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The Journal of Neuroscience, January 1, 2002, 22(1):239-247
Growth-Associated Protein-43 Is Required for Commissural Axon
Guidance in the Developing Vertebrate Nervous System
Yiping
Shen1, 2,
Shyamala
Mani2,
Stacy L.
Donovan2,
James E.
Schwob1, and
Karina F.
Meiri1
1 Department of Anatomy and Cellular Biology, Tufts
University School of Medicine, Boston, Massachusetts 02111, and
2 Programs in Cell and Molecular Biology and Neuroscience,
State University of New York Upstate Medical University, Syracuse, New
York 13210
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ABSTRACT |
Growth-associated protein-43 (GAP-43) is a major growth cone
protein whose phosphorylation by PKC in response to extracellular guidance cues can regulate F-actin behavior. Here we show that 100% of
homozygote GAP-43 ( /) mice failed to form the anterior commissure
(AC), hippocampal commissure (HC), and corpus callosum (CC) in
vivo. Instead, although midline fusion was normal, selective fasciculation between commissural axons was inhibited, and
TAG-1-labeled axons tangled bilaterally into Probst's bundles.
Moreover, their growth cones had significantly smaller lamellas
and reduced levels of F-actin in vitro. Likewise, 100%
of GAP-43 (+/) mice with one disrupted allele also showed defects in
HC and CC, whereas the AC was unaffected. Individual GAP-43 (+/) mice
could be assigned to two groups based on the amount that PKC
phosphorylation of GAP-43 was reduced in neocortical neurons. In mice
with ~1% phosphorylation, the HC and CC were absent, whereas in mice
with ~10% phosphorylation, the HC and CC were smaller. Both results
suggest that PKC-mediated signaling in commissural axons may be
defective. However, although Probst's bundles formed consistently at
the location of the glial wedge, both GAP-43 (/) and GAP-43 (+/+)
cortical axons were still repulsed by Slit-2 in vitro,
precluding failure of this deflective signal from the glial wedge as
the source of the phenotype. Nonetheless, the data show that a
functional threshold of GAP-43 is required for commissure formation and
suggests that failure to regulate F-actin in commissural growth cones
may be related to inhibited PKC phosphorylation of GAP-43.
Key words:
anterior commissure; hippocampal commissure; corpus
callosum; GAP-43; F-actin; PKC; Slit-2
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INTRODUCTION |
Communication between the
telencephalic hemispheres occurs through three major commissures: the
corpus callosum (CC), the hippocampal commissure (HC), and the anterior
commissure (AC) (Abbie, 1940 ). Mutational and genetic analyses have
identified several environmental cues that guide axons through the
commissures during development (Tear, 1999) and have also shown a
crucial requirement for actin regulation. Thus, genetic deletion of
F-actin binding proteins such as myrisotylated alanine-rich C kinase
substrate (MARCK) (Stumpo et al., 1995), MacMARCKs (Chen et al.,
1996), ankyrinB (Scotland et al., 1998), Mena
(Lanier et al., 1999), and p190RhoGAP (Brouns et al., 2000) all prevent
commissure formation. However, in these instances, defects in midline
fusion may also contribute to the phenotype. Thus, how guidance cue
receptors are coupled to cytoskeletal effectors within commissural
growth cones during midline crossing remains little understood
(Mueller, 1999).
Growth-associated protein-43 (GAP-43) is a single copy gene that is a
major neuronal substrate of protein kinase C (PKC) and, like Mena,
MARCKs, MacMARCKs, and p190rhoGAP, modulates F-actin in growth cones
(He et al., 1997; Oestreicher et al., 1997). Like MARCKs also,
regulation of F-actin depends on its phosphorylation status:
PKC-phosphorylated GAP-43 is spatially segregated to motile and
advancing lamellas and filopodia (Dent and Meiri, 1998) and stabilizes
actin filaments in vitro by removing them from the polymerization-depolymerization cycle (He et al., 1997). In contrast, unphosphorylated GAP-43 is enriched in retracting or collapsing lamellas (Dent and Meiri, 1992) and inhibits F-actin polymerization in vitro (He et al., 1997). Phosphorylation of GAP-43 by PKC
in growth cones can be stimulated by Ig superfamily cell adhesion molecules (IgSF-CAMs), such as L1, that have been implicated in commissural axon guidance (Kamiguchi et al., 1998; Demyanenko et al.,
1999). Neurites require GAP-43 to respond to IgSF-CAM-mediated signals
in vitro (Meiri et al., 1998).
