Role of Thalamic Axons in the Expression of H-2Z1, a Mouse Somatosensory Cortex Specific Marker

Yorick Gitton, Michel Cohen-Tannoudji1 and Marion Wassef

CNRS UMR 8542, Régionalisation Nerveuse, niveau 8, Ecole Normale Supérieure, 46 rue d'Ulm, 75230 Paris Cedex 05 and , 1 Unité de Biologie du Développement, CNRS URA 1960, Institut Pasteur, Paris, France


    Abstract
 Top
 Abstract
 Introduction
 Background
 Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
In the H-2Z1 mouse line, postnatal expression of the lacZ containing transgene in the cerebral cortex is restricted to layer IV neurons of the somatosensory area. We have used H-2Z1 embryos in previous heterotopic transplantation experiments to investigate the chronology of determination of areal identity. From the onset of neurogenesis, the cortex was regionalized in domains fated to express or not the somatosensory area-specific transgene. Determination occured 1 day later. In the present study, we show that, in vivo, H-2Z1 expression coincides with invasion of the cortical plate by thalamic afferents. We therefore investigated the role of thalamic innervation in the onset of H-2Z1 expression. For this purpose, we examined the pattern of H-2Z1 expression in perinatal cortical explant, in reeler mutant and MaoA deficient mice, or in animals which had received neonatal lesions affecting the somatosensory cortex or the thalamocortical projection. We found that, around birth, a switch occurs in the control of H-2Z1 expression: whereas H-2Z1 expression developed autonomously in embryonic parietal cortex in the absence of thalamic fibers, a transient requirement for a thalamic axon derived signal was observed postnatally. This property has interesting implications for the plasticity of cortical areas in development and evolution.


    Introduction
 Top
 Abstract
 Introduction
 Background
 Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
The mammalian neocortex is subdivided into functionally specialized areas that are anatomically distinguishable based on differences in cytoarchitecture and connections. Experimental manipulations and molecular approaches have provided evidence that areal identity is based on a set of properties which are acquired progressively during development.

Broad regional differences in gene expression and in the chronology of differentiation have been observed early in cortical development. Whether and how the early regionalization relates to the later parcellation of the cerebral cortex into distinct areas is still poorly understood. Several regional molecular markers have been described. Their expression is not, in general, confined by areal boundaries. It was shown to be specified early in cortical development (Levitt, 1984Go; Barbe and Levitt, 1991Go; Arimatsu et al., 1992Go; Ferri and Levitt, 1995Go; Cohen-Tannoudji et al., 1994Go; Levitt et al., 1997Go; Nothias et al., 1998Go; Gitton et al., 1999Go) even if the proportion of neurons expressing the regional trait is influenced by local cues (Arimatsu et al., 1999Go). Similarly, the establishment of area-specific projection profiles is a multistep process. It is influenced by locally available cues as well as by intrinsic area-specific properties. The relative importance, or at least experimental accessibility, of each step depends on the chronology of cerebral cortex development which is very different in rodent and primate. Indeed, in primate, visual areas differ in their capacity to develop early patterned projections [(reviewed by Kennedy and Dehay (Kennedy and Dehay, 1997Go)]. In rodent, layer V neurons first develop a transient exuberant efferent projection. This was interpreted as an indication that common cortical properties operate at this stage [reviewed by O'Leary and Koester, (O'Leary and Koester, 1993Go)]. The acquisition of the final area-specific pattern of efferent connections is thus delayed in rapidly developing species. The chronology of cortical development and the size of the brain in primate leaves the impression that the initial exuberant step is marginal and that it may be more related to the fine tuning of mature connections, rather than being related to the mechanisms of areal specification (Kennedy and Dehay, 1997Go). In these slowly developing species, area-specific connections tend to be established from the outset. It is probable that the relative importance of similar mechanisms varies between species. The cytoarchitectural features of sensory areas appear late (Stanfield et al., 1982Go) [reviewed by Levitt et al. (Levitt et al., 1997Go)] and are readily modified by pharmalogical or other experimental manipulations affecting afferent input (Van der Loos and Woolsley, 1973Go; Dawson and Killakey, 1987Go; Rakic et al., 1991Go; O'Leary and Stanfield, 1989Go; Killakey et al., 1989, 1994; Schlaggar and O'Leary, 1991Go; Schlaggar et al., 1993Go; Bennett-Clarke et al., 1994Go; Fox et al., 1996Go) or by mutations modifying the cross-talk between thalamic afferents and their cortical targets (Cases et al., 1995Go, 1996Go; Welker et al., 1996Go; Iwasato et al., 1997Go; Abdel-Majid et al., 1998Go).

Even if some area-specific characters are determined early, a long period, ~1 week in the mouse, is necessary for the generation of the complete set of cortical neurons. Areal identity has to be maintained and deployed during this period. The maintenance process is revealed by variations in proliferation characteristics observed between areas which result in areal differences in the timetable of laminar production of neurons contrasting with a relative homogeneity within areas (Dehay et al., 1993Go; Polleux et al., 1997Go). Signals involved in the maintenance of a given areal phenotype are in general unknown except in the case of the limbic phenotype. All cells in the limbic cortex express a specific protein called limbic system associated membrane protein (LAMP) (Levitt, 1984Go). It has been shown that specific components of the extracellular matrix are necessary to maintain limbic cortex identity during development: transforming growth factor {alpha} (TGF{alpha}) associated with collagen type IV acts as a signal and is active prior to the final cell cycle. It is capable of inducing the expression of a limbic phenotype (LAMP expression) in cortical progenitors grown in vitro (Ferri and Levitt, 1995Go; Eagleson et al., 1997Go) without interfering with proliferation (Ferri et al., 1996Go).

The somatosensory area contains a topographically organized representation of sensory receptors present on the body surface. In rodents, the somatosensory cortex layer IV neurons form discrete aggregates around bundles of thalamic axons arranged in a somatotopic pattern (Woolsey and Van der Loos, 1970Go). The body map can be directly visualized with routine neuro-anatomical procedures — either cytoarchitectonic stains or preferential labeling of thalamic fibers (histochemistry for acetylcholinesterase, cytochrome oxidase or succinate dehydrogenase, or immunodetection of the borrowed neurotransmitter serotonin). In addition, the only area-specific marker described to date, H-2Z1, is a somatosensory marker. H-2Z1 is an enhancer trap transgenic mouse line in which the lacZ reporter is specifically expressed postnatally in the somatosensory cortex where it is restricted to a subset of layer IV neurons (Cohen-Tannoudji et al. 1992Go, 1994Go).

In previous studies, we used expression of the H-2Z1 transgene as an intrinsic marker of somatosensory identity in transplantation and explantation experiments of fragments of embryonic day (E) 10.5–15.5 cortex (Gitton et al., 1999Go). We found that expression of a somatosensory-specific marker is regionalized at the onset of neuronal production (E11.5) and is no longer plastic one day later. Although the finding that cortical regionalization is determined early in development was consistent with what is known in general about regional specification in the anterior neural tube (Barbe and Levitt, 1991Go; Itasaki et al., 1991Go), it was difficult to reconcile this observation with what is known about the plasticity of the somatosensory area body representation and the observation that H-2Z1 expression follows the ingrowth of thalamocortical axons in the somatosensory area.

