1 Institut Cochin, GDPM, INSERM U567, CNRS 8104, Université Paris V, CHU
Cochin, 24 rue du Faubourg Saint Jacques, 75014 Paris, France
2 CRBM/CNRS FRE2593, 1919 Route de Mende, 34293 Montpellier Cedex, France
3 Institut Pasteur, Unité des Bactéries Anaérobies et
Toxines, 28 rue du Dr Roux, 75724 Paris Cedex 15, France
Author for correspondence (e-mail:
bloch-gallego{at}cochin.inserm.fr)
Accepted 9 March 2004
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SUMMARY |
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Key words: Nuclear translocation, Hindbrain, Chemotropic molecules, Rho GTPases, Mice, Collagen assays, In situ hybridization, GST-RBD-Rhotekin
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Introduction |
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Intracellular signaling cascades involved in axon guidance and neuronal
migration remain poorly characterized. In vivo, RhoB has been involved in the
migration of neural crest cells (Liu and
Jessell, 1998; del Barrio and
Nieto, 2002
), whereas Rac and Cdc42 GTPases have been previously
implicated in various axon growth and guidance processes in the nematode
(Lundquist et al., 2001
) and
Drosophila (Hakeda-Suzuki et al.,
2002
; Kim et al.,
2002
; Kim et al.,
2003
). The function of Rho GTPases has been most extensively
studied in fibroblasts in vitro, showing that Cdc42, Rac1 and RhoA are key
modulators of the cytoskeletal dynamics that occur after a cell adhesion
event, and/or during cell migration (Clark
et al., 1998
; Hall,
1998
; Nobes and Hall,
1999
; Kaibuchi et al.,
1999
). Remodeling of the cytoskeleton in response to netrin 1 has
been studied in, among others, Swiss 3T3 fibroblasts. When expressing
exogenous Dcc, addition of netrin 1 triggers actin reorganization and a
lasting activation of Cdc42 and Rac1 in these cells
(Li et al., 2002a
). In N1E-115
neuroblastoma cells, both Rac1 and Cdc42 activities are required for netrin
1/Dcc-induced neurite outgrowth, whereas downregulation of RhoA, and its
effectors Rock1 and Rock2, stimulates the ability of Dcc to induce neurite
outgrowth (Li et al., 2002a
).
Nonetheless, few studies have reported the role of Rho GTPases during
development of the central nervous system through parallel in vivo and in
vitro analyses in vertebrates.
We have attempted to characterize small Rho GTPases that might participate in axon guidance and nuclei migration of developing PCN. We have combined in vivo analysis of Rho GTPases expressed during the development of PCN and in vitro assays with PCN, using LRN/ION explants. This model allows the analysis of the initiation of neurophilic migration, a process that can be divided into two distinct steps: axon outgrowth and nuclear translocation (i.e. cell body migration). We report that Rhob is strongly expressed in the marginal and submarginal migratory streams in the dorsal hindbrain, whereas Rhoa, Rac1 and Cdc42 all show a very similar expression pattern, with a high expression in the ventricular and subventricular zones. Pharmacological blockades in collagen matrix assays suggest that Cdc42/Rac1 and RhoA/RhoB/RhoC have distinct roles on axon outgrowth and nucleokinesis. Cdc42/Rac1 inhibition strongly decreases axon outgrowth whereas RhoA/B/C inhibition favors axon extension. In addition, RhoA/B/C but not Rac/Cdc42 are strictly required for nucleokinesis to occur. Activation of endogenous RhoA/B/C GTPases and expression of Rock effectors are found in the early migrating stream of PCN, and also in later migrating LRN and ION. In addition, we find through pharmacological experiments that the Rock signaling pathway is necessary for nucleokinesis to occur during neurophilic migrations.
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Materials and methods |
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In situ hybridization and RNA probes
In situ hybridization was carried out on cryosections according to Myat et
al. (Myat et al., 1996).
Murine Cdc42, Rac1, Rhoa, Rhob, Rock1 and Rock2 IMAGE cDNA
clones were obtained from HGMP (Cambridge, UK). Brn3b and
Tag1 cDNA clones have been described previously
(Bloch-Gallego et al., 1999
;
Backer et al., 2002
). No signal
was obtained when using the sense probes.
