Coexistence of Widespread Clones and Large Radial Clones in Early Embryonic Ferret Cortex

Marcus L. Ware1,2, Sohail F. Tavazoie2, Christopher B. Reid1,2 and Christopher A. Walsh1,2,3

1 Division of Neurogenetics, Department of Neurology, Beth Israel Deaconess Medical Center, Harvard Institutes of Medicine, 77 Avenue Louis Pasteur, Boston, MA, , 2 Program in Neuroscience and , 3 Program in Biological and Biomedical Sciences, Harvard Medical School, Boston, MA, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cell lineage analysis in rodents has shown that the cerebral cortex is formed from both widespread and large radial clustered clones representing partly distinct lineages and producing differing cell types. Since previous cell lineage analysis of the ferret cortex using retroviral libraries showed that most neurons labeled at E33–E35 formed widespread clones, we determined whether clones labeled earlier in neurogenesis showed a greater tendency to form coherent radial clones. Clones labeled at E27–E29 occasionally consisted of widespread multineuron clones (13% of PCR-defined clones), but commonly consisted of small clusters of two to four neurons (65%). Moreover, 6/21 hemispheres contained a single, much larger (6–150 cells) radial cluster. Although large clusters were observed in 28% of experiments, they contained many neurons, accounting for 38% of retrovirally labeled cells. The large clusters showed at most few widely scattered sibling cells, either by histological analysis or by PCR analysis, suggesting that radial and widespread clones coexist but are lineally separate at early stages of corticogenesis. Coexistence of large radial and widespread neuronal clones appears to be an evolutionarily conserved mechanism for cortical neurogenesis.


    Introduction
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Studies of cell lineage and migration in cortical neurons have demonstrated a striking diversity of clonal and migratory patterns. Morphological studies (Rakic, 1971Go), electron microscopic studies (Rakic, 1972Go; Rakic et al., 1974Go), and in vitro analysis of cerebellar cultures (Edmondson and Hatten, 1987Go) have long shown that radial glia provide a substrate for radially migrating neurons. However, studies of cell lineage using retroviral vectors (Price and Thurlow, 1988Go; Walsh and Cepko, 1988Go, 1992Go) or X-inactivation mosaic analysis (Tan and Breen, 1993Go; Tan et al., 1995Go) have provided a picture of diverse migratory routes, including evidence for both directly radial and less direct patterns. Direct studies of cortical cell migration in explant or slice cultures (O'Rourke et al., 1992Go; Fishell et al., 1993Go) have also shown the predominantly radial routes of some cells, but the strikingly non-radial routes of other cells. What is the mechanistic basis for these two widely differing clonal and migratory patterns?

Several lines of evidence suggest that cortical neurons derive from a heterogeneous population of progenitor cells with very different migratory properties and progeny. Retroviral studies suggested that distinct progenitors give rise to pyramidal and non-pyramidal neurons (Parnavelas et al., 1991Go), but this idea was extended with the observation that clones of pyramidal and non-pyramidal neurons showed distinct patterns of clonal organization (Mione et al., 1994Go). Although some clones expressed both pyramidal and non-pyramidal features, especially shortly following retroviral labeling (Lavdas et al., 1996Go), pyramidal neurons (defined either by electron microscopy, or by the expression of glutamate as a neurotransmitter) tended to be found in large radial clusters, whereas non-pyramidal neurons tended to be found in non-radial, widespread patterns (Mione et al., 1994Go, 1997Go). The notion that distinct progenitors preferentially produce widespread clones of mainly GABAergic neurons, or radial clones of mostly glutamatergic neurons, has recently been very elegantly extended and refined using highly unbalanced chimeras and X-inactivation mosaics (Tan et al., 1998Go). Large radial clones consisted of >90% glutamatergic neurons, whereas widespread ‘horizontal' clones contained 51–92% GABAergic (average = 75%) neurons, with the remaining cells glutamatergic (Tan et al., 1998Go). The general notion that cortical clones consist of two fundamentally distinct types both topographically and physiologically appears able to reconcile most of the existing data from retroviral lineage studies, retroviral library analysis, and in vitro time-lapse studies (see Discussion). Although large radial clones only form up to 70% of cortical neurons and perhaps less and are apparently absent from far lateral neocortex (Tan et al., 1995Go), they include most pyramidal neurons and form a potential source of positional information as first proposed by Rakic (Rakic, 1978Go, 1988Go).

One of the most remarkable recent discoveries regarding the origins of cortical neurons is the observation that a substantial fraction of GABAergic, inhibitory cortical interneurons actually originates outside of the cortex altogether, in the proliferative zone that gives rise to the striatum. This area is called the lateral ganglionic eminence (LGE) (Anderson et al., 1997Go; Tamamaki et al., 1997Go). Neurons that express Dlx-1 and Dlx-2 arise here, and migrate through the striatum and into the overlying cerebral cortex (Anderson et al ., 1997Go). Interestingly, transplantation studies of striatal precursors into the cortex had previously shown that the striatal cells could take on cortical fates (Fishell, 1995Go). The formation of inhibitory neurons outside the cortex is especially intriguing because the same proliferative region, the SVZ of the striatum, is a persistent source of inhibitory interneurons for the olfactory bulb (Alvarez-Buylla, 1990Go; Luskin, 1993Go; Reid et al., 1999Go). This suggests that inhibitory interneurons destined for large portions of the forebrain could arise from a common progenitor pool. The origin of some cortical neurons from the LGE begs the question whether all widespread/inhibitory cortical clones derive from the LGE, or whether widespread clones have more than one origin (e.g. from both the VZ of the LGE and the VZ of the cortex proper). In chimera experiments (Tan et al., 1998Go) most, though not all, experiments with widespread clonal patterns also showed labeled cells in the underlying striatum, suggesting that the striatum is the major, if not only, source of widespread clones. This question is difficult to address with current techniques, since there is no simple in vivo method that selectively labels just the striatal VZ or cortical VZ progenitors in clonal fashion.

Since evolutionary models (Rakic, 1988Go) have suggested potential phylogenetic expansion of the cortex by addition of radial modules, the question also arises whether similar patterns of widespread and radial clones are conserved among species with larger cerebral cortices. Cell lineage studies in monkey (Kornack and Rakic, 1995Go) have demonstrated both radial and horizontal patterns of retrovirally labeled neurons. However, very large radial clusters have not been reported in primates, and in ferrets (members of the order Carnivora), retroviral library analysis of cortical clones at midto late corticogenesis demonstrated striking numbers of widespread clones that covered large portions of the cortical mantle with little evidence for radial clustering (Reid et al., 1997Go). The average multineuron clone labeled at E33–E35 in ferret covered 40% of the cortex, while the radial clusters of labeled neurons were seen quite rarely. This may, however, be a reflection of the relatively later stage of development at which the labeling was performed.

