Download P-GAP-43 Is Enriched in Horizontal Cell

Cerebral Cortex 2006;16:i121--i131
doi:10.1093/cercor/bhj171
P-GAP-43 Is Enriched in Horizontal Cell
Divisions throughout Rat Cortical
Development
Asymmetric cell divisions are correlated to neurogenesis in the
mammalian cortex and occur often with a horizontal orientation of
cell division. However, the molecular mechanisms of spindle
orientation or asymmetric cell divisions are not well understood in
the developing mammalian central nervous system. Here we show
a new molecular marker for horizontally dividing precursors in the
mammalian telencephalon. The antibody 2G12 directed against
phosphorylated serine of growth associated protein 43 (GAP-43)
labels postmitotic neurons and a subset of cells in mitoses in the
developing rat telencephalon. 2G12 immunoreactivity was found at
a high frequency in mitotic cells dividing parallel to the ventricular
surface throughout neurogenesis (embryonic day 13--17) in the
cerebral cortex and ganglionic eminence. Interestingly, we detected
the same predominance of 2G12 immunoreactivity in horizontally
dividing cells in the subventricular zone, the second proliferative
layer that has recently been involved in the generation of neurons.
Moreover, 2G12 immunostaining is no longer detectable in mitotic
cells of the ventricular zones at E21, the onset of gliogenesis in rat
telencephalon. These data imply GAP-43 phosphorylation in the
phase of neuronal commitment during M-phase and present to our
knowledge the first molecular correlate to horizontally dividing
precursors in mammalian neurogenesis.
Keywords: asymmetric cell division, basal progenitor, GAP-43,
subventricular zone, symmetric cell division, 2G12-immunoreactivity
Introduction
A key question in developmental biology is how different cell
types can arise from a population of common precursors. A wellknown mechanism to generate 2 different daughter cells is the
asymmetric distribution of cell fate determinants (for recent
reviews, see Fishell and Kriegstein 2003; Roegiers and Jan 2004;
Gotz and Huttner 2005). Intrinsic fate determinants are often
unequally distributed along an apicobasal polarity axis, and
depending on the inheritance of such fate determinants, the
progeny acquires different cell fates. The vertebrate pseudostratified neuroepithelium still retains some aspects of apicobasal polarity (Aaku-Saraste and others 1997), and also in
mammalian, central nervous system (CNS) development is the
orientation of cell division linked to the fate of the daughter
cells. Chenn and McConnell (1995) showed first that mitotic
cells with a cleavage plane parallel to the ventricular surface
(VS), a horizontal cell division, give rise to 2 morphologically
and behaviorally different cells, with only the basal daughter
cells leaving the proliferative layer and supposedly differentiating into a neuron (Chenn and McConnell 1995). Since the
discovery of radial glia as the major neuronal precursor cell type
(Malatesta and others 2000, 2003; Miyata and others 2001;
Noctor and others 2001), the importance of horizontal cell
Ó The Author 2006. Published by Oxford University Press. All rights reserved.
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Stefan H. Stricker1, Karina Meiri2 and Magdalena Götz3
1
Center for Molecular Medicine, Department of
Developmental Genetics, 1030 Vienna, Austria, 2Department
of Anatomy and Cellular Biology, Tufts University School of
Medicine, Boston, MA, USA and 3Institute for Stem Cell
Research, GSF—National Research Center for Environment
and Health, 85764 Neuherberg, Germany and Department of
Physiological Genomics, Institute of Physiology, University of
Munich, 80633 München, Germany
division was further supported by the observation that dividing
radial glial cells maintains their radial process during cell
division (Miyata and others 2001, 2004; Tamamaki 2002).
Thus, the basal daughter cell of a horizontal cell division remains
attached to the basement membrane and often differentiates
directly into a neuron, whereas the apical daughter remains
a precursor and regrowths a radial process (Morest 1970b; Miyata
and others 2001; Nadarajah and others 2001). However, it has
also been observed that the cell that inherits the radial process
remains a radial glial cell, and the daughter cell loosing the
process leaves the ventricular zone (VZ) and differentiates into
a neuron (Noctor and others 2001; Fishell and Kriegstein 2003;
Miyata and others 2004). Taken together, this evidence suggests
that a horizontal cell division is predominantly asymmetric
(producing a precursor cell and a neuron).
As recently demonstrated, vertically oriented cell divisions
can be either symmetric or asymmetric as determined by the
equal or unequal distribution of the apical membrane patch to
the 2 daughter cells (Kosodo and others 2004; Gotz and Huttner
2005). These experiments further support the notion that
asymmetric inheritance of fate determinants located in the
apical membrane patch may be crucial to determine the
unequal fate of daughter cells in the developing cortex.
However, it is not clear how the orientation of cell division is
regulated in mammalian neurogenesis, and the identity of the
key fate determinants is also not yet unraveled.
