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Previous Article | Next Article
The Journal of Neuroscience, October 15, 2002, 22(20):8981-8991
Influence of the Embryonic Preplate on the Organization of the Cerebral Cortex: A Targeted Ablation Model
Y. Xie1, E. Skinner1, C. Landry2, V. Handley1, V. Schonmann1, E. Jacobs1, R. Fisher1, and A. Campagnoni1 1 Developmental and Molecular Neuroscience Group, Neuropsychiatric Institute, University of California at Los Angeles, School of Medicine, Los Angeles, California 90024-1759, and 2 Psychiatric Institute, University of Wisconsin, Madison, Wisconsin 53719
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
Transgenic mice were generated to permit the targeted ablation of cortical preplate cells at the time they are born. In these mice, the 1.3 kb golli promoter of the myelin basic protein gene was used to drive the herpes simplex virus thymidine kinase (TK) transgene in cortical preplate cells. Heterozygous transgenic pairs were bred, and pregnant dams were treated with ganciclovir at embryonic days 11-12 to ablate preplate cells at the time the preplate was forming. This paradigm exposed control (TK
) and experimental (TK+) littermates to exactly the same conditions. Embryological ablation of preplate cells led to an early disruption of the radial glial framework and subplate structure in the developing cortex and dramatically altered the cellular lamination and connectivity of the cortical plate. The disturbed radial glial network contributed to an impaired radial migration of neurons into the cortical plate from the ventricular zone. The cortical plate became dyslaminated, and there was a substantial reduction in short- and long-range cortical projections within the cortex and to subcortical regions. Cell death within the cortical plate and the proliferative zones was substantially increased in the ablated animals. After birth, a cortical lesion developed, which became exacerbated with the secondary onset of hydrocephaly in the second postnatal week. The results underscore the critical importance of the preplate in cortex formation, mediated through its guidance of the formation of radial glial scaffolding, subsequent neuronal migration into the incipient cortical plate, and the final arrangement of its vertical organization and cellular connectivity.
Key words: preplate; cortex; development; transgenic;
-galactosidase; HSV-thymidine kinase; ablation; cell death
INTRODUCTION
The cortical preplate is believed to play an important role in the formation of the cerebral cortex, and it has been the subject of considerable investigation. Early neurons proliferating within the neuroepithelial zone migrate into restricted quadrants of the telencephalic vesicle to form the cortical primordial plexiform layer, or preplate, between embryonic day 11 (E11) and E13 in the mouse. Subsequently, at approximately E14, newly born neurons migrate along radial glial fibers from the ventricular zone (VZ) into the transient preplate structure. During this process, they split the preplate into a peripheral marginal zone (MZ), destined to become cortical layer I, and a deep intermediate zone (IZ), destined to become the cortical subplate. They occupy a new layer between these two zones to form the cortical plate (CP) (Bayer et al., 1991
; Allendoerfer and Schatz, 1994
An attractive approach to test the role of preplate cells in cortical development is to ablate them at very early stages and then examine the consequences of the ablation on cortical development. A variety of methods, including irradiation (Roper, 1998
To investigate the role of the preplate in the formation of the cortex, we generated transgenic mice using the golli promoter of the myelin basic protein (MBP) gene to drive expression of the herpes simplex virus thymidine kinase (HSV-TK) gene. We showed previously that this promoter targets
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Figure 1. Cortical preplate neurons genetically targeted for ablation and the early impact of genetic ablation on embryonic cerebral cortex. A, B, Photomicrographs of preplate target neurons containing brown immunoreactivity for
MATERIALS AND METHODS
Generation of golli-HSV-TK transgenic mice
Construction of the vector for golli-TK transgenic line
The golli-TK plasmid was created by first cloning the 1.3 kb EcoRI-BamHI fragment of the golli promoter into EcoRI-BamHI site of pGEM3ZfFounders