In the present paper we examined the role of thalamocortical interactions in H-2Z1 expression. We provide evidence that the expression of H-2Z1 by layer IV neurons, which occurs autonomously in embryonic parietal cortex fragments grown in the absence of thalamic fibers, becomes dependent on thalamic fibers shortly after birth. This dependency is expected to allow a perfect matching between the autonomously determined somatosensory area and the thalamocortical projection.


    Background
 Top
 Abstract
 Introduction
 Background
 Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
Origin of the H-2Z1 Somatosensory Marker

The H-2Z1 marker was serendipitously obtained in the course of the in vivo analysis of cis-acting regulatory sequences of two genes coding for major histocompatibility class I molecules, H-2K and ß2-microglobulin. Surprisingly, when linked to Escherischia coli lacZ coding sequences, H-2K and ß2-microglobulin regulatory sequences were not competent to drive a regulated expression of the fusion transgenes. Instead, these transgenes were highly sensitive to position effect, their expression being the reflection of promoter and/or enhancers elements present in the vicinity of their integration site. Each H-2/lacZ and ß2-microglobulin/lacZ transgenic line is characterized by a unique but reproducible pattern of lacZ expression (Cohen-Tannoudji et al., 1992Go). Altogether these transgenic lines represent a reservoir of genetically marked cell populations that has proven useful for various developmental studies (Soriano et al., 1995Go, 1997Go; Oukka et al., 1996Go).

One of the H-2/lacZ transgenic lines, H-2Z1, has been extensively studied because of its striking pattern of expression. When assayed for ß-galactosidase (ß-gal) expression, postnatal day (P) 7 cerebral cortex of H-2Z1 mice presents in the parietal region a patch of stained cells that exactly coincides with the primary somatosensory area (SI). Another group of positive cells, located in a more ventral region, corresponds to the secondary somatosensory area (SII). On whole mounted stained cortex, the parcelled organization of the SI area is outlined by the distribution of ß-gal-positive cells into small round units. This is particularly evident in the posteriomedial barrel subfield (PMBSF) region where five rows of barrrels are easily recognizable. In the somatosensory cortex, H-2Z1 expressing cells are located in layer IV. These cells are non-GABAergic interneurons with a polyhedral shape and a main apical dendrite (Gitton et al., 1999Go). In addition to these cells, a small population of ß-galpositive cells are scattered in deeper layers of all cortical regions.

Early Determination of H-2Z1 Expression In Vivo and In Vitro

In order to determine when the parietal cortex becomes engaged and determined to express H-2Z1, we compared the behaviour of fragments of occipital or parietal embryonic H-2Z1 cortex in various experimental conditions in vivo and in vitro.

In Vivo Transplantation Experiments (Cohen-Tannoudji et al., 1994Go)

Fragments of E13.5–15.5 H-2Z1 cortex were transplanted into the cerebral cortex of newborn non-transgenic hosts. After a survival time which allowed the grafts to reach the equivalent of P7 (maximum expression of the transgene in vivo), the hosts were fixed by perfusion and their brains were treated in toto for the detection of ß-gal activity. Two types of grafts were performed: parietal to occipital or occipital to parietal. More than 80% of the transplants maintained their presumptive expression of H-2Z1, i.e. parietal grafts expressed ß-gal whereas occipital grafts did not. Similar results were obtain when the embryonic H-2Z1 cortex was grafted into the postnatal cerebellum. Retrograde axonal tracing confirmed that the grafts were integrated in the host cortex. The result of the in vivo transplantation study indicated that, beginning from E13.5, fragments of parietal or occipital cortex maintain their regional characteristics and the expression of an area-specific transgene when transplanted in ectopic locations even in the absence of thalamic afferents.

In Vitro Development of Cortical Explants (Gitton et al., 1999Go)

We determined when the capacity to express H-2Z1 becomes regionalized in the cortical anlagen and tested whether it remains plastic under the influence of signals produced by embryonic cortical or pericortical structures. As early as E11.5, the parietal cortex is engaged in a somatosensory identity and expresses H-2Z1 in vitro in the absence of extracortical afferents. Expression of the transgene was partially modifiable under the influence of ectopic cortical cues at E11.5. Beginning from E12.5, expression of H-2Z1 in parietal cortex transplants or explants was not modified by ectopic cortical or basal telencephalic cues, whereas H-2Z1 was never expressed in occipital cortex transplants. These observations indicate that the capacity to express an area-specific character is acquired around E11.5.


    Materials and Methods
 Top
 Abstract
 Introduction
 Background
 Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
Animals

H-2Z1 (Cohen-Tannoudji et al., 1992Go) and ß-geo ROSA-26 (Friedrich and Soriano, 1991Go) (purchased from Jackson Laboratories, Bar Harbour, USA) transgenic embryos were recovered from crosses between heterozygous H-2Z1 transgenic males and (C57Bl/6J x CBA)F1 females (Iffa Credo, Lyon, France) and ROSA-26 homozygous males and OF-1 females (Iffa Credo) respectively.

The reelerOrl allele is maintained on a BALB/c background. As H-2Z1 somatosensory-specific expression is hardly vizualized in this genetic background (unpublished observations), the reeler mutation was progressively brought into the (C57Bl/6J x CBA)F1 genetic background of the H-2Z1 transgene by backcrossing reelerOrl animals with H-2Z1 (C57Bl/6J x CBA)F1 mice. After two or three rounds of backcross, animals were intercrossed and the brains of offspring were processed for ß-galatosidase activity either in toto or in tangential sections in combination with serotonin immunostaining (P5–P7) or cytochrome oxidase histochemistry (from P9 on). The defective cerebellum was used to detect homozygous reeler brains.

The MaoA-deficient mice resulted from the insertion of an H-2K/ interferon-ß transgene into the X-linked MaoA gene (Cases et al., 1995Go). Homozygous MaoA mutant females, maintained on a C3H or a C57Bl/6 genetic background, were mated to H-2Z1 (C57Bl/6J x CBA)F1 males. Brains of offsprings were assayed for ß-gal histochemistry.

Non-transgenic OF-1 embryos and pups were also used. The day of vaginal plug detection was considered E0.5, parturition generally occurred late on E18.5. Therefore E18.5 or E19.5 were considered P0. Newborn P0–P1 pups used for experimental manipulations were anaesthetized by cooling on ice. All other postnatal animals were anaesthetized irreversibly with an overdose of ether or chloroform before any further processing, perfusion or dissection. Embryos were recovered in chilled phosphate-buffered saline (PBS) and killed by decapitation. For histo-or immuno-cytochemistry animals were perfused through the ascending aorta with 0.12 M phosphate buffer (pH 7.2–7.4) containing 4% paraformaldehyde or 2% paraformaldehyde/0.2% glutaraldehyde. Perfusion lasted 10–15 min and the total fixation time varied between 15 and 30 min. Embryos and explants still attached on the membranes were fixed by immersion. Explants were detached from the membranes before further processing (see below).

