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Volume 17, Number 6,
Issue of March 15, 1997
pp. 2018-2029
Copyright ©1997 Society for Neuroscience
Cell Fate Specification and Symmetrical/Asymmetrical Divisions in
the Developing Cerebral Cortex
Maria C. Mione,
John F. R. Cavanagh,
Brett Harris, and
John G. Parnavelas
Department of Anatomy and Developmental Biology, University College
London, London WC1E 6BT, United Kingdom
 ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
Two different modes of cell division are adopted by progenitor
cells to generate the neurons and glia of the cerebral cortex: they
either divide symmetrically to generate other progenitors or a pair of
postmitotic cells or divide asymmetrically to generate both a
progenitor and a postmitotic cell. In this study we used a lineage
marker, the BAG retrovirus, in embryonic day 16 rats in combination
with bromodeoxyuridine (BrdU) to identify patterns of cell generation
in the cerebral cortex, and investigated the relationship between the
phenotype of cells and the history of their lineages. The location,
phenotype and birth order of clonally related cells were studied in the
subsequent 3 weeks. Only pyramidal neurons and/or astrocytes formed
discrete clusters in which several generations of family members were
present, whereas nonpyramidal neurons were found exclusively in pairs
or as single cells. Analysis of BrdU levels in these cells showed that
nonpyramidal neurons were originally part of larger clones and were
found dispersed in the neocortex because of tangential migration of
their progenitors, dispersion of postmitotic cells, or death of clonal
relatives. These results suggest that both symmetrical and asymmetrical
division can be adopted by progenitor cells to generate cortical
neurons and glial cells and that cell extrinsic events contribute to
the isolation of nonpyramidal neurons.
Key words:
neocortex;
development;
retrovirus;
cell birthdate;
BrdU;
rat;
neurons;
glia
INTRODUCTION
The neurons and glia of the mammalian cerebral
cortex are generated by proliferating neuroepithelial cells in the
telencephalic ventricular and subventricular zones (Boulder Committee,
1970 ). As neurogenesis proceeds, the mitotic potential of progenitor cells becomes gradually restricted, resulting in more postmitotic cells
being produced at the later stages of corticogenesis (Takahashi et al.,
1995). The two main classes of cortical neurons, the pyramidal and
nonpyramidal cells, are produced throughout the period of neurogenesis
(Miller, 1985), with no apparent relation between time of birth and
neuronal subtype.
Lineage studies in the rat cortex (Parnavelas et al., 1991; Grove et
al., 1993; Luskin et al., 1993) demonstrated that clusters of clonally
related neurons were composed of cells of the same phenotype. However,
a large number of clonally related cells do not migrate in tandem along
the same radial glial fibers, and clonal relatives may be found at some
distance from each other (Walsh and Cepko, 1992, 1993). Different types
of cells were present in dispersed clones, but cells that migrated
together invariably showed the same phenotype (Reid et al., 1995). One
interpretation of these findings is that widespread clones are
generated by migrating progenitors that give rise to committed
precursors in different locations in the proliferative zone (Reid et
al., 1995). Alternatively, concerted migration may expose sibling cells
to the same cues, which may influence the development of cell
phenotype.
In invertebrates, cell fate specification is highly correlated with
birth order. The most common mechanism used to establish different
fates for sibling cells is through the asymmetrical localization of
cell determinants during mitosis (for review, see Doe and Spana, 1995).
Is the asymmetrical localization of determinants relevant to the
production of different cell types in the cortex as it is in the
Drosophila CNS? A number of studies, including population
studies on the generation of cortical cells (reviewed in Caviness et
al., 1995) and lineage studies with recombinant retroviruses (Kornack
and Rakic, 1995), have shown that cells undergoing either symmetrical
or asymmetrical divisions, in which daughter cells adopt different
fates, coexist in the ventricular zone neuroepithelium throughout
corticogenesis.
To gain information on the organization of cortical cell lineages, we
used a lineage marker in combination with bromodeoxyuridine (BrdU) to
analyze the pattern of cell generation in the cerebral cortex of the
rat. We found that cells born soon after the incorporation of
retrovirus and BrdU were almost exclusively pyramidal neurons of layers
V and IV, whereas labeled nonpyramidal cells of all layers, as well as
pyramidal neurons of the upper layers, were generated during the
subsequent cell cycles; glial cells were the last to be born. Analysis
of the clusters of clonally related cells in the cortex of 2-week-old
rats provides evidence for both asymmetrical and symmetrical divisions
coexisting in the same lineage during the generation of pyramidal
neurons, with a prevalence of asymmetrical divisions in radial arrays
and of symmetrical divisions in horizontal arrays. In contrast, labeled
nonpyramidal neurons were found either as isolated cells or pairs of
clonally related neurons, and their low content of BrdU indicated that they were part of larger clones.
