receptor tyrosine kinases – gene products that mediate cellular recognition in many embryonic systems. Individual members of this family are expressed during primate corticogenesis in pronounced gradients and/or well-defined compartments with distinct boundaries. Together, these results suggest that at least two modes of grouping cells within the neocortex exist: the graded patterning of cells across its full anteroposterior extent and the parcellation of cells into defined domains. Moreover, emergence of molecular differences between regions of the cortical plate, prior to the arrival of afferent and formation of efferent connections, suggests that the initial cellular parcellation in the telencephalon is cell-autonomously regulated. This initial independence from peripheral influences supports the existence of an intrinsic protomap that may function both to differentially attract and respond to specific afferents, thus predicting the functional map of the mature cortex.


    Introduction
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The primate cerebral cortex consists of more than a billion neurons that must be precisely organized and interconnected during development in order to function properly throughout life. The parcellation of neurons into distinct areas is one strategy used to organize the cortex. Such cortical areas, originally described at the turn of the century as well-defined histological units in the adult human brain (Brodmann, 1909Go), are now known to correspond to functional domains (Peters, 1984). For example, areas with cells dedicated to the processing of visual information are cytoarchitectonically distinct and anatomically separate from areas devoted to the processing of other types of sensory inputs. In addition, hierarchies exist within each sensory domain, such that lower-ordered areas receive more direct input from the periphery and are more simple in terms of information processing, while higher-ordered areas are intricately connected and integrative in nature (Goldman-Rakic, 1988Go; Mountcastle, 1997Go). In spite of these heterogeneities, cortical areas of all complexity levels form characteristic patterns of neural connections, both within the cortex and between it and other parts of the nervous system (Felleman and Van Essen, 1991Go). Thus, stereotyped patterns of cellular organizations and connections, established during the development of the cortex, result in the highly structured, precisely functioning cerebral cortex.

The elucidation of the developmental processes responsible for the formation of cortical areas has been a challenge to developmental neurobiologists and many aspects of their genesis are still unclear. While it is widely accepted that both intrinsic programs and environmental factors play roles (Rakic, 1988Go; O'Leary et al., 1994Go), the timing and extent to which each influence impacts the formation of cortical areas are still obscure. One possibility is that intrinsic cellular programs bias cortical cells toward particular areal fates early in development, with extrinsic influences shaping these biases as development proceeds. This idea, that cells of the embryonic neocortex are inherently different from one another, resulting in early, region-specific cellular heterogeneities, is based upon the finding that the majority of postmitotic neurons retain their relative positions following their migration from the ventricular zone to the overlying cortical plate (Rakic, 1972Go), leading to the postulation of the radial unit and protomap hypotheses (Rakic, 1988Go; Rakic et al., 1991Go). The protomap hypothesis postulates that region-specific or intersecting gradients of morphoregulatory molecules within the embryonic cerebral wall may guide and attract specific afferent systems to appropriate cortical regions where they can interact with responsive sets of cells.

To better understand the contribution of intrinsic, cell-autonomous factors in cortical specification, we sought to identify and characterize molecular differences between presumptive functional domains early in cortical development. To this end, we selected the embryonic monkey cortex for several reasons. Firstly, the primate cortex is complex in its areal make-up, providing a rich diversity of functional identities. Secondly, gestation in primates is lengthy, with a proportionally large amount of time devoted to the development of the nervous system. This temporal expansion results in the generation of presumptive target cells within the cortex (Rakic, 1974Go) in the absence of afferent or efferent innervation of these cells (Rakic, 1976Go, 1977Go; Shatz and Rakic, 1981Go), effectively separating cell-intrinsic factors from cell-extrinsic influences. Thirdly, the rhesus monkey cortex has a large surface area, providing exquisite spatial resolution and the opportunity to examine distinct embryonic regions along the cortex's tangential axes and zones spanning the width of the developing cerebral wall. Thus, we set out to identify and characterize molecular differences between regions of the embryonic neocortex.

We have focused on two groups of molecules: transcription factors and EphA receptors. We selected transcription factors because they are proteins that bind to DNA and activate gene expression. As such, they are capable of controlling cellular identity by regulating cascades of gene expression (Simeone et al., 1992Go; Boncinelli et al., 1993Go; Bulfone et al., 1995Go). Our results demonstrate that the transcription factors TBr-1, Lhx-2, Emx-1, and a novel POU domain-containing gene, clone 10, are differentially expressed within the forming primate forebrain, with their expression forming gradients across the neocortex. Next, we examined the EphA receptor family, a group of cell surface-bound tyrosine kinases important in mediating cellular recognition (Cheng et al., 1995Go; Drescher et al., 1995Go; Orioli et al., 1996Go; Frisen et al., 1998Go). These receptors are also differentially expressed within the embryonic monkey cortex, with some of them present in well-defined compartments with sharp boundaries. Intriguingly, some of these EphA-positive compartments correspond to prospective functional domains, and moreover, distinct components within a single functional domain are molecularly distinct. Thus, in combination, our analysis reveals molecular heterogeneities within the developing forebrain, prior to the formation of precise connections between the neocortex and other parts of the nervous system.


    Materials and Methods
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Surgical Procedures

Timed pregnant rhesus monkeys were obtained from the Yale primate breeding colony (New Haven, CT) and the New England Regional Primate Center (Southborough, MA). Cesarean sections were performed 40, 65, 80 or 95 days following the estimated day of conception, as described previously (Rakic, 1972Go, 1977Go). Briefly, pregnant females were sedated with 5–10 mg/kg ketamine and 0.2 mg/kg atropine sulfate, an i.v. catheter was introduced for fluid administration, and the abdomen was sterilely prepared. Heart rate and respiration were monitored throughout the procedure, which was performed under isofluorane/oxygen inhalation anesthetic. A midline incision was made, the uterus was incised, the chorioallantoic membrane was punctured and the embryo was delivered. Finally, the mother's uterus and abdominal walls were sutured and her health was closely monitored for several days. Three animals at each embryonic age were examined.