In mice, failure to express GAP-43 gives rise to a severe in
vivo phenotype: >95% of GAP-43 (/) mice die perinatally,
with abnormalities of axonal pathfinding reflected in failure to form topographic maps in somatosensory, visual, and auditory cortices (Maier
et al., 1999). Axon guidance at the optic chiasm is also disrupted
(Strittmatter et al., 1995; Kruger et al., 1998; Sretavan and
Kruger 1998). Here we investigated telencephalic commissure formation in GAP-43-deficient mice that were generated from targeted CJ7 embryonic stem cells in isogenic 129S3/imJ mice (genetic
designation+Mgf-SlJ; JAX stock number 002448, Bar
Harbor, ME) and backcrossed for 8-12 generations with C57BL/6N.
Homozygote GAP-43 (/) mice failed to form the AC, HC, and CC.
Significantly midline fusion was normal. Moreover, growth cones from
commissural axons had smaller lamellas and reduced F-actin
immunoreactivity. None of the GAP-43 (+/+) mice from either genetic
background (129S3/imJ or C57BL/6N) showed any commissural defects,
precluding congenital abnormalities as the source of the phenotype.
Furthermore, the severity of commissural dysgenesis in individual
GAP-43 (+/) mice was directly proportional to the amount that
phosphorylation of GAP-43 was reduced. The results suggest that the
functional threshold of GAP-43 requires its phosphorylation by PKC in
commissural growth cones.
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MATERIALS AND METHODS |
Histology and immunocytochemistry. Anesthesia by
halothane inhalation was followed by perfusion with PBS, pH 7.4, followed by Bouin's fixative. Brains were post-fixed in Bouin's,
embedded in paraffin, and sectioned at 14 µm. Staining used Nissl and
GAP-43 immunohistochemistry used 7B10 [the pan-GAP-43 monoclonal
antibody (mAb)] or 2G12 (the phospho-specific mAb) (Dent and Meiri,
1998). For TAG-1 immunohistochemistry with 4D7 (Developmental
Studies Hybridoma Bank, University of Iowa, Iowa City, IA), brains were fixed in 4% paraformaldehyde (PFA) and cryosectioned at 14 µm. Sections were blocked in PBS containing 10% serum according to the 20 antibodies and permeabilized with 0.02% digitonin. The 20 FITC- and
Texas Red-conjugated antibodies were from Vector Laboratories
(Burlingame, CA).
Dye tracing. Brains from postnatal day 0 (P0) animals
fixed in 4% PFA were double dye labeled with a single crystal of
Fast DiA
[4-(4-dilinoleyamino)stryryl)-N-methyl-pyridinium iodide, 4-chlorobenzenesul-fonate] and Fast DiI
(1,1'-dilinoleyl-3, 3,3',3'-tetramethyl-indocarbocyanine 4-cholorobenzenesulfonate] (Molecular Probes, Eugene, OR)
implanted into the cortical layer of frontal cortex and the occipital
cortex, respectively (Ozaki and Wahlsten, 1992). Labeled brains were
incubated at 37°C for 30 d and sectioned sagittally at 50 µm.
Dissociated cultures. Fetuses were dissected from timed
pregnant GAP-43 (+/) females [embryonic day 15.5 (E15.5); noon of day on which plug is found is E0.5] and kept in cold sterile Gray's solution while a modified multiplex PCR was performed on tail samples
(Maier et al., 1999). After genotyping, neocortices from identified (/) and (+/+) mice were cut into 3 × 4 mm pieces using a McIllwain tissue chopper, rinsed in
Ca2+ and
Mg2+-free HBSS, and digested with
trypsin EDTA. Serum-containing culture medium (DMEM plus 10%
horse serum plus 0.1% penicillin-streptomycin) was added, and cells
were dispersed by trituration. Dissociated cells were recovered from
the 400 rpm pellet, resuspended in culture medium, and plated onto
poly-D-lysine-coated eight-well Lab-Tek (Naperville, IL) plates at low density (800-1000 cells per well). Cultures were grown in 5% CO2 at 37°C. After
24 hr in vitro, dissociated cortical neurons were fixed with
prewarmed 4% paraformaldehyde in PBS as described previously (Dent and
Meiri, 1998) and then double labeled with anti-TAG 1 antibody, followed
by biotin-labeled secondary antibody and streptavidin-labeled FITC
(Vector Laboratories). Rhodamine phalloidin (Molecular Probes) was
applied for 40 min before cells were coverslipped with Vectashield
mounting medium (Vector Laboratories). Quantitation of area and
documentation of immunoreactivity with TAG-1 and phalloidin was done
blindly on growth cones selected as the microscope stage was moved in the same random pattern around each well.