Transgenic mouse lines were identified by Southern blot analyses on BSTEII digests of isolated tail DNA using a probe specific for exon 1 of the golli-MBP gene. The insertion of a single copy of the golli-TK transgene was determined using Southern blot analyses to compare the intensities of the hybridization signals of the TK probe with those from a probe of a known single copy gene, MBP. The double-transgenic golli-TK/lacZ mice were generated by crossing hemizygous (TK/+) mice to transgenic mice (1E2) homozygous for theAnalysis of the transgenic mice
Genotyping by PCR
The golli-TK transgene was identified by PCR using the MGTB sense primer (within golli-MBP promoter) 5'-CTGAGCTTCACGACCCCGGAACATAGT and the TK3P antisense primer 3'-GTCATGCTGCCCATAAGGTATCGCG. The double-transgenic mice (golli-TK/lacZ) were identified using the MGTB sense primer and theDrug treatment
Ganciclovir (Cytovene-IV tm; Roche Laboratories, Nuffy, NJ) was prepared at 0.25 mg/ml in sterile water. Timed-pregnant females were given intraperitoneal injections of ganciclovir (20 µg/gm) on E11 and E12 (day of insemination, day 0.5). Two injections (~8-12 hr apart) were given each day. In some experiments, ganciclovir was administered by two injections on each E16-E17 and E17-E18 and two injections on each E16, E17, and E18.Tissue preparation
Postnatal day 1 (P1), P7, and P14 animals were anesthetized with halothane (Halocarbon Laboratories, North Augusta, SC) and perfused intracardially with 4% paraformaldehyde in PBS. For embryonic time points (E11-E18), timed-pregnant females were anesthetized and killed by cervical dislocation. Embryos were removed, and the brains were dissected. All tissues were postfixed either overnight or for 1 hr (histochemistry) with the 4% paraformaldehyde in PBS. The fixed tissue was then cryoprotected in sucrose, frozen in OCT, and sectioned (20 µm). Sections were mounted on Superfrost Plus slides (Fisher Scientific, Pittsburgh, PA) and processed for histochemical and immunohistochemical examination.Frozen sections were removed from
Immunohistochemistry
Frozen sections were air dried and rinsed in PBS before all immunohistochemical procedures. For immunofluorescence, sections were incubated in 2% BSA in 0.1 M PBS and 0.03% Triton X-100 for 1 hr at RT to block nonspecific staining. The sections were then incubated with the primary antibody at 4°C for 48-72 hr. Bound primary antibody was detected by incubation with the appropriate secondary antibody labeled with fluorescein (FITC) or Texas Red (1:200; Jackson ImmunoResearch, West Grove, PA) for 1-2 hr at RT. After washing in PBS, the slides were coverslipped in Aquamount and analyzed with a Leica (Nussloch, Germany) DMR microscope. A similar protocol was followed for immunohistochemistry with biotinylated secondary antibodies except that, before blocking, the sections were treated with 0.3% H2O2 in methanol to quench endogenous peroxidase activity. After incubation with the appropriate secondary antibody, the avidin-biotin-peroxidase system (Vectastain Elite ABC) was used as recommended by the manufacturer (Vector Laboratories, Burlingame, CA) and developed in DAB color solution. The sections were then dehydrated, cleared, and coverslipped with Permount. The following antibodies were used in this study: polyclonal rabbit anti-Bromodeoxyuridine birth dating and detection
Timed-pregnant female mice were given intraperitoneal injections of 100 µg/gm body weight of 5-bromo-2'-deoxyuridine (BrdU) in sterile PBS. After BrdU injection, pups were harvested at specific ages, and the brains were processed as described above. Cryostat sections were incubated in 2N HCl for 30 min at 37°C, rinsed in 0.1 M sodium borate, pH 8.3, and then incubated overnight with anti-BrdU monoclonal antibody (Becton Dickinson, San Jose, CA) at 1:100. Detection of the antibody was performed as described above.Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling in situ detection procedures
Frozen sections were air dried, rinsed in PBS, and incubated in 0.3% H2O2-methanol solution to block endogenous peroxidase activity. Cell permeability was increased by incubating the sections in 0.1% Triton X-100 in 0.1% sodium citrate for 2 min on ice at 4°C. The sections were rinsed in PBS and incubated with the terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) reaction mixture for 1 hr at 37°C [In Situ Cell Death Detection Kit; peroxidase (POD); Roche, Indianapolis, IN]. The sections were then examined with epifluorescence or treated with the Convertor-POD reagent and peroxidase substrate according to the instructions of the manufacturer (Roche).Quantitative measurements