Tissue Preparation

Embryonic monkey brains were dissected and split into hemispheres. Each hemisphere was then placed on a thin layer of embedding media on a microscope slide and frozen by placing the slide on dry ice and sprinkling dry ice powder over the sample. Upon full freezing, each sample was transferred to –80°C and stored. On the first day of each in situ hybridization, the tissue was brought to –20°C and cryostat sections of 10–20 µm were cut and thaw-mounted onto silanated slides.

In Situ Hybridizations

In situ hybridizations were performed according to a previously published procedure (Donoghue et al., 1996Go). Briefly, slides containing freshly cut embryonic monkey brains were incubated in the following series of solutions at room temperature (RT): (i) 4% paraformaldehyde, pH 7 for 10 min, (ii) phosphate-buffered saline (PBS) for 10 min, (iii) 0.75% glycine/PBS twice for 3 min each, (iv) PBS for 5 min, (v) 0.1 M triethanolamine (TEA) buffer for 5 min, (vi) 0.1 M TEA containing 500 µl acetic anhydride for 10 min, (vii) 0.1 M TEA for 5 min, (viii) 50, 70, 95 and 100% ethanol for 2 min each, (ix) chloroform for 5 min, (x) 100% ethanol twice for 2 min each. Probes were diluted in hybridization solution and denatured at 100°C for 2 min. Hybridization solution, containing probe (3 x 106 in a volume of 120 µl), was then spread over each section and a coverslip was placed over this solution and sealed. Slides were then incubated in a humidified chamber at 65°C for at least 16 h. Following hybridization, slides were incubated in the following series of solutions: (i) 2 x SSC for 15 min at RT, (ii) 0.5 x SSC for 5 min at RT, (iii) 0.1 x SSC for 20 min at 65°C, (iv) 1 x RNase buffer for 5 min at 37°C, (v) 20 µg/ml RNase A in 1 x RNase buffer for 30 min at 37°C, (vi) 1 x RNase buffer for 30 min at 37°C, (vii) 2 x SSC for 30 min at RT, (viii) 0.1 x SSC twice for 10 min at 65°C, (ix) 0.1 x SSC for 30 min at RT, (x) 50, 70, 95 and 100% ethanol for 2 min each at RT. Following exposure to film, slides were dipped in NTB2 nuclear track emulsion (Kodak), exposed for ~1 month at 4°C, developed, lightly counterstained with hematoxylin and bisbenzamide, coverslipped in glycerol, and photographed with either dark-field, fluorescent or bright-field optics.

Generation of Primate Antisense Probes

The human TBr-1 clone was the generous gift of J. Rubenstein (University of California, San Francisco). The human Emx-1 cDNA was kindly provided by E. Boncinelli (H.S. Raffaele, Milan). The monkey Lhx-2 and Clone 10 clones correspond to each cDNA's 3'-most ends and were isolated in a differential screen (M.J. Donoghue and P. Rakic, unpublished). Human cDNAs, corresponding to EphA6 and EphA7, were generous gifts from Nick Gale at Regeneron Pharmaceuticals, Inc. (Gale et al., 1996Go) . EphA3 was generated from embryonic monkey brain RNA by reverse transcription-polymerase chain reaction (RT-PCR) and subsequent cloning and characterization (Sambrook et al., 1989Go). Each of these templates were then linearized and antisense RNA probes were generated by in vitro transcription (Melton et al., 1984Go). The quality of the RNA probes was then confirmed by polyacrylamide gel electrophoresis followed by autoradiography.


    Results
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The developing cerebral wall in primates contains several transient embryonic zones (Sidman and Rakic, 1973Go, 1982Go; Rakic, 1976Go, 1977Go; Kostovic and Rakic, 1990Go; Shatz et al., 1990Go). These zones include: (i) the ventricular zone (VZ), with its dividing neural progenitor cells; (ii) the subventricular zone (SVZ), which acts early in corticogenesis as a secondary neuronal progenitor compartment and holding area for postmitotic cells from the VZ, and later in development as the major source of glia; (iii) the intermediate zone (IZ), through which migrating neurons traverse along radial glial processes; (iv) the subplate zone (SP), thought to be essential in orchestrating proper thalamocortical connectivity; (v) the cortical plate (CP), the initial condensation of postmitotic neurons that will become the characteristic six-layered structure of the mature cortex, generated in a stereotyped inside-first, outside-last manner; and (vi) the marginal zone (MZ), the most superficial, cell-sparse layer, important in the establishment of the laminar organization of the cortex. Although these zones exist in other mammalian species, they are especially broad and pronounced in primates, enabling their visualization within the width of the cerebral wall at each developmental stage.

We examined patterns of gene expression at four embryonic ages (E) in the macaque monkey forebrain: E40, E65, E80 and E95 of the 165 day gestational period of the rhesus monkey (Fig. 1Go). These ages were selected because they correspond to times at which distinct developmental processes are occurring: cell proliferation is intense in the VZ and the first postmitotic neurons, comprising the forming MZ, are just starting to be generated at E40 (Fig. 1A,BGo). By E65, however, the CP is present with cells that will differentiate into future deep cortical laminae (V and VI). However, the CP is not innervated by either afferent or efferent inputs at this age (Fig. 1CEGo). At E80, in contrast, the CP is more substantial, with cells that will contribute to layer IV already generated and with the only layer still missing in the most areas being layer II/III. Moreover, afferent and efferent connections are being forged between the CP and other parts of the nervous system at E80 (Fig. 1F,GGo). Finally, E95 corresponds to a final stage of corticogenesis, involving the waning of neurogenesis and presence of substantial thalamocortical as well as incipient corticocortical connections (Fig. 1H,IGo). Thus, the major stages of corticogenesis can be examined by studies of molecular expression at these ages.