Collagen gel cocultures. E15.5 fetuses were dissected and
genotyped as before. Neocortex was separated from cortex and the area
of the neocortex previously described as giving rise to callosal axons
(Ozaki and Wahlsten, 1998), cut into 200 µm3 cubes with a McIllwain tissue
chopper, and kept in cold L-15 medium with 0.6% glucose until
embedding in collagen. HEK 293 cells stably transfected with
Slit-2 or vector alone (a generous gift of Dr Y. Rao, Washington
University, St. Louis, MO) were premixed with collagen gel [900
µl of Vitrogen 100 (Cohesion, Palo Alto, CA), 100 µl of 100× DMEM
(Life Technologies, Gaithersburg, MD), and 23 µl of 1 M NaHC03]. Approximately
5-6 µl of gel was dotted onto eight-well Lab-Tek slides, gelled in a
37°C incubator for 15-20 min, and then overlaid with 100 µl of
collagen gel. Tissue pieces were placed in the collagen gel at a
distance of 100-300 µm from the cell-gel mix. After the collagen
gelled, 500 µl of culture medium consisting of DMEM with GlutaMAX,
10% horse serum, and 0.1% penicillin-streptomycin was added to each
well. Explants were cultured for 24 hr in a humidified
CO2 incubator, and live cultures were
photographed using differential interference contrast optics. Cultures
were fixed after 48 hr. To quantitate the response of the explants,
explants from the 24 hr photographs were bisected parallel to the
orientation of the transfected cells, and neurites were counted. An
attractive response was defined as >70% of neurites in the hemisphere
adjacent to the transfected cells, whereas a repulsive response was
defined as >70% of the neurites in the hemisphere distant from the
transfected cells. A response was scored as neutral when there was no
difference in the distribution of neurites between the two hemispheres.
Explants that did not send out neurites were not included.
Microscopy. Laser-scanning confocal microscopy of both
immunostained and dye-labeled sections used an MRC 1024 microscope (Bio-Rad, Hercules, CA) equipped with a single krypton-argon laser and
T1 and T2A filters using identical parameters for direct comparison between samples. Dissociated cortical neurons were viewed under FITC
and rhodamine optics at 60× magnification using a Nikon (Tokyo, Japan)
Diaphot 200 microscope and ND4 and ND2 filters. Cultures were
photographed with a Hamamatsu (Hamamatsu City, Japan) 4742-95 CCD
camera under identical conditions, and images were saved as TIFF files
using Openlab (Improvision Inc., Lexington, MA) software. All
post-photography manipulations of images were identical. Area measurements and densitometry F-actin labeling of TAG-1 immunoreactive growth cones with rhodaminated phalloidin used IP Lab (Scanalytics Inc., Fairfax, VA).
Western blot analysis. Forebrains from P0 mice were rapidly
dissected, homogenized in 10 mM Tris, 2 mM DTT, 1 mM PMSF, and 5 mM EDTA, pH7.4, at 4°C, and centrifuged at
100,000 × g for 30 min. Microsomal proteins were
separated by 10% PAGE, transferred to polyvinylidene difluoride
membrane, and incubated with either 7B10 or 2G12. Specific
immunoreactivity was detected with
125I-labeled anti-mouse IgG (specific
activity 18 µCi/µg) and quantitated by PhosphorImager
analysis (Molecular Dynamics, Sunnyvale, CA) using Imagequant software
(He et al., 1997).
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RESULTS |
GAP-43 (/) mice fail to form telencephalic commissures
The telencephalic commissures have distinct roles and
developmental histories: the largest is the CC, which interconnects the
neocortex; the HC interconnects the archicortex, principally the
hippocampus; and the AC interconnects paleocortex, components of the
amygdala, interhippocampal gyrus, and temporal neocortex (Fig.
1a,c,e).
At P0, no axon bundles crossing the midline could be detected in
Nissl-stained, midsagittal sections from GAP-43 (/) brains (Fig.
1b). Instead, CC and HC axons formed neuroma-like whorls
(Probst's bundles) on either side of the midline, which could be
detected in either Nissl-stained horizontal sections (Fig.
1d) or anti-TAG-1 immunolabeled coronal sections (Fig.
1f). The phenotype resembles human type 1 callosal
agenesis, which is considered to be an axon guidance defect (Kamiguchi
et al., 1998). The diverse origins of the AC give rise to more complex behavior (Velut et al., 1998). The anterior branch, which is derived from the anterior olfactory nucleus and anterior piriform cortex, was
immunolabeled with TAG-1, like the CC and HC, and likewise formed
Probst's bundles (Fig.
2f). In contrast, the
posterior branch, which is derived from piriform and temporal cortices, was not immunolabeled with TAG-1 (Fig. 2d) and did not form
Probst's bundles, instead defasciculating and descending
posteroventrally (Fig. 2a).
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Figure 1.
a, b,
Nissl-stained parasagittal sections at P0. a, GAP-43
(+/+) showing corpus callosum (cc), hippocampal
commissure (hc), and anterior commissure
(ac). b, GAP-43 (/) showing no
commissures. c, d, Nissl-stained
horizontal sections at P0. c, GAP-43 (+/+) showing CC
and HC. d, GAP-43 (/) showing that midline
commissural axons tangle into Probst's bundles
(pb). Asterisks indicate
inappropriate fasciculation of axons in the corpus striatum.
e, f, Coronal sections at P0 labeled with
the anti-TAG1 mAb 4D7, followed by TRITC-labeled secondary antibody.
e, GAP-43 (+/+) showing CC and HC. f,
GAP-43 (/) showing no midline crossing (Probst's bundles
themselves are located more anteriorly than this section illustrates).