Immunohistochemistry and antibodies
Immunohistochemistry on cryosections was performed in phosphate buffered
saline (PBS) containing 2 g/l gelatin, 0.05% Triton X-100 and 0.2 mg/ml sodium
azide, and immunochemistry on collagen assays was performed in PBS, 1% normal
goat serum (NGS), 0.1% Triton X-100 and 0.2 mg/ml sodium azide. The following
primary antibodies were used in collagen assays: rabbit polyclonal anti-GFP
(1:400; Molecular Probes, Eugene, OR) and mouse monoclonal anti-class III
ß-tubulin (Tuj1; 1:2000; Jackson ImmunoResearch, West Grove, PA). These
primary antibodies were revealed using secondary antibodies raised from goat,
directed against mouse or rabbit IgG and conjugated to Alexa 488 (1:400;
Molecular Probes) or Cy3 (1:200; Jackson ImmunoResearch).
In situ detection of active Rho GTPases
GST (glutathione S-transferase)-RBD (Rho binding domain)-Rhotekin was
provided by M. A. Schwartz (State University of New York) and prepared as
described in Li et al. (Li et al.,
2002b). After fixation and cryosectioning of embryos, active
RhoA/B/C activity was detected through the binding of their GST-RBD-Rhotekin
effector, as described by Li et al. (Li et
al., 2002b
) with some modifications. Cryosections were saturated
in 0.3% Triton X-100 and 5% NGS for 10 hours, and GST-proteins, which had been
previously detached from the beads with 0.05 M Tris (pH 8) and 3 mg/ml
Gluthation, were incubated over-night at 4°C. After post-fixation with 2%
paraformaldehyde for 10 minutes and several washes in PBS/0.3% Triton X-100,
sections were saturated with 0.1 M lysine and incubated with a polyclonal
anti-GST antibody (Oncogene). As a control of the binding of GST-coupled
proteins, we used a non-relevant protein corresponding to the extracellular
domain of an adhesion molecule that is upregulated during differentiation and
fusion of muscle cells, GST-M-Cadherin. The EcoRV fragment of
M-cadherin corresponding to nucleotides 750-1765 was cloned in the pGEX5X
vector. The GST-M-cadherin fragment was produced as described in Li et al.
(Li et al., 2002b
). At least
three animals were examined for each condition tested.
To evaluate RhoA activity on explants of brainstem, E11 explants of rhombic lips were incubated for 2 hours in presence of netrin 1, then washed and lyzed in 50 mM Tris (pH 7.2), 1% Triton X-100, 500 mM NaCl, 10 mM MgCl2, 1 mM PMSF and protease inhibitors cocktail (Sigma-Aldrich, St Louis, MO). Cleared lysate was incubated with 25 µg of GST-fusion protein, containing the RhoA-binding domain of Rhotekin (GST-RBD-Rhotekin), attached to beads (Sigma) for 40 minutes at 4°C. The beads were then washed four times in 50 mM Tris (pH 7.2), 1% Triton X-100, 150 mM NaCl, 10 mM MgCl2 and 0.1 mM PMSF and protease inhibitors cocktail before the addition of Laemmli buffer. Fractions were analyzed by western blotting with RhoA antibody (1:400; Santa Cruz Biotechnology, Santa Cruz, CA). Owing to poor antibody specificity in western blots, RhoB presence could not be tested.
Western blots
Lysates were prepared from E11 or E12 rhombic lip, and E13 ventro-medial
parts excluding floor plate, and used as samples. Freshly dissected tissues
were dissociated in lysis buffer [50 mM Tris (pH 7.5), 120 mM NaCl, 20 mM NaF,
1 mM EDTA, 6 mM EGTA, 1% NP-40, 0.5 mM orthovanadate, 0.2% protease inhibitor
cocktail and 0.1 mM PMSF]. Total protein (30-40 µg) was loaded on a 7.5%
SDS-polyacrylamide gel and transfered onto nitrocellulose Hybond ECL
(Amersham, UK). After saturation in TBST with 5% milk, membranes were
incubated with the following antibodies: mouse monoclonal anti-Cdc42 (1:300;
BD Transduction Laboratories, Lexington, KY); mouse monoclonal anti-Rac1
(1:1000; BD Transduction Laboratories, Lexington, KY); mouse monoclonal
anti-RhoA (1:400; Santa Cruz, CA); mouse monoclonal anti-Rock1 (1:100, BD
Transduction Laboratories); mouse monoclonal anti-Rock2 (1:1000; BD
Transduction Laboratories); and anti-cNetrin 1 raised from goat (1:500;
R&D Systems, Minneapolis, MN). Primary antibodies were revealed by
incubation with HRP-conjugated anti-mouse IgG (1:25,000; Jackson
ImmunoResearch), or biotin-conjugated rabbit anti-goat secondary antibody
(1:5000; Vector, Burlingame, CA) and HRP-conjugated streptavidin (1:1000,
Amersham).
