Yale University School of Medicine, Section of Neurobiology, 333 Cedar Street, New Haven, CT 06510, USA
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Abstract |
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Introduction |
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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, 1988; O'Leary et al., 1994
), 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, 1972
), leading to the postulation of the radial unit and protomap hypotheses (Rakic, 1988
; Rakic et al., 1991
). 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, 1974) in the absence of afferent or efferent innervation of these cells (Rakic, 1976
, 1977
; Shatz and Rakic, 1981
), 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., 1992; Boncinelli et al., 1993
; Bulfone et al., 1995
). 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., 1995
; Drescher et al., 1995
; Orioli et al., 1996
; Frisen et al., 1998
). 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.
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Materials and Methods |
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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, 1972, 1977
). Briefly, pregnant females were sedated with 510 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 1020 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., 1996). 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., 1996) . EphA3 was generated from embryonic monkey brain RNA by reverse transcription-polymerase chain reaction (RT-PCR) and subsequent cloning and characterization (Sambrook et al., 1989
). Each of these templates were then linearized and antisense RNA probes were generated by in vitro transcription (Melton et al., 1984
). The quality of the RNA probes was then confirmed by polyacrylamide gel electrophoresis followed by autoradiography.
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Results |
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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. 1). 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,B
). 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. 1CE
). 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,G
). 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,I
). Thus, the major stages of corticogenesis can be examined by studies of molecular expression at these ages.
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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., 1995) is present throughout primate corticogenesis (Fig. 2
). At E40, TBr-1 is expressed within the developing telencephalon (Fig. 2A
) and this expression is restricted to postmitotic cells (Fig. 2B
). Thus, cells of the VZ are TBr-1 negative, while more superficial cells populating the newly formed marginal zone are TBr-1 positive (Fig. 2B
). 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, D
). 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. 2D
). TBr-1 expression is restricted to cells of the CP and MZ at E95, as neurogenesis and neuronal migration have all but ceased (Fig. 2E
). 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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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,F). 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,D
).
TBr-1 expression is restricted to particular telencephalic compartments in the monkey, as it is in rodents (Bulfone et al., 1995). 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. 2
and data not shown). This division of gene expression supports the prosomeric model of forebrain organization (Puelles and Rubenstein, 1993
; Rubenstein et al., 1994
).
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., 1992). 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. 3
). At E40, Emx-1 is expressed by both proliferating and differentiated cells (Fig. 3B
) and this expression pattern is expanded as the cerebral wall expands at E65, present in all cellular zones (Fig. 3D
). By E95, however, while expression within the CP is obvious, levels in other embryonic zones have significantly decreased, consistent with their demise (Fig. 3E
).
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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. 3C and data not shown).
Lhx-2
The primate Lhx-2 gene, a LIM-homeodomain-containing gene product (Xu et al., 1993), 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. 4A
) and is at highest levels within the proliferating cells of the VZ (Fig. 4B
). At E65, levels are high within the VZ and SVZ but are low within the CP (Fig. 4C
). Nonetheless, defined regions of the caudal CP express Lhx-2 at E65 (Fig. 4D
). By E95, Lhx-2 is expressed throughout the CP (Fig. 4E
)
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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,E 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,B). Nonetheless, within the monkey neocortex this gene is expressed within the VZ (Fig. 5B
). Furthermore, Clone 10 is expressed selectively by cells of the VZ and SVZ, but not other zones at E65 (Fig. 5C,D
). 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,D
). Expression is robust at E95, with high levels of Clone 10 expression in both dividing and differentiated cells, again with a posterior bias (Fig. 5F
).
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Clone 10 is expressed by all sectors of the telencephalon and, similar to Emx-1, displays no distinctions between embryonic compartments (Fig. 5C). 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,E
). 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 5C
).
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, 1999). 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. 6A). Within the CP, EphA3 expression displays a distinctive pattern: narrowly defined posterior regions contain cells that express high levels of EphA3 (Fig. 6A
). 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. 6C
). 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. 6C
). Finally, at E95 EphA3 expression is restricted to the CP.