The present study was undertaken to determine clonal relationships at earlier stages of ferret neurogenesis using the retroviral library technique. Rodents are very difficult to approach surgically at early stages, although this may soon change with improved technology (Olsson et al., 1997Go). The larger ferret embryo is somewhat easier to mark with retroviruses at early stages. We have successfully labeled the ferret cortex with retroviral libraries at E27–E29, when some of the prelate and subplate cells are still being generated, and before most cortical plate cells are generated. We find that clonal patterns at E27–E29 are markedly different compared to E33–E35 injections. Occasional widespread clones are still seen, but are relatively less common, and instead small (two to four neurons), tightly clustered clones are frequent. Most strikingly, we occasionally observed very large, radial clusters that contained up to 150 cells which are similar in their overall appearance to the radial clones described by Tan (Tan et al., 1998Go). The large clusters account for a small portion of all clones, but a significant fraction (38%) of all retrovirally labeled cells. Interestingly, we did not see evidence that large radial clones and widespread clones were derived from a common progenitor, suggesting that theses two clonal types may diverge at a very early stage of development, consistent with the possibility that they emerge from distinct geographic regions of the neuraxis. Some of these data have been previously presented in abstract form (Ware et al., 1997aGo).


    Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Alkaline Phosphatase (AP)-encoding Amphotropic Retroviral Library

The preparation and composition of the amphotropic retroviral library used for these experiments is described elsewhere (Reid et al., 1997Go). In brief, an ecotropic retroviral library that infects only rodent cells (Reid et al., 1995Go; Walsh, 1995Go), and that contains 400–2000 retroviral constructs, was repackaged to form an amphotropic virus by infection of the amphotropic producer cell line CR7, a subclone of the {Psi}CRIP producer line (Danos and Mulligan, 1988Go) with the ecotropic library. Producer cells (>20 000 colonies) were then grown in G418 to select for neomycin-resistant producer colonies. The amphotropic library then was isolated as the supernatant removed from confluent plates of producer cells, showed a titer of 2 x 106 cfu/ml, and was concentrated x50 before use.

Animal Surgery

All animal housing and experimentation were performed according to protocols approved by the IACUC of Beth Israel Deaconess Medical Center and Harvard Medical School. Timed-pregnant ferrets were purchased from Marshall Farms (North Rose, NY, USA). Pregnancies were timed from the day after breeding (E0). Birth usually occurred on E42. Pregnant mothers were anesthetized with Nembutal or inspired halothane. The uterus was exposed at embryonic days 27 (E27), E28 or E29 by a midline incision and transilluminated to facilitate identification of fetal skull landmarks. Concentrated amphotropic AP-encoding virus (2–8 µl ) containing 80 mg/ml polybrene (Sigma, St Louis, MO, USA) and 0.05% trypan blue was injected through the uterine wall and fetal membranes into the lateral ventricles of each fetus using pulled glass capillary pipettes (Drummond Instruments). The accuracy of the injection was monitored by direct observation, since the trypan blue caused the ventricular system to appear blue. Incisions were closed with sutures and staples and animals were returned to their cages to allow development to continue normally. Kits were born vaginally, and nursed by the operated mother. On postnatal day 0 (P0), P8, or P24 the kits were killed by an overdose of Nembutal and perfused with 2–4% paraformaldehyde in 2 mM MgCl2 and 1.25 mM EGTA in 0.1 M PIPES buffer (pH = 7.2).

Histology and Analysis of Clones

Brains were removed and submerged in fixative overnight at 4°C, then transferred to 30% sucrose in phosphate-buffered saline (PBS) at 4°C until they sank. Brains were sectioned at 100 µm thickness using a Bright cryostat. Sections were mounted onto gel-coated glass slides and processed for AP activity according to protocols presented elsewhere (Cepko et al., 1995Go). Labeled cells were detected by microscopic examination of tissue sections, and cell morphology and location were recorded by photography and/or camera lucida drawings. AP staining of cell bodies and processes allowed identification of most labeled cells as presumptive neurons or glia by morphological criteria (Fig. 1Go). Rostral– caudal location was determined by counting frontal section number, using the rostral extreme of the forebrain as 0, and multiplying by the section thickness (generally 100 µm).



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Figure 1.  Morphology of retrovirally infected cells. Retrovirally encoded AP allowed for the morphological identification of many labeled neurons. Labeled cells were present in all layers of the cortex and included cell types produced during both earlier and later stages of development. Many of the cells observed were singly labeled cells (as in AE) or in small cell clusters (as in F). AC Cerebral cortical neurons labeled at E27 and analysed at P0 (experiment 6, Table 1Go). D and F Cells labeled at E29 and analysed at P24 (experiment 20, Table 1Go). E Cell labeled at E28 and analysed at P0 (experiment 9, Table 1Go). A Radially migrating cell (presumably a neuron) crossing the VZ/IZ border. B An embryonic Cajal–Retzius cell in layer I. C Young fusiform neuron with a long descending axon. D Layer II/III basket cell. E Immature neuron in layer VI with descending processes. F Cluster of four bipolar neurons in layer III of the medial cortex. Scale bar is 50 µm.

 
Clonal Analysis with PCR

Tissue analysis was performed by preparing DNA samples from labeled cells for amplification by PCR, as presented elsewhere (Walsh and Cepko, 1992Go; Walsh, 1995Go). Briefly, coverslips were removed in a 50 ml centrifuge tube filled with sterile water. Small tissue fragments (~100 µm x 200 µm x 200 µm) containing the nucleus of each labeled cell were dissected using a fresh razor blade edge for each cell. Tissue fragments were digested in 10 µl of proteinase K (0.2 mg/ml) in 1 x PCR buffer (2.5 mM MgCl2, 50 mM Tris buffer, pH = 8.3, 25 mM KCl, 0.5% Tween-20) at 65°C for 4–24 h. Each well was covered with 30 µl of mineral oil to prevent evaporation. Samples were then heated to 85°C for 20 min to inactivate proteinase K and then 95°C for 5 min to denature the DNA. A nested PCR protocol was employed to increase the sensitivity and specificity of amplification and is described elsewhere (Walsh and Cepko, 1992Go; Walsh, 1995Go). At least 10% of all PCR reactions were negative controls, consisting either of unlabeled tissue, or reagents alone. No experiments contained false PCR-positives.

Analysis of PCR Products

The PCR products from the second PCR reaction were separated on a 3%/1% NuSieve/Seakem agarose gels to determine tag sizes. Each tag was then digested with CfoI, RsaI, AluI, MseI, and MspI. Finally, digested samples of similar predigest size were run side by side on agarose gels to allow direct comparison of restriction fragment sizes. Cells from which a PCR product of indistinguishable size and restriction enzyme digestion pattern was amplified were interpreted as arising from a common progenitor; cells with distinguishable tags were interpreted as having arisen from separate progenitors.