Whereas the basal end-feet of dividing neuroepithelial cells
or radial glia is attached to the basement membrane and the
daughter cell inheriting this contact may get special signals
from there (for review, see Fishell and Kriegstein 2003), the
apical pole of neuroepithelial and radial glial cells is characterized by adherens junctions (Shoukimas and Hinds 1978; AakuSaraste and others 1996; Manabe and others 2002; Kosodo and
others 2004). Interestingly, disruption of adherens junctions
affects the symmetry of neuroepithelial cell division in Drosophila (Lu and others 2001; Tepass 2002) and in mammals
(Chenn and Walsh 2002). Moreover, mammalian homologues of
Par3 and Par6, important mediators of asymmetric cell divisions
in flies and worms, are found at the apical adherens junctions
(Manabe and others 2002; Takekuni and others 2003), as is bcatenin, an important mediator of wnt- and cadherin signaling
(Chenn and Walsh 2002). These molecules are excellent
candidates for cell fate determinants that may become unequally distributed in a horizontal cell division.
Besides the molecular identity of intrinsic fate determinants,
asymmetric cell divisions require a machinery to properly
orient the cleavage plane of cell division that is determined by
the orientation of the mitotic spindle (Strome and Wood 1983).
Interestingly, the mitotic spindle is rotating for a longer time in
metaphase cells that will divide horizontally, suggesting that
this, more rare orientation of cell division, is specifically
regulated (Haydar and others 2003). So far, nothing is known
about the molecular mechanism that might influence the
orientation of cell division in vertebrate neuronal precursor
cells. We therefore aimed to find a molecule restricted or
enriched in horizontally dividing neural precursors independent of the stage of development.
Materials and Methods
Animals
The rat strain Wistar was used for all experiments. The animals were
time mated. The time of sperm detection was considered embryonic day
(E) 1. The animals were anesthetized with CO2 and sacrificed at
different stages of development by cervical dislocation. For the analysis
of the orientation of the cell division in wildtype and GAP-43–/– (Maier
and others 1999) littermates, sagittal cryostat sections were used. The
day of plaque detection was considered as E0.
Organotypic Slice Cultures and Dissociated Cell Cultures
The cortex was dissected from E14 Wistar rat embryos, and the
hippocampus was removed from each hemisphere. For slice cultures,
frontal sections were cut at 300-lm thickness with a McIlwain tissue
chopper and cultured in Dulbecco’s modified eagle medium and 10%
fetal calf serum on a filter inset (Millipore) in a 6-well plate at 37 °C (5%
CO2) for 1 day. Slices were then fixed for 30 min at room temperature in
50% Bouins fixative and washed 3 times in phosphate-buffered saline
(PBS). Dissociated cell cultures of the cortex of E14 Wistar rats were
prepared as described previously (Heins and others 2001) and fixed
after 1--4 days in vitro for 15 min in 50% Bouins fixative at room
temperature.
Immunocytochemistry
For in vivo analysis, the brains were dissected and fixed 2 h at room
temperature in 50% Bouins fixative, washed in PBS, and cryoprotected
at 4 °C in 30% sucrose overnight. Frontal sections of 50- to 60-lm
thickness were cut with a freezing microtome. For immunostaining
with 2G12 or 7B10, sections or cultures were incubated 30 min in 0.01%
digitonin (Gasser and Laemmli 1987), followed by overnight incubation
at 4 °C in 2G12 (hybridoma supernatant) (Meiri and others 1991; Brittis
and others 1995) or 7B10 (Meiri and others 1991) diluted 1:10 in
0.05% digitonin and 10% normal goat serum (NGS) in PBS. After washes
in PBS, sections or slice cultures were incubated 2 h and the dissociated
cell cultures 1 h at room temperature with the secondary antibody
(a-mouse, Jackson Immunoresearch Laboratories, Inc., Bar Harbor, ME)
diluted 1:100 with 0.05% digitonin and 10% NGS in PBS. After washing in
PBS, the samples were incubated 10 min at room temperature in 5-lg/mL
propidium iodide (PI) diluted in PBS with 25 U RNaseA. Mounting in
glycerol containing mounting medium followed after washes in PBS.
Optical Analysis
All analyses were done on confocal sections. Mitotic phases were
determined by eye using PI staining of the nuclei as a morphological
marker (Figs 1f and 2a,b). With an image analysis software (ImageJ), the
angle of cell divisions was measured by a line drawn between the
dividing chromatids and a reference line parallel to the VS. The
reference line along the VS was drawn at low magnification (340) to
exclude smaller surface variations. The orientation of cell division was
assured by analyzing single cell divisions in different z axis values. The
fluorescence intensity of the dividing cells and the background of each
image were determined as follows: First the background of each picture
was measured as the mean intensity of nonmitotic cells. This value NF
was used as normalization factor in order to compare different sections.
Fluorescence intensity of a dividing cell was quantified by drawing
a selection surrounding the cell body. The brightness of this cell was
measured as an integral CI (sum of the values of all pixels of a selection).
To determine the local background, larger area including the cell was
measured (CI+) and calculated as (CI+) – (CI). This divided by the
i122 2G12 Labels Horizontal Cell Divisions
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Stricker and others
difference of the measuring area of CI+, AC+, with that of CI, AC, is the
mean intensity of this adjacent background Bg.