ß-Galactosidase Histochemistry

Whole brains, explants or sections were fixed, washed in PBS + 0.1% Triton X100 (PBT) before overnight or more incubation with X-gal (4-chloro-5-bromo-3-indoyl-ß-D-galactopyranoside) at 30°C as previously described (Cohen-Tannoudji et al., 1994Go). To visualize the deepest sites of signal, some dissected cortical hemispheres were dehydrated and cleared in a 2:1 solution of benzylbenzoate/benzylalcohol (Merck).

Cytochrome Oxidase Activity

Fixed postnatal brains were cryoprotected in 30% saccharose in PBS. Sections were cut in the transversal or tangential planes at 25 or 60 µm thickness on a freezing microtome. They were incubated freely floating for 90 mn in 300 µM D+saccharose in 0.12 M phosphate buffer (pH7.2–7.4) and reaction for 2–3 h at 37°C in 300 µM D+saccharose containing 24 µM cytochrome C, 700 µM DAB (di-aminobenzidine) and catalase (500 U/ml, Sigma). The reaction was stopped by rinses in 300 µM saccharose followed by distilled water.

Immunocytochemistry

Frozen sections were rinsed and processed freely floating, and were incubated overnight in rat monoclonal anti-serotonin (1/50, Harlan-Seralab). Biotinylated anti rat IgG was used as secondary antibody (1/200, Jackson Lab) followed by streptavidin–biotin–peroxidase complex (1/400, Amersham). Peroxidase activity was detected with DAB/H2O2. After several rinses, the sections were treated for the detection of ß-gal activity.

Organotypic Cultures

All products were purchased from Gibco BRL, unless otherwise specified. Embryos were recovered from killed mice and their brains were dissected out and collected individually in Ca2+,Mg2+-free PBS containing 33 mM glucose and 50 µg/ml penicillin/streptomycin solution (PBSG). From E13.5 on, the transgenic embryos were selected among those displaying FDG-positive clusters of ß-gal activity in the pons (Cohen-Tannoudji et al., 1994Go). Meningeal tissue was removed manually from telencephalic vesicles. Washing and subsequent dissections were performed in L-15 medium supplemented with 5% heat-inactivated horse serum. Coronal slices, 400 µm thick, were obtained from embryonic brains embedded in 3% agar in PBS using a vibrating slicer, or cut freehand with a scalpel blade from perinatal telencephalic vesicles.

Thalamocortical cocultures were obtained by either of two following ways. First, four to six transversal slices through the presumptive parietal cortex were sectioned with a scalpel blade. Then, pieces of the presumptive ventrobasal thalamus were dissected and recombined with parietal explants. The donors of thalamic tissue were either OF-1 embryos or non-transgenic embryos from the same litter. Second, one or two thalamocortical slices were directly sectioned according to a plane respecting the connection between both structures, with a 55° angle from the sagittal plane (Agmon and Connors, 1991Go).

The explants were laid alone or recombined, on a 0.4 µm Biopore® membrane (Millipore, Bedford, MA, USA) floating in a 35 mm Petri dish and cultured at 37°C in a humidified atmosphere containing 5% CO2. Explants were floated on 1 ml of the following culture medium 1:1 DMEM/F12 with 2mM L-glutamine, 33 mM D-glucose, 3 mM sodium bicarbonate, 10 mM HEPES (pH 7.4) buffer, 50 UI/50 µg/ml of penicillin/ streptomycin supplemented with 10% and 5% heat-inactivated fetal calf serum (Biological Industries, Israel) and horse serum. After 24 h in vitro, the antibiotic concentration was lowered to 5 UI/5 µg/ml. Medium was changed every 2 days until the transgenic cortex reached the equivalent of P5–P7.

Cortical, Thalamic and Upper Lip Lesions

P0–P1 H-2Z1 pups were anaesthetized by hypothermia on ice. Cortical lesions were performed by aspiration or electrocoagulation. A triangular incision was made in the skin on the right side of the head and a flap of skull was opened in the vicinity of the mediolateral suture separating frontal and occipital bones. Roughly 0.5 mm3 of brain tissue was aspired with a glass pipette (0.8 mm i.d., Clark Electromedical Instruments, UK). Alternatively a pair of tungsten electrodes was inserted through two adjacent holes made in the skull of the right side. Four 40 ms pulses at 60 V were delivered using a BTX square electroporator. The skull flap was closed and the lips of the skin incision were glued with cyanoacrylate. The pups were warmed before being returned to their mothers. The brains were fixed between P5 and P9. Some P5 brains were stained in toto for ß-gal activity. Tangential 60 µm thick cryosections from transgenic cerebral hemispheres were immunoreacted for serotonin and assayed for ß-gal activity. The P9 sections were reacted first for cytochrome oxidase then for ß-gal activity.

A nichrome electrode was inserted into the left thalamus through a hole made in the skull, while a ground electrode was laid on the exposed neck tissues, as described previously (Lebrand et al., 1996Go). A 20–30 mA current was delivered for 30 s. Fixation and processing were performed at P5 and P7 and P9. The upper lip of the snout was electrically coagulated. Fixation and processing were perfomed at P7.


    Results
 Top
 Abstract
 Introduction
 Background
 Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
Development of H-2Z1 Expression

The development of H-2Z1 expression was followed in postnatal brains reacted in toto. In addition, the relative positions of H-2Z1 expressing cells and the thalamocortical somatosensory projection were examined on double-stained coronal or tangential sections. Thalamic axons were identified on the basis of their transient serotonin immunoreactivity and H-2Z1 positive cells were stained for ß-gal histochemistry.

At P2, the somatotopic body representation can already be recognized on tangential sections stained for serotonin immunoreactivity. Five rows corresponding to the vibrissae rows and additional immunoreactive clusters devoted to the representation of the snout, limbs and body were detected. In addition, a dense innervation present in the adjacent auditory cortex was serotonin immunoreactive (Lebrand et al., 1996Go). At the same stage, H-2Z1 was expressed in two convergent lines of cells in the parietal cortex (Fig. 1AGo). The first line overlay the representation of the dorsal part of the head; the other corresponded to the secondary somatosensory area where serotonin innervation was less dense (not illustrated).



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Figure 1.  Development of H-2Z1 expression. P2–P7 brains were processed in toto for X-gal histochemistry. The right cerebral hemispheres are disposed such that the ‘musculus' representations remain upward (see G for orientation: a, anterior; d, dorsal). The expression domain overlaps both primary and secondary somatosensory areas (SI and SII). The body representation develops progressively in SI, starting in the snout (s) at P2 (A), progressing posteriorly to the PMBSF where large mysticial vibrissae are represented (P3, asterisk in C), and dorsally to the lower jaw (j) domain at P4 (D). Forelimb (f) and hindlimb (h) domains then appear by P5–P6 (D,E). Although less intensely stained, the trunk territory (t) follows at P7 (F). Overall, the onset of transgene expression in both trigeminal and spinal-related territories parallels the gradient of cortical maturation, as sketched by dotted lines delineating the frontiers of the X-gal-positive cells at successive stages (P7, G). ob, olfactory bulb.

