Department of Animal and Plant Cell Biology, Faculty of Biology, University of Barcelona, Diagonal 645, Barcelona 08028, Spain
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Abstract |
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Introduction |
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Although tangential migration in the intermediate and subventricular zones is likely to occur through axon fascicles (O'Rourke et al., 1992, 1995
), in the developing cerebral cortex migration of postmitotic neurons from the ventricular zone to layer I mainly takes place via a specialized class of cell, the bipolar radial glia (Rakic, 1972
; Hatten, 1990
; Misson, 1991
; Misson et al., 1991
). Thus, a central issue in corticogenesis concerns the mechanisms involved in the regulation of radial glia cell identity and in the organization of their three-dimensional scaffold, with their endfeet terminating in layer I. Experiments in vitro have shown that the identity of radial glia is regulated by diffusible signals present in forebrain differentiating cells, although the source of these signals has not been determined (Hunter and Hatten, 1995a
,b
). Another crucial issue for cortical histogenesis is the mechanisms that control the directional movement of migrating neurons from the ventricular zone to layer I, which are responsible for the generation of the typical laminar arrangement of neurons in the cerebral cortex. While some specific surface proteins expressed by either radial glia or migrating neurons, such as astrotactin and the D4 epitopes (Fishell and Hatten, 1991
; Cameron and Rakic, 1994
; Anton et al., 1996
; Zheng et al., 1996
), play a role in the gliding of migrating neurons over the glial surface, no explanation has been given for what might attract migrating neurons to layer I. Recent data on the development of nematodes (Wadsworth et al., 1995
), the murine brain stem (Serafini et al., 1996
; Block-Gallego et al., 1998; Yee et al., 1999
; Alcántara et al., 2000
), and the cerebral cortex and rostral migratory pathway to the olfactory bulb (Hu and Rutishauser, 1996
; Hu et al., 1999; Wu et al., 1999
) indicate that directional migration may rely on chemoattractive and chemorepellent signals, similar to those used in the guidance of developing axons. Since migrating cells in dissociated cell cultures appear to move randomly along radial glia (Edmunson and Hatten, 1987; Hatten, 1990
), it is likely that directional migration towards layer I in vivo relies on specific cues intrinsic to the cortex.
The cells of CajalRetzius (CR) in layer I are generated slightly earlier than the subplate cells, and they are the first neurons to differentiate in the developing cortex (Marín-Padilla, 1971, 1972
, 1984
, 1988
; Edmunds and Parnavelas, 1982
; Parnavelas and Edmunds, 1983
; Derer, 1985
; Derer and Derer, 1990
, 1992
; Del Río et al., 1995
, 1997
). Most likely, these early neurons are transient and disappear by cell death at the end of migration (Derer and Derer, 1990
; Del Río et al., 1995
, 1996
). For many years the function of these pioneer neurons has remained unknown. The mouse mutant reeler shows abnormal migration and positioning of migrating neurons in the cerebral cortex and other brain areas (Caviness and Sidman, 1973
; Caviness, 1982
; Goffinet, 1980
, 1992
; Derer and Nakanishi, 1983
). Studies showing that reelin the gene disrupted in the reeler mutation is expressed mainly by CR cells and that inhibition of its encoded protein disrupts histotypic reaggregation cultures, have indicated a role for CR cells in migration (D'Arcangelo et al., 1995
; Hirotsune et al., 1995
; Ogawa et al., 1995
; Alcántara et al., 1998
). Moreover, recent data have identified a number of Reelin receptors and associated signal transduccion proteins (Howell et al., 1997
; Sheldon et al., 1997
; Hiesberger et al., 1999
; D'Arcangelo et al., 1999
; Senzaki et al., 1999
). However, the mechanisms by which the lack of Reelin and CR cell disfunction might lead to abnormal cortical migration are not understood. For instance, since in the reeler mutant mouse the preplate does not split appropriately into a true layer I and the subplate, it has been proposed that migratory deficits in reeler mice may result from the abnormal patterning of these pioneer neurons, which prevents the normal settling of cortical plate neurons within the preplate (Goffinet, 1992
; D'Arcangelo et al., 1997
; Hoffarth et al., 1995
). To gain more information on the developmental functions of CR cells in vivo we have selectively ablated this neuronal population in newborn mice, a developmental stage which is long after the splitting and differentiation of preplate neurons. We show that ablation of CR cells in newborn mice disrupts neuronal migration of late-generated neurons destined to layers IIIII. Furthermore, we show that the ablation of CR cells accelerates the transformation of radial glial cells into mature astrocytes, suggesting a role for CR cells in the regulation of the identity and function of radial glia.