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Figure 1.  Hematoxylin-stained sagittal sections of embryonic rhesus monkey brains. Representative images of brains from E40 (A,B), E65 (CE), E80 (F,G) and E95 (H,I) monkeys in which the boxes in (A), (C), (F) and (H) correspond to the images in (B), (D,E), (G) and (I) respectively. Low-power views of an E40 head (A) and E65 (C), E80 (F) and E95 (H) brains, sectioned parasagittally and stained with hematoxylin, with higher-power views of either the cerebral wall (D) or regions around the cortical plate (E,G,I) are shown. In all sections presented in this paper, anterior (a) is to the left, posterior (p) is to the right, dorsal (d) is up and ventral (v) is down (see coordinates in H). At E40, the telencephalic compartment (T) sits at the anterior-most tip of the folded neural tube (A) and consists of dividing cells within VZ and newly differentiated cells within the MZ (B). By E65, the neocortex is becoming morphologically differentiated, consisting of a VZ, SVZ, IZ, SP, CP and MZ. At E80, all of the above-mentioned embryonic zones exist within the neocortex, with the CP becoming more substantial. By E95, proliferative zones have diminished in size and the CP is fully populated. Presumptive cortical layers are indicated to the right of panel I. VZ, ventricular zone; MZ, marginal zone; SVZ, subventricular zone; IZ, intermediate zone; SP, subplate zone; CP, cortical plate; I–VI, cortical layers I–VI; GE, ganglionic eminence; Th, thalamus; C, caudate; P, putamen; H, hippocampus.

 
Here, we present the patterns of expression of a selection of transcription factors and receptor tyrosine kinases. Four aspects of each gene's expression will be discussed: (i) the embryonic zones in which each gene is expressed, (ii) the patterning of gene expression within the cortical, (iii) the selective expression of gene products in particular presumptive cortical laminae, and (iv) the restriction of expression to particular telencephalically derived compartments.

Transcription Factors in the Macaque Monkey Forebrain

The transcription factors discussed below were selected for these studies either because of intriguing neocortical expression patterns in other species (TBr-1 and Emx-1) or because they were isolated in our laboratories in a screen for gene products that are differentially expressed along the neocortex's anteroposterior axis during macaque monkey corticogenesis (Lhx-2 and Clone 10). While all of these factors are present throughout cortical development, their expression at three stages is presented here: the start of corticogenesis (E40), a time corresponding to mid-corticogenesis (E65) and the end of cortical neurogenesis (E95).

T-brain-1

T-brain-1 (TBr-1), a brachyury-like transcription factor, originally cloned and characterized in rodents by Rubenstein and colleagues (Bulfone et al., 1995Go) is present throughout primate corticogenesis (Fig. 2Go). At E40, TBr-1 is expressed within the developing telencephalon (Fig. 2AGo) and this expression is restricted to postmitotic cells (Fig. 2BGo). Thus, cells of the VZ are TBr-1 negative, while more superficial cells populating the newly formed marginal zone are TBr-1 positive (Fig. 2BGo). This pattern of expression is maintained at E65; embryonic zones containing differentiated cells, such as the SVZ, IZ, CP and MZ, express TBr-1, while cells of the VZ are negative (Fig. 2C, DGo). Interestingly, compared with expression patterns in the neocortex of embryonic mice, TBr-1 expression within the monkey SVZ is very pronounced, either because of the larger size of this zone or a difference in the timing or make-up of the SVZ (Fig. 2DGo). TBr-1 expression is restricted to cells of the CP and MZ at E95, as neurogenesis and neuronal migration have all but ceased (Fig. 2EGo). Thus, TBr-1 is an early marker of postmitotic cortical neurons and thus, expression is observed in embryonic zones that contain differentiated cells.



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Figure 2.  T-brain-1 (TBr-1) expression in the developing rhesus monkey brain. In situ hybridizations using an antisense probe to TBr-1 that has been hybridized to sections of either an E40 head (A,B), or E65 (C,D) or E95 (E,F) brains from rhesus monkey embryos, similar to those shown in Figure 1Go. Low-power views of entire brains are shown in panels (A), (C) and (E), while higher-power views of the cerebral wall (B,D) or CP (F) are also included. Much of the forebrain is positive for TBr-1 at E40 (A) and this positive signal corresponds to differentiated cells within the MZ (B). By E65, all compartments containing postmitotic neurons are TBr-1 positive, including the SVZ, IZ, CP and MZ (C,D). At E95, the CP is TBr-1 positive and other compartments are negative, corresponding to their demise (E). Furthermore, anteroposterior differences are distinct; TBr-1 is present throughout the thickness of the CP anteriorly but is limited to the deepest strata posteriorly (E,F). Finally, restriction to a subgroup of telencephalically derived structures is apparent with TBr-1 expression; the neocortex, olfactory bulb and hippocampus are positive, while the neostriatum is negative (C,E and data not shown). All abbreviations are given in the legend to Figure 1Go.

 
TBr-1 expression is also patterned along the neocortex's anteroposterior axis; levels are high and uniform anteriorly and lower and more restricted posteriorly (Fig. 2C,EGo). In particular, TBr-1 is expressed by cells of all presumptive layers in its anterior-most regions but is restricted to cells that will give rise to the deepest cortical layers posteriorly (Fig. 2D,FGo). While these regional differences are most apparent at E95 (Fig. 2EGo), they are also present within the E65 CP (Fig. 2DGo), suggesting that programs intrinsic to the neocortex are directing this expression.

TBr-1 also displays presumptive laminar-specific expression. While present within all presumptive laminae anteriorly, TBr-1 expression is strong in cells that will populate future layers V and VI, and weak in cells that will comprise future layers II/III within the posterior neocortex (Fig. 2D,FGo). Again, while selective expression to specific strata of the CP is most obvious at E95, these differences are also present at E65; expression is uniform rostrally and within the deepest aspects of the CP caudally (Fig. 2C,DGo).

TBr-1 expression is restricted to particular telencephalic compartments in the monkey, as it is in rodents (Bulfone et al., 1995Go). The neocortex, olfactory bulb and hippocampus are TBr-1 positive, but the ganglionic eminences, as well as its differentiated offspring, the striatum and pallidum, are TBr-1 negative (Fig. 2Go and data not shown). This division of gene expression supports the prosomeric model of forebrain organization (Puelles and Rubenstein, 1993Go; Rubenstein et al., 1994Go).