 
Between P3 and P7 individual barrels became progressively discernible by serotonin immunostaining, first in the posteromedial (PM) then in the anteromedial (AM) barrel subfields (BSF) of the somatosensory cortex. The density of H-2Z1 positive cells increased progressively in a dorso-anterior direction following the maturation gradient of the somatosensory cortex (Fig. 1BGGo). At P4 (Fig. 1 CGo) the snout and lower jaw representations were covered by a dense population of H-2Z1 positive cells which were also detected on the barrelfield in a gradient decreasing from nasal to temporal. Although the domain of H-2Z1 expression seemed to develop in a reproducible way, there was a variability of ~1 day between litters and the stages were not exactly correlated with age.

At P5–P6 (Fig. 1D,EGo) the number of blue cells was still low over the periphery of the PMBSF. The distal parts of the limb fields were detectable and a scarce population of blue cells appears in the body field.

By P7 (Fig. 1FGo) the whole body representation could be detected on X-gal-reacted brains, and the body and proximal lower limb fields were still faintly labelled.

Comparison of transverse and tangential sections double-stained for serotonin immunoreactivity and ß-gal histochemistry indicated that the blue cells formed a continuous line slightly above the bottom of the barrels (Fig. 2AGo). An additional population of H-2Z1 positive cells was distributed in the bottom and in the deep part of the barrel walls. This distribution explains why, despite the fact that H-2Z1 cells were not absent from the raphes which separate the barrels, the barrel organization is detectable in brains reacted in toto and in tangential sections (Fig. 2BGo).



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Figure 2.  Distribution of the H-2Z1-positive cells in relation to barrels. Co-localization of transgene-expressing cells and serotonin-immunoreactive thalamocortical afferents in the somatosensory cortex of a P7 H-2Z1 brain. (A) Coronal section through the PMBSF. The X-gal-positive cells (blue) form a continuous array (star). They accumulate in the barrels (arrow) here visualized with serotonin immunochemistry (brown). (B) Tangential section through the PMBSF. The pattern of blue cells depends on the radial level of the section (arrowheads in A). They are distributed more or less uniformly (stars) or accumulate around and within the brown serotonin-positive clusters at more superficial levels (arrowheads). Scale bar: 75 mm in (A), 150 mm in (B).

Figure 3.  Neonatal switch in the capacity to develop ß-gal expression in vitro. Slices were dissected from H-2Z1 parietal cortices just before and after birth and maintained in vitro. E17.5 explants developed a row of transgene expressing cells (A, arrowheads), reminiscent of layer IV-expressing cells in vivo. In contrast, P0–P2 explants (P0, B) hardly developed any detectable signal. P3–P4 (P4, C) slices maintained expression of ß-gal. P7 slices maintained their initial strong level of transgene expression for a 4 day incubation (D). Scale bar: 450 µm in (A); 220 µm in (B); 300 µm in (C); 200 µm in (D).

Figure 4.  H-2Z1 expression in reeler and MaoA mutants. One-week-old wildtype (A) reeler (A,B) or MaoA (C,D) H-2Z1 pups were analyzed for ß-gal activity. The brains in (A,B) have been cleared. In rl/rl brains (B), the expression level is weaker than in wildtype (A), and the overall shape of the domain appears shrunked dorsoventrally. The H-2Z1 expression domain lacks the limb and body representations (the arrow marks the forelimb territory). Furthermore, blue cells of deep layers origin become located superficially in rl/rl and are scattered over the whole cortical surface. (C,D) H-2Z1 expression is fairly normal in MaoA mutants. The level of ß-gal activity is somewhat lower than in wildtype. Besides the slight reshaping of the expression domain, the most salient feature of mutated brains is that the subdomains of SI are not detectable (C,D). In particular, the PMBSF lacks barrels, some of the blue cells being detectable as ill-defined rows (arrows). In the spinal domain, the limbs and trunk representations form a single continuous territory. The expression domain of older brain displays no refinement (D ). The region separating SI from SII appears filled with supernumerary transgene-expressing cells (D, arrowhead).

 
The data show that the expression of the H-2Z1 somato-sensory marker by cortical neurons lags behind the development of serotonin-positive thalamic terminals. At P2, as serotonin already spans the whole S1 area, the AMSBF is the sole domain to be consistently covered by blue cells (notwithstanding the SII area). At P3–P4, while serotonin material distribution refines, the expression of the transgene heads caudally, with the front wave reaching the caudalmost barrels. From P5 on, while the serotonin-labelled axon terminals acquire their mature distribution, transgene expression develops toward dorsomedial territories, catching up with the fibre distribution.

Loss of the Autonomous Control of H-2Z1 Expression in Late Embryonic/Early Postnatal Cortical Explants

In previous experiments (Gitton et al., 1999Go), we observed that early (E12.5–16.5) cortical explants developed a regionalized expression of H-2Z1 when cultured in vitro for 9–13 days, further demonstrating the existence of an early autonomous component in areal specification. Similarly, most E17.5 parietal explants expressed H-2Z1 in vitro (Fig. 3AGo; see Table 1Go). In contrast, late embryonic H-2Z1 explants (E18.5) showed a greater variability in their capacity to develop H-2Z1 expression in vitro. Moreover, none of 41 slices of newborn (P0–P1) and P2 parietal cortex maintained for 5–7 days in vitro expressed ß-gal (Fig. 3BGo). At later stages, after the onset of H-2Z1 expression, the density of H-2Z1 positive cells is maintained in vitro but the density of expressing cells does not increase (Fig. 3C,DGo). A scarce population of blue cells was detected in parietal slices dissected at P3 and maintained for 4 days in vitro; the density of blue cells was increased in P4 (Fig. 3CGo) but the staining was weaker than that of the contralateral cortex fixed at the time of explantation (not shown) and did not reach the level of embryonic-derived explants (Fig. 3AGo). P7 explants (Fig. 3DGo) maintained a high level of ß-gal expression in vitro for 4 days.


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Table 1 ß-Galactosidase activity in late H-2Z1 parietal explants
 
Because the failure to develop H-2Z1 expression in vitro parallels the differentiation of thalamocortical axons terminal arbors, we wondered whether thalamic axons could be required for the maturation or survival of H-2Z1 expressing cells. Several types of thalamus/cortex coculture were investigated. Because the survival of late thalamic explants was poor, thalamic explants were dissected from the same stage as cortical explants (E16.5–P0) or from younger stages (E13.5 or 14.5 thalamus with E18.5–P0 cortex). Thalamic explants seemed to provide a trophic support to cocultured cortical explants as judged by their increased volume and uniform aspect compared to controls (not shown). Coculture of E16.5, E17.5 H-2Z1 cortex with a thalamic explant increased the intensity of ß-gal staining regardless of the presence of thalamocortical connections. When thalamic explants were removed from E16.5–17.5 thalamus/ cortex cocultures at a stage corresponding to P1, the expression of ß-gal in H-2Z1 cortical explants was not markedly modified compared to undisturbed cocultures. In contrast, coculture with thalamus at various stages did not increase the proportion of E18.5 cortical explants expressing ß-gal nor did it induce ß-gal expression in P0–P1 explants.