No callosal or hippocampal commissures ever formed in GAP-43 (/)
mice (n = 12). Scale bars, 500 µm.
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Figure 2.
a, b, Nissl-stained
coronal sections at P0. a, GAP-43 (+/+) showing both
anterior (ab) and posterior (pb)
branches of the anterior commissure. b, GAP-43 (/)
showing only posterior branches. Neither branch crossed the midline.
c, f, Coronal sections at P0.
c, GAP-43 (+/+) visualized by differential interference
contrast showing the anterior and posterior branches.
d, Same section after TAG-1 immunohistochemistry to show
that only the anterior branch is labeled with TAG-1. e,
GAP-43 (+/+) after TAG-1 immunohistochemistry showing TAG-1-labeled
(anterior branch) axons crossing the midline. f, GAP-43
(/) after TAG-1 immunohistochemistry showing TAG-1-labeled
(anterior branch) axons formed Probst's bundles
(arrows). Scale bar: a, b,
500 µm; c, d, 100 µm;
e, f, 250 µm.
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Failure to express GAP-43 prevents formation of the telencephalic
commissures completely. In GAP-43 (+/+) mice between P7 and P21, the CC
increased in area by 1.74-fold, the HC by 2.8-fold, and the AC by
1.5-fold. In contrast, no axons crossed the midline in any of the
GAP-43 (/) mice up to P21. Quantitation used three mice at each
time point.
GAP-43 (/) commissural growth cones have reduced lamellal area
and levels of F-actin in vitro
Total absence of GAP-43 had two effects on TAG-1-labeled
neocortical (presumed callosal) growth cones, which are illustrated in
Figure 3. First, although GAP-43 (/)
TAG-1-labeled cultures extended neurites normally in dissociated
cultures, the surface areas of their growth cones were reduced from
60.36 ± 4.76 µm2 [SEM;
n = 54 GAP-43 (+/+) growth cones from 10 random fields from four independent cultures] to 41.06 ± 6.6 µm2 [SEM; n = 56 GAP-43
(/) growth cones from 10 random fields from four cultures]. This
reduction of 32% was statistically significant (p < 0.001; two-tailed t test).
Second, levels of F-actin were reduced from an average pixel intensity
of rhodaminated phalloidin of 136.5 ± 9.4 [SEM;
n = 45 GAP-43 (+/+) growth cones from 10 random fields
from four independent cultures] to 64.8 ± 7.5 [SEM; n = 27 GAP-43 (/) growth cones from eight random
fields from four independent cultures]. This reduction of 57% was
also statistically significant (p < 0.001;
two-tailed t test). In contrast, no consistent differences
were seen in the pixel intensities of rhodamine phalloidin between
TAG-1-labeled GAP-43 (+/+) or (/) cell bodies. Hence, GAP-43 is
required to maintain F-actin levels in dissociated callosal growth
cones in culture.
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Figure 3.
Growth cones from dissociated cortical cultures
from GAP-43 (+/+) (a-d) or GAP-43 (/)
(e-h) mice double labeled with TAG-1
(a-h) and rhodamine phalloidin (a'-h')
after 24 hr in vitro. Arrows indicate the
growth cone used for measuring both lamellal area and pixel intensity
of rhodamine phalloidin labeling with IP Lab (Scanalytics Inc.).
Lines bisecting neurites indicate limit of growth cones
used for measurement. Scale bar, 100 µm.
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GAP-43 (/) axons respond normally to Slit-2
Axon guidance across the corpus callosum requires that axons are
deflected toward the midline. One source of repulsive cues is the glial
wedge, located bilaterally to the midline, which secretes Slit-2 (Shu
and Richards, 2001). In GAP-43 (+/+) mice, the glial wedge was highly
immunoreactive with GFAP antibody, visualized with tetramethylrhodamine
isothiocyanate (TRITC) labeled secondary antibody (Fig.
4a). In contrast, although the
glial wedge was present in GAP-43 (/) mice, it was very poorly
immunoreactive with GFAP and instead labeled with anti-RC1 antibody, a
marker of immature glia (Fig. 4b,c,
green, arrows). GAP-43 (/) axons that fail to
cross the midline consistently formed Probst's bundles in the area of
the glial wedge, partially obscuring it at this level (Fig.