Bg =
ðCI + Þ – ðCIÞ
:
ðAC + Þ – ðACÞ
To obtain the signal of only the cell as accurate as possible, the
product of AC and Bg was subtracted from CI and divided by NF to
normalize it to the other pictures. The outcome is the improved integral
of the cell iCI; it was measured 2 times independently, and the values
were averaged. The mean fluorescence intensity (mFI) is iCI divided
through AC.
iCI =
CI – ððACÞ3ðBgÞÞ
;
NF
mFI =
iCI
:
AC
For some analysis, an intensity cutoff was defined. Cells with a mFI
larger than this cutoff were defined as 2G12 immunoreactive. The
cutoffs for sections of different stages were defined independently
because they derived from different experiments. In comparison with
the negative control and the positive evaluation by eye, the cutoffs were
determined such that approximately a third of telophase cells was 2G12
immunoreactive.
Western Blot Analysis
To detect GAP-43 protein in embryonic brains, E13.5 forebrains and
E21.5 cortices were dissected on ice, homogenized in H buffer (10 mM
Tris pH 7.4, 2 mM dithiothreitoc (DTT), 5 mM ethylenediaminetetraacetic acid, 1 mM phenylmethyl sulphonyl fluoride (PMSF), and
centrifuged at 3000 g for 5 min. The supernatant was then further
centrifuged at 100 000 g for 30 min to produce a membrane pellet and
cytoplasmic (soluble) fraction. Protein from the membrane pellet was
solubilized in Laemmli sample buffer containing 5 mM DTT, whereas
protein from the supernatant was first precipitated with trichloracetic
acid before solubilization in sample buffer. Protein concentrations
were determined using the bicinchonimic acid assay (Pierce, Rockford,
Illinois) and equal amounts of protein were separated on an 8% gel.
Following western blotting onto polyvinylidene flouride membrane,
total GAP-43 immunoreactivity was detected with the 7B10 monoclonal
antibody, whereas phosphorylated GAP-43 immunoreactivity was detected with the 2G12 monoclonal antibody. Specific 7B10 immunoreactivity was revealed with horse radish peroxidase (HRP)-conjugated
secondary antibody followed by avidin-HRP and visualized with the
Pierce supersubstrate detection method, whereas 2G12 immunoreactivity was detected with a biotin-labeled secondary antibody followed
by avidin-HRP before chemoluminescence.
Results
The Monoclonal Antibody 2G12 Labels a Subset of
Mitotic Cells in Telophase during Neurogenesis
To identify molecules enriched in horizontal cell divisions, we
examined labeling of precursor cells in mitosis during neurogenesis of the developing cerebral cortex. For example,
immunoreactivity with the monoclonal antibody 2G12 was
previously detected in ventricular cells of the embryonic cortex
(Brittis and others 1995), but it had not been examined whether
2G12 labels all or only a subset of mitotic cells. To address this
question, we used microtome sections of embryonic rat brain
(E13--17) stained with 2G12 and PI to detect cells in different
mitotic phases. The sections were analyzed using confocal
microscopy (Figs 1--3). Immunoreactivity was mainly seen in the
cell body and processes (Figs 1b--e and 3a; see also Brittis and
others 1995), suggesting that the 2G12 antigen is cytoplasmatic
Figure 1. The monoclonal antibody 2G12 labels a subset of mitotic cells on the VS and on the SVZ. Micrographs depict 2G12 immunoreactivity (green) and PI fluorescence (red) in
sections of E15 (a), E21 (b), and E17 (c--f) rat forebrain. The different forebrain regions are indicated in (a) as Ctx, Cortex; GE, ganglionic eminence; and Hip, hippocampus. After
neurogenesis, ventricular staining of 2G12 is no longer detectable, but 2G12 immunoreactivity still is seen due to differentiated neurons (b). Note that 2G12-immunopositive cells
during neurogenesis are located in the VZ in (d) and SVZ in (d, arrow in c); at these stages, only mitotic cells are immunopositive for 2G12 (c--f). (f) Cells in a later phase of mitosis
show higher mFI than early ones; examples of mitoses depicted in (f) are taken from (e). Note that not all cells in telophase are 2G12 immunoreactive.
Cerebral Cortex 2006, V 16 Supplement 1 i123
Figure 2. Most ventricular but not subventricular cells divide vertically. Micrographs depict PI fluorescence (red) in sections of E17 (a) and E21 (b) rat cortex. (a) Yellow lines
indicate the VS (long line) and the orientation of the dividing chromatids of the 3 cells in telophase (short line). Note that the cell in the middle divides horizontally (13°) and the outer
ones vertically (65° and 68°). (b) Yellow line indicates the VS, arrows depict 2 dividing cells of the SVZ that are depicted enlarged in (b`, b``). Note that SVZ cells are dividing
horizontally with respect to the VS. (c, d) Quantitative analysis of the angle of cell division of all dividing cells; mitotic cells of the VZ divide mostly vertically (c; E13 Ctx, n = 35; E15
Ctx, n = 56; E17 Ctx, n = 273; E17 GE, n = 101; E21 Ctx, n = 21) and cells of the SVZ horizontally (d; E15, n = 48; E17, n = 51; E21, n = 56) in all tested stages.
or located in internal membranes. As previously observed
(Brittis and others 1995), 2G12 immunoreactivity is seen on
the VS of cortex, hippocampus, and ganglionic eminence (GE)
(Fig. 1a), and in later stages of neurogenesis in processes of the
differentiated neurons (Fig. 1b, see also Brittis and others
1995). Analysis of DNA labeling by PI showed that all 2G12immunoreactive cells at the VS were in M-phase of the cell cycle
(Fig. 1c--e), and the immunoreactivity increased from pro- to
telophase (Fig. 1f ). In the course of the analysis of mitotic
cells, we noted that only a subset of telophase cells at the VS
were 2G12 immunoreactive (Figs 1e and 3a). Notably, 2G12immunoreactive, mitotic cells were also detected in the second
proliferative layer of the cortex, the subventricular zone (SVZ)
(Fig. 1c,d, arrow), where cells undergo mitoses at abventricular
positions (Smart 1973). Mitotic staining with 2G12 was found
throughout neurogenesis (E13--17) but ceased thereafter
(E21; compare Fig. 1a with Fig. 1b).