As we were unable to obtain H-2Z1 expression in late cortical explants cocultured with thalamic explants, the in vitro culture sytem used was not suitable to dissect the mechanisms underlying the observed change in cortical differentiation which occurs around birth. Therefore, we turned to the in vivo analysis of the pattern of H-2Z1 expression in mice where the cross-talk between thalamic fibers and layer IV neurons was modified by genetic or experimental manipulations.

In Vivo Experiments Aimed at Interfering with Thalamocortical Interactions

We chose two mutations: reeler and monoamine oxidase A (MaoA)-deficient mice. In reeler, the abnormal positioning of layer IV neurons could alter the timing and efficiency of their interactions with thalamic axons. In MaoA–/– mutants the function of thalamic axons is impaired due to an excess of extracellular serotonin. We also disturbed the thalamocortical interplay by using different kinds of lesions. First, upper-lip coagulation was performed at birth in order to prevent the patterned remodelling of the PMBSF by thalamic afferents (Van der Loos and Woolsley, 1973Go). Second, we changed the balance between thalamic fibers and their cortical targets by making lesions either in the somatosensory cortical territory or in the ventrobasal thalamus.

Reeler mutants

The Orleans reeler mouse mutant resulted from a spontaneous mutation caused by the insertion of an L1 sequence in the coding region of the reelin gene (Takahara et al., 1996Go). The reelerOrl allele is maintained on a BALB/c background. The BALB/c genetic background modified the expression of H-2Z1: although the outline of the layer IV somatosensory-specific H-2Z1 labeling was maintained, the number of deep layer non-somatosensory neurons expressing lacZ was markedly increased. This cumbersome effect on H-2Z1 expression was progressively eliminated by backcrossing reelerOrl animals with H-2Z1 mice. After several rounds of backcross, animals were intercrossed and the brains of offspring were processed for ß-gal activity either in toto or on tangential sections in combination with serotonin immunostaining (P5–P7) or cytochrome oxidase histochemistry (from P9 on). At P5, the brains of rl/rl mutant were consistently smaller than those of wildtype littermates. The relative surface of the somatosensory domain was maintained in the mutant brain but the overall density of blue cells in the somatosensory area was always lower than in wildtype (Fig. 4A,BGo). Some isolated H-2Z1 blue cells were scattered over the whole cortical surface and probably corresponded to layer V–VI ß-gal-positive cells. The barrels were extended in the radial dimension and their diameters were reduced. The head or ‘trigeminal' domain appeared normal in P5–P7 reeler brains. However, the ‘spinal' domain of H-2Z1 expression was missing, in particular the anterior limb representation which is always well detectable in P7 wildtype. The possibility that these cells were not detected on whole mounts due to their deeper position was ruled out by clearing the reeler and wildtype brains.

MaoA mutants

MaoA-deficient mice resulted from the integration of an interferon-ß transgene into the MaoA gene (Cases et al., 1996Go). P7 pups were obtained from crosses between MaoA–/– females maintained on C3H or C57BL/6 genetic backgrounds and H-2Z1 males. The MaoA gene is located on chromosome X. Therefore, all males derived from these crosses inherited a MaoA mutated allele from their mother and were deficient in MaoA enzymatic activity; their female littermates were heterozygous and phenotypically normal. Except for a few animals in which the somatosensory pattern was obscured by expression of the H-2Z1 transgene in deep cortical layers, the outline and intensity of H-2Z1 expression seemed slightly delayed and was similar in males and females. The H-2Z1 domain was uniform in males (Fig. 4C,DGo) even though no barrel organization could be detected. It was clear that the MaoA mutation did not interfere with the capacity to develop H-2Z1 expression.

Upper Lip Coagulation

Upper lip coagulations were performed unilaterally on newborn mice in order to disrupt the thalamocortical organization. H-2Z1 expression was examined at P7 on brains reacted in toto with X-gal. As expected, the barrel organization was disrupted in the PMBSF of the contralateral cortex (Fig. 5AGo). Nevertheless the intensity of X-gal staining was identical in the experimental and control hemispheres (Fig. 5AGo).



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Figure 5.  Lesion-induced changes in H-2Z1 expression pattern. The affected (left) and control (right) hemispheres were dissected from P7 brains reacted in toto with X-gal. d, dorsal; v, ventral. (A) Upper lip coagulation at birth: the intensity of ß-gal staining is similar on both hemispheres. The PMBSF (star) contralateral to the lesion lacks row or barrel-like organization and appears as a uniform array of blue cells. The spinal domain is unaffected. (B) aspiration of cortical tissue at birth yields, besides a normal SII, a P3–P4 like domain of transgene expression. Blue cells were restricted to part of the snout representation and to the dorsal head representation (arrowhead, compare with P3–P4 Fig. 1B,CGo). (C) Tangential section through the lesioned parietal cortex of another mouse, immunoreacted for serotonin. In this as well as in all other sections of the same brain, the ß-gal-expressing cells were absent from the spinal domain (below the dotted line, arrow). In (B,C) the open arrowheads indicate the lesion sites, the dotted lines mark the limit of the H-2Z1 domain spared by the ablation. (D) Electrolytic lesion of the thalamus at birth wipes out most of blue cells in SI ipsilateral cortex (star), only sparing a variable fraction of dorsal SI and of SII positive cells (arrowhead). Scale bar: 200 mm in (C).

 
Parietal Cortex Lesions

Ablations of parietal cortex tissue were performed at P0–P1 by aspiration of a small volume of cortical tissue. The control and lesioned hemispheres were compared at P5, P7 and P9. When reacted in toto with X-gal, the brains displayed the same intensity of transgene expression on both sides but parts of the H-2Z1 body representation were always missing (Fig. 5BGo). In particular, the spinal part of the representation was absent on the operated side. The head representation was shifted dorsally and was truncated: only the most temporal barrels — the first to develop normally — were represented. The relative positions of the thalamocortical projection field and the blue H-2Z1 domain were reconstructed from double-stained tangential sections treated for the detection of thalamic fibers (by serotonin immunostaining or cytochrome oxidase histochemistry) and of H-2Z1 expressing cells (by X-gal staining ) (Fig 5CGo). As already reported (Ito and Seo, 1983Go), thalamic axon body representation was relatively unaffected, except for occasional distorsions around the scar (open arrowhead in Fig. 5B,CGo). The H-2Z1 expressing cells were always found in the thalamic somatosensory domain overlying a cluster of thalamic terminals. However, they did not always belong to the expected part of the body representation. When part of the blue body representation was missing, there was a bias towards the accumulation of H-2Z1 positive cells over the early maturing parts of the somatosensory cortex which could not be accounted for simply by the location of the lesions. Conversely, although the thalamic fiber representations of the body or lower jaw were easily distinguished; these late developing thalamic projections were in general devoid of associated blue cells.