4b,c). In contrast, glia within the Probst's
bundles were immunoreactive with GFAP. The results raise the question of whether the Probst's bundles arise at this location because callosal axons fail to respond to Slit-2-mediated deflective signals supplied by the glial wedge. Indeed, explants of E15.5 GAP-43 (+/+)
neocortex were strongly repulsed by Slit-2 when they were cocultured
with transfected cells in collagen gels in vitro
(n = 14 explants) (Fig. 4d, black
bars). However, GAP-43 (/) explants were also strongly
repulsed by Slit-2 under similar conditions (n = 14 explants) (Fig. 4d, white bars). The results
therefore suggest that, although GAP-43 (/) callosal axons
consistently form Probst's bundles in the vicinity of the glial wedge,
with consequent failure to cross the midline, this defect cannot be attributed to failure of the GAP-43 (/) axons to respond to Slit-2-mediated signals.
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Figure 4.
a, Coronal confocal section of
E17.5 GAP-43 (+/+) cortex labeled with GFAP mAb, followed by TRITC
showing bilateral localization of the glial wedge
(gw; arrows), which is highly
immunoreactive with GFAP. b, c, Similar
sections of GAP-43 (/) cortex double labeled with GFAP, followed by
TRITC and RC1 followed by FITC. Note that the glial wedge
(gw; arrows) is detected by RC1
immunoreactivity but is very poorly immunoreactive with GFAP. The
Probst's bundles (pb) are consistently localized
in the vicinity of the glial wedge and are immunoreactive with GFAP.
Scale bar, 125 µm. d, Quantitation of the responses of
each explant culture to Slit-2-expressing HEK 293 cells suspended in a
collagen gel capsule. A response was defined as attractive
(A) when >70% of the total neurites grew in the
hemisphere adjacent to the cell-gel capsule, whereas a response was
defined as repulsive (R) when >70% of the total
neurites grew in the hemisphere distant from the cell-gel capsule. A
response was defined as neutral (N) when neurites
in neither hemisphere were >70% of total. Black bars
represent GAP-43 (+/+) cultures (n = 14), and
white bars represent GAP-43 (/) cultures
(n = 12). Dashed bars represent
GAP-43 (+/+) cultures (n = 4) cocultured with HEK
293 cells expressing vector alone as control.
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Absence of GAP-43 affects selective fasciculation rather
than axon elongation
Implanting Fast DiI and Fast DiA
into frontal cortex and occipital cortex, respectively, demonstrated
that callosal axons at the midline of GAP-43 (+/+) mice show rough
topographic organization in both the anteroposterior and dorsoventral
axes (Fig. 5a), consistent with selective fasciculation between commissural axons that derive from
neighboring areas, as has been reported previously (Ozaki and Wahlsten,
1992). In contrast, no labeled fibers reached the midline in GAP-43
(/) mice as expected (Fig. 5b). Similarly, when
parasagittal sections were taken at the distance from the midline at
which Probst's bundles are found in the (/) mice, topographic
orientation was maintained in the GAP-43 (+/+) sections. In contrast a
similar section through GAP-43 (/) Probst's bundles revealed
mixing of dye-labeled axons and significant misrouting: axons exited
the bundles in trajectories that invaded anterior territories
inappropriately (Fig. 5d). Thus, GAP-43 is also required for
selective fasciculation to maintain topographic organization of
commissural axons.
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Figure 5.
Parasaggital sections of GAP-43 (+/+)
(a, b) and GAP-43 (/)
(c, d) mice that have been labeled in
frontal cortex with a crystal of DiI (green) and
in occipital cortex with a crystal of DiA (red).
a, c, Sections at the midline showing
appropriate topographic organization of GAP-43 (+/+) callosal axons
(a) but no GAP-43 (/) axons crossing the
midline (c). b, d,
Sections taken at the distance from the midline in which Probst's
bundles are found in the (/) mice (d). Again,
topographic organization of label is seen in the GAP-43 (+/+) section
(b) but abnormal mixing and projection of axons
in the GAP-43 (/) section (d). Scale bar, 500 µm.
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Commissural agenesis is not attributable to a
congenital abnormality in the GAP-43 (/) mice
Callosal dysgenesis has been described in several of the inbred
129 substrains often used to make knock-out mice, although not in
the129S3/imJ substrain from which our mice were derived (Livy and
Wahlsten, 1991; Magara et al., 1999; Wahlsten et al., 1999). The
congenital abnormalities differ from the GAP-43 phenotype: the HC and
the AC are normal in affected adults (Livy and Wahlsten, 1991), and the
trait is recessively inherited (see below). To conclusively verify that
the phenotype we described above reflects genetic deletion of GAP-43
rather than congenital abnormalities in the 129S3/imJ substrain that
persists despite backcrossing, we repeated the experiments in the
founder 129S3/imJ colony. None of the GAP-43 (+/+) mice
(n > 25) showed any abnormalities in telencephalic
commissures. Thus, the commissural phenotype described here cannot be
attributed to congenital defects in the 129S3/imJ substrain.