MATERIALS AND METHODS
Retrovirus and BrdU injections. The  BAG
retrovirus, which carries the lacZ reporter gene (Price et al., 1987),
was injected into the telencephalic ventricles of rat embryos at
embryonic day (E) 16 as described previously (Mione et al., 1994). The
day in which a vaginal plug was found in pregnant rats was considered as E1. The injections of 0.5-1 µl of retroviral suspension, at a
dilution of 105 colony-forming units/ml, containing
polybrene (0.005%) and fast green (0.01%), were made through a 33 gauge needle. After closure of the abdominal wall, pregnant rats were
given injections of BrdU (50 mg/kg, i.p., dissolved in sterile saline
containing 0.007 N NaOH). A number of studies suggest that in dividing
mammalian cells, retroviral integration takes place at the first
mitosis after infection (Miller et al., 1990; Roe et al., 1993;
Hajihosseini et al., 1994). Because of the unknown and probably
variable length of time required for the incorporation and expression
of the reporter gene (Cepko et al., 1993) and the asynchronous pattern
of cell division of cortical progenitor cells (Takahashi et al., 1994), it was necessary to make BrdU available for the entire length of the
cell cycle during which the integration of the reporter gene was
presumably taking place. In addition, the intervals between BrdU
injections were designed to reduce the variability in the amount of
BrdU taken up by asynchronously cycling cells. Five injections of BrdU
were performed at 3.5 hr intervals. Some brains were examined 48 hr
after the injections of retrovirus and BrdU, and all  -galactosidase
(-gal+) cells were found to be labeled with BrdU.
Histology. The brains of injected rats were examined 3 d (at E19), 6 d (at birth, P0), or 20 d (2 weeks postnatally,
P14) after the injections. Animals (including embryos) were perfused through the heart with 4% paraformaldehyde in 0.1 M
phosphate buffer, pH 7.4. Brains were dissected out and postfixed in
the same solution for a total of 2 hr. Brains of 12 E19 rat embryos (from 3 different mothers), 5 newborn animals, and 14 2-week-old rats
were cryoprotected, embedded in OCT (Miles), and frozen in liquid
nitrogen. Serial coronal sections (7 µm thick for embryos and newborn
rats, and 15 µm thick for postnatal rats) were cut with a cryostat
and collected on poly-L-lysine coated slides. Sections were
immunostained for -gal using a polyclonal -gal antiserum (a gift
of Dr. J. Price, Smith Kline Beecham, Harlow, United Kingdom; Williams
et al., 1991), at a dilution of 1:1000. Incubation in primary antibody
was followed by donkey anti-rabbit biotinylated antiserum (Amersham,
Amersham, United Kingdom), and streptavidin Texas Red (Amersham) as
third layer, both diluted 1:250. After treatment with 2 N HCl for 1 hr
to expose single strands of DNA containing BrdU (Nowakowski et al.,
1989), sections were incubated overnight with a mouse monoclonal
anti-BrdU antibody (Sigma, Poole, UK), diluted 1:1,000, and the
reaction was successively revealed using a goat anti-mouse
FITC-conjugated antiserum (Sigma). Sections were coverslipped with
Citifluor (Canterbury, UK) and viewed with a Leica fluorescence
microscope equipped with a drawing tube.
-gal+ cells were mapped in camera lucida drawings of
serially arranged sections. Labeled cells were assigned to individual clusters (a group of closely spaced -gal+ cells
contained within 540 µm in both the rostrocaudal and mediolateral planes) or pairs (two-cell clusters) or classified as isolated -gal+ cells (cells separated from other labeled cells by
at least 540 µm in all planes). Five clusters or pairs, of the 104 examined, had -gal+ cells that spanned a distance of
more than 540 µm (marked with asterisks in Tables 1-4). For these
clusters, the absence of other labeled cells in the vicinity and the
spread along the main axis of the cluster (i.e., lateral for horizontal
clusters and vertical for radial clusters) suggested that all the cells
in these expanded clusters were clonally related. We restricted our
analysis to brains with a low infection rate (for discussion see Mione
et al., 1994); in these brains, all -gal+ cells found in
the cerebral cortex from the frontal to the occipital pole were
studied. However, only clusters, pairs, or isolated -gal+ cells located within AP coordinates bregma +3.2 mm
to  5.8 mm (Paxinos and Watson, 1986) are reported here, because of
the documented deviation of the glial fibers from the radial
orientation in the more posterior and lateral areas of the neocortex
(Misson et al., 1991).
Brains of four 2-week-old rats were sectioned with a Vibratome. Coronal
sections, 100 µm thick, were collected in serial order and incubated
overnight in X-Gal to reveal -gal+ cells (Sanes et al.,
1986). Brains containing labeled cells were processed for electron
microscopy and flat-embedded in Araldite as described previously
(Luskin et al., 1993). After classification, performed as described for
cryostat sections, a number of -gal+ cells in selected
clusters, pairs, or isolated cells were cut from the Vibratome
sections, mounted on Araldite stubs, and sectioned with an
ultramicrotome. Several adjacent sections through the same cell were
obtained and processed for GABA, glutamate, and BrdU-immunohistochemistry as described previously (Mione et al., 1994).