Neonatal Thalamic Lesions Prevented H-2Z1 Expression

Lesions of the ventrobasal thalamus were produced unilaterally by electrocoagulation in P0–P1 H-2Z1 pups. The pups were killed at P6–P7 and their brains were processed in toto for ß-gal histochemistry. A large domain where H-2Z1 expression was missing was observed in the ipsilateral parietal cortex (n = 3; Fig. 5DGo). The remaining blue cells adopted a normal arrangement as compared to the contralateral side. The observed decrease in H-2Z1 expression was not a direct effect of the lesions. In some cases, the lesions were performed too laterally; they spared the thalamus but were placed directly in the parietal cortex. In these cases ß-gal expression in the parietal cortex almost abuts the limit of the lesion. These experiments indicate that, in vivo, thalamic axons are necessary for the onset of H-2Z1 expression.

In summary, the data suggest that H-2Z1 expression is controlled by both early intrinsic and late thalamocortical dependent influences.


    Discussion
 Top
 Abstract
 Introduction
 Background
 Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
Transplantation and explantation experiments have demonstrated that H-2Z1 is expressed autonomously in parietal cortex explants dissected before any contact occurred with thalamic axons (Cohen-Tannoudji et al., 1994Go; Gitton et al., 1999Go). In the present study we provide evidence that, around birth, thalamocortical afferents are required for H-2Z1 expression in the somatosensory cortex. We show that the thalamic signals involved in the development of the somatosensory cortex barrel organization are not necessary for H-2Z1 expression. In addition, we find that somatosensory thalamic axons can reorganize outside the somatosensory cortex after cortical ablation at birth but that the competence to express H-2Z1 is limited to a restricted parietal domain of the cortical primordium.

Evidence for a Perinatal Switch in the Control of H-2Z1 Expression

Whereas H-2Z1 expression developed readily in explants dissected from embryonic parietal cortex and maintained in culture in the absence of extracortical afferents, it failed to start in explants dissected perinatally. Several observations point to a switch in the control of H-2Z1 expression and a transient dependence on thalamic axons. In vivo, the progressive development of H-2Z1 expression follows the ingrowth of thalamic afferents in layer IV (Agmon et al., 1993Go; Molnar et al., 1998Go). In reeler and MaoA mutants as well as in lesioned cortex, H-2Z1 expression correlates with the presence of thalamic axon terminals. More decisively, removal of thalamic afferents by neonatal electrocoagulation of the ventrobasal thalamus prevents H-2Z1 expression. These observations indicate that the neurons which normally express the H-2Z1 transgene switch to a differentiation stage where they require the presence of thalamic axons to trigger H-2Z1 expression. A higher number of layer IV neurons express H-2Z1 in cortical explants dissected at P7 compared to those explanted at P4, suggesting that once H-2Z1 expression starts, thalamic axons are not required for its maintenance.

The cortical ablation experiments were intended to show whether thalamic fibers can recruit supernumerary H-2Z1 expressing cells when the size of the somatosensory area is reduced. At birth, thalamic fibers were unable to induce H-2Z1 expression in neighbouring neurons in order to compensate for the ablated H-2Z1 neurons. This suggests that the putative somatosensory cortex is well delimited: in normal situations, all the cells which are competent to express H-2Z1 are gathered into the somatosensory area. On the other hand, the blue cells spared by the ablations in general meet an early fate in the somatosensory representation, suggesting a mechanism of first come, first served and a lack of intrinsic internal regionalization in the somatosensory area. This is consistent with the previously observed plasticity of the body representation (Van der Loos and Woolsley, 1973Go; Killakey et al., 1989 Killakey et al., 1994; Shlaggar and O'Leary, 1991).

It is striking that the development of H-2Z1 expression in embryonic explants bypasses the requirement for signals from the thalamic axon. The abrupt change in the developmental properties of the cortex observed around birth could be elicited by earlier signals provided by thalamocortical axons when they invade the cortex starting at E16.5 (Agmon et al., 1993Go; Molnar et al., 1998Go) or, alternatively, by hormonal modifications related to parturition. The perinatal signal, whatever its origin may be, places the final expression of an early autonomous regionalization process under the control of peripheral afferents.

Do Thalamic Axons Control the Survival or Differentiation of H-2Z1 Neurons?

Whether thalamic axons are necessary for neuronal survival or merely for H-2Z1 expression is still unclear. It would not seem unlikely that layer IV neurons undergo programmed cell death when their main afferents are eliminated at the onset of synaptogenesis. Nevertheless, the available data point to a dependence of layer IV neurons on thalamic afferents to differentiate specific features like the characteristic distribution of GABAA receptor subunits (Paysan et al., 1997Go) but not for survival. Indeed, layer II/III neurons rather than layer IV die by apoptosis when NMDA receptors are blocked transiently between P3 and P14 in the rat (Ikonomidou et al., 1999Go) or when the thalamus is ablated by neonatal lesions in rat or hamster (Wise and Jones, 1978Go; Windrem and Finlay, 1991Go). The intriguing possibility that layer IV neurons could later change fate when deprived of thalamic afferents neonatally, has been proposed (Windrem and Finlay, 1991Go) to explain the discrepancy between the layers affected by cell death soon after thalamic lesions and those surviving in adult animals. Although layer IV neurons seem to survive neonatal thalamic lesions in other rodent species, our observations in mouse and those of Paysan et al. (Paysan et al., 1997Go) in rat indicate that they fail to express some characteristic features when deafferented. Previously unsuspected, this late control allows a precise matching between the body representation conveyed by somatosensory afferents and a somatosensory area expressing distinct physiological properties. Better than adjustment by cell death, the modulation of target gene expression by afferents allows for both developmental and evolutionary plasticity.

Different Thalamic Signals Control Barrel Organization and H-2Z1 Expression in the Somatosensory Cortex

We found no correlation between H-2Z1 expression in the somatosensory cortex and the development of barrel organization. Lesion of the periphery (upper lip electrocoagulation) which interfered with thalamic organization or excessive amounts of extracellular serotonin thought to mediate strong presynaptic inhibitory effects upon thalamocortical transmission in MaoA mutants (Cases et al., 1996Go) [see, however, Rhoades et al. (Rhoades et al., 1998Go)] prevented the formation of barrels but had no effect on the extent of H-2Z1 expression. This indicated that, if thalamic axons are indeed involved in setting up H-2Z1 expression, this area-specific function is distinct from their more general cytoarchitectural capacity to orient layer IV neuron dendrites (Schlaggar and O'Leary, 1993). In this respect, it is perhaps interesting to note that the H-2Z1 positive neurons are not oriented towards the core of the barrels. Their main dendrite can be outlined in fresh sections treated with the soluble ß-gal substrate FDG and is oriented radially, extending towards the pial surface (Gitton et al., 1999Go).

The density of H-2Z1 expressing cells in brains reacted in toto with X-gal was consistently decreased in reeler mutants, compared to their wildtype littermates. The reeler phenotype is classically described as an inversion of cortical layers preserving the specificity of their afferent and efferent connections. The most relevant aspect of the reeler phenotype, as concerns H-2Z1 expression, was probably that distinct layers did not form properly in the mutant. Cells destined to layer IV and their afferent axon terminals were scattered in the radial dimension as observed by X-gal staining or by serotonin immunocytochemistry. Even if the exact mechanism remains obscure, it is likely that short-range cell–cell interactions, possibly modulated by thalamic axons, are involved in the development of the competence of layer IV neurons to express H-2Z1. The disruption of tissue architecture in reeler mutants could affect the efficiency of interactions between cortical neurons resulting in a decrease in the number of H-2Z1 expressing cells.