Collagen assays
Collagen assays were performed as previously described
(Causeret et al., 2002), using
rhombic lip explants from E11 or E12 mouse embryos facing cNetrin 1-secreting
cells (Kennedy et al., 1994
).
After 60-72 hours in a 5% CO2, 37°C, 95% humidity incubator,
collagen assays were fixed in 4% paraformaldehyde and immunolabeled with Tuj1
antibody for visualization of neuronal processes. Cell nuclei in explants and
migrating cells were visualized with DAPI (1 µg/ml, Vector). F-actin
staining was performed with rhodamine-conjugated phalloidin (1:200, Molecular
Probes) (Gallo and Letourneau,
1998
).
Drug application
Several drugs were used to inhibit specific pathways involving precise Rho
GTPases. Lethal toxin from Clostridium sordellii strain VPI9048
[LT-9048 (Humeau et al.,
2002)] was used at a final concentration of 1 ng/ml to inhibit
Rac1 and Cdc42. TAT-C3 [a kind gift from Jacques Bertoglio
(Sauzeau et al., 2001
)] was
used at a final concentration of 20 µg/ml to inhibit RhoA, RhoB and RhoC
signaling. Y-27632 (Calbiochem, San Diego, CA)
(Ishizaki et al., 2000
) was
used at various concentrations from 5-100 µM to inhibit Rock1 and Rock2
effectors. These drugs were diluted in the collagen culture medium and applied
from the first day in culture. We checked, by western blot, that none of them
affected netrin 1 secretion by quantifying netrin 1 protein in the supernatant
of cells cultured in presence of working concentrations of drugs.
In vitro ADP-ribosylation
Control rhombic lips treated in vivo with TAT-C3 were washed with PBS, and
then lyzed with ADP-ribosylation buffer [50 mM triethanolamine/HCl (pH 7.5),
100 mM KCl, 5 mM MgCl2, 10 mM dithiothreitol, 1 mM EDTA, 0.3 mM GDP
and 10 mM thymidine] containing leupeptin (1 µg/ml), pepstatin (1
µg/ml), 1 mM PMSF and 0.5% Triton X-100 (Marvaud et al., 2002). The
extracts were centrifuged (1000 g, 5 minutes) and the
supernatant (50 µg of total protein) was ADP-ribosylated in vitro in a
final volume of 20 µl ADP-ribosylation buffer containing 2.5 mM of
[32P]NAD (NEN-Du Pont de Nemours, Boston, MA; 20,000 dpm/pmol) and
10-7 M TAT-C3. After incubation at 37°C for 30 minutes, samples
subjected to SDS-PAGE, and the radioactive Rho bands were visualized and
quantified by PhosphorImager (Amersham) and the IQMac program.
Scoring of cell migration and quantification of leading process outgrowth
For quantification of cell migration and axon outgrowth in collagen assays,
pictures of each explant were taken using a Leica fluorescent microscope and a
Photometrics coolsnap fx monochrome CCD camera (Roper Scientific,
Duluth, GA). The blue channel allowed visualization of cell nuclei staining
with DAPI, the red channel allowed visualization of Tuj1 staining with a
Cy3-conjugated secondary antibody and the green channel allowed visualization
of anti-GFP staining with an Alexa 488-conjugated secondary antibody.
Thereafter, the surface covered by Tuj1 immunostaining outside of the explant
was measured using the Metamorph Area Analysis program (Universal Imaging
Corporation, Downingtown, PA). We only considered proximal and distal regions
of the explant (as defined in Fig.