Cortical thickness. E18, P1, and P4 control and ablated brains (five to nine brains in each age) were used for this analysis. Coronal sections of constant thickness (20 µm) were taken rostrally, starting from the lateral ventricle. Every sixth section was transferred to a slide, yielding a total of 15 sections per brain (with each n equaling the pooled average of the 15 measurements per section). This sampling procedure was used in all subsequent measurements. Minimal cortical depth was measured between the pial surface and the bottom of the cortex using a 10× objective. Data were statistically analyzed with SPSS (Chicago, IL) software. BrdU-labeled cells. Pregnant mice at E15 were injected with BrdU, and embryonic brains of control (n = 5) and ablated (n = 8) pups were harvested 1.5 hr after injection. BrdU-labeled cells in each section were counted within a 6.0 × 102 mm2 cortical area with a 40× objective in similar brain regions for all animals. The percentage of the total number of BrdU-labeled cells found within the intermediate zone and cortical plate was calculated, and statistical analysis was made using SPSS software. TUNEL-labeled nuclei. E15, E18, and P1 control and ablated brains (8-12 brains in each age) were used for this quantitative analysis. TUNEL-labeled nuclei in each section were counted in a 2.4 × 10
RESULTS
Generation of a transgenic mouse that expresses HSV-TK in cortical preplate and subplate neurons
We identified previously a promoter element in the myelin basic protein gene capable of targeting
Previously, we established the specificity of the golli promoter using golli-lacZ mice (Landry et al., 1998
We wanted to take advantage of the toxicity of ganciclovir to kill TK-expressing cells at an early stage in cortical development, during formation of the cortical preplate and the birth of cortical pioneer neurons. The drug kills dividing cells and it is cleared from the system within hours, so we administered ganciclovir by four injections over E11 and E12. As shown in coronal views in Figure 1, C and D, this resulted in a significant loss of lacZ-expressing preplate neurons. Note that the lateral regions, in which the preplate first develops, were relatively less affected than the midline regions, consistent with the known timing and progression of preplate neurogenesis. Although not shown, we also observed a substantial reduction in calretinin+ Cajal-Retzius cells in the ablated animals.
Histological abnormalities are evident in the development of the cortex shortly after ganciclovir treatment
Cresyl violet histological analyses were performed on the "ablated" brains of fetuses, neonates, and early postnatal transgenic animals treated with ganciclovir at E11-E12. Histological differences in the cerebral walls between the ablated and control fetuses were evident as early as 3 hr after the fourth injection on E12. Three-layered structures of the cerebral wall, corresponding to a neuroepithelial proliferative zone, preplate, and a subpial "marginal" layer, could be observed by this time. Compared with the controls, it also appeared that the preplate was thinner in both the lateral regions and midline regions of the ablated animals (Fig. 1, compare counterstaining in C, D).
At later ages, particularly by E18 and beyond, significant histological abnormalities were evident in the cortices of the ablated animals. Figure 2 illustrates some of these in P4 mice. It was apparent that the subplate layer was indistinct and that the cortices of the ablated mice were thinner and dyslaminated. The cortical disorganization of the ablated mouse made it difficult to assign layers. Cellularity in layers V-VI appeared to be significantly reduced, and the presence of prominent pyramidal neurons in layer V of controls was not evident in the ablated cortex. We saw no evidence of heterotopia in the ablated cortices.
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Figure 2. Cresyl violet staining of control (TK
Other alterations in the brains of the ablated animals included a malformed corpus callosum and increased ventricular size. At P4, the incipient white matter region between gray matter and the ventricle was much narrower in the ablated brain than that of controls, and, by P7, the corpus callosum in the ablated mice, located beneath the neocortical subplate region, was very thin. Slightly larger lateral ventricles were evident in the ablated mice by late embryonic and neonatal life, and these continued to enlarge with age.