Orientation of Cell Divisions in VZ and SVZ
To examine the orientation of cell division of 2G12-immunopositive and -immunonegative mitotic cells, we measured the
angle of cell division during (E13, E15, and E17) and after
neurogenesis (E21) in the rat cerebral cortex. The orientation
of the dividing chromatids was used as an indicator of how
cytokinesis will be orientated. The exact angle of the dividing
chromatids (labeled with PI, Fig. 2a, red) in regard to the VS
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was measured with the image analysis software (Fig. 2a, see
Materials and Methods for details). According to the previous
work (Chenn and McConnell 1995; Heins and others 2001;
Estivill-Torrus and others 2002; Weissman and others 2003), we
classified a cell division as horizontal when the angle between
the chromatids and the VS was smaller than 30°, as oblique
when the angle exceeded 30° but not 60°, and as vertical with
an angle larger than 60°. This analysis revealed a predominance
of vertical cell divisions at all stages analyzed (Fig. 2c). In
contrast to earlier reports on cell division in ferret cortex
(Chenn and McConnell 1995), we found no significant change
in the proportion of vertically dividing cells during development, consistent with more recent data obtained in the mouse
cerebral cortex (Weissman and others 2003). Notably, this
predominance of vertically oriented cell divisions in the VZ was
also observed in other brain regions, such as in the ventral
telencephalon, the GE (n = 101, E17, Fig. 2c).
Next, we examined the orientation of cell division in the
second proliferative zone, the SVZ. This zone is defined as precursors that undergo little to no interkinetic nuclear migration
and divide at some distance from the ventricle (Smart 1973;
Haubensak and others 2004; Miyata and others 2004; Noctor
and others 2004). Because Smart (1973) suggested that SVZ
cells may also orient their cell division parallel to the VS, we also
measured the angle of cell division with respect to the VS as
described above. SVZ cells were defined as mitotic cells located
Figure 3. Horizontally dividing cells are mostly 2G12 immunoreactive. (a) Micrograph depicts PI fluorescence (red) and 2G12 immunoreactivity (green) in sections of E17 rat cortex.
Note that only the horizontally dividing cell is 2G12 immunoreactive. (b) Quantitative analysis of the angle of cell division of cortical mitotic 2G12-immunoreactive cells (n = 35, E13;
n = 76, E15; n = 273, E17). (c) Quantitative analysis of the angle of cell division of mitotic 2G12-immunoreactive cells of the GE (n = 58). Note that horizontally dividing cells are
mostly 2G12 immunoreactive at all stages and regions of the brain examined, whereas only a smaller proportion of vertically dividing cells is 2G12 immunoreactive. (d) Graph
depicting the angle of cell division (x axis) versus the 2G12 immunoreactivity of all 384 analyzed cells dividing during neurogenesis on the VS. Note that most cells divide vertically,
but most of the horizontally dividing cells are 2G12 positive.
at a distance of 5 or more cell diameters from the VS (Fig. 2b,d).
In contrast to the pronounced majority of vertically oriented
cell divisions at the VS in the VZ, vertically oriented mitoses
were a minority (20--30%) in the SVZ, where most mitoses were
oriented horizontally or oblique throughout embryonic development (Fig. 2d). Interestingly, the orientation of mitosis became
more randomized at later stages when neurogenesis stops and
glial precursors dominate in the SVZ (Fig. 2d, n = 56, E21).
The Monoclonal Antibody 2G12 Labels a High Fraction
of Horizontally Dividing Mitotic Cells in VZ and SVZ
Next, we combined the measurements of the angle of cell
division with the quantitative assessment of the 2G12-immunofluorescence intensity for each cell in telophase (Fig. 3a). In
comparison with the negative control, we defined the fluorescence intensity above which cells were regarded as positive (for
details, see Material and Methods). Interestingly, a higher proportion of horizontally dividing cells was 2G12 immunoreactive
than of cells dividing vertically (Fig. 3b). Notably, the correlation
between the orientation of cell division and 2G12 immunoreactivity was observed during the entire neurogenesis (E13--17;
Fig. 3b). At all stages, a high proportion of the rare horizontally
dividing cells was 2G12 immunoreactive (70--60%), whereas
only a small part (20--30%) of vertically oriented telophase cells
was 2G12 positive (Fig. 3d). As described above, we also
detected 2G12-immunopositive cells in the SVZ (Fig. 1c,d).
Indeed, when we examined the angle of cell division in correlation to 2G12 immunoreactivity in the SVZ, we observed a
similar correlation as in the VZ. Also in the SVZ, horizontally
dividing cells were most frequently 2G12 immunoreactive
(78% E17, n = 32).