Quantification of BrdU labeling in -gal+
cells. Cells labeled for BrdU and -gal were examined with a
Leica TCS 4D confocal microscope, and the digitized images were
evaluated by computer using a standard image analysis program (PC
Image, Foster Findlay Associates, Newcastle, UK). For each preparation,
the maximum degree of BrdU labeling was taken to be that of the nuclei
of layer V neurons, which are born at E16 (Bayer and Altman, 1991) and
corresponded to 70-100% immunofluorescent nuclear area.
-gal+ cells displaying such high BrdU nuclear labeling
were considered to be born immediately after the incorporation of
retrovirus (and BrdU). Accordingly, cells with less than 50%
immunofluorescent nuclear area were classified as born after a further
division; cells with less than 25% immunofluorescent nuclear area were
classified as born after 2 cell divisions, and cells with less than
12% immunofluorescent nuclear area were classified as born after 3 or
more divisions. Quantification of BrdU levels was carried out in
optical sections of labeled cells, followed by three-dimensional
reconstruction. In the vast majority of cases, the entire nucleus of
double labeled cells was contained within one section and available for
such analysis without further manipulation. This approach offered
several advantages compared with classical birthdate studies with
[3H]thymidine autoradiography followed by grain counting
(see Acklin and van der Kooy, 1993). First, it was possible to
visualize both the somata and processes of -gal-labeled cells, which
was necessary for phenotypic classification. Second, it reduced the
problems connected with assessing the degree of labeling from a single thin section, as is often done in [3H]thymidine
autoradiography. The reliability and consistency of this approach were
evaluated previously in cultures of embryonic cortical cells, where
clonally related cells remain closely apposed and are easy to study
(Mione et al., 1996). In cultures exposed to both retrovirus and BrdU
for 6 hr, and examined 48 hr after the double exposure, we found that
BrdU was clearly detectable in all clonally related cells at least up
to the 16-cell stage, equivalent to 4 cell divisions (not shown), thus
confirming the reliability of the double labeling protocol.
Preliminary experiments were carried out in these cultures to evaluate
the degree of BrdU immunostaining in cells generated after one, two, or
three cell divisions. Briefly, primary cultures of E16 rat cortices
were prepared as described previously (Mione et al., 1996). Cultures
were exposed to 103 colony-forming units of BAG retrovirus
on their second day in vitro for a total of 2 hr, and to 30 min pulses of 10 5 M BrdU every 3.5 hr for a total of 12 hr. Cultures were fixed 1, 2, or 3 d after exposure to BAG
retrovirus and immunostained for -gal and BrdU as described above.
All members of the 21 clones found in these cultures were evaluated for
their degree of BrdU immunoreactivity. The results were as follows: in
single-cell clones (n = 5), BrdU levels were between
50% and 100%; in 8 of 10 two-cell clones, BrdU levels were between
25% and 50% in both cells. In the other eight clones, the amount of
BrdU immunoreactivity was consistent with the number of generations, as
deduced from the number of cells in that lineage. There was evidence of
cell loss in four clones.
RESULTS
We examined 719 -gal-labeled cells in the cortices of 18 two-week-old rats given injections of retrovirus at E16 and found that
they were distributed either in clusters of three or more cells, pairs,
or single cells. These cells formed 64 discrete clusters, 40 pairs, and
130 single -gal+ cells. The location, phenotypes, and
percent of BrdU-immunoreactive nuclear area of all cells are shown in
Tables 1-4. Often, clonally related cells were distributed in radial
or horizontal arrays, similar to what has been reported for the primate
cortex (Kornack and Rakic, 1995). The analysis of the degree of BrdU
labeling of these cells revealed the occurrence of asymmetrical
divisions in the radial arrays, and of symmetrical divisions in the
horizontal clusters.
Horizontal clusters
The majority of cells within these clusters resided in the
same layer, usually II/III (Fig.
1A,B). In the 18 brains examined, 23 clusters, representing 36% of all cortical clusters, could be
classified as horizontal (Table 1). In cryostat-cut
sections stained with -gal antiserum, all cells within the
horizontal clusters had morphological features of pyramidal neurons
with similar size and staining pattern. In semithin sections, all
clonally related cells of the horizontal clusters were immunoreactive
for the neurotransmitter glutamate, a marker of cortical pyramidal neurons (Fig. 1C,D).
Fig. 1.
Horizontal clusters. A,
Camera lucida drawing showing the location of -gal+
cells of cluster 18 (Table 1). These cells were all located in layers
II and III and distributed within two consecutive 100-µm-thick Vibratome sections. Glutamate immunoreactivity was used as a marker of
pyramidal neurons. Two of the cells of this cluster are displayed in
B, and one (arrow) is shown in adjacent
semithin sections (C-E): unstained (C)
or after immunostaining for glutamate (D) or BrdU (E). The other cells of this cluster were all
immunoreactive for glutamate and displayed similar low levels of BrdU
immunoreactivity. None of the cells was immunoreactive for GABA, a
marker of nonpyramidal neurons. Four neighboring neurons
(1-4) are shown in all three sections as
landmarks. Asterisks mark the same blood vessel.