Other structures within the brain did not appear to be affected by the ganciclovir treatment. Unlike the neocortex, the regional topology of other forebrain structures of the ablated brain, such as the striatum and septum, was intact. Interestingly, cortico-fugal fiber tracts running through the striatum were significantly smaller in diameter, consistent with the perturbed subplate cells and their role in pioneering these connections. Similarly, there was a clear correlation of the affected areas of the ablated brains with the timing of cortical development.
Treatment of pregnant females with ganciclovir at middle to late embryonic stages in cortical development (e.g., E16, E17, and E18) resulted in no obvious histological abnormalities as late as 28 d of age (data not shown). Thus, the formation of the lesion correlated with ganciclovir treatment during genesis of the cortical preplate and the birth of subplate neurons in the mouse brain.
Immunohistochemical studies confirm the histological findings and reveal other abnormalities in the ablated (TK+) brains
Additional evidence of abnormal cortical organization was obtained by immunostaining with several neuronal markers, including NF-M, golli proteins, and srPLP. In postnatal brain, golli antibody stains fibers as well as cell bodies within the cortex. As shown in Figure 3, A and B, golli+ fiber staining in the cortex is significantly reduced in the ablated brain. Compared with the controls (Fig. 3A), a significant reduction in density of neuronal fibers stained for golli proteins was observed in the upper cortical layers (II-IV) of the ablated brains (Fig. 3B). In view of the fiber loss revealed by golli immunostaining, we also examined the ablated animals by immunostaining for NF-M. Figure 3, C and D, shows staining of a portion of the P1 cortex, close to the midline, with anti-NF-M. Again, a reduction in the number of fibers was evident in the ablated brains. One of the more striking features of the NF-M staining was the appearance of Probst bundles in the cingulate cortices (Fig. 3D, arrows). Probst bundles are aggregations of fibers that derive from axons that fail to cross the midline after their initial extension to the medial hemispheric walls (das Neves et al., 1999
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Figure 3. Evidence of fiber reduction-misdirection and cellular abnormalities in the ablated cerebral cortex at P1. A, C, E, Wild-type control mice; B, D, F, ablated littermate mice. A, B, Photomicrographs of golli protein immunoreactivity in wild-type and transgenic ablated mice. Note the near-complete absence of golli-like immunoreactivity in the ablated cortex. C, D, Photomicrographs of the NF-M immunoreactivity in wild-type and ablated mice. Note the substantial reduction of NF-M expression in the subcortical white matter structures of the corona radiata, corpus callosum, and internal capsule of the ablated brain. Abnormal Probst bundles (see arrows) were also observed in the cingulate cortex of the ablated animals. E, F, Photomicrographs of srPLP immunoreactivity in wild-type and transgenic ablated mice. Note that strings of srPLP-immunoreactive cells found in the intermediate and subventricular zones of the control case were disrupted in the ablated animals. V , Lateral ventricle. Scale bars: A, B, E, F, 40 µm; C, D, 80 µm.
Figure 3, E and F, shows a portion of the subventricular zones (SVZs) of the control and ablated brains at P1, immunostained with anti-srPLP, a product of the myelin PLP gene expressed in neurons (Bongarzone et al., 1999
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Figure 4. Morphological evidence for impaired neuronal migration and disrupted radial glial fiber in ablated animals. A-D, After ganciclovir treatment, dams were injected with BrdU at E13 and analyzed 48 hr later (E15). In control (B) and ablated (D) brains, BrdU was detected immunohistochemically (red), and adjacent sections were stained with cresyl violet. Numerous BrdU-labeled cells had migrated into the CP and IZ by this time in the control, but significantly fewer cells had migrated to these regions in the ablated brains. E, F, E15 animals were injected with BrdU, and the distribution of labeled cells was compared after 1.5 hr in control and ablated brains. Again, migration into the upper layers was much more apparent in the control (E) than in the ablated (F) animal. The sections were double stained with a marker for radial glia, RC2. Labeling for radial glia appeared to be less intense and less extensive between the ventricular and pial surfaces in the ablated mice than in the controls. G-I, At E13, radial glial fibers were visualized by immunostaining with RC2. Whereas straight and regular radial fibers extending between the two surfaces could be seen in control sections (G), there appeared to be reduced numbers of frequently truncated or malformed radial glia in the ablated animals (I). H is an adjacent section of the control animal stained with cresyl violet. Scale bars: A-D, 40 µm; E, F, 30 µm; G-I, 20 µm.