2A). For quantification of nuclear migration we measured the
surface covered by DAPI labeling outside the explant toward the netrin 1
source. We also evaluated the number of nuclei leaving the explants, using
standard area count in the Integrated Morphometry Analysis function of
Metamorph. In some cases we also calculated the migration/outgrowth ratio,
which corresponds to the surface covered by migrating nuclei, divided by the
surface covered by growing axons in the proximal quadrant for each explant.
Areas were expressed in mm2 and migration/outgrowth ratios were
presented using arbitrary units and normalized to one in control conditions.
Averages and distributions were analyzed. Differences were considered as
significant when P<0.05 using a non-parametric Mann-Whitney
test.
|
Electroporation and videomicroscopy experiments
E12 rhombic lip explants were transfected with pEGFP-N1 (Clontech, Palo
Alto, CA), as a reporter construct, using electroporation. Using a
microcapillary glass pipette, 2 µl of plasmid DNA [2.5 µg/µl in
25 µM EDTA and 1 mM Tris (pH 8)] tied with 0.025% fast-green
(Sigma-Aldrich) were injected into the rhombencephalon (before dissection),
and three pulses of 100 V were then applied to each side of the injected
hindbrain using an Intracell electroporator and CUY610 electrodes (Nepa Gene,
Chiba, Japan). Videomicroscopy experiments were performed using an inverted
Zeiss microscope and a Hamamatsu camera. Collagen assays were performed in
phenol red-free medium. Images were acquired from the second day of culture
and at a rate of one every 8 minutes. Quantification of axons and nuclei
velocities was obtained using the tracking function in Metamorph.
Confocal microscopy
Confocal fluorescence microscopy was performed by using a Leica Microsystem
confocal microscope (SP2) equipped with a 40xFluar oil immersion
objective (numerical aperture 1.25), He-Ne (ex=543 nm) lasers.
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Results |
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Cdc42/Rac1 are required for initial extension of the PCN leading process whereas nuclear migration depends on RhoA/B/C activity
To study the role of Cdc42, Rac and RhoA/B/C GTPases in both axon outgrowth
and nuclear translocation of PCN, we used pharmacological approaches in a
collagen assay that had been previously set up
(Causeret et al., 2002). In
this assay, E11 or E12 rhombic lip explants were faced with netrin 1-secreting
cells in collagen gel matrix. Under those conditions, initial extension of
neurites out of the explant toward the netrin 1 source occurred from the first
day in culture (DIC). Nuclear migration developed on the second DIC, in the
same orientation as axons (i.e. toward the netrin 1 source). Cultures were
maintained for 3 DIC, and then both axon outgrowth and nuclear migration were
analyzed (Fig. 2A,C)
(Causeret et al., 2002
).
Quantifications revealed the surfaces covered by Tuj1-positive neurites and by
migrating nuclei toward the netrin 1 source after 3 DIC.
We tested the effects of blocking Cdc42, Rac and RhoA/B/C GTPases to establish whether they could be involved in axon outgrowth and/or nuclei migration of PCN. We first tested the ability of various concentrations of lethal toxin (LT-9048) to inhibit Rac1 and Cdc42 in PCN explants, and determined that an application of 1 ng/ml was an optimal concentration to analyze the effects of LT-9048 without any toxicity (as was observed at 10 ng/ml, data not shown). Application of 1 ng/ml LT-9048 in culture resulted in a 54% reduction of axon outgrowth at E11 after 3 DIC (compare proximal quadrants in Fig. 2A with 2B; quantifications in Fig. 2G; P<0.001, n=24 control and treated explants). The outgrowth showed a 63% decrease at E12 (data not shown; P<0.001; n=21 for control; n=19 for LT-treated explants). In addition, phalloidin staining was used to visualize F-actin in control and LT-9048-treated explants. Whereas control growth cones revealed F-actin-rich structures by labelling with rhodamine-conjugated phalloidin (Fig. 2I), these actin structures were severely affected in LT-9048-treated axons that lacked phalloidin staining at their distal tip (Fig. 2J).