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Expression of EphA3 is within the boundaries of the neocortex at E65 (Fig. 6A), but has spread to encompass the ganglionic eminence by E80 (Fig. 6C
) and this expanded expression is maintained at E95 (Fig. 6E
). 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. 7A). 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. 7C
), a region that corresponds to the future visual cortex (Dehay et al., 1996
; Rakic, 1976
; Kostovic and Rakic, 1984
) 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. 7E
).
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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 8A), EphA7 is expressed more extensively throughout the region of the CP (Fig. 8B
). 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. 8C
). 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 EphA6s, it is more extensive in two respects: (i) the anterior border of EphA7 expression is more rostral than EphA6s; 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 EphA6s; however, it is considerably more extensive, both along the cortex's anteroposterior axis and within the cerebral wall.
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In contrast to EphA6, whose expression becomes restricted to the neocortex, EphA7 is expressed within the striatum as well at E80 (Fig. 8C). Thus, similar to EphA3, gene expression that was restricted to particular telencephalically derived structures at E65 becomes expanded as development proceeds.
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Discussion |
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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. 2C with previous work by Bulfone and co-workers (Bulfone et al., 1995
)]. 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, 1985; Pankratz and Jackle, 1990
; Sanes, 1993
), Moreover, gradients of gene expression within the nervous system have been shown to be essential for its proper function (Simeone et al., 1992
; Drescher et al., 1995
; Yoshidaet al., 1997
; Frisen et al., 1998
) In fact initially weak gradients can be enhanced in the course of development and transformed into compartments with sharp boundaries (Gierer and Muller, 1995
).
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. 9). Interestingly, the border between these fields (see arrows in Fig. 9
) designates the boundary between visual and nonvisual cortical space.
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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, 1985, 1988
; Frantz et al., 1994
; Algan and Rakic, 1997
). 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, 1990; McConnell et al., 1994
). Furthermore, since the Eph receptor tyrosine kinases mediate cellular recognition in many developmental systems (Cheng et al., 1995
; Drescher et al., 1995
; Donoghue et al., 1996
; Wang and Anderson, 1997
), they may act similarly within the cortex (Castellani et al., 1998
; Donoghue and Rakic, 1999
). However, whether these gene products mediate repulsive interactions, as has commonly been assumed (Gale and Yancopoulos, 1998
), or attractive processes, as has been hinted at recently (Castellani and Bolz, 1997
; Castellani et al., 1998
) 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, 1988; Rubenstein et al., 1994
). 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, 1991; Arimatsu et al., 1992
; Boncinelli et al., 1993
; Cohen-Tannoudji et al., 1994
; Bulfone et al., 1995
; Na et al., 1998
; Nothias et al., 1998
; Gitton et al., 1999
; Rubenstein et al., 1999
). 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., 1993
; Eagleson et al., 1997
; Ferri and Levitt, 1993
; Kennedy and Dehay, 1993
; Polleux et al., 1997
). Furthermore, cell lineage, transplantation and selective elimination studies demonstrate significant predetermination of fates, both areally and phenotypically (Luskin et al., 1988
; McConnell and Kaznowski, 1991
; Parnavelas et al., 1991
; Algan and Rakic, 1997
; Tan et al., 1998
). Finally, within the visual system, specification occurs in the absence of mature patterns of visual activity (Horton and Hocking, 1996
; Algan and Rakic, 1997
; Meissirel et al., 1997
; Snider et al., 1998
; Wallace et al., 1997
; Khachab and Bruce, 1999
). 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, 1988
; Rakic et al., 1991
). 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, 1991; Eagleson and Levitt, 1999
; Gitton et al., 1999
). 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, 1985
; Sur et al., 1988
; Roe et al., 1990
). Likewise, regulation of specific thalamic input diminishes the size of the target areas (Rakic, 1988
; Rakic et al., 1991
). 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.
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Notes |
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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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