    Conclusion
 Top
 Abstract
 Introduction
 Background
 Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
It has been determined that the parietal cortex early on expresses H-2Z1 — a somatosensory-specific marker expressed postnatally. We show here that, around birth, the cell interactions which govern the autonomous expression of H-2Z1 in layer IV are modified, bringing H-2Z1 expression under the control of thalamic afferents. Thus, whereas region-specific cell–cell interactions produce neurons which are competent to express area-specific traits, their actual expression in vivo depends on late thalamocortical interactions. This results in the precise alignment of the domain expressing ‘somatosensory cortex properties' (i.e. H-2Z1 positive) with the thalamocortical projection field. This plasticity also opens the possibility of forming new cortical domains during development or evolution (Rakic, 1988Go, Rakic et al. , 1991Go; Dehay et al., 1996Go).


    Notes
 
The authors thank Isabelle Seif for providing MaoA-deficient mice, Cecile Lebrand for her demonstration of thalamic electrocoagulation, Joelle Adrien for lending the electrocoagulator and Henry Kennedy for critical reading of the manuscript. We aknowledge the skilful technical assistance of Rosette Goiame.This work was supported by grants from the EC (ERB BIO 4CT960146) and HFSP (RG83/96) to M.W.

Address correspondence to Marion Wassef, Régionalisation Nerveuse, CNRS UMR 8542, niveau 8, Ecole Normale Supérieure, 46 rue d'Ulm, 75230 Paris Cedex 05, France. Email wassef{at}wotan.ens.fr.


    References
 Top
 Abstract
 Introduction
 Background
 Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
Abdel-Majid RM, Leong WL, Schalkwyk LC, Smallman DS, Wong ST, Storm DR, Fine A, Dobson MJ, Guernsey DL, Neumann PE (1998) Loss of adenylyl cyclase I activity disrupts patterning of mouse somatosensory cortex. Nat Genet 19:289–291.[ISI][Medline]

Agmon A, Connors BW (1991) Thalamocortical responses of mouse somatosensory (barrel) corex in vitro. Neuroscience 41:365–379.[ISI][Medline]

Agmon A, Yang LT, O'Dowd DK, Jones EG (1993) Organized growth of thalamocortical axons from the deep tier of terminations into layer IV of developing mouse barrel cortex. J Neurosci 13:5365–5382.[Abstract]

Arimatsu Y, Miyamoto M, Nihonmatsu I, Hirata K, Uratani Y, Hatanaka Y, Takiguchi-Hayashi K (1992) Early regional specification for a molecular neuronal phenotype in the rat neocortex. Proc Natl Acad Sci USA 89:8879–8883.[Abstract]

Arimatsu Y, Ishida M, Takiguchi-Hayashi K, Uratani Y (1999) Cerebral cortical specification by early potential restriction of progenitor cells and later phenotype control of postmitotic neurons. Development 126:629–638.[Abstract/Free Full Text]

Barbe MF, Levitt P (1991) The early commitment of fetal neurons to the limbic cortex. J Neurosci 11:519–533.[Abstract]

Bennett-Clarke CA, Leslie MJ, Lane RD, Rhoades RW (1994) Effect of serotonin depletion on vibrissa-related patterns of thalamic afferents in the rat's somatosensory cortex. J Neurosci 14:7594–7607.[Abstract]

Cases O, Seif I, Grimsby J, Gaspar P, Chen K, Pournin S, Muller U, Aguet M, Babinet C, Shih JC, DeMaeyer E (1995) Aggressive behavior and altered amounts of brain serotonin and norepinephrine in mice lacking MAOA. Science 268:1763–1766.[ISI][Medline]

Cases O, Vitalis T, Seif I, De Maeyer E, Sotelo C, Gaspar P (1996) Lack of barrels in the somatosensory cortex of monoamine oxidase A-deficient mice: role of a serotonin excess during the critical period. Neuron 16:297–307.[ISI][Medline]

Cohen-Tannoudji M, Morello D, Babinet C (1992) Unexpected position-dependent expression of H-2 and beta2-microglobulin/lacZ transgenes. Mol Reprod Dev 33:149–159.[ISI][Medline]

Cohen-Tannoudji M, Babinet C, Wassef M (1994) Early determination of a mouse somatosensory marker. Nature 368:460–463.[ISI][Medline]

Dawson DR, Killakey HP (1987) The organization and mutability of the forepaw and hindpaw representations in the somatosensory cortex of the neonatal rat. J Comp Neurol 256:246–256.[ISI][Medline]

Dehay C, Kennedy H, Bullier J, Berland M (1988) Absence of interhemispheric connections of area 17 during develoment in monkey. Nature 331:733–747.

Dehay C, Horsburgh G, Berland M, Killackey H, Kennedy H (1989) Maturation and connectivity of the visual cortex in monkey is altered by prenatal removal of retinal input. Nature 337:265–267.[ISI][Medline]

Dehay C, Giroud P, Berland M, Smart I, Kennedy H (1993) Modulation of the cell cycle contributes to the parcellation of the primate visual cortex. Nature 366:464–466.[ISI][Medline]

Dehay C, Giroud P, Berland M, Killackey H, Kennedy H (1996) Contribution of thalamic input to the specification of cytoarchitectonic cortical fields in the primate: effects of bilateral enucleation in the fetal monkey on the boundaries, dimensions, and gyrification of striate and extrastriate cortex. J Comp Neurol 367:70–89.[ISI][Medline]

Eagleson KL, Lillien L, Chan AV, Levitt P (1997) Mechanisms specifying area fate in cortex include cell-cycle-dependent decisions and the capacity of progenitors to express phenotype memory. Development 124:1623–1630.[Abstract/Free Full Text]

Ferri RT, Levitt P (1995) Regulation of regional differences in the differentiation of cerebral cortical neurons by EGF family–matrix interactions. Development 121:1151–1160.[Abstract/Free Full Text]

Ferri RT, Eagleson KL, Levitt P (1996) Environmental signals influence expression of a cortical areal phenotype in vitro independent of effects on progenitor cell proliferation. Dev Biol 175:184–190.[ISI][Medline]

Fox K, Schlaggar BL, Glazewski S, O'Leary DD (1996) Glutamate receptor blockade at cortical synapses disrupts development of thalamocortical and columnar organization in somatosensory cortex. Proc Natl Acad Sci USA 93:5584–5589.[Abstract/Free Full Text]

Friedrich G, Soriano P (1991) Promoter traps in embryonic stem cells: a genetic screen to identify and mutate developmental genes in mice. Genes Dev 5:1513–1523.[Abstract]

Gitton Y, Cohen-Tannoudji M, Wassef M (1999) Specification of somatosensory area identity in cortical explants. J Neurosci 19:4889–4898.[Abstract/Free Full Text]

Itasaki N, Ichijo H, Hama C, Matsuno T, Nakamura H (1991) Establishment of rostrocaudal polarity in tectal primordium: engrailed expression and subsequent tectal polarity. Development 113: 1133–1144.[Abstract]

Ito M, Seo ML (1983) Avoidance of neonatal cortical lesions by developing somatosensory barrels. Nature 301:600–602.[ISI][Medline]

Ikonomidou C, Bosch F, Miksa M, Bittigau P, Vockler J, Dikranian K, Tenkova TI, Stefovska V, Turski L, Olney JW (1999) Blockade of NMDA receptors and apoptotic neurodegeneration in the developing brain. Science 283:70–74.[Abstract/Free Full Text]

Iwasato T, Erzurumlu RS, Huerta PT, Chen DF, Sasaoka T, Ulupinar E, Tonegawa S (1997) NMDA receptor-dependent refinement of somatotopic maps. Neuron 19:1201–1210.[ISI][Medline]

Kennedy H, Dehay C(1997) The nature and nurture of cortical development. In: Normal and abnormal develoment of cortex (Galaburda AM and Christen Y, eds), pp. 25–56. New York: Springer-Verlag.