    Results
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The ferret's short gestation period (41–42 days), coupled with a protracted period of cortical neurogenesis (35 days), makes this species ideal for the study of early cortical development. Neurogenesis in the ferret cerebral cortex begins at E19 and continues until P14 (where P0 = E42) (Jackson et al., 1989Go). The preplate is formed from E19–E29, and the first cortical plate neurons, which reside in layer VI, appear on E25 (Jackson et al., 1989Go). In this study, retrovirus was injected when preplate neurons and early cortical plate neurons are born, and labeled hemispheres were analysed before neurogenesis was complete. Because the postnatal survival of injected animals was low, many ferrets were analysed at P0–P8, while labeled progenitors are still proliferating and their progeny are still migrating. The age at injection and analysis also biases labeling toward neurons as glial production in the ferret occurs later in development (Jackson et al., 1989Go). The active proliferation and migration at the time of analysis produced a consistent underestimation of the number of cells generated and of the final distance between migrating sibling cells. To correct for distances between sibling cells caused by analysis at different ages, we have converted all distances to percentages of the rostral–caudal length of the cortex at the time of analysis.

Spatial Distribution of Retrovirally Labeled Cells

In each of the 21 hemispheres successfully injected and AP-stained, retrovirally labeled cells most commonly consisted of dispersed single labeled cells and/or small clusters of two to four labeled cells (Table 1Go). Single labeled cells refer to labeled cells with a nearest neighbor of greater than 3% of the rostral–caudal cortex, and small cell clusters are labeled cells within 3% of the rostral–caudal cortex (Fig. 1Go). As in previous studies (Austin and Cepko, 1990Go; Parnavelas et al., 1991Go; Walsh and Cepko, 1992Go; Mione et al., 1994Go; Reid et al., 1995Go), cells within small clusters in this study contained two to four phenotypically similar cells usually within the same laminae of the cortex, suggesting similar birthdates. The single cells and small clusters generally resembled the pattern of cell labeling described previously in the mouse (Luskin et al., 1988Go; Austin and Cepko, 1990Go), the rat (Price and Thurlow, 1988Go; Walsh and Cepko, 1988Go; Parnavelas et al., 1991Go; Luskin et al., 1993Go) and ferret after E29 labeling (Reid et al., 1997Go). However, compared to retroviral labeling at E33–35 ages in ferret, small clusters of retrovirally labeled neurons were notably more common in the present material.


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Table 1 Overview of experiments
 
Besides the pattern of single cells and small cell clusters, we also occasionally observed strikingly large radial clusters of closely associated cells in considerably larger numbers than previously observed in retroviral studies in the rat, mouse, ferret, or primate. Based on the morphology of cells in these clusters as well as the early age of injection and analysis, we have identified many of the cells within these radial clusters as neurons (Fig. 2Go), although morphological criteria cannot rule out the inclusion of a few glia in these clusters. The radial pattern was markedly different from the more scattered pattern of AP labeling, since radial clusters contained many more cells than the small cell clusters and covered multiple layers of the cortex (Figs 2 and 3GoGo). Although the large radial clusters were somewhat uncommon (occurring in only 6/21 experiments as a single cluster in each experiment) given their striking nature and resemblance to the large radial clusters described previously (Tan et al., 1998Go), we have analysed them in some detail.



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Figure 2.  Hemispheres contained labeled neurons in large radial clusters. A Camera lucida drawing of a radial cluster labeled at E29 and analysed at P8 (experiment 12, Table 2Go). The large radial cluster consists of 150 cells and has a diameter of 9% of the rostral–caudal cortex. Radial cluster includes mature neurons in the subplate and all layers of the cortical plate as well as migrating neurons. B Camera lucida drawing of a radial cluster labeled at E29 and analysed at P8 (experiment 11, Table 2Go). The radial cluster contains 25 mature neurons and has a diameter of 7% of the rostral–caudal length of the cortex (measured at approximately layer III/IV). C Picture of layer V pyramidal cell with long apical dendrites ascending towards the pial surface, located within the radial cluster in A. D Picture of a layer III pyramidal cell with an ascending dendrite. Arrows in A and C indicate the same cell; arrowheads in B and D indicate same cell. Radial cluster diameters measured at approximately layer III/IV of the cortex. Scale bar in A and B is 500 µm, scale bar in C and D is 50 µm.

 


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Figure 3.  Camera lucida plots of large radial clusters. The location of cells within each radial cluster is indicated by filled circles. A Drawing of radial cluster in experiment 1. This radial cluster is located in the medial frontal cortex and contains 20 labeled cells. Cells drawn are within a rostral–caudal distance of 300 µm. B Drawing of radial cluster in experiment 14. This radial cluster is located in the frontal cortex and contains 10 labeled cells. All cells drawn are within a rostral–caudal distance of 400 µm. C Drawing of radial cluster in experiment 8. This radial cluster is located in the frontal cortex and contains 30 cells. All cells drawn are within a rostral–caudal distance of 200 µm. D Drawing of radial cluster from experiment 18. This radial cluster is located in the medial occipital cortex and contains six labeled neurons. All cells drawn are within a rostral–caudal distance of 200 µm. The clonal relationship of these neurons was confirmed by PCR analysis (see Table 3Go, clone 15). Scale bars are 1 mm.

 
Analysis of Radial Clusters

Large radial clusters are most clearly distinguished from the smaller clusters of retrovirally labeled cells observed here and in previous studies by the number of cells per cluster (Table 2Go). Radial clusters identified in this study showed 6–150 cells per cluster with an average of 40 cells per cluster. The large numbers of labeled cells in these clusters gave them a density and radial coverage that was not so clearly seen in the smaller clusters seen in other retroviral studies. Moreover, the large numbers of labeled neurons in each cluster caused them to account for a substantial fraction of all retrovirally labeled cells observed in this study: the six radial clusters accounted for 241 of 637 (38%) of the labeled cells in this study (Table 1Go).


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Table 2 Analysis of radial clusters
 
Despite the large numbers of neurons contained in the radial clusters, they were mainly confined to small cortical regions. Large clusters showed diameters of 1–9% of the rostral–caudal cortex (measured at approximately layer III/IV, which represented the radial midpoint of radial clusters in the cortex), with an average diameter of 5% of the rostral–caudal length of the cortex. Thus the diameter of the larger clusters is similar to the diameter of smaller neuronal clusters observed in this study and previous retroviral studies (Figs 2 and 3GoGo), but the large clusters contain a much denser population of labeled cells in the same region. Moreover, the large radial clusters were quite coherent with sharp borders, and did not show an obvious tendency towards containing widespread sibling cells in the near vicinity: outside of the border of the large clusters, the density of scattered labeled cells dropped off rapidly.

Another feature that distinguished the large radial clusters observed here from the smaller clusters observed in most earlier retroviral studies is that the large radial clusters contained cells in many different layers of the cortex (Figs 2 and 3GoGo), indicating that progenitors producing radial clusters are mitotic throughout most of cortical neurogenesis. Moreover, migrating cells were present in three of six radial clusters (experiments 1, 8, and 12). Because these radial clusters were analysed while the later-born upper layer neurons were being generated, the presence of young migrating presumptive neurons in these radial clusters suggests that radial cluster progenitors were mitotic shortly before analysis, as late as P8. Thus, despite the absence of the radial pattern of labeling after later injections (E33–E35), progenitors producing radial clusters are apparently actively mitotic during the later stages of development, as late as P8.