Interestingly, the surface covered by migrating nuclei upon lethal toxin treatment represented 101% of control at E11, and 113% at E12 (Fig. 2C,D,E; not significant, P=0.94 and P=0.53, respectively). The number of migrating nuclei was also evaluated using the Integrated Morphometry Analysis function (Metamorph) at E11 (243±27 in control and 281±21 in the presence of 1 ng/ml lethal toxin, P=0.28; Fig. 2F). As a consequence, the migration/outgrowth ratio was significantly different in control explants and in explants treated with lethal toxin (Fig. 2H; 82% increase, P<0.001). Altogether, these data show that Rac/Cdc42 inhibition through application of 1 ng/ml lethal toxin specifically altered axon outgrowth without modifying nuclear migration toward the netrin 1 source.
To assess the specific requirement of RhoA/B/C in PCN neuronal migration
and axon outgrowth, we treated rhombic lip explants in collagen assays with
the toxin C3 fused with a TAT domain in order to optimize intracellular
penetration of the toxin (Sauzeau et al.,
2001). We first controlled TAT-C3 efficiency on ION explants
through ADP-ribosylation assays (see Materials and methods, and
Fig. 3A). In vitro
ADP-ribosylation of neurons pre-treated with TAT-C3 showed that the
radiolabeled Rho band was markedly decreased, indicating that TAT-C3
efficiently modified Rho substrate in neurons. Application of 20 µg/ml of
TAT-C3 in a collagen assay resulted in a significant reduction of nuclear
migration outside E11 (Fig.
3C,D) or E12 (data not shown) rhombic lip explants (at E11, 75%
reduction, n=19 control explants, n=13 TAT-C3-treated
explants, P<0.001; at E12, 71% reduction, n=12 control
explants, n=17 TAT-C3-treated explants, P<0.001),
combined with a potentiation of axon outgrowth
(Fig. 3B,E,F). Tuj1-positive
neurites appeared less fasciculated (compare
Fig. 3B with
Fig. 2A); they lost their
preferential orientation, extending similarly towards and away from the netrin
1 source (Fig. 3E, and compare
proximal and distal quadrants in Fig.
3B). In addition, TAT-C3 treatment affected axonal morphology. A
detailed analysis of axonal morphology was performed after GFP electroporation
that allowed the visualization of individual processes. Control axons showed a
straight morphology (Fig.
4A,C), whereas TAT-C3-treated explants exhibited tortuous axons
(Fig. 4B,E). The deviation from
a straight line was measured to quantify the trajectories of the axons from
the drawings of 11 control and TAT-C3-treated axons
(Fig. 4D).
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|
RhoA/B/C are in a GTP-bound active state in the rhombic lips and in the early migratory stream of PCN
We have focused on the regulation of the migratory process during
development of PCN that appeared, from expression patterns and inhibition
experiments, to be regulated by RhoA/B/C GTPases. As the Rho GTPases cycle
between a GDP-bound inactive state and a GTP-bound active state
(Ridley, 1997), we aimed to
measure Rho GTPase activation. We first analyzed the amount of active RhoA
GTPase in E11 rhombic lips using pull-down assays. Rhombic lips contained a
low but detectable amount of active RhoA-GTPase
(Fig. 5A). We aimed to get a
precise localization of active RhoA/B/C GTPases during PCN migration. For this
purpose, we adapted the assay initially developed for studying in situ Rho
GTPase activation in optic tectal cells
(Li et al., 2002b
). We
analyzed the localization of active RhoA/B/C GTPases at key stages of the
migration, when ION and LRN initiate their migration (at E11 and E12,
respectively), and at E13, when ION stop their migration ventrally, whereas
LRN continue migrating and cross the floor plate. In situ binding assays, with
the Rho-binding domain (RBD) of the specific Rho effector Rhotekin fused to
glutathione-S-transferase (GST) (which could only bind active
RhoA/B/C-GTPases), were performed on cryosections at different stages of
embryonic development (Fig.