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Materials and Methods |
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Newborn (P0) mice (n = 67) of the NMRI strain (Iffa Credo, Lyon, France) were anesthetized by hypothermia and ether and fixed to a stereotaxic frame. The skull overlying the right parietal/occipital cortex was carefully removed without damaging the meninges or main blood vessels. Then, a vibratome slice of solid 3% agar (400 µm thick) was soaked in the -amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate receptor (AMPA/KA-R) agonist domoic acid (DA; 2 mM in 0.1 M phosphate buffer; Sigma, Poole Dorset, UK). The agar slice was then trimmed and held directly in contact with the exposed cortical surface for 1 min. The agar slice was carefully removed and the brain surface was rinsed several times with saline solution. Thereafter, the skull was repositioned, the skin was sutured, and the animals were returned to their mothers. Newborn mice from the same litters were either unoperated (n = 24) or operated as described above, but with agar slices soaked in vehicle solution lacking domoic acid (n = 16; sham-controls). Other newborn mice (n = 8) were injected intracortically with 0.20.5 µl of DA solution in deep cortical layers through a Hamilton syringe, and thereafter perfused at P4.
Histology and Immunocytochemistry
After ether anaesthesia, the animals were transcardially perfused at different ages (P2P16) with 4% paraformaldehyde dissolved in phosphate buffer. Parallel series of coronal sections (50 µm thick) were obtained with a vibratome. Two series from each animal were always processed for Nissl staining and for calretinin-immunostaining (Del Río et al., 1995) to monitor the cytoarchitectonics of the neocortex and the ablation of CR cells, respectively. The other series of sections were immunostained for the visualization of microtubule-associated protein 2 (MAP2, 1:2000; clone SMI52; Sternberger-Meyer), calbindin D-28k (1:3000; Swant) or
-aminobutyric acid (1:2000; Incstar). Briefly, after blocking with 10% normal serum, sections were incubated overnight with the primary antibody and subsequently with biotinylated secondary antibodies (1:200, 2 h) and the avidinbiotinperoxidase complex (ABC complex, 1:200, 2 h). Peroxidase activity was developed with 0.03% diaminobenzidine (DAB) and 0.01% hydrogen peroxide. Sections were then mounted onto gelatinized slides and coverslipped. In order to monitor the distribution and morphology of radial glial cells and astrocytes, sections from lesioned and control mice (P2P13) were also immunostained with the Rat-401 mab [which recognizes nestin; dilution 1:5 (Hockfield and McKay, 1985
)] and with rabbit anti-glial fibrillary acidic protein (GFAP) antibodies (dilution 1:1000; Dako) essentially as described above. Additional sections from P0P2 unlesioned animals, perfused and fixed as above, were incubated with rabbit antibodies directed to the glutamate receptor subunits GluR1, GluR2/3 and GluR4 (1:10002000; Chemicon). These sections were further processed with biotinylated secondary antibodies, the ABC complex and DAB as above. Immunocytochemical controls in which the primary antibodies were replaced by preimmune normal serum did not reveal immunolabeling.
For electron microscopy, lesioned and unlesioned mice (P2P4, n = 7) were perfused with 1% paraformaldehyde and 1% glutaraldehyde dissolved with phosphate buffer, postfixed with 2% osmium tetroxide, and embedded in Araldite as described (Del Río et al., 1995). Thin sections were mounted onto formvar-coated slot grids, stained with lead citrate and examined in a Hitachi microscope.