Next, we examined whether the correlation between 2G12
immunoreactivity and the orientation of cell division could also
be observed in other regions of the developing brain. Indeed,
also in the ventral telencephalon, the GE, almost all horizontally
dividing cells were strongly 2G12 immunoreactive, whereas
only 20% of the vertically dividing precursors were 2G12
positive (n = 58, Fig. 3c). Taken together, 2G12 immunoreactivity is found in most cells dividing parallel to the VS in the VZ
and SVZ of the telencephalon throughout neurogenesis but
disappears in mitotic cells thereafter (E21).
2G12 Immunoreactivity in Mitotic Cells Is Dependent on
Cell Contacts
In order to get some first insights into the signals that regulate
2G12 in mitotic cells, we examined 2G12 immunoreactivity
Cerebral Cortex 2006, V 16 Supplement 1 i125
in slice cultures, where cell contacts are typically preserved,
as opposed to dissociated cell cultures, where cell contacts
are disrupted and only reestablished after some days in vitro.
In slices from E15 rat cortex cultured for 1 day, many
2G12-immunoreactive mitotic cells were detectable lining the
VS (Fig. 4a,b) and also at SVZ positions (data not shown). In
contrast, no 2G12-positive mitoses were detectable when
cortical cells from the same developmental stage were cultured
as dissociated cells for 1 day in vitro (Fig. 4c,d). These data
suggest that the intact intrinsic cytoarchitecture, that is, orderly
cell contacts, maintained in cortical slice cultures, is required
for 2G12 immunoreactivity.
GAP-43 Identity of the 2G12 Antigen in Mitotic CNS
Precursor Cells
2G12 has been shown to recognize a phosphoepitope of GAP43 (Meiri and others 1991). GAP-43 has been mostly described
not only in developing neurons but also in neural precursors
(Kanazir and others 1996). We therefore examined whether the
mitotic GAP-43 staining can also be detected with other
antibodies against the GAP-43 molecule. 2G12 immunoreactiv-
ity in differentiating neurons coincides with staining by the
antibodies 7B10 or 10E8 that also recognize GAP-43 (Fig. 5a,b)
(Meiri and others 1991). These antibodies in turn show no
ventricular staining in mitotic cells (Fig. 5b, arrows), in contrast
to 2G12. Thus, 2G12 immunostaining in mitotic cells might
either be due to recognition of a similar phosphoepitope on a
different antigen or reflect the different affinities or accessibility
of the epitopes recognized by the different GAP-43 antibodies.
To clarify this further, we performed western blot analysis of
lysates from forebrain of E13 and E21. We chose these ages because E13 cortex contains exclusively the mitotic immunoreactivity of 2G12, as hardly any neurons have yet been generated.
On the contrary, the 2G12 immunoreactivity in mitotic cells has
vanished by E21, when staining is exclusively detectable in
postmitotic neurons. Interestingly, several antibodies directed
against GAP-43 (data not shown) give a signal in the insoluble
membranous fraction at the early developmental stage (E13),
when hardly any neurons are present (Fig. 5c,d). The signal
increases notably during development as mitotic cells represent
a small fraction of cells in contrast to postmitotic neurons at
later stages. Taken together, these data suggest that GAP-43 is
Figure 4. Immunoreactivity to 2G12 in vitro is dependent on intact cell contacts. Micrographs depict PI fluorescence (red) and 2G12 immunoreactivity (green) in organotypic slice
cultures (a, b) and dissociated cell cultures (c, d) of E15 rat cortex after 1 day in vitro. Note that 2G12 immunoreactivity in mitotic cells is detectable only in organotypic slice cultures
(a), where cell contacts are still intact, but not in dissociated cell cultures (c), where cell contacts are disrupted.
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Figure 5. Identity of the 2G12 antigen and orientation of cell division in GAP-43–/–. (a, b) Micrographs depict PI fluorescence (red) and 7B10 immunoreactivity (green) in sections of
E15 rat cortex. Note that 7B10, an antibody against GAP-43, does not reveal immunoreactivity in cells in mitoses (arrows and insets in b). (c, d) Western blot analysis of soluble and
membrane fractions from E13 rat forebrains, where no postmitotic neurons are yet present (left 2 lanes, 20 lg), and from E21 rat cortex, where only postmitotic neurons are
present, and no 2G12 immunoreactivity in mitotic cells is detectable (right 2 lanes, 70 lg) probed with the 7B10 monoclonal antibody that recognizes GAP-43. The specific band of
the GAP-43 monomer migrates at around 52 kD (arrow) and is detected at both ages. Additional multimers migrate at higher molecular weights. (d) Duplicate blot of soluble and
membrane fractions from E13 rat forebrains (left 2 lanes, 20 lg) and E21 rat cortex (right 2 lanes, 70 lg) probed with the 2G12 monoclonal antibody that recognizes phosphorylated
GAP-43 reveals a band that comigrates with the GAP-43 monomer at both ages. (d) Quantitative analysis of the angle of cell division of ventricular cells in the GAP-43–/– cortex at
E14 (GAP-43+/–, n = 130; GAP-43–/–, n = 262). Note that cells divide with similar orientations in the presence or absence of GAP-43.