Commissure formation is also defective in heterozygote GAP-43
(+/) mice
The telencephalic commissures were also abnormal in the GAP-43
(+/) mice. Both CC and HC were totally absent in 39% of mice (16 of
41) examined at P0, P7, and P21 (Fig.
6c) and was reduced in the
remainder (25of 41) (Fig. 6a,b). The reduction in
the cross-sectional area of the HC-CC at the midline was significant
at each time point (p < 0.01; two-tailed
t test) (Fig. 6d). The rate of increase was also
reduced, suggesting that HC and CC axons grow more slowly or that they
were misrouted (Fig. 6d). However, DiI labeling of CC and HC
axons showed no evidence that they were rerouted through the AC
(results not shown). In contrast, the midline cross-sectional area of
the AC was not affected in the GAP (+/) mice.
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Figure 6.
a-c, Nissl-stained parasagittal
sections close to the midline of GAP-43 (+/) forebrains at P0 showing
corpus callosum (cc), hippocampal commissure
(hc), and anterior commissure (ac). The
severity of phenotypes in the (+/) ranges from reduced size of CC and
HC (a, b) to complete absence of both
commissures (c). Note that the anterior
commissure is normal in all cases. Scale bar, 250 µm.
d, The area of the CC-HC at midsagittal levels was
measured at P0, P7, and P21 in GAP-43 (+/+) ( ) and those GAP-43
(+/) ( ) mice with commissures. Results presented as mean ± SEM; n  3 in each case. There was a
significant reduction in size of the HC and CC at both P7 and P21
(p < 0.05).
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The severity of the callosal phenotype in GAP-43 (+/) mice
correlates with amount of PKC phosphorylated GAP-43
Both levels of GAP-43 expression and its phosphorylation by PKC
were significantly reduced in the GAP-43 (+/) mice, and, moreover,
there was a strong correlation between the reduction and severity of
the commissural phenotype. Levels of GAP-43, detected with the
pan-GAP-43 mAb 7B10 (data not shown), or PKC-phosphorylated GAP-43
detected with the phospho-specific mAb 2G12 (Fig.
7), were lower in most areas of the
forebrain and brainstem (Fig. 7b,c). Phosphorylated GAP-43 was lowest when the CC and HC were absent (Fig.
7c). In contrast, expression and phosphorylation of GAP-43 in the AC appeared normal, as did the AC itself (see below). We quantitated GAP-43 expression and phosphorylation in anterior cortex of
two litters of mice by Western blotting and used
125I-labeled secondary antibody to detect
specific immunoreactivity, as we had done previously (He et al., 1997).
In addition, we measured the midline cross-sectional area of the CC and
the AC in the same mice (Fig. 7d). The GAP-43 (+/) mice
expressed only 27.3 ± 3.5% of (+/+) levels of GAP-43 protein.
However, they could be assigned to two distinct groups on the basis of
the levels of phosphorylation that were independent of the litter from
which they were derived. In the first group, phosphorylated GAP-43 was
between 1.3 and 1.8% of wild type (n = 4); these
animals were all acallosal. In the second group, phosphorylated GAP-43
was between 4.4 and 10.6% of wild type (n = 6); here
the CC was between 19.9 and 63.7% of normal. The area of the AC
averaged 93.7 ± 0.25% of (+/) (n = 10 from
both litters). The results show that a functional threshold of GAP-43
is required for CC formation. The threshold strictly parallels the
amount of phosphorylated GAP-43 in anterior cortex, the source of
callosal axons.
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Figure 7.
a-c, Parasaggital sections
at P0, from GAP-43 (+/+) (a) and (+/)
(b, c) mice labeled with the
anti-phospho-GAP-43-specific mAb 2G12, followed by FITC mice and
arranged according to severity of callosal phenotype (CC).
Arrows indicate position of the CC. There is less PKC
phosphorylated in both cortex and CC in GAP-43 (+/) compared with
(+/+). On the other hand, phosphorylation in the AC appears normal.
Scale bar, 500 µm. d, Reduction in callosal area of
GAP-43 (+/) mice correlates with the amount of phosphorylated GAP-43
in anterior cortex. White bars indicate area of AC,
hatched bars indicate area of CC, and black
bars indicate the amount of PKC-phosphorylated GAP-43 in
anterior cortex quantitated from Western blots by
125I-labeled secondary antibody, followed by PhosphorImager
analysis. Note that the quantitative results are presented on a log
scale.
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DISCUSSION |
Our results here show that GAP-43, a protein whose phosphorylation
in response to extracellular signals can regulate actin dynamics in
growth cones (Dent and Meiri 1992, 1998; He and Meiri, 1997; Rosner and
Vacun, 1999), is required for formation of the major interhemispheric
projection pathways in the telencephalon. Both homozygotes and
heterozygotes are affected, and the phenotype is the most severe thus
far described for telencephalic commissure formation. Together with our
previous results showing that GAP-43 (/) mice also have profound
defects in thalamocortical ingrowth resulting in failure to form
topographic maps in the cortex (Maier et al., 1999), they pinpoint
GAP-43 as a major contributor to cortical assembly in vertebrates.