The large radial clusters were observed in several different areas of the cortex (Figs 2 and 3GoGo), though the small number of clusters observed does not allow a comprehensive analysis of their topographic distribution. Three of six radial clusters identified were in the frontal cortex, two of six were in the parietal cortex, and one of six was in the occipital cortex (Table 2Go). Additionally, three of six radial clusters were in the medial cortex, while three of six were more laterally, though none was in the extreme lateral-most portion of the neocortex. Although the small number of radial clusters labeled here did not allow us to determine if the radial clusters occurred preferentially in different areas of the cortex, it did not appear that radial clusters were restricted to a single region of the cortex.

Clonal Analysis by PCR

To determine the clonal relationship of the AP-labeled cells, we performed PCR amplification of retrovirally encoded DNA tags. Cells containing the same tag were interpreted as siblings derived from a single retroviral infection. Given the complexity of the library [100–400 tags represented roughly equally, with as many as 2000 potential tags (Reid et al., 1997Go)], the possibility of clones derived from two different progenitors coincidentally infected with the same tag is <5% for experiments with fewer than four clones and <40% for experiments with fewer than eight clones (Walsh and Cepko, 1992Go). PCR amplification was successfully performed on four cerebral hemispheres infected with retrovirus at E27 and E29 representing 23 clones (Table 3Go, Fig. 4Go). The PCR efficiency of experiments included in this study ranged from 45 to 86%. Experiments with PCR efficiency of less than 40% were excluded from further analysis, since they showed a large number of tags seen in single cells only. Because PCR analysis is less than 100% successful, the number of sibling cells per clone is generally underestimated. Since the precise degree of underestimation could not be determined for each clone, we did not apply a numerical correction. However, on average the clone size is expected to be between 1.2 and 2.2 times higher than the number indicated.


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Table 3 PCR analysis of labeled hemispheres
 


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Figure 4.  Camera lucida plots of widespread multineuron clones. Widespread clones appear to consist of neurons in different functional areas of the brain. AF Camera lucida drawings of coronal slices of a hemisphere containing two widespread clones. The open circles represent clone 5 (Table 3Go, experiment 5), and the filled circles represent clone 6 (Table 3Go, experiment 5), although one cell (5400 µm) is not illustrated. Distances indicated below each drawing represent the rostral–caudal distances of cells from the rostral tip of the olfactory bulb. Scale bar is 1 mm.

 
PCR analysis of four experiments (one injected at E27 and analysed at P0, and three injected at E29 and analysed at P24) showed a predominance of small clustered clones. Three of 23 clones (13%) consisted of single cells, 15 (65%) were small clusters containing two or three PCR-positive cells within a single cluster. Three clones (13%) were widespread, defined as having siblings separated by at least 10% of the rostral–caudal length of the cortex. Finally one clone (4%) was a large radial cluster, with six PCR-positive neurons in a single radial cluster (Table 3Go). Of the PCR-positive cells, three of 54 cells (6%) were from single-cell clones, 29 (54%) were from small clustered clones, 16 (30%) were from widespread clones, and six (11%) were from the large radial clone (Table 3Go, Fig. 4Go). Although proportionately far fewer progenitors at E27–E29 produce widespread clones than at E33–E35, widespread clones still account for about one third of all retrovirally labeled cells even after early injections, and still show dispersion over wide cortical areas. In contrast, small clustered clones and large radial clusters account for a much higher proportion of retrovirally labeled neurons than after later injections. PCR analysis of the large radial cluster in experiment 18 (Fig. 3DGo) verified that cells in this cluster were all clonally related, and that this cluster was not part of a widespread clone. Since PCR analysis is based on a small number of cells, it does not exclude the possibility that other large radial clones may have widely dispersed siblings.

PCR analysis confirmed that sibling cells in small clusters usually occurred within the same layer or in adjacent layers, as observed in previous retroviral studies. Of the mature two-cell clusters analysed by PCR (Table 3Go), nine of 14 (64%) showed identical laminar location. Although morphology could only be studied incompletely, small clusters appeared to contain cells with similar morphology as well. This observation is consistent with previous findings in later injections in the rat (Walsh and Cepko, 1992Go; Reid et al., 1995Go). Clusters of deep-layer neurons also showed similar morphological and laminar fate, with one clone composed of a layer VI neuron and a subplate neuron (Table 3Go, clone 17), and another composed of a layer V neuron and a layer VI neuron (Table 3Go, clone 20). The similarities between small clusters generated early in development with those generated later suggests that the processes that produce small clusters in earlier development persist to produce similar patterns later in development. Interestingly, however, these deeper-layer clusters did not obviously contain sibling cells in upper layers of the cortex in the same or different cortical regions, suggesting that the clusters may arise from a progenitor that does not continue dividing throughout neurogenesis.


    Discussion
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In this report, we used an amphotropic retroviral library to determine whether cortical lineage patterns observed with retroviral injections during the late preplate stage and early cortical plate stage are similar or different to patterns previously observed after later injections. Consistent with findings with later injections (Reid et al., 1997Go), we observed that clones labeled at E27–E29 commonly consisted of small clusters of neurons, single neurons and widely dispersed multineuron clones. In addition, however, we observed a striking radial pattern of clonal dispersion in a small but significant number of experiments. The radial pattern is seen only after early progenitor labeling, and radial progenitors appear to coexist with progenitors of single cell clones, small clustered clones, and widespread clones.

Radial and Non-radial Clones

Analysis of highly unbalanced chimeras (Tan et al., 1998Go) and previous retroviral experiments (Parnavelas et al., 1991Go; Mione et al ., 1994Go, 1997Go) has suggested that large radial clusters and widespread clones are lineally distinct, and several lines of evidence in this study confirm that. First, in several experiments (experiments 8, 11, 12) the large radial cluster accounts for the majority of the labeled cells in the entire experiment, suggesting that the progenitor(s) that produce the large radial cluster give rise to few if any widely scattered cells. Second, large radial clusters were only labeled in a minority (6/21) of experiments, with most experiments showing only labeled single neurons or small clusters of neurons; since progenitors of large radial clusters are rarely labeled, the progenitors of scattered neurons or small clusters usually seem to be distinct from progenitors of large clusters. Third, large radial clusters, when labeled, generally had sharp borders without obvious widely scattered neurons near the edges. Fourth, limited PCR analysis also showed that a large cluster found in an experiment with many scattered labeled cells and small clusters of cells (experiment 18) nonetheless was clonally distinct from other labeled cells.