F, A possible family tree for this cluster. To obtain
five neurons with <12% BrdU-immunoreactive nuclear area, the
progenitor cells must have divided symmetrically at least three times
(corresponding clonal size, 8 cells) after incorporation of retrovirus
and BrdU. Only five cells were found, which suggests that at least one
postmitotic [line ending above the ventricular zone
(VZ)] and one progenitor cell (line ending
within the VZ) were lost (through death or migration) from this
cluster. Scale bars: A, 1 mm;
B-E, 20 µm.
[View Larger Version of this Image (47K GIF file)]
Table 1.
Cell-type composition, location, and degree of BrdU
labeling in horizontal clusters of -gal+
cells
 |
 |
 |
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Data from 36 cortical hemispheres of 2-week-old rats
given injections of retrovirus and BrdU at E16 are reported here.
Cells were studied either in double-immunostained cryostat sections or
ain adjacent semithin sections. Horizontal
clusters are defined as groups of -gal+ cells located
predominantly in the same layer and within 540 µm in both
rostrocaudal (R-C) and mediolateral (M-L) dimensions. Clusters
extending for >540 µm are marked with an asterisk.
b
Clusters including one cell located in a
different layer. Cortical areas (FPM, fronto-parieto-motor cortex;
FPSS, fronto-parieto-somatosensory cortex; AC, anterior cingulate
cortex) were defined according to Paxinos and Watson (1986); laminar
location was assigned according to Krieg (1946). Cells were classified
as pyramidal neurons (Py) according to morphological features, which
included size and shape of the cell body and dendritic processes,
axonal morphology when applicable, and nuclear size and shape (Peters
and Jones, 1984). In semithin sections, cells were classified on the
basis of their immunoreactivity for glutamate. For the level of BrdU
immunoreactive nuclear area:  = 25-50%; = 12-25%; = <12%; ? = unknown.
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Analysis of BrdU labeling indicated that clonally related cells located
in the same layer were born at the same time, typically four divisions
after the incorporation of retrovirus and BrdU for layer II/III neurons
(Fig. 1E). Five of these clusters contained a single
early-born pyramidal neuron (BrdU labeling, ~50%) located in layer
V, as well as several late-born pyramidal cells in layers II and III.
In almost all horizontal clusters, there was evidence of cell loss. For
example, a level of BrdU immunoreactivity of <12% found in
-gal+ cells of layers II and III was compatible with 4 cell divisions after retrovirus and BrdU incorporation, which is
equivalent to a final number of 16 cells, or 9 cells in the clusters
that included 1 early-born neuron. However, no clusters matched the
expected size: a representative family tree of a horizontal cluster
including the missing cells is shown in Figure
1F.
Radial clusters
These clusters were composed of cells located in different layers
and arranged radially with little lateral displacement. Twenty-one
clusters (representing 33% of all cortical clusters found in 18 brains) showed such a distribution. These clusters were composed
exclusively of pyramidal neurons (Table 2) located in
different layers, from layer V to layers II and III. Cells within these
clusters, visualized with -gal immunohistochemistry, showed
different morphology and size, corresponding to the features of deep
layer and upper layer pyramidal neurons (Fig.
2A-D). In the clusters examined with
postembedding immunohistochemistry, cells were immunoreactive for
glutamate. Various levels of BrdU labeling were displayed by the
pyramidal neurons of the radial clusters, higher (~50%) for cells
located in the infragranular layers (Fig. 2D) and
progressively lower for cells located in more superficial layers (Fig.
2B,C). There was also evidence of cell loss,
although the extent of it was less pronounced than in the
horizontal clusters. A representative family tree of a radial cluster
is shown in Figure 2E.
Fig. 2.
Radial clusters. A, Camera lucida
drawing showing the location of -gal+ cells of cluster
18 (see Table 2). Cells were located in different layers, from layers V
to II, but displayed very little lateral displacement.
B-D, Laser scanning confocal micrograph of cells located in layer II (B), layer IV (C), or
layer V (D). -gal+ cells were stained
(red) with a polyclonal -gal antiserum, followed by
streptavidin-Texas Red, whereas BrdU immunoreactivity
(green) was detected with a monoclonal antiserum
followed by FITC. Coexistence appears yellow. All cells
displayed morphological features of pyramidal neurons, with visible
basal and/or apical dendrites. The cell depicted in D
had a prominent apical dendrite (dotted lines) in the
adjacent section. The levels of BrdU immunoreactivity were higher in
the cell located in layer V (D, immunoreactive nuclear
area, ~50%) and progressively lower for the cell located in layer IV
(C, immunoreactive nuclear area, 12-25%) and II
(B, immunoreactive nuclear area, <12%).
E, A possible family tree of this cluster. To obtain
five neurons with these levels of BrdU, the progenitors must have
undergone three asymmetrical and one symmetrical division after the
incorporation of retrovirus and BrdU to give rise to postmitotic
daughters during the first three divisions and two terminal postmitotic
cells from the last symmetrical division. Glial clusters usually showed
a clear radial alignment; the cells were grouped in several subunits
and distributed along different layers. F-H, Examples
of confocal micrographs of clonally related glial cells stained with
-gal+ antiserum and Texas Red. In F and
G, small clusters of -gal+ astrocytes can
be recognized by their thin, spider-like processes. In
H, an oligodendrocyte, part of cluster 7 (see Table 3),
shows the characteristic parallel processes. The levels of BrdU
immunoreactivity (here visualized with FITC) were very low in all glial
cells, indicating that their progenitors must have divided several
times after retroviral and BrdU incorporation before these cells were born. Scale bars: A, 1 mm; B-H, 20 µm.