Cerebral Cortex 2006, V 16 Supplement 1 i127
present at E13 in neural precursors, suggesting that the mitotic
2G12 immunoreactivity is due to phosphorylated GAP-43.
Because the above results suggested that the serine 41-phosphorylated form of GAP-43 is present preferentially in
horizontally dividing precursors, we examined its functional
relevance in mice bearing a targeted disruption of GAP-43
(Maier and others 1999). However, the orientation of cell
division of mitotic cells at the ventricle was no different in
E14 GAP-43–/– mice compared with heterozygous littermates
(Fig. 5e). Thus, 2G12 is enriched in horizontally dividing
precursors but is not required for the orientation of these
cell divisions.
Discussion
The orientation of cell division is part of the machinery
regulating the mode of cell division—the decision whether to
generate 2 equal daughters in a symmetric division or 2 unequal
daughters in an asymmetric cell division. Here we describe, to
our knowledge for the first time, a molecular correlate to
horizontal cell divisions, in both the VZ and SVZ of the
developing rat telencephalon. The phosphorylated form of
GAP-43 recognized by the monoclonal antibody 2G12 is
upregulated during M-phase and is found in a larger proportion
of mitoses oriented parallel to the VS than those oriented
vertically. This is the case throughout neurogenesis, although
no 2G12 immunoreactivity is detectable any longer in M-phase
when gliogenesis starts after E21 in the rat telencephalon.
Interestingly, 2G12 immunoreactivity in dividing cells depends
critically on the intact tissue polarity, and no 2G12 signal is
detected in dividing dissociated cells. These data further
support the notion that 2G12 labels mitoses that orient in
respect to a certain polarity that is missing in dissociated cell
cultures.
A Novel Molecular Correlate to Horizontal Cell Division
during Neurogenesis
Asymmetric distribution of fate determinants arranged along
the apicobasal polarity of neural precursors is a well-established
mechanism for neural fate specification in Drosophila ( Jan YN
and Jan LY 2001). Also in vertebrate neurogenesis, evidence is
accumulating that asymmetric distribution of fate determinants
may be important during neurogenesis (for recent review, see
Gotz and Huttner 2005). Live observations of cell divisions in
slice or whole mount preparations of the developing cerebral
cortex or retina have reported that horizontal cell division
oriented perpendicular to the apicobasal axis leads to the
generation of 2 distinct daughter cells, the basal cell differentiating into a neuron and the apical cell remaining a precursor
(Chenn and McConnell 1995; Miyata and others 2001, 2004;
Haubensak and others 2004). Notably however, other authors
could not observe asymmetric cell division during neurogenesis
(Lyons and others 2003), or the relation between the orientation of the cell division and the final progeny was not apparent
(Noctor and others 2001, 2004). However, all authors agree on
the predominance of vertically oriented cell divisions during
neurogenesis, in pronounced contrast to neuroblast division in
Drosophila, where almost all neuroblasts divide horizontally
(Schober and others 1999). In a variety of mammalian species,
only a small minority (10--20%) of precursors divides in parallel
to the VS (apical) during neurogenesis and a similar minority
(20--30%) of cell divisions is oriented with an oblique angle to
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the VS (Chenn and McConnell 1995; Heins and others 2001;
Estivill-Torrus and others 2002; Weissman and others 2003).
The molecular determinants specifying these cell divisions in
mammalian neurogenesis and regulating their special orientation as opposed to the vertically oriented default orientation are
not known at all. Here we demonstrate a proportional increase
in 2G12 immunoreactivity in correlation to the angle of cell
division. Although 70% of horizontally dividing mitoses are
2G12 positive, only 50% of the oblique cell divisions are 2G12
positive and this further decreases to 20% in vertically oriented
cell divisions. Thus, within the population of cells dividing in
a certain orientation, 2G12 immunreactivty increases consistently from vertically to horizontally oriented cell divisions.
Interestingly, Haydar and others (2003) observed in cortical
slices prepared during neurogenesis that the rotation of metaphase spindle takes significantly longer for the spindles assuming a vertical orientation that leads to horizontally oriented cell
divisions than that of spindles assuming a horizontal orientation
leading to vertically oriented cell divisions. These data suggest
that the vertical orientation of cell division is the default
orientation, and deviation from this default orientation of cell
division may require additional signals. These may involve the
phosphoepitope of GAP-43 recognized by the monoclonal 2G12
antibody because the correlation between 2G12 immunoreactivity and the orientation of cell division was found as a general
effect in different brain regions (such as the ventral telencephalon) as well as in VZ and SVZ precursors. Thus, although the
phosphoepitope of GAP-43 is not required for horizontal cell
divisions to occur because GAP-43–/– mice exhibit normal
orientations of mitotic cells on the VS, this epitope may still
play a role in specifying this mode of cell division together with
other molecular signals.