While large radial clones and widespread clones may be two distinct extreme patterns, the small clonal clusters appear to be less distinct in their identity, and may relate both to widespread clones or to radial clones (Fig. 5Go). For example, small clusters have been commonly seen as ‘subunits' of widespread clones in rat (Reid et al., 1995Go), and widespread clones in ferrets also can contain some neurons that are individually clustered (Table 3Go, and see Reid et al., 1997). On the other hand, some small clustered clones seem likely to represent products of the same progenitor types that produce large labeled clusters after earlier labeling. Birthdating studies have suggested that, once cortical plate formation begins, a large number of cortical progenitors may become extinguished after relatively few cell cycles; these observations suggest that cortical clone size may change radically when labeling is performed at slightly different ages (Takahashi et al., 1997Go). Therefore, smaller clusters of retrovirally labeled neurons may be heterogeneous with regard to their derivation and progeny, representing portions of clones that, if labeled earlier, would form either large radial or widespread clones (Fig. 5A, BGo). This interpretation appears to fit nicely with the observation of small or large clusters of retrovirally labeled cells that are often homogeneous anatomically or neurochemically (Mione et al., 1994Go, 1997Go).



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Figure 5.  Schematic summary of a model of cortical neurogenesis based on lineage data from many sources. Two basic clonal types are shown, though there may be more. Large radial clustered clones (blue) form neurons, predominantly pyramidal, in most or all cortical layers. After early retroviral labeling (or using chimera analysis or X-inactivation mosaics) the large radial clusters can be seen (A), but labeling late in neurogenesis produces only small clusters of labeled cells (B). Widespread progenitors (purple) are formed in part in the LGE (A), though additional sources of widespread clones are not ruled out. They form mainly inhibitory interneurons that seem to take very complicated migratory trajectories to the cortex. After later labeling (B), these widespread clones are still clearly seen.

 
The overall appearance of the large radial clusters and widespread clones in the ferret suggest that they may represent the same fundamental biological mechanism in Carnivora as previously observed in rodents. Although the radial columns described by Tan (Tan et al., 1998Go) contain larger numbers of neurons than the radial clusters described here, the gross appearance of radial columns labeled by the two techniques is quite similar. The radial columns observed by Tan (Tan et al., 1998Go) contained neurons in quantal multiples of 600, suggesting that a single precursor in the cerebral vesicle produces 600 labeled neurons, or the result of 10 cell cycles, in the mature mouse cortex. The radial patterns that we observed, with an average of 40 cells per radial cluster, consisted of far fewer cells than the radial clones observed in chimera experiments. Our largest radial cluster contained 150 cells, the result of at least eight cell cycles. Our injections were made into the ferret at E27–E29, and since preplate development begins at E20, it is possible that we labeled single progenitors several cell cycles after the onset of preplate development. Moreover, most of the injected ferrets were harvested from P0 to P8, while the cortical plate cells were still being generated. Thus, in most experiments, we harvested animals before labeled progenitors had completed proliferation.

The retrovirally labeled clusters do not form simple quantal multiples in terms of cell number. However, the integration of retroviruses into daughter cells of infected cells makes retrovirally labeled clones subject to large variability, and tends to be subject to ‘lineage tree' extinction (Takahashi et al., 1997Go) effects that limit the size of labeled clones as well. Therefore, we believe that the differences in the number of cells seen in these experiments may be primarily due to the timing of injections and analysis, in addition to differences between ferret and mouse cortical development. The radial clusters here may represent a subset of clones described in unbalanced chimera experiments. Given the differences in these two techniques, it is particularly appealing that we have identified radial clusters that may be the same patterns as the radial clusters observed in chimera experiments.

Although the very large radial clusters have not been observed previously with retroviral labeling, smaller radially aligned clones or up to 23 neurons have been labeled at later stages of corticogenesis the rat (Walsh and Cepko, 1988Go; Luskin et al., 1993Go; Mione et al., 1997Go) and in the monkey (Kornack and Rakic, 1995Go). In monkeys, hemispheres labeled between E63 and E68, when 40% of cortical neurogenesis is complete, contained arrays of two to six cells with 54% of the total arrays composed of only two cells. Similarly, E16 injection of retrovirus into the developing rat cortex produced clones showing radial migration in up to seven neurons in different layers of the cortex (Mione et al., 1997Go). Interestingly, only pyramidal neurons were seen in these radial clones, whereas non-pyramidal neurons were found in pairs or as single neurons. The radial clones observed in the rat and monkey are similar to parts of the larger radial clusters observed here. Taken together, these findings suggest that radial clones are present in the cortex of a number of species and that the pattern of cell proliferation and migration involves biases at different times in development towards symmetric and asymmetric division.

Given that a substantial proportion of inhibitory interneurons of the cerebral cortex are formed in the lateral ganglionic eminence (Anderson et al., 1997Go; Tamamaki et al., 1997Go), and given that inhibitory interneurons are more commonly found in the widespread clones, the most parsimonious model (though probably too simple) is that widespread clones are preferentially if not exclusively derived from the LGE and/or other sources outside of the cortical VZ/SVZ. The observation that widespread and large radial clones are mainly if not exclusively lineally distinct, even at the earliest stages of cortical neurogenesis, is most consistent with the derivation of widespread and radial clones from two different, non-overlapping sources. However, this remains uncertain and it is still possible that some widespread cortical clones derive from the cortical ventricular zone as well.

Radial and Non-radial Migration

The observation that there are distinct lineages for widespread and radial clones appears to resolve a number of studies of migration into the cortex. Time-lapse imaging has shown a variety of radial and non-radial migratory routes between the VZ and the cortex. For example, there is rapid dispersion of cortical cells in the ventricular zone of the cerebral cortex (Fishell et al., 1993Go; Walsh and Cepko, 1993Go; Neyt et al., 1997Go; O'Rourke et al., 1997Go), and there is radial and non-radial migration in the intermediate zone (O'Rourke et al., 1992Go, 1995Go, 1997Go). However, virtually all non-radially migrating cells in the layers beneath the cortex are postmitotic (Neyt et al., 1997Go; O'Rourke et al., 1997Go). The derivation of widespread clones from the LGE would produce a population of non-radially migrating cells in the layers beneath the cortex that might be expected to be postmitotic, since they are far from the respective proliferative zone. Taken together, these studies show that patterns of cell migration change markedly over time, that there are many migratory pathways into the developing cortex, and that cortical precursors sometimes derive from distant sites.

Relationship to Genetic Defects in Neuronal Migration

It remains a remarkable fact that two sets of progenitor cells give off similar-appearing neurons that appear to migrate through the same terrain of the subcortical intermediate zone according to completely different rules. These differences may arise simply from the location of the progenitors (i.e. in the cortical VZ versus the LGE), or from the extensive interconnection of cortical VZ cells by gap junctions (Bittman et al., 1997Go), but more likely reflects two distinct genetic programs. For example, LGE progenitors are characterized by expression of Dlx-1, 2, and other family members, whereas the cortical VZ cells express Emx-1, 2, and other distinct transcription factors (Rubenstein and Lai, 1999). These transcription factors may control very distinct sets of downstream targets in the form of adhesion molecules that control differential adhesion to radial glial cells. For example, neuronal outgrowth in distinct neuronal populations shows opposite effects in response to applied netrins (Shirasaki et al . 1996Go), and neuronal migration in C. elegans also shows cell-specific effects of netrin homologues (Hedgecock et al., 1990Go). Similar guidance cues in vertebrates may have differential effects on distinct populations that ultimately reach the cerebral cortex.

