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The Journal of Neuroscience, October 1, 1999, 19(19):8454-8463
The Neuronal Architecture of Xenopus Retinal Ganglion
Cells Is Sculpted by Rho-Family GTPases In Vivo
Maureen L.
Ruchhoeft1,
Shin-ichi
Ohnuma2,
Lisa
McNeill1, 3,
Christine E.
Holt2, and
William A.
Harris2
1 Biology Department, University of California at San
Diego, La Jolla, California 92093-0357, 2 Department of
Anatomy, Cambridge University, Cambridge, CB2 3DY, United Kingdom, and
3 Xcyte Therapies, Seattle, Washington 98134
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ABSTRACT |
Dendritogenesis, axonogenesis, pathfinding, and target recognition
are all affected in distinct ways when Xenopus retinal ganglion cells (RGCs) are transfected with constitutively active (ca),
wild-type (wt), and dominant negative (dn) Rho-family GTPases in
vivo. Dendritogenesis required Rac1 and Cdc42 activity.
Moreover, ca-Rac1 caused dendrite hyperproliferation. Axonogenesis, in
contrast, was inhibited by ca-Rac1. This phenotype was partially
rescued by the coexpression of dn cyclin-dependent kinase
(Cdk5), a proposed effector of Rac1, suggesting that Rac1 activity must
be regulated tightly for normal axonogenesis. Growth cone
morphology was particularly sensitive to dn-RhoA and wt-Cdc42
constructs. These also caused targeting errors, such as tectal bypass,
suggesting that cytoskeletal rearrangements are involved in target
recognition and are transduced by these pathways.
Key words:
RhoA; Rac1; Cdc42; axonogenesis; dendritogenesis; GTPase
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INTRODUCTION |
Studies in fibroblasts have
demonstrated that Rho-family GTPases regulate the actin cytoskeleton.
Changes in cell morphology occur after the microinjection of Cdc42,
which induces filopodia; Rac1, which induces lamellipodia; and RhoA,
which induces stress fibers (Hall, 1998 ). Recent studies have
identified these GTPases as molecules involved in axon and dendrite
formation during neuronal development (Luo et al., 1997; Tapon and
Hall, 1997).
Work on neuritogenesis in vitro suggests that RhoA
negatively influences growth cone and neurite formation. Wild-type
(wt)-RhoA microinjection (Kozma et al., 1997) or exposure to
lysophosphatidic acid (an activator of RhoA) (Jalink et al., 1994;
Tigyi et al., 1996) causes growth cone collapse and neurite retraction
in PC12 and N1E-115 cells. Also, the trituration of C3 exoenzyme (an
inhibitor of RhoA activity) into dorsal root ganglion cells stimulates
neurite outgrowth (Jin and Strittmatter, 1997), and both dominant
negative (dn)-RhoA and C3 exoenzyme microinjection promote filopodia
and lamellipodia formation in N1E-115 cell growth cones (Kozma et al.,
1997). In contrast, the overexpression of wt-Cdc42 and Rac1 induces
N1E-115 cell filopodia and lamellipodia formation (Kozma et al., 1997),
and expression of constitutively active (ca)-Cdc42 and Rac1 promotes
hippocampal neuron dendritic development (Threadgill et al., 1997). In
addition, chicken Rac1B overexpression in retinal cells increases
neurite number and branching, whereas dn-Rac1B inhibits neuritogenesis
(Albertinazzi et al., 1998).
These GTPases also have been examined in vivo, where it is
possible to look at axon and dendrite formation separately. ca- and
dn-Cdc42 reduce sensory neuron dendrites in the fly, whereas mutant
Rac1 proteins do not affect dendritogenesis (Luo et al., 1994).
However, when expressed in mouse Purkinje cells, ca-Rac1 causes smaller
but more numerous dendritic spines (Luo et al., 1996). Defects in axon
initiation, extension, and pathfinding have been observed also. ca- and
dn-Rac1 and Cdc42 all inhibit axonogenesis in fly sensory neurons (Luo
et al., 1994), and ca-Rac1 greatly reduces the number of axon terminals
in mouse Purkinje cells (Luo et al., 1996). In agreement with these
observations, ca-Rac1 and Cdc42 inhibit axon extension in fly motor
neurons (Kaufmann et al., 1998). However, dn-Rac1-expressing cells did extend axons, with a subset of neurons exhibiting pathfinding defects.
Loss of Rac1 activity also has been implicated in pathfinding errors in
Caenorhabditis elegans, because defects were observed after
the loss of unc-73, an activator of Rac1 (Steven et al., 1998). In
contrast, activated MIG-2, a novel Rac1/Cdc42-like molecule in C. elegans, causes axon pathfinding errors (Zipkin et al., 1997).
These findings highlight the importance of Rho-family GTPases in axonal
and dendritic growth in vivo, but the findings are not
always consistent among different systems. To try to develop a complete
picture of the functions of these GTPases in a single cell type, we
expressed wild-type and mutant RhoA, Rac1, and Cdc42 in developing
Xenopus retinal ganglion cells (RGCs). We report here that
RhoA, Rac1, and Cdc42 have distinct effects on dendrite formation, axon
initiation and extension, growth cone morphology, and target selection,
which suggests specific roles for these small GTPases during RGC
process development in vivo.