The commissural phenotype is independent of the genetic background of
the mice and reflects the targeted deletion at the GAP-43 locus:
congenital callosal dysgenesis occurs in several inbred mouse strains,
including some 129 substrains, namely 129/Ola,129P3/J, 129/ReJ, and
129sv/ev (Livy and Wahlsten, 1991; Livy et al., 1997; Magara et al.,
1999; Wahlsten et al., 1999). It has never been described in the
129S3/svImJ substrain, from which the CJ7 embryonic stem cells were
derived. Nor have we encountered it in >25 wild-type 129S3/svImJ mice
or in >75 wild-type 129S3/svImJ//C57BL/6N 8-12 generation
backcrosses, in which 99.9% of the genome derives from the C57BL/6N
background and in which the mutation is maintained. Current models
suggest that at least three separate loci are involved in congenital
callosal dysgenesis (Livy and Wahlsten, 1991; Magara et al., 1999;
Wahlsten et al., 1999) and that at least four of the recessive alleles
(from two of the putative three loci) are required for a phenotype to
become evident (Livy and Wahlsten, 1991). In addition, the action of
genetic modifiers causing phenotypic variation among the isogenic sibs
has been seen in other mouse strains (Bulman-Fleming and Wahlsten,
1991). In contrast, the GAP-43 phenotype requires only one affected
allele. Moreover, the penetrance is 100% in both homozygotes and
heterozygotes, significantly more than that seen in the congenitally
affected mice. Thus, the abnormalities described here are attributable to disruption of the GAP-43 gene. However, we cannot rule out, and
indeed it seems highly likely, that genetic modifiers contribute to the
reduction of GAP-43 levels in the heterozygotes.
Partial depletion of GAP-43 in growth cones with antibodies or
oligonucleotides in vitro resulted in unstable lamellas and a reduction of F-actin (Aigner and Caroni, 1993; Frey et al., 2000),
suggesting that a functional threshold of GAP-43 is required for
regulation of F-actin growth cone function. Our results, showing that
both lamellal area and F-actin levels were also significantly reduced
in TAG-1-labeled GAP-43 (/) commissural growth cones, confirm those
data. Moreover, the GAP-43 heterozygotes strongly suggest that
PKC-phosphorylated GAP-43 is a major functionally relevant form
in vivo. Thus, the HC and CC mice were absent in mice with
only 1% of normal phosphorylation and significantly smaller in mice
with only 10% of normal phosphorylation. Because the phosphorylation
status of GAP-43 determines whether it will stabilize F-actin
(phosphorylated GAP-43) or prevent F-actin polymerizing (unphosphorylated GAP-43; see introductory remarks) (He et al., 1997;
Rosner and Vacun, 1999), we hypothesize that the effects on
phosphorylation and F-actin are causally related. However, we cannot
rule out that unphosphorylated GAP-43 also has indirect effects on
F-actin via phosphatidyl inositol bis phosphate, which also
contribute to the phenotype (Laux et al., 2000; Tejero-Diez et al.,
2000).
The close correlation between reduced levels of PKC-phosphorylated
GAP-43 and the severity of the phenotype in GAP-43 (+/) mice further
implicates PKC in the regulation of commissural axon guidance. Direct
activation of PKC in growth cones concurrently increases elaboration of
motile lamellas and levels of phosphorylated GAP-43 within them (Rosner
and Vacun, 1999). These effects on growth cone dynamics are prevented
by inhibitors of PKC and F-actin polymerization (Rosner and Vacun,
1999), suggesting that regulation of GAP-43 by PKC phosphorylation may
modulate F-actin behavior in commissure growth cones in
vivo, as it does in vitro (He et al., 1997).
Like GAP-43, Mena, MARCKs, and p190RhoGAP interact with F-actin and are
enriched in growth cones. However, the commissural defects seen when
they are genetically deleted have been attributed, in part, to
inhibition of neural tube closure and midline fusion (Stumpo et al.,
1995; Brouns et al., 2000). Midline fusion was normal in our GAP-43
(/) mice (Y. Shen and K. F. Meiri, unpublished results), as
well as the guidance receptor mutants (Fazeli et al., 1997; Bergemann
et al., 1998), indicating that callosal agenesis does not
require fusion to be disrupted and suggesting that the two effects
might be independent. In fact, the severity of the phenotype closely
parallels the extent of protein distribution in growth cones. For
instance, GAP-43, MARCKs, and p190rhoGAP, in which penetrance is 100%,
are all highly enriched throughout growth cones (Meiri et al.,
1988; Wiederkehr et al., 1997; Brouns et al., 2001), whereas Mena
and ankyrinB, in which penetrance is only
~50%, are limited to the tips of filopodia and sites of contact,
respectively (Scotland et al., 1998; Lanier et al., 1999).