Cell Counts
All cell counts were performed in the parietal region of the neocortex containing the primary somatosensory cortex, using a millimetric eyepiece and an oil-immersion x40 objective. All the following quantifications were performed in both the lesioned cortex and in different control tissue, including the contralateral cortex and the cortex of unlesioned and sham-operated animals (five animals each, four sections per animal, 24 counts/section). To estimate the percentage of CR cells ablated, the number of calretinin+ CR cells in layer I present in 500 µm horizontal bands were counted at P2P4. To calculate the density of radial glia, the number of radial glial vertical processes appearing in horizontal stripes (125 µm long) in layers IIIII and VI, were counted in both nestin- and GFAP-immunoreacted sections (P2, P4). Mature, GFAP+ perikarya displaying stellate morphology were also counted in 62500 µm2 samples. Data are presented for layer IIIII, but similar relative differences were found in layers VI. The number of pyknotic nuclei per 62 500 µm2 samples was counted in Nissl-stained sections from P2P4 animals separately in layers VIb, VIaIV and in the cortical plate.
5-Bromodeoxyuridine (BrdU) Labeling and Analysis
Fourteen time-pregnant females (NMRI strain) received two injections of BrdU (50 mg/kg body wt) on E16 and E16.5 (Soriano and Del Río, 1991) to label neurons fated to layers IIIII (Angevine and Sidman, 1961
; Caviness, 1982
). Newborn (P0) animals were lesioned with DA as above, after which they were perfused with 4% paraformaldehyde at P6 (n = 8) and P20 (n = 6). Unlesioned (n = 15) and sham-operated (n = 11) animals from the same litters were processed in parallel. After paraffin embedding, the brains were cut (10 µm thick) and the sections were immunostained for the visualization of BrdU using a monoclonal antibody and a nickel-enhanced DAB peroxidase reaction (Soriano and Del Río, 1991
). Sections were then counterstained with hematoxylin and coverslipped.
The quantitative distribution of BrdU+ cells in the cortex was determined from photomicrographs covering the entire cortical depth of the somatosensory area (500 µm thick bands), which were subdivided into 14 equidistant horizontal bands from layer IIIII to layer VIb. The numbers of BrdU+ cells per bin were represented as percentages (five animals per condition; four or five samples each).
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Results |
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As identified with antibodies against the calcium-binding protein calretinin, a cell marker that stains murine CR cells throughout their life (Del Río et al., 1995a, 1996; Soriano et al., 1994
), CR cells showed a typical subpial localization in layer I in perinatal mice, forming a conspicuous band of labeled neurons. CR cells have a distinctive morphology, with large horizontal perikarya and one or two thick horizontal dendrites (Fig. 2A,C
). Earlier studies showed that some pioneer preplate neurons, such as subplate cells, could be selectively ablated by injections of glutamate receptor agonists due to the early, preferential expression of these receptors in these older cortical neurons (Ghosh et al., 1990
; Ghosh and Shatz, 1992
). To determine whether a similar approach could be used to ablate CR cells, we examined the distribution of AMPA/KA-R subunits in newborn mice by immunostaining. GluR1, and to a lesser extent GluR2/3 and GluR4 subunits, were all heavily expressed in layer I, labeling neurons that resembled CR cells (Fig. 1C
). Weaker staining was present in the subplate (layer VIb) and in occasional neurons in layer V extending apical dendrites to upper layers. However, labeling was very weak in the cortical plate neurons lying below layer I, and spindle-shaped cells resembling migrating neurons, or cell bodies and apical processes of radial glia, were never stained.
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Consistent with these observations, counts of pyknotic cells at P2 and P4 revealed that DA lesions did not significantly increase (ANOVA; Scheffé's test) the number of pyknotic, dead cells either in the cortical plate or in the infragranular layers and the subplate-layer VIb [e.g. at P2, cortical plate, lesioned cortex (L) = 2 ± 0.9, contralateral control cortex (CL) = 2 ± 1.0, unlesioned control animals (C) = 2 ± 0.7; layers IVVIa, L = 8.25 ± 0.9, CL = 6.75 ± 1.2, C = 5,75 ± 0.8; layer VIb, L = 2 ± 0.7, CL = 3 ± 1.0, C = 3.2 ± 0.8 (mean ± SEM)]. Immunostaining for the neural antigens MAP2 (Fig. 1F), calretinin (Fig. 2B,D
), calbindin and GABA (not shown) confirmed that different neuronal populations through layers VIbII displayed healthy shapes. Taken together, the above findings indicate that the local application of domoic acid did not lead to the degeneration of developing neurons in the cortical plate or infragranular layers, including the subplate.