[View Larger Version of this Image (86K GIF file)]
Glial clusters
Twenty clusters included cells with glial morphology. In most of
these clusters, cells were distributed radially. Twelve radial clusters
were composed exclusively of astrocytes, and six included both
astrocytes and pyramidal neurons (Table 3). Astrocytes
were arranged in subclusters, each composed of several cells that were radially aligned, sometimes spanning the entire thickness of the cortex. These cells were readily identified as such, both in
cryostat-cut sections stained with -gal antiserum (Fig.
2F,G) and in semithin sections stained for GFAP (not
shown). They were also characterized by very low, sometimes
undetectable levels of BrdU immunoreactivity (Fig.
2F,G). In mixed clusters that included both neurons
and astrocytes, pyramidal neurons always had higher BrdU labeling than
did their sibling astrocytes (Table 3). We also encountered two
clusters that contained oligodendrocytes (identified on the basis of
the parallel orientation of their processes, see Fig. 2H), one of which also included a number of
astrocytes. However, the levels of BrdU immunoreactivity were too low
to establish a hierarchy of birth order between the two cell types.
Table 3.
Cell-type composition, location, and degree of BrdU
labeling in clusters containing -gal+ glial
cells
|
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| |
 |
|
Data from 36 cortical hemispheres of 2-week-old rats
given injections of retrovirus and BrdU at E16 are reported here.
Cells were studied either in double-immunostained cryostat sections or
ain adjacent semithin sections. Glial clusters
are defined as groups of -gal+ cells located within 540 µm in both rostrocaudal and mediolateral dimensions, which are
composed exclusively of, or contain, glial cells. A cluster extending
for >540 µm is marked with an asterisk.
b
Clusters that include cells located in
different layers.
c
Clusters that include cells with different
phenotype. Cortical areas were defined according to Paxinos and Watson
(1986); laminar location was assigned according to Krieg (1946). Cells were classified as pyramidal neurons (Py), astrocytes (A), or oligodendrocytes (O) according to morphological features, which included size and shape of the cell body and cellular processes, axonal
morphology when applicable, and nuclear size and shape (Peters and
Jones, 1984; Peters et al., 1991). For the level of BrdU-immunoreactive
nuclear area: = 25-50%; = 12-25%; = <12%;  = unlabeled; ? = unknown.
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Two-cell clusters
Most -gal+ neurons were found in pairs. We assumed
that cells within these clusters were clonally related because their
separation was less than 540 µm in the mediolateral and rostrocaudal
dimensions, and were often located in the same layer as if they had
been generated by the final division of their immediate precursors.
Accordingly, it was common to find that both cells in these clusters
showed similar degrees of BrdU immunoreactivity. This suggested that two-cell clusters were generated by symmetrical divisions, although only a few of these divisions had taken place immediately after the
incorporation of retrovirus. In fact, the level of BrdU
immunoreactivity found in these cells (Fig.
3A,B) suggested that most two-cell clusters
were part of larger clones (Table 4).
Fig. 3.
Two cell clusters and isolated cells. An example
of a two-cell cluster, composed of nonpyramidal (A) and
pyramidal (B) neurons (cluster 21 in Table 4). Confocal
micrographs showing -gal immunoreactivity in red
(Texas Red) and BrdU immunoreactivity in green (FITC). Coexistence is shown in yellow. The pyramidal cell was
characterized by the presence of dendritic spines along the apical
dendrite, whereas the nonpyramidal neuron showed intense staining of
its many dendrites. BrdU levels were low in both cells, suggesting that
they may have been generated by the same division or the same order of
divisions after the incorporation of retrovirus and BrdU by their
ancestor. C, D, Examples of isolated
-gal+ cells, double-immunostained for -gal (Texas
Red) and BrdU (FITC) in the cortex of 2-week-old rats given injections
of retrovirus and BrdU at E16. Only isolated pyramidal neurons, like
the cell depicted in C, were characterized by high
levels of BrdU immunoreactivity. The majority of isolated
-gal+ cells, however, was nonpyramidal neurons
(D), which displayed low levels of BrdU
immunoreactivity. Scale bars, 20 µm.
[View Larger Version of this Image (159K GIF file)]
Almost one-half of the two-cell clusters were composed of nonpyramidal
neurons. In cryostat-cut sections stained with -gal antiserum,
nonpyramidal neurons showed extensive dendritic staining (Fig.
3A), allowing for unequivocal classification; in
Araldite-embedded sections, these neurons were immunoreactive for GABA.