Impairment of neuronal migration in the ablated brains
The apparently reduced cellularity in the cerebral cortex and the apparent disruption of cells migrating out of the subventricular zone suggested the possibility that neuronal migration from the proliferative zones into the cortical plate during development might be impaired as a consequence of the ablation. To investigate this possible impairment, we performed BrdU incorporation experiments to visualize the migration of BrdU-labeled cells in control (TK
Additional evidence for impaired neuronal migration was obtained by short-term labeling of E15 mice. Figure 4 shows BrdU incorporation experiments combined with RC2 immunostaining for radial glial fibers 1.5 hr after injection of control and ablated animals with BrdU at E15. In the E15 control brain (Fig. 4E), BrdU-labeled cells were distributed throughout the proliferative zones, intermediate zone, and even into the cortical plate. Many BrdU cells could be seen along RC2+ fibers. In contrast, in the ablated mice (Fig. 4F), the BrdU+ cells were primarily confined to the proliferative zone, and some had migrated into the intermediate zone. A quantitative assessment of these data showed that, after 1.5 hr, 16.0 ± 0.4% (mean ± SEM) of the labeled cells were found in the IZ or CP of the control animal, in contrast to 4.4 ± 0.8% of the labeled cells in the ablated animals.
Also, it appeared as if the radial glial network was disrupted in the ablated animals. There appeared to be fewer radial glia, and many fibers seemed to terminate prematurely along the radial network. This is illustrated more clearly for E13 animals in Figure 4, G and H. In this case, the radial glial fibers have been labeled in red with RC2. As can be seen, there was considerable alteration in the number and morphology of the radial glial fibers between the control (TK
Thus, the results suggested that ablation of preplate neurons resulted in a reduction in the numbers and morphologies of the radial glia, accompanied by impaired migration of cortical neurons out of the proliferative zone into the growing cortical plate. The impaired migration could be attributable to the lack of sufficient numbers of glial fibers along which newly born neurons could migrate.
Increased cell death in the cortex and SVZ/VZ of the ablated brains
Several findings implied that cell death might be increased in the ablated cortices during embryonic and neonatal life. Impaired migration of cortical neurons from the VZ/SVZ suggested that there might be greater turnover of those cells within the VZ/SVZ. Also, the disrupted radial glial network might misdirect those neurons that did exit the VZ/SVZ within the cortical layers. This and the absence of a clear subplate could explain the loss of cortical connections and lead to the death of those neurons in the absence of synaptic activity. For these reasons, we examined TUNEL to compare cell death in the control and ablated brains. TUNEL-positive labeled nuclei were observed in the ablated animals throughout cortical development. Figure 5 shows views of control and ablated cortices at E15 (Fig. 5A,B) and P1 (Fig. 5C,D) after TUNEL staining (dark brown) and cresyl violet counterstaining (blue). Figure 6 shows measurements of the density of TUNEL+ nuclei in the cortices of normal and ablated mice from E15 to P1. Even at E15, an increase in TUNEL-labeled nuclei was evident in the SVZ/VZ of the ablated animals. Interestingly, there was a sharp increase in cell death in the SVZ/VZ and cortical plate between E18 and P1, and even more cell death throughout the cortex was observed at later ages in postnatal ablated animals. Figure 5 illustrates the substantial increase in TUNEL+ nuclei (dark brown) throughout the cortical plate and the SVZ in the ablated animal (Fig. 5D) compared with control (Fig. 5C) at P1. In the ablated animals the TUNEL-labeled nuclei appeared in clusters of variable size at P1. Thus, during embryonic development, increased cell death occurred within the VZ/SVZ of the ablated mice, and, at older ages, this increased cell death continued within this proliferative zone but extended to the cortical plate as well.