In contrast, the number of CR cells in layer I identified by calretinin-immunostaining was dramatically reduced following such excytotoxic treatment (46% at P2; 69% at P5; n = 6 animals each; Fig. 2), compared to the contralateral cortex. Interestingly, no significant differences were found between the number of CR cells present in the contralateral cortex (P2, 25.0 ± 1.6, 500 µm) and in sham-lesioned control animals (P2, 23.4 ± 1.3, 500 µm), indicating that the surgical procedure did not affect CR cell numbers. Furthermore, most of the calretinin-positive CR cells remaining in the DA-treated cortex had shrunken perikarya and atrophic dendrites (Fig. 2B,D
), which are characteristics of neuronal degeneration (Valverde and Facal-Valverde, 1987
; Valverde et al., 1989
; Del Río et al., 1995
). Indeed, at P4 the number of CR cells in the lesioned cortex with a healthy shape was only 9.3% of that in the contralateral hemisphere. Massive CR cell ablation was supported by electron microscopy, which showed the virtual absence of CR cells in the DA-treated cortex (not shown). We thus conclude that brief, local applications of domoic acid to the cortical surface led to a massive degeneration of CR cells, without affecting the survival of neurons in the remaining cortical layers.
Ablation of CajalRetzius Cells Perturbs Neuronal Migration of Late-generated Cortical Neurons
In the mouse, late-generated neurons destined to cortical layers IIIII become postmitotic at E15E17 (Angevine and Sidman, 1961; Smart and Smart, 1982
; Caviness, 1982
; Fairén et al., 1986). To determine whether the ablation of CR cells alters the migration of these neurons, pregnant dams were labeled with BrdU at E16 and their pups were treated with DA at P0 as above. As shown by BrdU-immunostaining, few of the neurons generated at E16 had already settled in the upper cortical plate at P0. At this age, when DA treatments were applied, most E16-generated neurons were still migrating in the white matter (formerly the intermediate zone) and in deep cortical layers V and VI (Fig. 3A
). By P6, migration of E16-generated neurons was complete in both unoperated and sham-lesioned littermates, and in the cortex contralateral to the DA-treated cortex. In all cases, BrdU+ cells at P6 were almost exclusively located in a thin band below layer I, corresponding to the upper half of layer IIIII (Fig. 3B
). In contrast, the distribution of BrdU+ neurons at P6 was dramatically altered in the DA-treated cortex, with many of them being located in the deep aspect of layer IIIII as well as in layers IV, V and VI (Fig. 3C,D
). Quantitative analyses of radial distributions of BrdU+ cells confirmed that the number of labeled neurons in the upper layer IIIII was reduced in DA-treated cortices to roughly half, when compared to the contralateral cortex and to control sham-lesioned animals (Fig. 4A
). Indeed, up to 52.5% of the BrdU+ cells in DA-treated cortices were sparsely distributed in layers VIIV. Sham-operated animals, did not reveal any alteration in the distribution of BrdU+ neurons, demonstrating that the surgical procedure did not alter cortical migration (Fig. 4A
). We thus conclude that ablation of CR cells in newborn mice perturbs neuronal migration of late E16-generated cortical neurons.
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In the DA-treated cortex the majority of BrdU+ neurons at P6, which were present in deep cortical layers, did not display the spindle or fusiform shapes characteristic for migrating cells. Instead, they had the large, round cell nuclei typical for differentiating neurons, which suggested that the ablation of CR cells arrested neuronal migration rather than merely slowing it. To substantiate this notion, the distribution of BrdU+ cells in DA-treated mice was analyzed at P21, long after migration had ceased in the murine neocortex (Angevine and Sidman, 1961; Smart and Smart, 1982
). The distribution of BrdU+ cells in the contralateral cortex of DA-treated animals was similar to that in sham-lesioned and unoperated animals, with most labeled neurons being present in layer IIIII (7784%, Figs 4B, 5A
). In the DA-treated cortex, in contrast, only 54% of the labeled BrdU+ cells were located in layer IIIII, with the remaining neurons distributed in the remaining layers, especially layers IV and V (Figs 4B, 5B
). Thus, although the abnormalities found at P21 were less dramatic than those observed at P6, the present findings show that the ablation of CR cells results in permanent alterations in the positioning of cortical neurons.