5B,C,D). Activated Rho was detected at E11 in the ventricular zone
(vz), in the most dorsal part of the hindbrain (arrow,
Fig. 5B), as well as in the
initial migratory pathway of PCN (mz and smz;
Fig. 5B). Scattered
GST-RBD-Rhotekin-positive cells (arrowheads,
Fig. 5C) were observed when
leaving the ventricular zone (vz; large arrow,
Fig. 5C) toward the early
migratory stream in the periphery of the dorsal hindbrain (short arrows,
Fig. 5C). At E12, active
RhoA/B/C-GTPases could be detected in throughout the migratory pathways of
caudal hindbrain, with GST-RBD-Rhotekin binding in the migratory streams (data
not shown). At E13, GST-RBD-Rhotekin proteins stained the whole marginal
stream that contains LRN and ECN migrating neurons (arrowhead,
Fig. 5D). Lower levels of
Rhotekin binding could be detected when ION ended their migration in the
vicinity of the floor plate, when ION stop their migration. A decreasing
gradient of activated RhoA/B/C was observed from ventrolateral ION to
ventromedially stopped ION (asterisk, Fig.
5D). However, significant levels of Rhob mRNA were still
expressed in ION ending their migration
(Fig. 1H). At E11 and E13, no
binding of a GST-tagged non-relevant protein (M-Cadherin) was observed
(Fig. 5E,F).
|
Rock1/2 are implicated downstream RhoA/B/C during PCN nuclei migration in response to netrin 1
Once the in situ localization of active Rho GTPases had been determined, we
aimed to characterize the Rho target(s) involved in nuclear migration. The
main effector proteins of RhoA GTPases are Rock1 (also named ROKß) (Amano
et al., 2000) and Rock2 (also named ROK)
(Leung et al., 1995
;
Matsui et al., 1996
;
Nakagawa et al., 1996
).
First, we analyzed Rock1/2 expression in the rhombic lips, and in the migratory pathways of ION and LRN in vivo. In situ RNA hybridization on hindbrain cryosections revealed a clear labeling of neurons located in the vz and subventricular zone (svz) at E11 for both Rock1 (Fig. 6A) and Rock2 (data not shown). Rock1 transcripts were also detected in the initial migratory streams (Fig. 6A'). At E13, a developmental stage allowing visualization and discrimination of both LRN/ECN and ION, Rock1 transcripts (Fig. 6B), as well as Rock2 transcripts (not shown), were strongly expressed in the marginal migratory stream that contains LRN and ECN, and at a lower level in late migrating and stopping ION.
|
We further investigated the Rho downstream signal transduction pathway that
leads to PCN nucleokinesis triggered by netrin 1 using a specific inhibitor of
Rock1/2 kinases, Y-27632 (Uehata et al.,
1997). This pharmacological reagent is a cell-permeant compound,
which is highly specific and efficient for inhibition of the catalytic
activity of Rock1/2 (Ishizaki et al.,
2000
). We faced E11 and E12 rhombic lip explants with netrin
1-secreting cells in the presence of 5-100 µM of Y-27632. We analyzed axon
pathfinding and nuclear migration in fixed collagen assays to quantify both
processes after 3 DIC. Quantification of nuclear migration toward the netrin 1
source revealed a gradual inhibition from 5 to 100 µM
(Fig. 7C). We then chose to
apply 20 µM Y-27632, which allowed a strong inhibition of nuclei migration
and was consistent with doses generally used in the literature (e.g.
Wylie and Chantler, 2003
). At
20 µM, the migration showed an 85% decrease at E11 (n=42 control,
n=24 Y-27632-treated, P<0.001;
Fig. 7B,C,E) and a 74% decrease
at E12 (n=24 control, n=18 Y-27632-treated,
P<0.001; data not shown). Neurons treated with 20 µM Rock
inhibitor exhibited a loss of fasciculation (compare
Fig. 7A with
Fig. 2A) and morphological
abnormalities similar to those observed in TAT-C3-treated PCN (compare
Fig. 7A with
Fig. 3B). Analyzing axon
polarity revealed that Y-27632 treatment also mimicked the effects of RhoA/B/C
inhibition but to a lesser extent. Although axon growth was significantly
increased in the distal quadrant (opposite to netrin 1-secreting cells),
compared with control conditions (distal quadrants in
Fig. 7A and
Fig. 2A), it did not reach the
values of axon outgrowth in the presence of TAT-C3 (compare
Fig. 7A,D and
Fig. 3B,E). Thus, Rock
inhibition impaired but did not completely abolish the orientation of PCN axon
outgrowth toward netrin 1.
|
Taken together, these results suggest that Rock1/2 act downstream RhoA/B/C to direct migration of PCN nuclei toward a netrin 1 source. In addition, Rho/Rock activation is required to allow correct attraction and fasciculation of axons.