Killackey HP, Dawson DR (1989) Expansion of the central hindpaw representation following fetal forelimb removal in the rat. Eur J Neurosci 1:210–221.[ISI][Medline]

Killackey HP, Chiaia NL, Bennett-Clarke CA, Eck M, Rhoades RW (1994) Peripheral influences on the size and organization of somatotopic representations in the fetal rat cortex. J Neurosci 14:1496–1506.[Abstract]

Lebrand C, Cases O, Adelbrecht C, Doye A, Alvarez C, El Mestikawy S, Seif I, Gaspar P (1996) Transient uptake and storage of serotonin in developing thalamic neurons. Neuron 17:823–835.[ISI][Medline]

Levitt P (1984) A monoclonal antibody to limbic system neurons. Science 233:229–301.

Levitt P, Barbe MF, Eagleson KL (1997) Patterning and specification of the cerebral cortex. Annu Rev Neurosci 20:1–24.[ISI][Medline]

Molnar Z, Adams R, Goffinet AM, Blakemore C (1998) The role of the first postmitotic cortical cells in the development of thalamocortical innervation in the reeler mouse. J Neurosci 18:5746–5765.[Abstract/Free Full Text]

Nothias F, Fishell G, Ruiz i Altaba A (1998) Cooperation of intrinsic and extrinsic signals in the elaboration of regional identity in the posterior cerebral cortex. Curr Biol 8:459–462.[ISI][Medline]

O'Leary DD, Stanfield BB (1989) Selective elimination of axons extended by developing cortical neurons is dependent on regional locale: experiments utilizing fetal cortical transplants. J Neurosci 9: 2230–2246.[Abstract]

O'Leary DD, Koester SE (1993) Development of projection neuron types, axon pathways, and patterned connections of the mammalian cortex. Neuron 10:991–1006.[ISI][Medline]

Oukka M, Colucci-Guyon E, Tran PL, Cohen-Tannoudji M, Babinet C, Lotteau V, Kosmatopoulos K (1996) CD4 T cell tolerance to nuclear proteins induced by medullary thymic epithelium. Immunity 4:545–553.[ISI][Medline]

Paysan J, Kossel A, Bolz J, Fritschy JM (1997) Area-specific regulation of gamma-aminobutyric acid type A receptor subtypes by thalamic afferents in developing rat neocortex. Proc Natl Acad Sci USA 94:6995–7000.[Abstract/Free Full Text]

Polleux F, Dehay C, Moraillon B, Kennedy H (1997) Regulation of neuroblast cell-cycle kinetics plays a crucial role in the generation of unique features of neocortical areas. J Neurosci 17:7763–7783.[Abstract/Free Full Text]

Rakic P (1988) Specification of cortical areas. Science 241:170–176.[ISI][Medline]

Rakic P, Suner I, Williams RW (1991) A novel cytoarchitectonic area induced experimentally within the primate visual cortex. Proc Natl Acad Sci USA 88:2083–2087.[Abstract]

Rhoades RW, Chiaia NL, Lane RD, Bennett-Clarke CA (1998) Effect of activity blockade on changes in vibrissae-related patterns in the rat's primary somatosensory cortex induced by serotonin depletion. J Comp Neurol 402:276–283.[ISI][Medline]

Schlaggar BL, O'Leary DD (1991) Potential of visual cortex to develop an array of functional units unique to somatosensory cortex. Science 252:1556–1560.[ISI][Medline]

Schlaggar BL, Fox K, O'Leary DD (1993) Postsynaptic control of plasticity in developing somatosensory cortex. Nature 364:623–626.[ISI][Medline]

Schlaggar BL, O'Leary DD (1994) Early development of the somatotopic map and barrel patterning in rat somatosensory cortex. J Comp Neurol 346:80–96.[ISI][Medline]

Soriano E, Dumesnil N, Auladell C, Cohen-Tannoudji M, Sotelo C (1995) Molecular heterogeneity of progenitors and radial migration in the developing cerebral cortex revealed by transgene expression. Proc Natl Acad Sci USA 92:11676–11680.[Abstract]

Soriano E, Alvarado-Mallart RM, Dumesnil N, Del Rio JA, Sotelo C (1997) Cajal–Retzius cells regulate the radial glia phenotype in the adult and developing cerebellum and alter granule cell migration. Neuron 18:563–577.[ISI][Medline]

Stanfield BB, O'Leary DD, Fricks C (1982) Selective collateral elimination in early postnatal development restricts cortical distribution of rat pyramidal tract neurones. Nature 298:371–373.[ISI][Medline]

Takahara T, Ohsumi T, Kuromitsu J, Shibata K, Sasaki N, Okazaki Y, Shibata H, Sato S, Yoshiki A, Kusakabe M, Muramatsu M, Ueki M, Okuda K, Hayashizaki Y (1996) Dysfunction of the Orleans reeler gene arising from exon skipping due to transposition of a full-length copy of an active L1 sequence into the skipped exon. Hum Mol Genet 5:989–993.[Abstract/Free Full Text]

Van der Loos H, Woolsley TA (1973) Somatosensory cortex: structural alterations following early injuries to sense organs. Science 179: 395–398.[ISI][Medline]

Welker E, Armstrong-James M, Bronchti G, Ourednik W, Gheorghita-Baechler F, Dubois R, Guernsey DL, Van der Loos H, Neumann PE (1996) Altered sensory processing in the somatosensory cortex of the mouse mutant barrelless. Science 271:1864–1867.[Abstract]

Windrem MS, Finlay BL (1991) Thalamic ablations and neocortical development: alterations of cortical cytoarchitecture and cell number. Cereb Cortex 1:230–240.[Abstract]

Wise SP, Jones EG (1978) Developmental studies of thalamocortical and commissural connections in the rat somatic sensory cortex. J Comp Neurol 178:187–208.[ISI][Medline]

Woolsey TA, Van der Loos H (1970) The structural organization of layer IV in the somatosensory region (SI) of mouse cerebral cortex. The description of a cortical field composed of discrete cytoarchitectonic units. Brain Res 17:205–242.[ISI][Medline]