Clonally related nonpyramidal cells displayed similar low levels of
BrdU immunoreactivity and were often located in the same layer. The laminar location was not necessarily the one expected for the low level
of BrdU immunoreactivity found in these cells (see Table 4). For
example, some clusters were observed in the infragranular layers (see
clusters 27, 36, and 37 in Table 4), with both cells displaying very
low levels of BrdU immunoreactivity. In contrast, pyramidal cells were
consistently found in the correct layer for the level of BrdU
immunoreactivity, both in two-cell clusters and in larger clusters (see
previous paragraphs). Five two-cell clusters composed of pyramidal
neurons were located in the infragranular layers and displayed high
levels of BrdU immunoreactivity (Table 4). This suggested that these
cells were also generated by symmetrical divisions of their immediate
precursors, and these terminal divisions had taken place immediately
after the incorporation of retrovirus and BrdU. Four two-cell clusters
were composed of one pyramidal and one nonpyramidal neuron either born
at the same time (see clusters 20 and 21 in Table 4 and Fig.
3A,B) or with the pyramidal neuron born earlier (clusters 18 and 19 in Table 4).
Isolated cells
One hundred thirty isolated -gal+ cells were found
in the cortex of 18 two-week-old rats given injections of retrovirus at E16. These cells were separated by at least 540 µm from any other -gal+ cell. Isolated -gal+ cells may be
the result of the integration of the reporter gene into the postmitotic
daughter of an infected progenitor cell (Hajihosseini et al., 1994) or
cells that had migrated away from their relatives. Alternatively,
isolated -gal+ cells may be the only survivors of larger
clones (or the only cells still expressing the reporter gene; Cepko et
al., 1993).
As a result, cells that had become postmitotic immediately after the
integration of retrovirus and BrdU were marked by high levels of BrdU
immunoreactivity (50-100% of immunofluorescent nuclear area). We
found that only 15 of the 130 isolated -gal+ cells found
in the cortex of 2-week-old rats were characterized by high levels of
BrdU immunoreactivity. Such cells were considered as clones and
represented 11% of the total number of isolated -gal+
cells. It was of interest that high levels of BrdU immunoreactivity, indicative of the incorporation of retrovirus and BrdU into progenitors immediately before the generation of postmitotic daughters, were found
only in isolated -gal+ pyramidal neurons (Fig.
3C). In cryostat-cut sections, 85% of the 87 isolated
-gal+ cells showed features of nonpyramidal neurons with
less than 12% of their nuclear area being BrdU immunoreactive,
supporting the notion that many isolated -gal+ cells
were originally part of larger clones (Fig. 3D). Similar findings were obtained from the study of the 43 isolated
-gal+ cells in semithin sections (Fig.
4A-D).
Fig. 4.
An isolated bipolar -gal+ cell from
the cortex of a 2-week-old rat is shown in A (Vibratome
section) and in B-D in adjacent semithin sections
(arrows), either unstained (B) or after
immunostaining for GABA (C) or BrdU (D).
This cell was GABA immunoreactive (a marker of nonpyramidal neurons),
as were many other isolated -gal+ cells at this age, and
displayed 12-25% BrdU-immunoreactive nuclear area. Three neighboring
neurons (1-3) and blood vessels
(asterisks) are shown as landmarks. Scale bars, 20 µm.
[View Larger Version of this Image (77K GIF file)]
We hypothesized that these isolated labeled cells with low levels of
BrdU could be the result of tangential migration or death/switching off
of the reporter gene in their clonal relatives. If isolated -gal+ cells were the result of epigenetic events,
including tangential migration or death of clonal relatives, then their
relative number should be much lower in brains examined shortly after
the injection of retrovirus, when migration of -gal+
cells has just started and the wave of cell death that affects postmitotic neurons is not at its peak (that is at 2-3 weeks of age,
Ferrer et al., 1992). We have indeed confirmed previous findings (Mione
et al., 1994) showing that isolated -gal+ cells
represented only 10% of the total number of clones 72 hr after the
injection of retrovirus and BrdU, as opposed to over 40% in 2-week-old
rats (Fig. 5). Moreover, whereas all isolated -gal+ cells present in E19 rat brains were heavily
labeled with BrdU, only a fraction of the isolated -gal+
cells present in 2-week-old rats were heavily labeled (Fig.
6).
Fig. 5.
Histogram showing the percentages of isolated
-gal+ cells over the total number of clusters and pairs
at different ages, after the injection of retrovirus and BrdU at E16,
and the proportion of these cells that displayed high levels of BrdU
immunoreactivity (dark bars). Isolated
-gal+ cells were studied in E19 rat embryos (3 d after
injections), in newborn rats, and in 2-week-old rats. Less than 10% of
the clones found at E19 were composed of a single cell, all of which displayed high levels of BrdU immunoreactivity. At later times after
the injections, isolated -gal+ cells increased in
number, possibly as a result of tangential migration and/or death of
clonal relatives. However, the number of single-cell clones (isolated
cells with high levels of BrdU immunoreactivity) remained constant and
represented ~10% of all clusters at any age examined. The results
obtained are pooled from at least five animals for each age
group.
[View Larger Version of this Image (31K GIF file)]
Fig. 6.