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Discussion |
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Rac1/Cdc42 activity is necessary for axon outgrowth, and RhoA/B/C is implicated in axon morphology/orientation and nuclear migration of PCN in vitro
We show that Cdc42 and Rac inhibition by lethal toxin results in severe
impairment of PCN axon outgrowth. Interestingly, 1 ng/ml lethal toxin did not
affect migration because migration occurred normally in shortened neurites,
indicating that it is not strictly dependent on previous long axon extension.
By contrast, PCN axon outgrowth could develop independently of nucleokinesis
as, following RhoA/B/C blockade with TAT-C3, axon outgrowth extensively
developed whereas nuclear migration toward netrin 1 was completely
blocked.
In addition, we report that TAT-C3 totally abolished the tropic effect of
netrin 1 on axon attraction, as axon outgrowth developed extensively and in a
similar fashion all around the explant. Thus, RhoA/B/C activity is absolutely
required to maintain an oriented axonal outgrowth of PCN toward a netrin 1
source. The requirement of RhoA/B/C for oriented axon outgrowth in response to
a chemoattractant may be specific to a particular biological system or to a
tropic molecule such as netrin 1. For instance, in cultured Xenopus
laevis spinal neurons, chemoattraction toward a source of brain-derived
neurotrophic factor is Cdc42 dependent, whereas chemorepulsion by
lysophosphatidic acid is RhoA dependent
(Yuan et al., 2003).
Altogether, these data indicate that: (1) axon outgrowth and migration of
PCN are not strictly dependent on each other because a shortened axon
extension allows nuclear translocation and axon outgrowth can develop without
nucleokinesis; (2) Rac1/Cdc42 are necessary for axon growth of PCN; and (3)
RhoA/B/C are necessary for nuclear migration, axon fasciculation and tropism
in response to netrin 1. During tangential migration of PCN, the activity of
both Rac and Rho has to be locally controlled in order to get coordinated axon
and nuclei pathfinding. This cellular process probably requires crosstalk
between Rho and Rac pathways, which could regulate spatio-temporally growth
cone and cell bodies dynamics, as has been reported in fibroblasts to
determine cellular morphology and migratory behavior (Sander et al., 1999), or
for chemorepulsion of spinal neurons from Xenopus laevis
(Yuan et al., 2003).
Rock1/2 act as key effectors in the RhoA/B/C signaling pathway for nuclear migration
We report evidence for the involvement of Rock1/2 in the regulation of
neuronal migration in response to netrin 1. We show here that upon
pharmacological blockade of Rock1/2 by Y-27632, the aspect of Y-27632-treated
axons was similar to the one observed after TAT-C3 treatment. Thus, upon
TAT-C3 or Y-27632 treatments, initiation of axon outgrowth and axon
fasciculation of PCN were modified, suggesting that Rho/Rock played a role in
the control of growth cone dynamics of various neurons, including PCN,
cultured chick neurons from dorsal root ganglion
(Fournier et al., 2003) and
cerebellar granule neurons (Bito et al.,
2000
).
Pharmacological experiments revealed that inhibition of Rock1/2 abolishes
PCN nuclear migration toward a netrin 1 source in collagen assays, confirming
that Rock1/2 are involved in nucleokinesis. Our data are consistent with
previous reports that describe Rock proteins as critical modulators of
Rho-mediated actin dynamics in several migratory processes in nematodes
(Spencer et al., 2001),
mammalian leukocytes and neural crest cells, and cancer cells
(Liu and Jessell, 1998
;
Itoh et al., 1999
;
Alblas et al., 2001
); however,
intracellular cascades involved in nucleokinesis were not characterized.
Nucleokinesis had been previously reported to be a process dependent on the
microtubule network, which would involve proteins interacting with
microtubules such as Lis1 and doublecortin
(Lambert de Rouvroit and Goffinet,
2001
). From mutant mice analyses, it has been proposed that the
reelin signaling pathway and Cdk5 could also regulate nucleokinesis (for a
review, see Walsh and Goffinet,
2000
). We report here the possible involvement of Rock in
nucleokinesis during tangential migration. Rocks are serine/threonine kinases,
with multiple functional domains involved in actomyosin assembly. Several
reports have analyzed the respective role of the different domains of
Rho-kinase in various cell lines (Amano et
al., 1999
; Chen et al.,
2002
; Riento et al.,
2003
). It will be informative to overexpress various mutant Rock
proteins in PCN to further characterize the involvement of their sub-domains
in the migratory process.