The 2G12 signal in horizontally dividing SVZ precursors was
particularly surprising as it sheds new light on the cell division
in the SVZ. Our quantitative analysis of the orientation of cell
division clearly showed that SVZ precursors do not divide
randomly during neurogenesis but rather are mostly oriented
parallel to the VS as previously suggested by Smart (1976). In
pronounced contrast to the mitoses occurring at the VS that are
predominantly vertical, those at abventricular positions divide
predominantly horizontally, at least during neurogenesis. Interestingly, the predominance of horizontally oriented cell
divisions observed in the SVZ at E15 vanished to an almost
random orientation of cell divisions by E21. Notably, this
random orientation of cell divisions is obviously different from
the predominant vertical orientation of cell division in the VZ
also at later gliogenic stages. Taken together, horizontal cell
divisions in the SVZ are correlated to the phase of neurogenesis
and disappear at the onset of gliogenesis. Indeed, the postnatal
SVZ of the cerebral cortex is a well-known source of glial cells
(Goldman 1995), whereas the embryonic SVZ is a source of
neurons (Tarabykin and others 2001; Malatesta and others 2003;
Haubensak and others 2004; Noctor and others 2004).
Interestingly, most of the neurogenic cell divisions in the SVZ
were observed to be symmetric in the sense that they give rise
to 2 postmitotic neurons—in pronounced contrast to the
mostly asymmetric neurogenic cell divisions in the VZ (Noctor
and others 2004). Thus, the horizontal orientation of SVZ cell
divisions and their 2G12 immunoreactivity may not be linked to
an asymmetry in the fate of the progeny. Alternatively, there
may be a yet unidentified asymmetry such as the generation of 2
distinct types of neurons (Shen and others 2002). Importantly,
however, recent data demonstrated asymmetric localization of
growth factor receptors on SVZ cells, clearly showing that
SVZ cells also polarize in regard to certain signals (Sun and
others 2005). It will be important to determine the nature of
these polarizing signals, as they obviously seem to differ from
the normal apicobasal cues. SVZ cells differ from VZ cells by the
lack of contact to the VS at the apical side as well as to the
basement membrane at the basal side (Haubensak and others
2004; Miyata and others 2004; Noctor and others 2004; Gotz and
Huttner 2005). One possibility of polarizing signals may be extra
cellular matrix components enriched in the intermediate zone
(Maier and others 1999) that is located basally on top of the SVZ.
In this regard, the maintenance of 2G12 immunostaining in
a subset of mitoses in cortical slice cultures is of interest
because also in this system signals from the ventricle as well as
the basement membrane are absent. As the cues regulating
2G12 immunoreactivity in horizontal cell divisions are present
in slice cultures, but absent in dissociated cell cultures, their
source is most likely within the cortical parenchyma itself. Thus,
2G12 immunoreactivity allows to monitor polarity cues instructing the deviation from the default orientation of cell
division, both in the VZ and SVZ, where the default orientation
seems to differ.
A Molecular Correlate to Asymmetric Cell Divisions
As most horizontal and oblique cell divisions are indeed
asymmetric in nature and generate one precursor cell and one
neuron (Chenn and McConnell 1995; Haubensak and others
2004; Kosodo and others 2004), 2G12 immunoreactivity also
appears to be correlated to asymmetric cell divisions. Interestingly, recent evidence suggests that also a third of all VZ
precursors dividing vertically distributes the apical membrane
patch asymmetrically and seems to generate a neuron and a
precursor cell (Haubensak and others 2004; Kosodo and others
2004; for review, see Gotz and Huttner 2005). This percentage
of neurogenic, asymmetric cell divisions among the vertically
oriented cell divisions (32%, (Kosodo and others 2004) is
reminiscent of the percentage of 2G12-immunopositive vertically oriented cell divisions (20--30%, Fig. 3), suggesting that the
2G12-positive vertically oriented cell divisions may indeed be
these that divide asymmetrically. In fact, 2G12 immunoreactivity in mitoses is tightly correlated to the phase of neurogenesis
(E13--20) in the rat cortex and is no longer detectable at E21
when gliogenesis takes over. Indeed, neurons arise often from
asymmetric cell divisions, although most glial precursors divide
symmetrically (Qian and others 1998, 2000). Further, in
agreement with the hypothesis that 2G12 immunoreactivity
relates to neurogenic fate decisions is the absence of 2G12
staining during S-phase of the cell cycle (Brittis and others
1995) and its gradual increase during M-phase as shown in our
quantitative analysis combined with PI staining. Thus, 2G12
immunoreactivity correlates to the phase of the cell cycle when
neuronal commitment occurs (McConnell and Kaznowski 1991).
Moreover, 2G12 immunoreactivity is maintained after mitosis
in young postmitotic neurons as demonstrated previously
(Brittis and others 1995). Notably, these young 2G12-immunoreactive neurons remain attached with their basal process to the
pial surface, as are 2G12-positive mitotic precursors (Brittis and
others 1995). These data were in fact among the first to show
that cells do not necessarily withdraw their basal process during
mitosis. Indeed, by now we know that the majority of
precursors during neurogenesis in the cerebral cortex are
radial glial cells that are attached via their radial processes to
the pial surface (Malatesta and others 2000; Hartfuss and others
2001; Miyata and others 2001; Noctor and others 2002;
Tamamaki 2002). Live imaging in cortical slices revealed that
radial glial cells do not withdraw their basal process during cell
division (Miyata and others 2001, 2004), as also detected by
other techniques in sections in situ (Gotz and others 2002;
Tamamaki 2002). Consequently, the basal daughter of a horizontally dividing radial glia cell is and remains attached to the
basement membrane and then moves toward the differentiating
cortical layers by somal translocation as first described by
Morest (1970a) and then observed live in slice preparations of
the developing cortex (Nadarajah and others 2001). 2G12 stains
these neurons connected to the pial surface by their radial
process during somal translocation (Brittis and others 1995).