In GAP-43 (/), mice the Probst's bundles were located at the glial
wedge, a bilateral structure thought to act as a source of deflective
signals that direct midline axons toward the midline (Shu and Richards,
2001). Nonetheless, GAP-43 (/) axons responded normally to Slit-2,
the proposed repulsive signal secreted by the glial wedge. Even so,
glial abnormalities may also contribute to the phenotype. GAP-43 is
first upregulated in neuronal precursors that are at least bipotential
(Sensenbrenner et al., 1997) and, when depleted, inhibits both neuronal
and glial differentiation (Mani et al., 1999). In fact, preliminary
evidence suggests that these glia may be immature (Shen and Meiri,
unpublished results). In this regard, the differing relationships
between commissural axons and their supporting glial pathways may also
explain why the AC but not the CC is spared in GAP-43 (+/) mice. In
the case of the CC, axons are actively guided across the midline by a
precise temporal coordination of midline fusion, glial wedge function, and sling formation (Shu and Richards, 2001). In contrast, in the case
of the AC, axon guidance is a substantially more passive process
because a defined glial channel is already in place (Pires-Neto et al.,
1998).
IgSF members, notably L1CAM and neural cell adhesion molecule
(NCAM), as well as basic FGF (bFGF), stimulate GAP-43
phosphorylation in growth cones (Meiri et al., 1998; Tejero-Diez et
al., 2000). Moreover, elimination of GAP-43 blocked neurite outgrowth
stimulated by either CAMs or bFGF, indicating its centrality in
mediating the effects of IgSF-type CAMs, at least in vitro
(Meiri et al., 1998). Interestingly, bFGF-mediated stimulation of
GAP-43 phosphorylation also increased its association with the actin
cytoskeleton (Tejero-Diez et al., 2000). Hence, the disruption in
selective fasciculation suggests that one component of the GAP-43
phenotype may be failure to transduce CAM-mediated signals. However,
although several IgSF members, most notably TAG1 and L1, have been
located in commissural axons (Fujimori et al., 2000) and functional
mutations of L1 and its intracellular signal transduction pathway
(which involves the FGF receptor 1) also lead to callosal
agenesis in humans (Kamiguchi et al., 1998), failure of IgSF-mediated
signaling alone seems unlikely to account for the GAP-43 phenotype: no
callosal defects have been reported in mice deficient in all NCAM
isoforms (Cremer et al., 1997), and callosal deficits in L1 mutant mice
are manifest as reduced size rather than agenesis (Demyanenko et al.,
1999). Thus, absent a significant combinatorial effect between IgSF
members in the callosum, our results suggest that regulation of GAP-43 phosphorylation by yet another receptor-mediated mechanism may also be
required for hemispheric communication across the HC and CC.
GAP-43 is required for midline crossing only in the
telencephalon. At the optic chiasm, retinal ganglion cells do
cross the midline, although subsequent guidance is impaired (Kruger et
al., 1998; Sretavan and Kruger, 1998). Likewise, axon crossing
through the habenular, posterior, and ventral spinal commissures was
normal (Shen and Meiri, unpublished results). Similarly, many neurons, especially in the periphery, are resistant to the absence of GAP-43 (Strittmatter et al., 1995). We do not yet understand why, nor why
GAP-43 expression does not reach a functional threshold in the
heterozygote cortex. Both results suggest the involvement of genetic
modifier(s). It will be important to understand how a functional
threshold of GAP-43 is achieved. Heterozygote deletions of the human
3q13.10-3q13.21 locus that encompasses the GAP-43 gene give rise to
callosal agenesis, together with severe mental retardation (Jenkins et
al., 1985; McMorrow et al., 1986; Okada et al., 1987; Genuardi et al.,
1994; Mackie Ogilvie et al., 1998). Our results suggest that a mutation
restricted to the GAP-43 gene itself will also cause predisposition to
a dominantly transmitted abnormal CNS phenotype.
| |
FOOTNOTES |
Received Aug. 15, 2001; revised Oct. 1, 2001; accepted Oct. 19, 2001.
This work was supported by National Institutes of Health (NIH) Grant
NS33118. S.L.D. was supported by NIH Grants NS31829 and IBN-9724102. We thank Dr. Y. Rao for Slit-2-transfected cells, Dr. D. Wahlsten for discussions, and Dr. T. J. Diefenbach for help
with microscopy of the cultures.
Correspondence should be addressed to Dr. Karina Meiri, Department of
Anatomy and Cellular Biology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111. E-mail: karina.meiri{at}tufts.edu.
S. Mani's present address: National Brain Research Centre,
International Center for Genetic Engineering and Biotechnology Campus, Aruna Asaf Ali Marg, New Delhi 110067, India.
| |
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ABSTRACT
INTRODUCTION