A number of single gene mutations disrupt the migration of neurons into the cerebral cortex and some may ultimately shed light on the differing genetic programs underlying the distinct migrational patterns of widespread and clustered clones. For example, in mice that carry mutations in the reelin gene (Caviness, 1982Go) or mdab1 (Gonzalez et al., 1997Go; Howell et al., 1997bGo; Sheldon et al., 1997Go; Ware et al., 1997bGo), the cortical preplate fails to split normally, with a consequent secondary disruption of the layers of the cortical plate. Reelin encodes a large secreted protein expressed only in the marginal zone of the cortex (D'Arcangelo et al., 1995Go); mdab1 encodes a protein that was first isolated via its binding to non-receptor tyrosine kinases such as Src and Abl (Howell et al., 1997aGo), and is homologous to the fly disabled gene that is so named because it interacts genetically with fly Abl in developing neurons (Gertler et al., 1993Go). mDab1 protein contains a phosphotyrosine binding (PTB) domain similar to domains in Shc and Numb that interact with transmembrane receptors. mdab1 is expressed in a complementary pattern to Reelin [in the migrating cortical plate neurons and not in the marginal zone (Howell et al., 1997bGo; Sheldon et al., 1997Go; Rice et al., 1998Go)] and Reelin expression is normal in mdab1 mutant mice (Gonzalez et al., 1997Go). Therefore, Reelin and mDab1 are thought to represent an extracellular ligand and intracellular adapter protein that may each bind to a common receptor, though that receptor is unknown at present. The Reelin-mDab1 signaling pathway appears to be critical to allowing cortical plate neurons to divide the preplate, and to allow later-born cortical plate neurons to migrate past earlier-born cortical plate neurons, although how this happens is also not clear. Mutations in the cdk-5 kinase gene (Ohshima et al., 1996Go; Gilmore et al., 1998Go), or its regulator, p35 (Chae et al., 1997Go), also cause inversion of the layers of the cortical plate, though some splitting of the preplate occurs in these mutants. Therefore, these proteins may represent part of a biochemical effector pathway for Reelin/mDab1, or may form a separate pathway.

It is unknown whether the several mouse mutations that alter cortical neuronal migration affect widespread clones or radial clones equally, or whether the mutations affect the two migratory patterns differentially. Since all of the known mutations affect the vertical or radial organization of the layers of the cortex, it seems unavoidable that the radial type of migration, i.e. that which occurs in relation to radial glial fibers, is affected. However, migration from the LGE occurs largely or completely independently of radial glial fibers. Hence, one possibility is that some of these genes may be required for normal migration of radial clones but expendable for non-radial migration from the LGE. These possibilities can be evaluated by direct analysis of clonal patterns in mutant mice.

A number of single gene mutations affect neuronal migration to the cortex in humans as well, though their modes of action are even less well understood. Some neuronal migration defects in humans are associated with lissencephaly (‘smooth brain') and can be caused by genes such as LIS1 (PAFAH1ß1) (Reiner et al., 1993Go) and doublecortin (des Portes et al., 1998Go; Gleeson et al., 1998Go). Although doublecortin is a likely substrate for c-Abl (des Portes et al., 1998Go; Gleeson et al., 1998Go), implying potential biochemical links to mDab1, there is accumulating evidence that PAFAH1ß1 and DCX proteins may regulate migration via actions on microtubules. PAFAH1ß1 has a strong homologue in Aspergillus nidulans (nudF), which is required for the translocation of nuclei along the fungal mycelium (Xiang et al., 1995Go), and which interacts genetically with other genes (nudA, nudC, nudE, etc.). These genes encode, among other things, the heavy chain of dynein, which in neurons represents a microtubule-based motor (Xiang et al., 1994Go; Morris et al., 1998Go). Furthermore, PAFAH1ß1 itself binds in part to microtubules and regulates microtubule dynamics (Sapir et al., 1997Go). Preliminary evidence (Gleeson, Lin, Flanagan, and Walsh, unpublished observations) also implicates DCX protein in regulating microtubule polymerization. Moreover, the phenotype of mouse mutations in PAFAH1ß1 is quite distinct from that of Reelin, mdab1, p35, or cdk5, consistent with the involvement of PAFAH1ß1 in different cellular mechanisms (Hirotsune et al., 1998Go). Given the dramatic changes that these mutations induce in the patterns of radial organization of cortical neurons, it will be interesting to determine whether these mutants show differential effects on the radially clustered versus widespread clonal patterns.

One neuronal migration disorder that seems to show an intriguing differential effect on different migratory patterns is the human syndrome of periventricular heterotopia, due to mutations in the X-linked gene encoding a large cytoplasmic actin-binding protein called filamin 1 (Fox et al., 1998Go). In PH, ‘heterotopic' neurons collect in the ventricular zone beneath the cerebral cortex (Eksioglu et al., 1996Go), and generally form large nodules. These neurons are highly developed and most of them are pyramidal in morphology (Eksioglu et al., 1996Go). Interestingly, the basal ganglia in PH have never been noted to show heterotopic neurons as would be expected if the migration of cortical neurons that are formed in the LGE were also arrested. This may imply that filamin 1 (FLN1) is selectively required for the highly radial migration that takes place between the cortical ventricular zone and the cortex, but may be expendable for the migration of cells from the LGE to the cortex that requires Dlx-1 or 2, and perhaps other downstream genes. However, careful review of human specimens, and analysis of an animal model with FLN1 mutations, will be required to determine whether there are specific defects in migration of specific clonal types, and whether that implicates interaction of filamin with receptor systems specific to radial versus widespread migration.


    Notes
 
The authors thank Brett Tallent and Lee Zhang for technical assistance with experiments. M.L.W. and C.B.R. were supported by grants from the NIGMS. C.A.W. was supported by NINDS 1RO1 NS32457, National Alliance for Research in Schizophrenia and Depression, and the National Alliance for Autism Research. C.A.W. was a scholar of the Rita Allen Foundation.

Address correspondence to C.A. Walsh, Division of Neurogenetics, Beth Israel Deaconess Medical Center/Harvard Medical School, Harvard Institutes of Medicine, 77 Avenue Louis Pasteur, Boston, MA 02115, USA. Email: cwalsh{at}caregroup.harvard.edu.


    References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Alvarez-Buylla A (1990) Commitment and migration of young neurons in the vertebrate brain. Experientia 46:879–882.[ISI]

Anderson SA, Eisenstat DD, Shi L, Rubenstein JL (1997) Interneuron migration from basal forebrain to neocortex: dependence on Dlx genes. Science 278:474–476.[Abstract/Free Full Text]

Austin CP, Cepko CL (1990) Cellular migration patterns in the developing mouse cerebral cortex. Development 110:713–732.[Abstract]

Bittman K, Owens DF, Kriegstein AR, LoTurco JJ (1997) Cell coupling and uncoupling in the ventricular zone of developing neocortex. J Neurosci 17:7037–7044.[Abstract/Free Full Text]

Caviness VS Jr (1982) Neocortical histogenesis in normal and reeler mice: a developmental study based upon [3H]thymidine autoradiography. Brain Res 256:293–302.[Medline]

Cepko C, Ryder EF, Austin CP, Walsh C, Fekete DM (1995) Lineage analysis using retrovirus vectors. Methods Enzymol 254:387–419.[ISI][Medline]

Chae T, Kwo YT, Bronson R, Dikkes P, Li E, Tsai LH (1997) Mice lacking p35, a neuronal specific activator of Cdk5, display cortical lamination defects, seizures, and adult lethality. Neuron 18:29–42.[ISI][Medline]

D'Arcangelo G, Miao G, Chen S, Soares H, Morgan J, Curran T (1995) A protein related to extracellular matrix proteins deleted in the mouse mutant reeler. Nature 734:719–723.