The phenotype and BrdU levels of 130 isolated
-gal+ cells found in the cortex of 18 two-week-old rats
given injections of retrovirus and BrdU at E16 are plotted here. A vast
majority of cells were nonpyramidal neurons with low levels of BrdU
immunoreactivity (light gray bars). Isolated
nonpyramidal neurons with intermediate levels of BrdU immunoreactivity
were also found. However, all isolated -gal+ cells with
70-100% BrdU-immunoreactive nuclear area, indicative of single-cell
clones, were pyramidal cells (dark bars).
[View Larger Version of this Image (45K GIF file)]
DISCUSSION
Analysis of BrdU levels in clonally related cells and
characterization of their phenotypes revealed that cortical pyramidal neurons may be generated through asymmetrical division (most early-born cells), symmetrical division (some early- and most late-born cells), and a combination of the two. Their distribution in clusters suggests that they follow a pattern of radial migration. In contrast,
nonpyramidal neurons, which were found exclusively as isolated cells or
pairs, were born relatively late within the lineages of progenitor
cells labeled at E16. Glial cells were the last to be born and formed discrete clusters. The present study provides new information about the
organization of cell lineages in the cerebral cortex and argues that
the composition of cortical clones as observed in lineage studies is
determined by both inherited and extrinsic mechanisms.
Symmetrical and asymmetrical divisions of cortical
progenitor cells
In all cases in which clonally related cells maintained a close
spatial relationship with each other, it was possible to analyze the
order of birth of sibling cells using the level of BrdU
immunoreactivity. The results were consistent with the laminar position
of the cells. Two types of clusters were seen, defined as radial and
horizontal, based on the spatial distribution of the constituent cells.
The family trees of radial neuronal clusters were indicative of
asymmetrical divisions of cortical progenitor cells, in which
postmitotic neurons were produced from early divisions.
The results presented here indicate that asymmetrically dividing
lineages give rise to pyramidal neurons in both infragranular and
supragranular layers. These cells project to different targets (for
review see O'Leary and Koester, 1993) and express different molecular
phenotypes (Frantz et al., 1994a,b). The absence of a lineage-dependent
laminar specification was suggested in previous studies with
recombinant retroviruses (Luskin et al., 1988; Walsh and Cepko, 1988;
Grove et al., 1993; Luskin et al., 1993; Mione et al., 1994), but other
authors (Fishell et al., 1990; Krushel et al., 1993) have hypothesized
the existence of lineages dedicated to the production of neurons for
either the infragranular or the supragranular layers. Such a hypothesis
is compatible with horizontal clusters, and with the finding that in
many two-cell clusters both neurons reside in the same or adjacent
layers (Table 4). In horizontal clusters, the location and low level of
BrdU immunoreactivity in clonally related cells suggest that their
progenitors have undergone some proliferative mitoses before giving
rise to postmitotic neurons. A similar pattern of distribution has
recently been described in the monkey cerebral cortex (Kornack and
Rakic, 1995) and was interpreted as being produced by symmetrically
dividing cells. On the basis of the levels of BrdU immunoreactivity and
the number of cells in these clusters, we envisaged that a larger
number of cells were lost from horizontal than from radial clusters
either through cell death (see Blaschke et al., 1996; Thomaidou et al., 1997) and/or dispersion.
Asymmetrical divisions have been related to the segregation of cell
fate determinants into only one of the newly generated progeny
(reviewed by Doe and Spana, 1995). The mechanism for this has been
studied intensively in Drosophila, where some lineages (e.g., the lineages of neuroblasts; Spana and Doe, 1995) are largely invariant. Recently, a mouse homolog of Drosophila Numb has
been found to be asymmetrically distributed during mitosis in cortical cell progenitors (Zhong et al., 1996). Moreover, m-Numb was found to
physically interact with Notch1, a transmembrane protein, that together
with Delta conveys cell-extrinsic mechanisms of fate determination
(reviewed by Artavanis-Tsakonas, 1995). Notch1 has recently been found
to be asymmetrically restricted to the apical cells in horizontally
cleaved mitoses (Chenn and McConnell, 1995).
Radial and nonradial migration
Results from our analysis of BrdU levels suggested that the vast
majority of isolated -gal+ cells and of two-cell
clusters were part of larger clones and that they had settled at some
distance from their clonal relatives. Dispersion of clonally related
cells during migration has been reported previously (Austin and Cepko,
1990; Walsh and Cepko, 1993), and Reid et al. (1995) have hypothesized
the existence of migrating progenitors that give rise to one to two
daughters in different locations in the ventricular zone to explain the observed periodical distance between subunits of the same clone. The
present findings of isolated -gal+ cells and pairs that
were born some divisions after the incorporation of the retroviral
lineage marker support the model proposed by these authors. According
to this model, our results suggest that subunits generated by the last
divisions of these migrating progenitors are composed predominantly of
nonpyramidal neurons.
In addition to the horizontal migration of progenitor cells in the
proliferative zones (Fishell et al., 1993; Reid et al., 1995),
displacement of some clonally related cells may be a result of
tangential migration of postmitotic neurons at the level of the
subventricular or intermediate zone. Evidence from both in vitro and in vivo studies suggests that migrating
neurons can slide from one glial process to another (Misson et al.,
1991) and may even ignore radial glial fibers during their migration to
the cortical plate (Roberts et al., 1993; O'Rourke et al., 1995).