Thus, 2G12 immunoreactivity is enriched in the horizontally
oriented subset of mitoses and seemingly persists in the basal,
neuronal daughters of these cell divisions. These data would
therefore be consistent with the interpretation that 2G12
immunostaining labels functionally asymmetric, neurogenic
cell divisions.
The Role of Phosphorylated GAP-43 in Cell Division
Given the enrichment of 2G12 immunoreactivity in horizontally
dividing neurogenic mitoses, the nature of the 2G12 antigen
is of great interest. The monoclonal antibody 2G12 was
raised against and clearly demonstrated to bind to a serinephosphorylated epitope (serine 41) of GAP-43 Meiri and others
(1991, 1998). However, the mitotic signal detected by 2G12 is
not detectable by other antibodies directed against GAP-43,
such as 7B10 or 10E8 (Fig. 5, see also Brittis and others 1995),
even though this may be explained by different fixation
requirements and affinities of these antibodies. Moreover, the
mitotic 2G12-immunopositive cells are detectable only in rat,
but not in mice, whereas 2G12 and other GAP-43 antibodies
label neurons and their processes in both species. Although this
may suggest that the mitotic 2G12 signal could be caused by
a yet unknown antigen recognized by 2G12, these data might
also be explained by a lower abundance of GAP-43 in VZ
precursors. Indeed, GAP-43 mRNA and protein are present in
precursors of the cerebral cortex of rats and humans, albeit at
lower levels than in neurons (Kanazir and others 1996; Esdar
and others 1999). Moreover, western blot and immunoprecipitation analysis of E13 rat cortex, a stage when hardly any
neurons are yet present, detected a clear GAP-43 signal with
2G12 and other antibodies directed against GAP-43, suggesting
that the mitotic 2G12 immunoreactivity is indeed the phosphoepitope of GAP-43. However, we cannot exclude the possibility that additional other epitopes are recognized by 2G12.
The signal detected by western blot in E13 cortex indicates
the presence of some GAP-43 that is phosphorylated by a serine
kinase during M-phase specifically in mitoses oriented horizontally or obliquely. Whereas protein kinase C is responsible for
the serine 41 phosphorylation of GAP-43 in axonal processes
(Meiri and others 1991) and a neural precursor cell line (Esdar
and others 1999), other proteins associated with the cytoskeleton, such as the intermediate filaments vimentin and glial
fibrillary acidic protein, are phosphorylated at specific serine
positions by cdc2-kinase exclusively during mitosis (Tsujimura
and others 1994; Kamei and others 1998). Noteworthy, the
cdc2-phosphorylated form of vimentin recognized by the
monoclonal antibody 4A4 is detected in all mitotic cells (Kamei
Cerebral Cortex 2006, V 16 Supplement 1 i129
and others 1998; Weissman and others 2003) in contrast to
serine 41--phsophorylated GAP-43 that is detectable mostly in
horizontally dividing precursors.
This difference further underlines the specificity of the
GAP-43 phosphorylation prompting the question as to its
function. Because GAP-43 is known to interact with the actin
cytoskeleton in neurons, its phosphorylation may play a specific
role during mitosis or cytokinesis. As a first step to elucidate
a functional role of GAP-43 during mitosis, we examined the
orientation of cell division in the GAP-43–/– mice (Maier and
others 1999). However, the number of horizontally or obliquely
dividing precursors was not significantly affected in the absence
of GAP-43, suggesting that either GAP-43 is not required for
spindle orientation or its absence may be compensated for by
other molecular mechanisms. However, the absence of GAP-43
does affect neurogenesis with a notable decrease in neuron
generation and maturation after GAP-43 deletion in vitro and in
vivo (Mani and others 2000, 2001; Shen and others 2004). Thus,
GAP-43 might play an important role in the differentiation
initiated immediately after cell division rather than the orientation of cell division.
Such an influence on cell fate could be achieved, for example,
by directing the transport of cell fate determinants via the
cytoskeleton. For example, dependent on its phosphorylation
state, lethal giant larvae mediates the transport of numb, an
inhibitor of Notch signaling, to the basal side of precursors and
thereby influences neurogenic fate decisions (Klezovitch and
others 2004). Similarly, the cdc2-kinase plays an important role
for the localization of apical fate determinants in Drosophila
neurogenesis (Tio and others 2001), prompting the speculation
that GAP-43 phosphorylation might be involved in this via its
interaction with the cytoskeleton. Moreover, the phosphorylation of GAP-43 increases binding to calmodulin and thereby
affects the Ca2+ concentration in the cells, a mechanism promoting cell cycle exit in various other cell types (Herget and
others 1993; Esdar and others 1999). Thus, the higher content
of the GAP-43 phosphoepitope in horizontally dividing cells
may provide a lead to identify key signaling components involved in asymmetric cell division in mammalian neurogenesis.
Notes
Conflict of Interest : None declared.
Address correspondence to Stefan H. Stricker, Center for Molecular
Medicine, Dr. Bohrgasse 9/4, 1030 Vienna, Austria. Email: magdalena.
[email protected].
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