Emx-1

Emx-1 was cloned as a mammalian homolog to the Drosophila empty spiracles gene and was shown to be expressed throughout most of the cerebral cortex of embryonic mice (Simeone et al., 1992Go). In keeping with these results in rodents, we detect this homeodomain-containing gene product throughout the forming macaque monkey forebrain. It is abundant at each age we examined, in all embryonic zones and within all cell populations (Fig. 3Go). At E40, Emx-1 is expressed by both proliferating and differentiated cells (Fig. 3BGo) and this expression pattern is expanded as the cerebral wall expands at E65, present in all cellular zones (Fig. 3DGo). By E95, however, while expression within the CP is obvious, levels in other embryonic zones have significantly decreased, consistent with their demise (Fig. 3EGo).



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Figure 3.  Emx-1 expression in the developing rhesus monkey brain. In situ hybridizations using an antisense probe to Emx-1 that has been hybridized to sections of either an E40 head (A,B), or E65 (C,D) or E95 (E,F) brains from monkey embryos, similar to those shown in Figure 1Go. Low-power views of entire brains are shown in panels (A), (C) and (E), while higher-power views of the cerebral wall (B,D) or CP (F) are also included. The entire forebrain is positive for Emx-1 at E40 (A) and hybridization is observed in both proliferating and differentiated populations (B). By E65, all embryonic zones are Emx-1 positive, including the VZ, SVZ, IZ, CP and MZ (C,D), while at E95, the CP is Emx-1 positive and levels in other compartments are significantly decreased, corresponding to the decrease in cells within them (E,F). Within the CP, emx-1 is most highly expressed in future layers II/III. Finally, Emx-1 is expressed in all telencephalically derived structures (C,F). All abbreviations are given in the legend to Figure 1Go.

 
Emx-1 is expressed in an anteroposterior gradient within the neocortex, again consistent its expression in the mouse (Simeone et al., 1992Go). In particular, Emx-1 expression is high posteriorly, with levels decreasing anteriorly, and this pattern is present both at E65 and E95 (Fig. 3C,EGo). Emx-1 expression also reveals a preference for particular presumptive laminae; levels are highest in cells that will populate layers II/III (Fig. 3FGo). Thus, in the occipital lobe (prospective primary visual cortex) Emx-1 has a complementary distribution to the TBr-1, which is expressed predominantly in layers V and VI (compare Figs 2 E and 3EGoGo).

The restriction of gene expression to particular telencephalic compartments that was observed with TBr-1 is also apparent in Emx-1's expression; Emx-1 is expressed by cells of all telencephalically derived structures except the neostriatum, including the neocortex, the hippocampus and the olfactory bulb by E65 (Fig. 3CGo and data not shown).

Lhx-2

The primate Lhx-2 gene, a LIM-homeodomain-containing gene product (Xu et al., 1993Go), was identified in a screen intended to identify gene products that are differentially expressed along the neocortex's anteroposterior axis (M.J. Donoghue and P. Rakic, unpublished). This gene was selected among several other candidates because of its posterior-greater-than-anterior pattern of expression in the initial screen. Lhx-2 is expressed at E40 (Fig. 4AGo) and is at highest levels within the proliferating cells of the VZ (Fig. 4BGo). At E65, levels are high within the VZ and SVZ but are low within the CP (Fig. 4CGo). Nonetheless, defined regions of the caudal CP express Lhx-2 at E65 (Fig. 4DGo). By E95, Lhx-2 is expressed throughout the CP (Fig. 4EGo)



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Figure 4.  Lhx-2 expression in the developing rhesus monkey brain. In situ hybridizations using an antisense probe to Lhx-2 that has been hybridized to sections of either an E40 head (A,B), or E65 (C,D) or E95 (E,F) brains from monkey embryos. Low-power views of entire brains are shown in panels (A), (C) and (E), while higher-power views of the cerebral wall (B,D) or CP (F) are also included. Much of the forebrain is positive for Lhx-2 at E40 (A) and hybridization is restricted to proliferating populations (B). By E65, the VZ and SVZ are strongly Lhx-2 positive and despite the fact that a substantial CP exists at E65, Lhx-2 is present at very low levels within it (C,D). Indeed, Lhx-2 is present at detectable levels posteriorly and undetectable levels anteriorly (D). At E95, the CP is Lhx-2 positive and levels in other compartments are significantly decreased, corresponding to the decrease in cells within them (E). Strikingly, anteroposterior differences are distinct; Lhx-2 is present in an posterior-high, anterior-low gradient within the E95 neocortex. Lhx-2 is most strongly expressed by cells that will populate future layers II/III (F). Finally, restriction to a subgroup of telencephalically derived structures is apparent with Lhx-2 expression; the neocortex, olfactory bulb and hippocampus are positive, while the neostriatum is negative (C,E and data not shown).

 
Lhx-2 expression displays a striking anteroposterior gradient, with levels within the E95 CP high posteriorly and diminishing in the anterior direction (Fig. 4EGo). Hints of this graded pattern of expression are present at E65, as Lhx-2 is only present within the caudal CP (Fig. 4C,DGo). Lhx-2 expression is also consistent with its being expressed within particular presumptive laminae. In particular, by E95 Lhx-2 expression is concentrated within future layers II and III (Fig. 4FGo).

Another feature of Lhx-2's expression within the developing primate brain is its restriction to defined telencephalic compartments. Just as TBr-1 expression is limited, Lhx-2 is expressed only within the neocortex, hippocampus and olfactory bulb, and is absent from the ganglionic eminence and its descendants, the differentiated striatum (Fig. 4C,EGo and data not shown).