Danos O, Mulligan RC (1988) Safe and efficient generation of recombinant retroviruses with amphotropic and ecotropic host ranges. Proc Natl Acad Sci USA 85:6460–6464.[Abstract]

des Portes V, Pinard J, Billuart P, Vinet M, Koulakoff A, Carrie A, Gelot A, Dupuis E, Motte J, Berwald-Netter Y, Catala M, Kahn A, Beldjord C, Chelly J (1998) A novel CNS gene required for neuronal migration and involved in X-linked subcortical laminar heterotopia and lissencephaly syndrome. Cell 92:51–61.[ISI][Medline]

Edmondson JC, Hatten ME (1987) Glial-guided granule neuron migration in vitro: a high resolution time-lapse video microscopic study. J Neurosci 7:1928–1934.[Abstract]

Eksioglu YZ, Scheffer IE, Cardenas P, Knoll J, DiMario F, Ramsby G, Berg M, Kamuro K, Berkovic SF, Duyk GM, Parisi J, Huttenlocher PR, Walsh CA (1996) Periventricular heterotopia: an X-linked dominant epilepsy locus causing aberrant cerebral cortical development. Neuron 16: 77–87.[ISI][Medline]

Fishell G (1995) Striatal precursors adopt cortical identities in response to local cues. Development 121:803–812.[Abstract/Free Full Text]

Fishell G, Mason CA, Hatten ME (1993) Dispersion of neural progenitors within the germinal zones of the forebrain. Nature 362:636–638.[ISI][Medline]

Fox JW, Lamperti ED, Eksioglu YZ, Hong S.E, Feng Y, Graham DA, Scheffer IE, Dobyns WB, Hirsch BA, Radtke RA, Berkovic SF, Huttenlocher PR, Walsh CA (1998) Mutations in filamin 1 prevent migration of cerebral cortical neurons in human periventricular heterotopia. Neuron 21:1315–1325.[ISI][Medline]

Gertler FB, Hill KK, Clark MJ, Hoffmann FM (1993) Dosage-sensitive modifiers of Drosophila abl tyrosine kinase function: prospero, a regulator of axonal outgrowth, and disabled, a novel tyrosine kinase substrate. Genes Dev 7:441–453.[Abstract]

Gilmore EC, Ohshima T, Goffinet AM, Kulkarni AB, Herrup K (1998) Cyclin-dependent kinase 5-deficient mice demonstrate novel developmental arrest in cerebral cortex. J Neurosci 18:6370–6377.[Abstract/Free Full Text]

Gleeson JG, Allen KM, Fox JW, Lamperti ED, Berkovic S, Scheffer I, Cooper EC, Dobyns WB, Minnerath SR, Ross ME, Walsh CA (1998) Doublecortin, a brain-specific gene mutated in human X-linked lissencephaly and double cortex syndrome, encodes a putative signaling protein. Cell 92:63–72.[ISI][Medline]

Gonzalez JL, Russo CJ, Goldowitz D, Sweet HO, Davisson MT, Walsh CA (1997) Birthdate and cell marker analysis of scrambler: a novel mutation affecting cortical development with a reeler-like phenotype. J Neurosci 17:9204–9211.[Abstract/Free Full Text]

Hedgecock EM, Culotti JG, Hall DH (1990) The unc-5, unc-6, and unc-40 genes guide circumferential migrations of pioneer axons and mesodermal cells on the epidermis in C. elegans. Neuron 4:61–85.[ISI][Medline]

Hirotsune S, Fleck MW, Gambello MJ, Bix GJ, Chen A, Clark GD, Ledbetter DH, McBain CJ, Wynshaw-Boris A (1998) Graded reduction of Pafah1b1 (Lis1) activity results in neuronal migration defects and early embryonic lethality. Nature Genet 19:333–339.[ISI][Medline]

Howell BW, Gertler FB, Cooper JA (1997a) Mouse disabled (mDab1): a Src binding protein implicated in neuronal development. EMBO J 16: 121–132.[Abstract/Free Full Text]

Howell BW, Hawkes R, Soriano P, Cooper JA (1997b) Neuronal position in the developing brain is regulated by mouse disabled-1. Nature 389:733–737.[ISI][Medline]

Jackson CA, Peduzzi JD, Hickey TL (1989) Visual cortex development in the ferret. I. Genesis and migration of visual cortical neurons. J Neurosci 9:1242–1253.[Abstract]

Kornack DR, Rakic P (1995) Radial and horizontal deployment of clonally related cells in the primate neocortex: relationship to distinct mitotic lineages. Neuron 15:311–321.[ISI][Medline]

Lavdas AA, Mione MC, Parnavelas JG (1996) Neuronal clones in the cerebral cortex show morphological and neurotransmitter heterogeneity during development. Cereb Cortex 6:490–497.[Abstract]

Luskin MB (1993) Restricted proliferation and migration of postnatally generated neurons derived from the forebrain subventricular zone. Neuron 11:173–189.[ISI][Medline]

Luskin MB, Pearlman AL, Sanes JR (1988) Cell lineage in the cerebral cortex of the mouse studies in vivo and in vitro with a recombinant retrovirus. Neuron 1:635–647.[ISI][Medline]

Luskin MB, Parnavelas JG, Barfield JA (1993) Neurons, astrocytes, and oligodendrocytes of the rat cerebral cortex originate from separate progenitor cells: an ultrastructural analysis of clonally related cells. J Neurosci 13:1730–1750.[Abstract]

Mione MC, Danevic C, Boardman P, Harris B, Parnavelas JG (1994) Lineage analysis reveals neurotransmitter (GABA or glutamate) but not calcium-blinding protein homogeneity in clonally related cortical neurons. J Neurosci 14:107–123.[Abstract]

Mione MC, Cavanagh JFR, Harris B, Parnavelas JG (1997) Cell fate specification and symmetrical/asymmetrical divisions in the developing cerebral cortex. J Neurosci 17:2018–2029.[Abstract/Free Full Text]

Morris NR, Efimov VP, Xiang X (1998) Nuclear migration, nucleokinesis and lissencephaly. Trends Cell Biol 8:467–70.[ISI][Medline]

Neyt C, Welch M, Langston A, Kohtz J, Fishell G (1997) A short-range signal restricts cell movement between telencephalic proliferative zones. J Neurosci 17:9194–9203.[Abstract/Free Full Text]