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Figure 5. Evidence for increased aptotic cell death in the cerebral cortex (Ctx) and VZ/SVZ of ablated mice. A, B, Photomicrographs showing increased TUNEL+ cerebral cortical cells (brown labeling) in E15 ablated (B) and littermate control (A). Tissue sections were counterstained with cresyl violet. Very few apoptotic cells were found in the control brains, but increased numbers of apoptotic cells were found in the subventricular, proximal intermediate zones and cortical plate of the ablated brains. C, D, Photomicrographs of TUNEL+ and counterstained apoptotic cells at P1 (C, wild-type control mouse; D, ablated littermate). Aptotic labeled profiles were dispersed widely from the subventricular and ventricular zones through the cortical plate in both groups. Developmental increases in apoptotic cells were also observed in both groups, but the increase was substantially greater in the ablated than the control mice. Scale bars: A, B, 40 µm; C, D, 80 µm.
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Figure 6. Increased cell death with the cerebral cortex of normal and ablated animals from E15 to P1. TUNEL+ nuclei were counted, and density measurements were performed on sections prepared from the brains as described in Materials and Methods. Error bars represent SEM. n = 8-12 animals analyzed in each group at each age.
End-stage lesions develop in the postnatal animals
TK+ pups born to dams treated with ganciclovir at E11-E12 were examined at many ages postnatally. During late embryonic life, the thickness of the cortex of the ablated animals was decreased relative to controls. This is illustrated in Table 1 in which cortical thickness for control and ablated mice is given for several ages in late embryonic and early postnatal life. This difference became more apparent, even on visual inspection, at later ages. After the first postnatal week, the ablated animals developed large cortical lesions that increased in size with age. Figure 7 illustrates the extent of these lesions in a comparison of Nissl-stained sagittal views of P14 and P20 TK+ brains. Note the massive cortical lesion at P20 and the thinned cortex of the ablated mice. Enlarged ventricles and decreased cortical thickness are clearly evident. By P24-P28, when the animals died, very little cortex remained in the TK+ brains.
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Table 1. Cortical thickness (in millimeters ± SEM) of control and ablated brains at several ages
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Figure 7. Development of postnatal lesion in HSV-TK+-ablated animals (parasagittal tissue sections; cresyl violet counterstain). A, Photomicrograph of brain from an ablated transgenic mouse at postnatal day 20. B, Photomicrograph of brain from an ablated mouse at postnatal day 14. C, Photomicrograph of brain from a control (TK
At approximately P10, the ablated pups appeared to become mildly hydrocephalic, as indicated by ventricular expansion and, in some cases, increased head size, which increased in severity with age. The enlarged ventricles and degenerated cortex was a constant feature of all ablated animals, but the incidence of increased head size was variable. In some cases, we found ablated animals with larger and smaller head sizes than the controls within the same litter. This supports the notion that the observed postnatal hydrocephaly is secondary to the degeneration of the cortex. The data in Figures 5 and 6 indicate that increased cell death is evident in the ablated animals from E15 on, and, there appears to be a large increase in cell death in the cortex between E18 and P1. This increased cell death continues postnatally and precedes the onset of hydrocephaly by ~10 d.
DISCUSSION
Our results support several conclusions. (1) Specific reductions of cortical preplate cells can be achieved in a transgenic model using the golli promoter of the MBP gene to drive HSV-TK during early corticogenesis in the mouse. (2) Embryological ablation of preplate cells leads to an early disruption of the radial glial framework and subplate structure in the developing cortex, which, in turn, dramatically alter the cellular lamination and connectivity of the cortical plate. (3) Preplate cell ablation results in a severe postnatal cortical lesion resulting from the embryological insult and aggravated by the onset of secondary hydrocephaly.
Advantages and selectivity of the genetic ablation approach
We exploited previous findings that the golli promoter element of the MBP gene can target expression of transgenes to the preplate at very early stages of corticogenesis (Landry et al., 1998
In studies using the HSV-TK gene, there is always the concern that "bystander" killing reduces the specificity of the ablation. In this instance, these concerns are minimal for several reasons. The susceptibility of adjacent cells to bystander killing has been directly related to the presence of gap junctions (to facilitate transfer of the toxic products of ganciclovir between cells) and the expression of connexins, the major components of gap junctions, by the cells. Whereas expression of the neuronal-specific connexin 36 has been reported in the developing mouse nervous system as early as E9-E10, it is not expressed in the telencephalic region giving rise to the cortical preplate (Gulisano et al., 2000
The phenotype of the ablated animals was confined to the cortex and completely consistent with an early disruption of preplate neurons. In this regard, the golli promoter-driven HSV-TK gene, like the lacZ gene, was expressed in neurological sites other than the incipient preplate (Landry et al., 1998
Thus, this approach is particularly suited to studying corticogenesis for several reasons. The model permits the ablation of cortical pioneer neurons earlier than previous approaches. The ablation is transient, so postmitotic cells will be unaffected and continue to differentiate into Cajal-Retzius and subplate neurons. Finally, individual variation among animals subjected to the ablation conditions is minimized because the expression of the "suicide" gene is genetically determined in the appropriate cells, and control fetuses lacking the gene are exposed to exactly the same conditions as the experimental fetuses.