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Radial glia cell identity has been shown to be dependent on diffusible factors present in the embryonic forebrain, with this activity ceasing by P6 (Hunter and Hatten, 1995a), coincident with the loss of CR cells (Derer and Derer, 1992
; Del Río et al., 1995
). To investigate whether the perturbed migration reported here could be due to alterations of the radial glia following CR cell ablation, sections from DA-treated animals were immunostained for nestin, which is present in radial glia (Hockfield and McKay, 1985
), and for GFAP, a marker of both radial glia and mature astrocytes. At P2P4, substantial numbers of radial glial cell processes could be seen extending from the ventricular surface to layer I, where they branch into several endfeet (Fig. 7A
). A marked loss in the number of radial glia apical processes was found 2 days after CR cell ablation, as identified with the two antibody markers, which was more pronounced at P4 (Fig. 7AD
). Quantitative analyses demonstrated that at this age both nestin+ and GFAP+ apical radial glial cell processes decreased to less than half in DA-treated cortices, compared to the contralateral cortex (Fig. 8A
). In contrast, no decrease in radial glia could be detected in the neocortex of sham-lesioned animals (Fig. 8A
). Consistent with these findings, radial glial cell fibers at subsequent stages (P6P8) were virtually absent from the lesioned cortex, while they were still present in the contralateral cortex and in control animals (not shown). These data indicate that the ablation of CR cells results in a reduction in the number of radial glia.
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In the DA-treated cortex there were many nestin+ transitional forms (Schmechel and Rakic, 1979), which suggested that ablation of CR cells may lead to a premature transformation of radial glia to astrocytes (Fig. 7B
). This assumption was supported by counts of GFAP+ mature astrocytes showing a dramatic 6-fold increase in DA-treated cortices, compared to the contralateral and unlesioned cortices (Fig. 8B
), whereas a 3-fold increase occurred in sham-operated animals (Fig. 8B
). This finding indicates that while the surgical procedure does not affect the number of radial glia, it does increase the numbers of GFAP+ astrocytes although not so dramatically as observed in the DA-treated cortex. Thus, the loss of radial glial fibers and the dramatic increase in GFAP+ astrocytes seen specifically after ablation of CR cells suggest that these neurons regulate both the radial glial cell phenotype and its transformation to mature astrocytes.
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Discussion |
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Previous studies have taken advantage of the early maturation of pioneer cortical neurons (namely subplate neurons) and of their expression of GluR, to inject locally excytotoxic GluR agonists to ablate these early neurons (Ghosh et al., 1990; Innocenti and Berbel, 1991
; Ghosh and Shatz, 1992
). The rationale for the present study was to ablate CR cells selectively by applying DA locally to the cortical surface for a very short period, to prevent diffusion of the excytotoxic agonist deep into the cortical layers including the subplate. Our results clearly show that this DA treatment does not result in increased cell death or lesioning of cortical layers including the cortical plate, layers V and VIa, and the subplate. In contrast, the local application of DA to the cortical surface results in a substantial loss of calretinin-immunoreactive CR cells in layer I (e.g. at P4 69%). Moreover, this percentage may represent an underestimation of the number of dead CR cells, since most calretinin+ neurons persisting in layer I shortly after the DA treatment resembled degenerating neurons (Valverde and Facal-Valverde, 1987
; Valverde et al., 1989
; Del Río et al., 1995
). The selectivity of the lesioning procedure for CR cells does not rule out the possibility that DA may diffuse to the tissue directly underneath layer I, in particular to the adjacent dense cortical plate. However, due to the absence or very low expression of AMPA/KA GluR subunits in the cortical plate, DA treatment may not have an effect on these neurons. Thus, at the concentration and exposure times used, local application of DA to the cortical surface of newborn mice results in the selective ablation of most CR cells in layer I.