Interestingly, Y-27632 treatment less severely affected axon attraction by
the netrin 1 source than did TAT-C3 treatment. The latter directly inhibits
RhoA/B/C GTPases, whereas Y-27632 acts as a specific inhibitor of one of the
targets of RhoA/B/C, the Rho effector Rock family of kinases
(Rho-kinase/Rok/Rock) (Uehata et al.,
1997). Several other proteins have been isolated as putative Rho
effectors on the basis of their selective interaction with the GTP-bound form
of Rho. These include, in addition to the Rock family comprising p160Rock
(Rock-I) (Ishizaki et al.,
1996
) and Rok/Rho-kinase/Rock-II
(Leung et al., 1995
;
Matsui et al., 1996
;
Nakagawa et al., 1996
),
protein kinase PKN (Amano et al.,
1996
; Watanabe et al.,
1996
), citron kinase (Madaule
et al., 1995
; Madaule et al.,
1998
; Madaule et al.,
2000
), and mammalian diaphanous homologs mDia1 and mDia2
(Watanabe et al., 1997
;
Alberts et al., 1998
). Studying
the possible involvement of these different effectors in axon morphology and
tropism will bring new insights into the responses of PCN mediated by the
small GTPases of the RhoA family proteins.
Requirement of a dynamic regulation of Rho GTPase activity for the proper development/positioning of PCN
During their migration, PCN repeatedly extend a leading process,
translocate their nucleus inside this leading process and retract their
trailing process. Consequently, cell bodies move through a series of jumps, as
reported in various neuronal systems
(Gilthorpe et al., 2002;
Polleux et al., 2002
). This
could result from a direct fast regulation of Rock activity, or it may be due
to the alternate presence of active/non active Rho proteins. The activity of
RhoA/B/C GTPases can be regulated either by downregulation of downstream
effectors such as Rock, or, more directly, by guanine nucleotide-exchange
factors (GEFs) and GTPase-activating proteins (GAPs). GEFs exchange the GDP on
an inactive GTPase for a GTP, whereas GAPs increase the intrinsic GTPase
activity, thus converting GTP-bound forms to GDP-bound forms
(Etienne-Manneville and Hall, 2002). During development, PCN migration can be
divided into successive steps, including the initiation of migration from the
rhombic lips, nucleokinesis through the migratory stream, and stopping of cell
bodies at their final location in the hindbrain. Thus, it remains to be
established which GAPs or GEFs are expressed and involved in regulating the
various phases of the migratory process.
For ION, contrary to other PCN populations, cell bodies do not cross the
floor plate. The intracellular mechanism that leads to the ending of nuclear
migration in the vicinity of the floor plate remains to be established, but it
possibly involves downregulation of Rho GTPases activity. It is noteworthy
that, whereas transcripts of Rho GTPases are present in ION located near the
floor plate, low levels of active Rho GTPases are detected in situ in ION that
reach the floor plate and stop. The extinction of Rho GTPase activity could be
due to the spatio-temporally restricted expression of a specific GAP when ION
reach the vicinity of the floor plate, or possibly could be due to the loss of
a GEF expression that would silence RhoA/B/C activity. Whether specific
regulators of Rho GTPase activity are expressed when PCN reach their
appropriate final location remains to be established. An interesting candidate
would be the GEF Trio. In C. elegans, both UNC-40/Dcc and UNC-73/Trio
participate in a signaling system that orients and polarizes neuroblast
migration (Honigberg and Kenyon,
2000). In addition, Trio is able to link RhoA and Rac/Cdc42
pathways (Bellanger et al.,
1998
; Blangy et al.,
2000
), and thus could be an appropriate coordinator of both axon
outgrowth and nucleokinesis during PCN migration.
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ACKNOWLEDGMENTS |
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Footnotes |
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* These authors contributed equally to this work
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