Clone 10

Clone 10 was isolated in a differential screen intended to identify molecules present at different levels along the embryonic monkey neocortex's anteroposterior axis, as described above for Lhx-2. However, sequence analysis suggests that Clone 10 is a rhesus monkey POU domain-containing gene product (M.J. Donoghue and P. Rakic, unpublished). Clone 10 is expressed within the E40 brain; however, levels are low within the neocortex and higher in more ventral regions of the telencephelon (Fig. 5A,BGo). Nonetheless, within the monkey neocortex this gene is expressed within the VZ (Fig. 5BGo). Furthermore, Clone 10 is expressed selectively by cells of the VZ and SVZ, but not other zones at E65 (Fig. 5C,DGo). This expression profile is consistent withClone 10 being restricted to dividing populations of cells. Clone 10 expression within the CP is low but apparent at E65, concentrated mainly within posterior region (Fig. 5C,DGo). Expression is robust at E95, with high levels of Clone 10 expression in both dividing and differentiated cells, again with a posterior bias (Fig. 5FGo).



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Figure 5.  Clone 10 expression in the developing rhesus monkey brain. In situ hybridizations using an antisense probe to Clone 10 that has been hybridized to sections of either an E40 head (A,B), or E65 (C,D) or E95 (E,F) brains from monkey embryos. Low-power views of entire brains are shown in panels (A), (C) and (E), while higher-power views of the cerebral wall (B,D) or CP (F) are also included. The E40 forebrain is positive for Clone 10 (A), with signal restricted to proliferating populations (B). By E65, the VZ and SVZ are strongly Clone 10 positive (C). Furthermore, despite the fact that a substantial CP exists at E65, Clone 10 is present at very low levels within it (D). At E95, the CP is Clone 10 positive and levels in other compartments are significantly decreased, corresponding to the decrease in cells within them (E). Clone 10 is most abundant in cells of future layers II/III (F). Strikingly, anteroposterior differences are distinct; Clone 10 is present in an posterior-high, anterior-low gradient within the E95 neocortex (E). Finally, Clone 10 is expressed throughout the neocortex, olfactory bulb, hippocampus, and neostriatum (C,E and data not shown).

 
Clone 10 displays an anterior-low, posterior-high pattern within the CP at E95 (Fig. 5EGo) and the foundation of this pattern is obvious already at E65 (Fig. 5CGo). Moreover, within the CP, Clone 10 is highest within the cells that will differentiate into future layers II/III (Fig. 5FGo). Thus, in several aspects, Clone 10 expression mirrors the expression of Lhx-2 (compare Figs 4 and 5GoGo).

Clone 10 is expressed by all sectors of the telencephalon and, similar to Emx-1, displays no distinctions between embryonic compartments (Fig. 5CGo). Interestingly, while Clone 10 is expressed by both dividing and differentiated cells within the neocortex, it is only expressed by the ganglionic eminence, the proliferative zone of the striatum, but not its differentiated structures (Fig. 5C,EGo). Finally, while Clone 10 and Lhx-2 share many features, they differ on their restriction to particular embryonic compartments; Clone 10 expression is widespread, while Lhx-2 expression is more limited (compare Figs 4C and 5CGoGo).

EphA Receptor Tyrosine Kinases in the Macaque Monkey Forebrain

The EphA family of receptor tyrosine kinases are widely and dynamically expressed throughout macaque monkey corticogenesis (Donoghue and Rakic, 1999Go). While some family members are present early in development, most are not expressed until later stages. This timing is consistent with their presence in embryonic zones that house postmitotic neurons, rather than those that correspond the areas of rapid proliferation. For this reason, we present here expression of three of these molecules (EphA3, A6 and A7) at E65, E80 and E95, ages when their expression and patterning are most striking.

EphA3

At E65 EphA3 is absent from the VZ and MZ, but is present at low levels within the IZ and is strongly expressed by cells of the SVZ (Fig. 6AGo). Within the CP, EphA3 expression displays a distinctive pattern: narrowly defined posterior regions contain cells that express high levels of EphA3 (Fig. 6AGo). In particular, EphA3 is expressed by cells within the ventral-most and dorsal-most regions of the posterior CP. Intriguingly, this pattern of expression is reminiscent of the future location of the extrastriate cortex, while the region of the prospective striate cortex within the occipital pole is devoid of EphA3 expression. At E80, similar to the earlier age, EphA3 is not expressed by cells within either the VZ or MZ with low but detectable levels present in the IZ and SP (Fig. 6CGo). However, EphA3 is strongly expressed by cells of the SVZ and CP at E80 with a discernible border between prospective striate and extrastriate areas (Fig. 6CGo). Finally, at E95 EphA3 expression is restricted to the CP.



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Figure 6.  EphA3 expression in the developing rhesus monkey brain. In situ hybridizations using an antisense probe to EphA3 that has been hybridized to sections of either E65 (A,B), E80 (C,D) or E95 (E,F) brains from monkey embryos. Low-power views of entire brains are shown in panels (A), (C) and (E), while higher-power views of the CP are shown in panels (B), (D) and (F). At E65, the SVZ and distinct areas of the CP are EphA3 positive; the dorsal and ventral aspects of the posterior CP express EphA3 (A). Within these regions, EphA3 is expressed throughout the forming CP (B). Strikingly, these regions of the CP corresponds to the area of the presumptive extrastriate, but not striate cortex. At E80, levels of EphA3 remain high within the SVZ and the most posterior parts of the CP, with much lower levels in more anterior regions of the CP (C). Within the CP region, however, EphA3 expression is present within the SP and the most superficial aspects of the CP, corresponding to future layer IV (D). At E95, the CP is EphA3 positive and levels in other compartments are undetectable (E). Moreover, EphA3 expression is localized to an intermediate level within the CP at E95, corresponding to the future layer IV (F). Finally, Finally, EphA3 is present within the neocortex, olfactory bulb and neostriatum but is excluded from the hippocampus (A,C).

 
Within each of the embryonic zones in which it is expressed, EphA3 is at highest levels within the posterior-most regions of the neocortex. This is apparent in the unique pattern of EphA3 gene expression observed at E65 (see Fig. 6AGo and above). Further, while EphA3 still marks the posterior portion of the neocortex, as development proceeds its pattern of expression is expanded; the anterior boundary is more rostral at E80 and E95 and expression is throughout the occipital lobe (Fig. 6C,EGo). Moreover, EphA3 expression corresponds to future layer IV at all embryonic ages we examined. At E65, when the generation of cells of prospective layer IV are just commencing, expression is within the most superficial aspects of the CP (Fig. 6BGo). By E80, when the number of cells that will eventually inhabit future layer IV are greater, expression is again restricted to this population of cells (Fig. 6DGo). By E95, when future laminae are slightly more well-defined, EphA3 expression is restricted to presumptive layer IV (Fig. 6FGo). Thus, EphA3 appears to mark future layer IV cells of the posterior regions throughout corticogenesis.