Ohshima T, Ward JM, Huh CG, Longenecker G, Veeranna, Pant HC, Brady RO, Martin LJ, Kulkarni AB (1996) Targeted disruption of the cyclin-dependent kinase 5 gene results in abnormal corticogenesis, neuronal pathology and perinatal death. Proc Natl Acad Sci USA 93:11173–11178.[Abstract/Free Full Text]

Olsson M, Campbell K, Turnbull DH (1997) Specification of mouse telencephalic and mid-hindbrain progenitors following heterotopic ultrasound-guided embryonic transplantation. Neuron 19:761–772.[ISI][Medline]

O'Rourke NA, Chenn A, McConnell SK (1997) Postmitotic neurons migrate tangentially in the cortical ventricular zone. Development 124:997–1005.[Abstract/Free Full Text]

O'Rourke NA, Dailey ME, Smith SJ, McConnell SK (1992) Diverse migratory pathways in the developing cerebral cortex. Science 258:299–302.[ISI][Medline]

O'Rourke NA, Sullivan DP, Kaznowski CE, Jacobs AA, McConnell SK (1995) Tangential migration of neurons in the developing cerebral cortex. Development 121:2165–2176.[Abstract/Free Full Text]

Parnavelas JG, Barfield JA, Franke E, Luskin MB (1991) Separate progenitor cells give rise to pyramidal and nonpyramidal neurons in the rat telencephalon. Cereb Cortex 1:1047–1057.

Price J, Thurlow L (1988) Cell lineage in the rat cerebral cortex: a study using retroviral-mediated gene transfer. Development 104:473–482.[Abstract]

Rakic P (1971) Guidance of neurons migrating to the fetal monkey neocortex. Brain Res 33:471–476.[ISI][Medline]

Rakic P (1972) Mode of cell migration to the superficial layers of fetal monkey neocortex. J Comp Neurol 145:61–84.[ISI][Medline]

Rakic P (1978) Neuronal migration and contact guidance in primate telencephalon. Postgrad Med J 54:25–40.[ISI][Medline]

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

Rakic P, Stensaas LJ, Sayre EP, Sidman RL (1974) Computer-aided three-dimensional reconstruction and quantitative analysis of cells from serial electron microscopic montages of fetal monkey brain. Nature 250:31–34.[ISI][Medline]

Reid CB, Liang I, Walsh C (1995) Systematic widespread clonal organization in cerebral cortex. Neuron 15:299–310.[ISI][Medline]

Reid CB, Tavazoie SF, Walsh CA (1997) Clonal dispersion and evidence for asymmetric cell division in ferret cortex. Development 124: 2441–2450.[Abstract/Free Full Text]

Reid CB, Liang I, Walsh CA (1999) Clonal mixing, clonal restriction, and specification of cekk types in the devloping rat olfactory bulb. J Comp Neurol 403:106–118.[ISI][Medline]

Reiner O, Carrozzo R, Shen Y, Wehnert M, Faustinella F, Dobyns W, Caskey C, Ledbetter D (1993) Isolation of a Miller-Dieker lissencephaly gene containing G protein beta-subunit-like repeats. Nature 364: 717–721.[ISI][Medline]

Rice DS, Sheldon M, D'Arcangelo G, Nakajima K, Goldowitz D, Curran T (1998) Disabled-1 acts downstream of Reelin in a signaling pathway that controls laminar organization in the mammalian brain. Development 125:3719–3729.[Abstract/Free Full Text]

Sapir T, Elbaum M, Reiner O (1997) Reduction of microtubule catastrophe events by LIS1, platelet-activating factor acetylhydrolase subunit. EMBO J 16:6977–6984.[Abstract/Free Full Text]

Sheldon M, Rice DS, D'Arcangelo G, Yoneshima H, Nakajima K, Mikoshiba K, Howell BW, Cooper JA, Goldowitz D, Curran T (1997) Scrambler and yotari disrupt the disabled gene and produce a reeler-like phenotype in mice. Nature 389:730–733.[ISI][Medline]

Shirasaki R, Mirzayan C, Tessier-Lavigne M, Murakami F (1996) Guidance of circumferentially growing axons by netrin-dependent and -independent floor plate chemotropism in the vertebrate brain. Neuron 17:1079–1088.[ISI][Medline]

Takahashi T, Nowakowski RS, Caviness VS Jr (1997) The mathematics of neocortical neuronogenesis. Dev Neurosci 19:17–22.[ISI][Medline]

Tamamaki N, Fujimori KE, Takauji R (1997) Origin and route of tangentially migrating neurons in the developing neocortical intermediate zone. J Neurosci 17:8313–8323.[Abstract/Free Full Text]

Tan SS, Breen S (1993) Radial mosaicism and tangential cell dispersion both contribute to mouse neocortical development. Nature 362: 638–640.[ISI][Medline]

Tan SS, Faulkner-Jones B, Breen SJ, Walsh M, Bertram JF, Reese BE (1995) Cell dispersion patterns in different cortical regions studied with an X-inactivated transgenic marker. Development 121:1029–1039.[Abstract/Free Full Text]

Tan SS, Kalloniatis M, Sturm K, Tam PP, Reese BE, Faulkner-Jones B (1998) Separate progenitors for radial and tangential cell dispersion during development of the cerebral neocortex. Neuron 21:295–304.[ISI][Medline]

Walsh C (1995) PCR-based techniques for utilizing retroviruses as cell lineage markers. In: Methods in molecular genetics, vol 4. Molecular Virology (Adolph KW, ed), pp 280–295. Orlando, FL: Academic Press.

Walsh C, Cepko CL (1988) Clonally related neurons show several patterns of migration in cerebral cortex. Science 255:1342–1345.

Walsh C, Cepko CL (1992) Widespread dispersion of neuronal clones across functional regions of the cerebral cortex. Science 255: 434–440.[ISI][Medline]

Walsh C, Cepko CL (1993) Widespread clonal dispersion in proliferative layers of cerebral cortex. Nature 362:632–635.[ISI][Medline]

Ware M, Reid C, Tavasoie S, Walsh C (1997a) Lineage analysis of early ferret cortex studied with an alkaline phosphatase (AP) retroviral library. Soc Neurosci Abstr 23:580.

Ware ML, Fox JW, Gonzalez JL, Davis NM, Lambert de Rouvroit C, Russo CJ, Chua SC Jr, Goffinet AM, Walsh CA (1997b) Aberrant splicing of a mouse disabled homolog, mdab1, in the scrambler mouse. Neuron 19:239–249.[ISI][Medline]

Xiang X, Beckwith SM, Morris NR (1994) Cytoplasmic dynein is involved in nuclear migration in Aspergillus nidulans. Proc Natl Acad Sci USA 91:2100–2104.[Abstract]

Xiang X, Osmani AH, Osmani SA, Xin M, Morris NR (1995) NudF, a nuclear migration gene in Aspergillus nidulans, is similar to the human LIS-1 gene required for neuronal migration. Mol Biol Cell 6:297–310.[Abstract]