It has been shown that degeneration of meningeal cells may lead to abnormalities in neuronal migration (Sievers et al., 1986; Hartmann et al., 1992
), and the alterations reported here may be due to injury of meningeal cells. However, the results in sham-lesioned animals showed that the surgical procedure did not modify either the number of CR cells, the distribution of BrdU-positive neurons or the density of radial glia. Furthermore, both the lesioned and sham-operated cortices display meningeal cells as identified by electron microscopy (unpublished observations). In addition, it has been reported that mechanical lesions to the perinatal layer I result in a prolonged persistence of radial glial cells and CR cells near the lesion (Rosen et al., 1994; Supèr et al., 1997a
). Thus, mechanical lesions have an opposite effect to the results described in this study. Taken together, these data rule out the possibility that the findings reported here on neuronal migration and radial glia regulation could be attributable to disruption or injury of meningeal cells, but they are a direct consequence of the disappearance of CR cells.
Although the lesioning procedure substantially increases the number of GFAP+ astrocytes, this increase is only half that observed after CR cell ablation. Similarly, the present findings are very unlikely to be due to a direct effect of GluR agonists on migrating cells since: (i) to our knowledge, there is no evidence for the presence of AMPA/KA-R in migrating cells (Miller et al., 1990; García-Ladona and Gombos, 1993
; Komuro and Rakic, 1993
; Bahn et al., 1994
; Gallo et al., 1995
) (see also present results); (ii) AMPA/KA-R agonists and antagonists do not alter migration in living slices (Komuro and Rakic, 1993
); (iii) normal migration is seen in the cortex after injection of GluR agonist in developing deep cortical layers (Ghosh et al., 1990
; Innocenti and Berbel, 1991
; Ghosh and Shatz, 1992
) (see also present observations); and (iv) a similar disruption of migration and cortical layering is reproduced after application of 6-OHDA, a toxin that degenerates CR cells through mechanisms other than AMPA/KA-R (Supèr et al., 1997b
). Thus, our data indicate that ablation of CR cells perturbs neuronal migration, even at very late stages of corticogenesis.
Alterations of Cortical Positioning Following Ablation of CajalRetzius Cells
The present study has shown that the ablation of CR cells in the neocortex of newborn mice results at P6 in a severe mispositioning of many late E16-generated cortical neurons, which are normally fated to the upper layer IIIII (Angevine and Sidman, 1961; Caviness, 1982
; Smart and Smart, 1982
) (see also present data);. In the DA-treated animals, more than half the neurons generated at E16 appear ectopically located in layer IV and in the deep cortical layers V and VI. These ectopic cells correspond to the neurons that were still migrating in the white matter and deep cortical layers at P0 (Fig. 3A
). In contrast, the BrdU-positive neurons that appear appropriately located in layer IIIII in experimental animals correspond most likely to the neurons that at the time of lesioning were near to, or had already reached, layer IIIII (Fig. 3A
). The preferential distribution of E16-generated neurons in deep cortical layers resembles the radial distribution of late-born neurons in the neocortex of the reeler mutant mouse (Caviness, 1982
; Hoffarth et al., 1995
). Although the gene disrupted in the reeler mutation is expressed primarily by CR cells, it is controversial whether the laminar alterations of the reeler phenotype are a consequence of very early abnormalities at the preplate stage, or due to malfunctioning of CR cells throughout prenatal and postnatal development (Hoffarth et al., 1995
; D'Arcangelo et al., 1997
). The present observations in postnatal animals point to the likelihood that normal CR cell function and reelin expression are essential for neuronal migration throughout corticogenesis, including the late migratory phase (Fig. 9
).