Expression of EphA3 is within the boundaries of the neocortex at E65 (Fig. 6AGo), but has spread to encompass the ganglionic eminence by E80 (Fig. 6CGo) and this expanded expression is maintained at E95 (Fig. 6EGo). Thus, EphA3 demonstrates expression that is restricted to particular telencephalically derived compartments early in development and is then spread throughout these compartments later.

EphA6

This receptor subtype is especially interesting because it has unique regional expression early in corticogenesis that remains stable throughout development. For example, similar to EphA3, EphA6 is present within the posterior-most region of the CP at E65 (Fig. 7AGo). However, EphA6 is expressed throughout the occipital lobe, in both presumptive striate and extrastriate regions, while EphA3 is present only within presumptive extrastriate regions. At E80, EphA6 expression remains restricted to a single domain of the E80 cortical wall (Fig. 7CGo), a region that corresponds to the future visual cortex (Dehay et al., 1996Go; Rakic, 1976Go; Kostovic and Rakic, 1984Go) while EphA3's expression has expanded significantly. Similar to patterns observed at E80, expression of EphA6 is tightly restricted to the posterior-most region of the cerebral cortex (Fig. 7EGo).



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Figure 7.  EphA6 expression in the developing rhesus monkey brain. In situ hybridizations using an antisense probe to EphA6 that has been hybridized to sections of either E65 (A,B), E80 (C,D) or E95 (E,F) brains from monkey embryos. Low-power views of entire brains are shown in panels (A), (C) and (E), while higher-power views of the CP are shown in panels (B), (D) and (F). At all ages examined, EphA6 expression is restricted to the posterior-most portion of the CP, corresponding with the future visual cortex (A,C,E). At E65, this region is strongly EphA6 positive (A) and silver grains are spread throughout the region of the forming CP (B). At E80, levels of EphA6 remain high the most posterior parts of the CP (C) and expression is localized to the deepest aspects of the CP, future layers V and VI, and the SP (D). By E95, the CP of the presumptive visual cortex remains EphA6 positive (E) and signal is localized to the SP (F). Finally, Finally, EphA3 is present only within the neocortex (A,C,E).

 
EphA6 is expressed by cells within the deepest strata of the CP at E65, corresponding to the future subplate zone and future deep cortical plate layers (Fig. 7BGo). Moreover, a single band of cells in the deep CP and SP is EphA6-positive at E80 (Fig. 7DGo). Finally, EphA3 continues to be tightly localized to the deepest CP and the SP zone at E95 (Fig. 7FGo). This pattern of future layer VI and SP expression is especially interesting since these are cortical regions that have been implicated in accomplishing proper guidance of afferent input.

Finally, EphA6 expression is restricted to the neocortex at all ages examined.

EphA7

At E65, EphA7 is also present in a well-defined posterior region of the CP; however, its pattern of expression is slightly expanded in comparison to EphA6. While its anteroposterior borders of expression are similar to EphA6's (compare Figs 7A and 8AGoGo), EphA7 is expressed more extensively throughout the region of the CP (Fig. 8BGo). At E80 EphA7 expression remains within the neocortex, mainly in the SP and CP, with low levels of expression within the posterior-most SVZ (Fig. 8CGo). Within the CP, there is differential expression, with levels highest posteriorly and lowest anteriorly. Thus, once again, while EphA7's expression pattern overlaps considerably with EphA6’s, it is more extensive in two respects: (i) the anterior border of EphA7 expression is more rostral than EphA6’s; and (ii) EphA7 is expressed in a broader range of cells within the CP region, including future layers V and VI, as well as SP cells. By E95 EphA7 expression includes cells within the anterior CP, although it is unclear what prospective cytoarchitectonic areas these patches correspond to. Thus, EphA7's expression is similar to EphA6’s; however, it is considerably more extensive, both along the cortex's anteroposterior axis and within the cerebral wall.



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Figure 8.  EphA7 expression in the developing rhesus monkey brain. In situ hybridizations using an antisense probe to EphA7 that has been hybridized to sections of either E65 (A,B), E80 (C,D) or E95 (E,F) brains from monkey embryos. Low-power views of entire brains are shown in panels (A), (C) and (E), while higher-power views of the CP are shown in panels (B), (D ) and (F). At E65, EphA7 expression mirrors EphA6’s, in that it is present within a defined portion of the posterior-most CP (compare Figs 7A and 8AGoGo). Moreover, positive signal is spread uniformly over the forming CP (B). At E80, levels of EphA6 remain high the most posterior parts of the CP (C) and expression is localized to the deepest aspects of the region of the CP, future layers V and VI, and the SP (D). By E95, the CP of the presumptive visual cortex remains EphA6 positive (E) and the signal in this domain remains localized to the SP (F). However, expression is now also present within regions of the anterior CP (E). Finally, Finally, EphA7 is present only within the neocortex and is excluded from other compartments (A,C,E).

 
EphA7 expression also demonstrates specificity to presumptive laminae throughout corticogenesis. At E65, EphA7 expression is consistent with it being expressed by cells that will constitute layers V and VI of the CP as well as the SP (Fig. 8BGo). By E80, this selectivity is apparent, with expression restricted to the deepest strata of the CP, future layers V and VI, as well as the SP (Fig. 8DGo). This pattern of expression is maintained at E95; EphA7 is expressed in SP and within the deepest layers of the CP (Fig. 8FGo).

In contrast to EphA6, whose expression becomes restricted to the neocortex, EphA7 is expressed within the striatum as well at E80 (Fig. 8CGo). Thus, similar to EphA3, gene expression that was restricted to particular telencephalically derived structures at E65 becomes expanded as development proceeds.