Early ablation of preplate cells causes a cascade of events leading to disturbed cortical development
In this study, the genetic ablation of significant numbers of preplate cells produced a fundamental perturbation of the developing cerebral cortex. Within hours of the last ganciclovir treatment, the ablated preplate was slightly thinner than controls. This was followed by a disturbance of the radial glial network, in terms of both the numbers and morphologies of the radial glia, probably through alterations of trophic and/or anchoring elements that guide its formation. Disruption of the radial glial network led to impaired neuronal migration from the ventricular zone into the growing cortical plate and the resultant dyslamination of the cortex. The extent to which migrating neurons reached their appropriate positions in the cortical plate might have depended on the integrity of the radial glia along which they migrated. There also was loss of a functional subplate resulting in the disruption of pioneering axons that establish cortico-cortico and cortico-fugal connectivity. This was manifest in fiber reduction within the cortex and disturbances of forebrain white matter structures, including the corpus callosum and internal capsule.
As noted, an immediate effect of preplate cell ablation was a disruption of the radial glial network and subsequent impairment of neuronal migration. This finding is consistent with the notion that Cajal-Retzius neurons, and perhaps subplate neurons, provide cues for the migrating neurons and support the radial glia scaffold for neurons migrating into the cortical plate from the germinal zone (Sheppard and Pearlman, 1997
It also has been suggested that Cajal-Retzius neurons may provide a "stop" signal that constrains migration of cortical plate neurons into the MZ (Ogawa et al., 1995
The effects of the ablation on reduced cortical fibers and decreased thickness-fiber density in white matter structures is likely to be a direct consequence of a disturbance in the cortical subplate, which has been postulated to play a key role in the formation of cortico-cortico and cortico-fugal connections (Allendoerfer and Schatz, 1994
Early ablation of subplate cells results in a severe postnatal lesion
The ablation of the preplate cells resulted in a later stage lesion and secondary, progressive hydrocephaly. It is likely that elevated cell death in the cortex and germinal zones contributed significantly to the thinning of the cortical plate and the formation of the lesion. We suggest that the marked increase in cortical cell death, particularly around birth, causes the degeneration of the cortex and the subsequent development of hydrocephaly, which may exacerbate the late-stage progression of the lesions in postnatal animals. We do not know the cause of the hydrocephaly, although it might result from cell debris retarding-blocking outflow of CSF. We were not able to identify a structural abnormality that would account for the hydrocephaly. We predict that the ablation conditions used in the present work can be "titrated" to produce less severe postnatal lesions and eliminate the secondary hydrocephaly.
We believe that the transgenic ablation model described in this report offers great potential value for elucidating the morphogenic mechanisms of cerebral cortex development. The results outlined clearly underscore the critical importance of the preplate for the formation of the cortical plate, mediated through its guidance of the formation of radial glial scaffolding, subsequent neuronal migration into the incipient cortical plate, and the final arrangement of its vertical organization and cellular connectivity.
FOOTNOTES Received April 2, 2002; revised May 23, 2002; accepted June 18, 2002.
This work was supported by National Institutes of Health Grants NS 33091 and NS 23022.
Correspondence should be addressed to Dr. A. T. Campagnoni, Room 47-448, Neuropsychiatric Institute, University of California at Los Angeles, School of Medicine, 760 Westwood Plaza, Los Angeles, CA 90024. E-mail: acampagnoni{at}mednet.ucla.edu.
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