Experiments with X-chromosome-linked mosaics (Tan and Breen, 1993; Tan
et al., 1995) also suggested the possibility of tangential migration to
explain the mixing of cortical cells with different genotypes, although
the intermingling of progenitor cells in the proliferative zone was
also considered (Rakic, 1995).
Radial and nonradial migratory routes may expose young neurons to yet
unknown but probably different cues that may be important for the
acquisition of a specific phenotype. Lineage experiments in the chick
optic tectum have provided evidence that different migratory paths are
related to the phenotypic choices of clonally related cells (Gray and
Sanes, 1991). Similar results have been obtained in the chick spinal
cord, where early circumferentially migrating cells develop features of
commissural interneurons (Leber and Sanes, 1995). Reese et al. (1995)
have reported that in the retina of X-inactivated transgenic mice, only
specific subtypes of retinal cells were displaced from the vertical
columns of cells with the same genotype. Cell type-specific migratory
routes may be a selective mechanism to sort out different phenotypes
generated by multipotential progenitors. The proportion of progenitors
and/or postmitotic cells that are engaged in nonradial migration is
between 12 and 30% (O'Rourke et al., 1992, 1995; Tan et al., 1995).
Moreover, O'Rourke et al. (1995) have provided evidence that the
tangentially migrating cells in the intermediate zone of newborn
ferrets are neurons. These data are compatible with the reported
proportion of nonpyramidal neurons present in the mammalian cerebral
cortex (15-30%; Parnavelas et al., 1977; Rockel et al., 1980; Lin et al., 1986; Meinecke and Peters, 1987).
Our findings that isolated -gal+ cells are predominantly
nonpyramidal neurons that have lost contact with their clonal relatives suggest that there may be a relationship between horizontal migration, either of young neurons or of their immediate precursors, and the
development of the nonpyramidal phenotype. Among the different cortical
cell types, nonpyramidal neurons show less distinctive regional
features than their neighbor pyramidal cells (DeFelipe and Farinas,
1992). In addition, many neurochemical and morphological aspects of the
nonpyramidal phenotype become evident relatively late in cortical
development (Parnavelas et al., 1978; Del Rio et al., 1992; Lavdas et
al., 1996). These aspects of the biology of interneurons are in
agreement with both a late birth, at least within lineages, and the
lack (or different complement) of cell-cell communication (through gap
junctions, for example) in horizontally migrating cells, that may be an
important determinant of cell identity in radially migrating clusters
of pyramidal neurons.
Order of birth of cortical cell types
The organization of cortical cell lineages in the mammalian brain,
as studied with lineage markers, has been hindered by the dispersal
and/or death of clonal relatives at various stages of their
development. The use of genetic tags to correctly assign labeled cells
to defined clones (Walsh and Cepko, 1992, 1993) has proved useful in
revealing the extent of dispersion of clonal relatives. However, this
approach has dealt with only a small sample of cells and provides no
information about the generation, migration, and differentiation in
cortical lineages. In an earlier study in adult rats (Mione et al.,
1994), we found that very large clusters (up to 100 cells) of
-gal+ cells were composed of glia, clusters of 2-23
-gal+ cells were composed of pyramidal neurons, whereas
nonpyramidal cells were typically found in pairs. More recently, when
we examined developing (1- to 3-week-old) rats, we observed a number of
mixed clusters composed of pyramidal and nonpyramidal neurons (Lavdas et al., 1996), which suggested the existence of common progenitors for
the two main classes of cortical neurons. Clusters composed of cells
with different phenotypes have also been reported by others (Price and
Thurlow, 1988; Walsh and Cepko, 1992; Luskin et al., 1993). Although
the number of mixed clusters was too low in the present study to draw
definitive conclusions about the order of birth of different cell
types, we noted that in mixed pyramidal/nonpyramidal clusters,
pyramidal cells were born earlier or at the same time as their sibling
nonpyramidal neurons; in mixed pyramidal/astrocyte clusters, the
neurons were always born earlier than the glial cells. Similarly, in
the population of labeled cells as a whole, all the early-born cells
were pyramidal neurons, whereas nonpyramidal neurons and glial cells
were characterized by progressively lower levels of BrdU. The
association of each class of labeled cells with a restricted and
distinct range of BrdU labeling suggests that in the mammalian cerebral
cortex, as in neural crest lineages (for review see Anderson, 1989),
the generation of different cell types from multipotential progenitors is a highly regulated process.
FOOTNOTES
Received June 3, 1996; revised Dec. 19, 1996; accepted Dec. 20, 1996.
This work was supported by the Wellcome Trust. We are grateful to Dr.
Jack Price for the generous gift of the -gal antiserum. We thank
Peter Boardman for technical assistance, and we thank Bagi Nadarajah
and Kate Whitley for help with the confocal microscope.
Correspondence should be addressed to Dr. Marina Mione, Department of
Anatomy and Developmental Biology, University College London, Gower
Street, London WC1E 6BT, UK.
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