    Discussion
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We examined the expression of several transcription factors and EphA receptors in the embryonic primate neocortex in a search for genes that are differentially expressed by cells of the embryonic primate cerebral wall. Taking advantage of the spatial and temporal resolution of the macaque monkey cerebrum, we highlight four aspects of gene expression that exist within the developing forebrain: (i) expression within particular embryonic zones of the cerebral wall; (ii) patterning of gene expression along the neocortex's anteroposterior axis and, in some cases, within prospective cortical areas; (iii) expression consistent with particular presumptive cortical laminae; (iv) expression within particular telencephalically derived compartments.

The fact that well-defined patterns of gene expression can be detected early in cortical development, prior to both afferent and efferent innervation, suggests that the expression of at least of some molecules is regulated by programs intrinsic to cortical cells. Furthermore, the maintenance of these early patterns of expression, for example TBr-1 and EphA6, demonstrates that such programs are stable during corticogenesis. In contrast to this stability, however, other patterns of gene expression change significantly as development proceeds, such as EphA3 and A7. Such refinements to initial patterns of support the concept that extrinsic factors can alter a cortical cell's molecular repertoire. Thus, the distinct yet dynamic patterns of gene expression that we observe within the developing primate cerebral wall are likely to reflect both intrinsically encoded cell specification as well as the influence of environmental signals.

Embryonic Zone Preference

We observed distinct patterns of gene expression in transient embryonic zones during the formation of the cerebral cortex. For example, TBr-1, EphA3, EphA6 and EphA7 expression was restricted to differentiated cells, whereas Lhx-2 and Clone 10 are predominantly expressed by progenitors and immature cortical cells. Finally, Emx-1 demonstrates no preference for a particular embryonic zone and is present throughout the developing neocortex. These differences in expression patterns are likely to directly reflect the differentiated state of cortical neurons. Interestingly, some of the genes we have examined here (i.e. TBr-1, Emx-1, Lhx-2 and clone 10) are likely to contribute to this differentiated state since some of them are transcription factors themselves. Nonetheless, together, these genes comprise convenient markers of cell populations as they form. Finally, TBr-1 expression is obviously within the SVZ in the forming primate neocortex, whereas expression within this zone was much less obvious in embryonic rodent brain [compare Fig. 2CGo with previous work by Bulfone and co-workers (Bulfone et al., 1995Go)]. This difference is likely to reflect the extended development of the primate cortex, both temporally and spatially.

Gradients versus Compartments

We observed two general categories of patterning of gene expression along the main axes of the embryonic neocortex: concentration gradients and distinct compartments. In the former group, Emx-1 is present in anterior-high, posterior-low pattern, while Lhx-2 and Clone 10 are expressed in opposing, posterior-high, anterior-low gradients. In contrast, compartments of gene expression are more consistent with our observations for TBr-1, EphA3, EphA6 and EphA7, as their expression is either present or absent, with no gradual decrease obvious.

What might underlie these distinct patterns? One possibility is that concentration gradients respond to the pressures of morphogenetic fields that encompass the full span of the neocortex, while compartments are regulated by factors that are more locally distributed. The idea that gradients of gene expression direct patterning along an anatomical axis is widely accepted in developmental biology (Wolpert, 1985Go; Pankratz and Jackle, 1990Go; Sanes, 1993Go), Moreover, gradients of gene expression within the nervous system have been shown to be essential for its proper function (Simeone et al., 1992Go; Drescher et al., 1995Go; Yoshidaet al., 1997Go; Frisen et al., 1998Go) In fact initially weak gradients can be enhanced in the course of development and transformed into compartments with sharp boundaries (Gierer and Muller, 1995Go).

The graded patterns of gene expression observed in the present study may play a role in the initial, broad organization of the cortex. In contrast, the compartments of expression we observe may act secondarily to specify cells within such an organized field, such as the designation of cortical areas. Such a model is supported by the timing of gene expression we report here: the molecules that are present in gradients within the neocortex are expressed by relatively immature cells, while those present in compartments are present in more differentiated populations. Thus, the specification of cells into functional domains within the cerebral cortex may require two steps: the assignment of a positional identity within a morphogenetic field by molecular gradients and the designation of cellular subpopulations at any given positional value by compartmentalized gene expression. Compartments of gene expression are most striking when viewed in concert. For example, TBr-1 and EphA3 are expressed in complementary patterns at E95, clearly indicating the presence of distinct cellular fields (Fig. 9Go). Interestingly, the border between these fields (see arrows in Fig. 9Go) designates the boundary between visual and nonvisual cortical space.



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Figure 9.  Boundaries between distinct compartments exist within the monkey neocortex. Adjacent sections of E95 monkey brains hybridized with either TBr-1 (top) or EphA3 (bottom) reveal compartments of gene expression with sharp borders. The point at which TBr-1 expression becomes restricted to deep layers and EphA3 expression is most prominent indicates a border between areas (see arrows). All cells posterior to this area are visual.

 
The idea that compartments may underlie local differences within a single structure is born out by the expression of the EphA genes we examined. EphA6 is expressed throughout development in a well-defined region of the forming cortical plate. In fact, this region corresponds to the presumptive visual cortex, including both the striate and extrastriate cortex. Thus, EphA6 appears to be a marker of a functional area prior to its morphological differentiation and its establishment of mature connections. In contrast, while also present in distinct compartments early in development, EphA3 is expressed in a more localized region of the posterior cortical plate, a region that corresponds only to the future extrastriate cortex leaving presumptive striate cortex devoid of this factor. Thus, the expression of these genes demonstrates hierarchies of gene expression, even within a single functional domain.

Presumptive Laminar-specific Gene Expression

Consistent with previous experimental studies, we find that cells of the cerebral cortex display signs of laminar specification early in their development (McConnell, 1985Go, 1988Go; Frantz et al., 1994Go; Algan and Rakic, 1997Go). Indeed, several of the genes we examined in this study demonstrate restricted expression that is consistent with their marking future cortical layers. For example, TBr-1 expression is consistent with its being expressed with future layers V and VI in the posterior regions of neocortex, while Lhx-2 and Clone 10 appear to mark future layers II/III. Finally, EphA3 expression is consistent with its being expressed within layer IV, the major target of thalamic inputs. Together, these patterns of gene expression demonstrate early-emerging heterogeneities among cortical cells. Some of these heterogeneities may have functional consequences, leading to the distinct properties of different populations of cells.