In contrast, the laminar alterations of late-generated cortical neurons detected at P21 following ablation of CR cells are not as dramatic as at P6, with lower numbers of BrdU+ neurons present in layers IV, V and VI. These findings suggest that, between P6 and P21, the neurons that are mispositioned are nevertheless able to migrate and reach the appropriate target layer IIIII. In the absence of radial glia, neurons could migrate using other favorable or adhesive substrates provided by, for instance, axon fascicles and other maturing neuronal elements, astrocytes or even blood vessels (O'Rourke et al., 1992, 1995
; Jankovsky and Sotelo, 1996; Lois et al., 1996
). In this vein, successful migration of cortical precursors grafted into the late postnatal neocortex has been reported recently (Sheen and Macklis, 1995). However, although this possibility cannot be ruled out, our cell counts indicate that most of the ectopic neurons at P6 that disappear from deep cortical layers at P21, do so most likely by neuronal cell death.
Regulation of Radial Glia by CajalRetzius Cells
How do CR cells affect cortical migration? The present study has shown that the ablation of CR cells in the developing neocortex in vivo results in a dramatic reduction in the numbers of radial glial cells, which is not attributable either to the surgical procedure or to the DA treatment (see above). Complementary transplantation experiments in which embryonic CR cells cultured with or grafted to cerebellar tissue induce a rejuvenation of Bergmann astrocytes into a radial glia phenotype (Soriano et al., 1997), give further support to this contention. In addition, emx2-deficient mice that have a reduced number of CR cells display severe abnormalities in the radial glia (Mallamaci et al., 2000
). Thus, taken together the above data indicate that CR cells are involved in sustaining the radial glia cell identity, necessary for cortical migration, as well as in the regulation of the radial glia-to-astrocyte pathway during normal corticogenesis (Fig. 9
).
During cortical development, radial glial cells are transformed into mature astrocytes at the end of the migration phase (Schmechel and Rakic, 1979; Hatten, 1990
). Our results show that, in addition to causing a reduction in radial glia, the ablation of CR cells leads to a dramatic 6-fold increase in the number of mature astrocytes, which is double that detected in sham-operated animals. This finding suggests that the loss of CR cells not only causes the disappearance of radial glial cells, but also promotes their transformation into astrocytes. The radial glia-to-astrocyte transformation is a bidirectional pathway, which has been shown to be mediated by diffusible factor(s) of 5060 kDa (Hunter and Hatten, 1995a
,b
), indicating that the radial glia phenotype may be controlled by specific soluble factors, similar to the regulation of other glial cell lineages, such as oligo-dendrocytes (Raff, 1988; Price, 1994). In a previous in vitro study we have shown that CR cells exert a radial glia-promoting activity through soluble factors (Soriano et al., 1997
), indicating that CR cells release the soluble signals that are essential for sustaining radial glia cell identity and function. Reelin is a secreted extracellular matrix protein involved in migration, and is highly expressed in CR cells. However, the radial glia scaffold persists in the reeler cortex although it shows a loose organization and is transformed into astrocytes sooner than in wildtype mice (Pinto-Lord et al., 1982
; Hunter and Hatten, 1995b
). This suggests that Reelin an essential protein for neuronal migration might be involved in the regulation of radial glia cell identity although it is probably not the essential promoting signal. One possibility is that Reelin may participate in the signaling pathway by activating or sequestering the key soluble factors, as other extracellular matrix proteins do for some cytokines (Lander, 1993
; Schlessinger et al., 1995
).
Conclusions
The onset of CR cells is coincidental with the emergence of radial glia (Misson et al., 1991; Del Río et al., 1995
), which suggests that CR cells are involved in inducing their differentiation in early corticogenesis. Thus, a picture of cortical development is emerging in which subplate neurons and CR cells are involved in the guidance and reshaping of neural connections (Allendoerfer and Shatz, 1994
; Del Río et al., 1997
; Supèr et al., 1998
). CR cells, in addition, may organize the radial glial scaffold, which supports the inside-out order of migration and cortical maturation. Thus, some abnormalities in migration leading to cortical malformations in humans might arise from structural or functional defects in CR cells.
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Notes |
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Address correspondence to Dr Eduardo Soriano, Department of Animal and Plant Cell Biology, Faculty of Biology, University of Barcelona, Diagonal 645, Barcelona 08028, Spain. Email: soriano{at}porthos.bio.ub.es.
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Footnotes |
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References |
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