Interestingly, EphA6's expression within the occipital neocortex is restricted to future layer VI and subplate cells. This expression pattern is especially intriguing in light of the fact that these strata are likely to play a role in establishing proper thalamocortical connectivity (Kostovic and Rakic, 1990Go; McConnell et al., 1994Go). Furthermore, since the Eph receptor tyrosine kinases mediate cellular recognition in many developmental systems (Cheng et al., 1995Go; Drescher et al., 1995Go; Donoghue et al., 1996Go; Wang and Anderson, 1997Go), they may act similarly within the cortex (Castellani et al., 1998Go; Donoghue and Rakic, 1999Go). However, whether these gene products mediate repulsive interactions, as has commonly been assumed (Gale and Yancopoulos, 1998Go), or attractive processes, as has been hinted at recently (Castellani and Bolz, 1997Go; Castellani et al., 1998Go) requires further study.

The Protomap Model of Cortical Specification

Our results support the idea that the cerebral wall develops by segmentation into discrete morphological units (Rakic, 1988Go; Rubenstein et al., 1994Go). Most striking in this regard is the exclusion of particular genes from distinct regions of the embryonic brain. For example, TBr-1, Lhx-2, EphA3 and EphA6 are expressed within the neocortex but are absent from the neostriatum, suggesting that regulatory programs are distinct within these two regions. Further restrictions are also obvious in the expression of these genes. For example, while the EphA genes are expressed only within the neocortex, TBr-1 and Lhx-2 are also present within the hippocampus and olfactory bulb.

Previous studies performed in rodents defined domain-specific molecular markers within the cortex (Barbe and Levitt, 1991Go; Arimatsu et al., 1992Go; Boncinelli et al., 1993Go; Cohen-Tannoudji et al., 1994Go; Bulfone et al., 1995Go; Na et al., 1998Go; Nothias et al., 1998Go; Gitton et al., 1999Go; Rubenstein et al., 1999Go). In addition, differential cell cycle kinetics and cellular properties in vitro also suggest that inborn heterogeneities may exist between cells of distinct cortical regions in both rodents and primates (Dehay et al., 1993Go; Eagleson et al., 1997Go; Ferri and Levitt, 1993Go; Kennedy and Dehay, 1993Go; Polleux et al., 1997Go). Furthermore, cell lineage, transplantation and selective elimination studies demonstrate significant predetermination of fates, both areally and phenotypically (Luskin et al., 1988Go; McConnell and Kaznowski, 1991Go; Parnavelas et al., 1991Go; Algan and Rakic, 1997Go; Tan et al., 1998Go). Finally, within the visual system, specification occurs in the absence of mature patterns of visual activity (Horton and Hocking, 1996Go; Algan and Rakic, 1997Go; Meissirel et al., 1997Go; Snider et al., 1998Go; Wallace et al., 1997Go; Khachab and Bruce, 1999Go). Taken together, these studies provided compelling evidence that a protomap of the future cortex exists early in development, indicating that developing cells may be informed, at least to some extent, of their eventual fates. Indeed, they are sufficiently different to attract selective sets of input with which they respond in particular ways and cooperatively engage in the formation of distinct cytoarchitectonic fields (Rakic, 1988Go; Rakic et al., 1991Go). Here, we extend previous studies, demonstrating that regional expression exists in the absence of patterned afferent input. Moreover, such regional expression is within embryonic zones or presumptive laminae consistent with their playing a role in guiding appropriate synapse formation.

However, it needs to emphasized that the prefix ‘proto’ implies that the areal, laminar and phenotypic identities of cortical cells are also highly plastic. For example, heterotopic transplantations demonstrate remarkable alterations in cellular properties following a change in the environment (Schlaggar and O'Leary, 1991Go; Eagleson and Levitt, 1999Go; Gitton et al., 1999Go). Moreover, rerouting of afferent input to the cortex results in a transformation of the target area so that it corresponds more closely to the source of its input rather than its original position (Frost and Metin, 1985Go; Sur et al., 1988Go; Roe et al., 1990Go). Likewise, regulation of specific thalamic input diminishes the size of the target areas (Rakic, 1988Go; Rakic et al., 1991Go). Together, these results argue that cortical cells, although initially programmed, are nevertheless flexible in the maintenance of that program and have the ability to adopt novel fates. Consistent with these findings, several patterns of gene expression in our study display significant changes as development proceeds. Thus, environmental changes, such as patterns of innervation, are likely to affect final cellular identities and, thus, patterns of gene expression as afferent fibers arrive and the cortex differentiates. Here, we demonstrate molecular differences between cells early in cortical development and prior to the formation of stable connections with other parts of the nervous system, supporting the idea that early acting intrinsic programs shape future cellular identities within the cortex. Future studies will determine the extent to which these programs influence subsequent development, as well as the specific roles that these genes have in the organization of the mature cerebral cortex.


    Notes
 
We thank Regeneron Pharmaceuticals, Inc., especially Nick Gale, for generously providing human Eph cDNAs, John Rubenstein for kindly providing the human TBr-1 clone and E. Boncinelli for his generous gift of the human Emx-1 cDNA. We thank Terri Beattie and Susan Morgenstern for providing animal care and John Rubenstein, Mihae Yun, Randy Johnson and Steve Holt for comments on this manuscript. M.J.D. is grateful to Nenad Sestan and Ladislav Mrzljak for assistance with embryonic primate neuroanatomy. This work was supported by a Life Sciences Research Foundation post-doctoral fellowship (M.J.D.) and grants from the National Institutes of Health (P.R.).

Address correspondence to Pasko Rakic, Yale University School of Medicine, Section of Neurobiology, 333 Cedar Street, SHM/B-E33, New Haven, CT 06510, USA. Email: pasko.rakic{at}yale.edu.


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