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MATERIALS AND METHODS |
Animals. Embryos were obtained by fertilizing eggs
from adult female Xenopus laevis stimulated by injection of
human chorionic gonadotropin (United States Biochemicals, Cleveland,
OH). Embryos were raised in 10% Holtfreter's (Holtfreter, 1943) at
temperatures between 14 and 27°C. Embryos were staged according to
Nieuwkoop and Faber (Nieuwkoop and Faber, 1967).
DNA constructs. Myc-tagged human wt-Rac1, Cdc42, and RhoA;
ca-V12 Rac1 and Cdc42; ca-V14 RhoA; and dn-N17 Rac1 and Cdc42 cDNAs were obtained from Dr. Alan Hall (University College London, UK) and
subcloned into pCS2, a eukaryotic expression vector designed by Dr.
David Turner (University of Michigan, Ann Arbor, MI), dn-N19 Rho was
obtained from Dr. Jerald Chun (University of California at San Diego)
and subcloned into pCS2-myc. All constructs were designed with one
myc-tag 5' to the gene and were sequenced after being subcloned into
CS2 to ensure that the genes were in-frame with the myc-tag. In
vitro transcription-translation was performed by using the TNT
Coupled Reticulocyte Lysate System kit (Promega, Madison, WI) to ensure
that appropriately sized proteins were produced. Briefly, template cDNA
was added to the cell-free transcription-translation lysate, and the
reaction was allowed to proceed. The lysate was spiked with translation
grade [35S]methionine (Amersham,
Arlington Heights, IL), and the translation products were run on an
SDS-PAGE gel. The proteins were visualized with autoradiography. The
control luciferase reporter plasmid, RSVL, has been described
previously (Holt et al., 1990). The green fluorescent protein (GFP)-myc
fusion cDNA in CS2 was a gift of Dr. David Turner. Expression plasmids
of Cdk5 and dn-Cdk5 were obtained from Dr. Anna Philpott (University of
Cambridge, UK) and have been described previously (Philpott et al.,
1997). Plasmid DNA was purified from E. coli by using Qiagen
Maxiprep kits (Hilden, Germany).
DNA transfection. DNA lipofections were performed as
described previously (Holt et al., 1990; Lilienbaum et al., 1995).
Briefly, retinal cells were transfected by microinjecting a 1:3 mixture by weight of DNA and the transfectant DOTAP (Boehringer Mannheim) into
the presumptive right eye of the anterior neural fold of stage 18-20
embryos. Embryos were cysteine-treated for 5 min to remove the jelly
coat [2% L-cysteine (Sigma, St. Louis, MO) in 10%
Holtfreter's, pH 8] and placed in a dish containing 5% Ficol (Sigma)
in 10% MMR [containing (in mM) 100 NaCl, 2 KCl, 1 MgSO4, 5 HEPES, 0.1 EDTA, and 2 CaCl2]. Borosilicate glass needles (FHC) were
pulled on an electrode puller (Sutter Instruments, Novato, CA), filled
with DNA/DOTAP, and mounted on a micromanipulator (Narishige, Tokyo,
Japan) attached to a Picospritzer II (General Valve, Fairfield, NJ).
Three to five extracellular injections of 5-10 nl each were made into
the developing eye primordium. After injection the embryos were
transferred to 10% Holtfreter's.
Antibodies. All embryos transfected with Rho-family GTPase
cDNA or with GFP-myc cDNA were stained with the anti-myc monoclonal antibody 9E10 (Santa Cruz Biotechnology, Santa Cruz, CA) diluted to
1:500. Embryos transfected with luciferase were stained with an
affinity-purified guinea pig anti-luciferase antibody used at 1:100.
Donkey anti-mouse CY3-conjugated secondary was used at 1:100.
Horseradish peroxidase (HRP)-conjugated goat anti-mouse and goat
anti-guinea pig secondary antibodies from Jackson Laboratories (Bar
Harbor, ME) were used at 1:500.
Immunocytochemistry and histology for vibratome-sectioned
embryos. In general, the embryos were allowed to develop to stage 40 (~45 hr at room temperature), fixed overnight at 4°C in 4% paraformaldehyde in 0.1 M PO4 buffer,
rinsed several times with PBS [containing (in mM) 136.9 NaCl, 2.7 KCl, 81 Na2HPO4,
and 1.5 KH2PO4, pH 7.4],
and pigment-bleached overnight on a light box in methanol and 10%
hydrogen peroxide. After several rinses in PBS and blocking solution
[PBS with 5% normal goat serum (Core Cell Culture Facility,
University of California at San Diego), 0.2% Fraction V bovine serum
albumin (Sigma), and 0.5% Triton X-100 (Sigma)] the embryos were
incubated overnight at 4°C in primary antibodies diluted in block,
rinsed in block, incubated overnight in secondary antibodies diluted in
block, and rinsed again. Then the embryos were incubated in 0.5%
diaminobenzidine (Sigma) for 30 min, after which 1% hydrogen peroxide
was added to catalyze the peroxidase reaction. Embryos were post-fixed
in 1% glutaraldehyde for 30 min, embedded in gelatin/albumin [1.5% gelatin (Fisher, Pittsburgh, PA), 45% albumin (Sigma) in 0.9% saline], hardened with glutaraldehyde, and sectioned at 50 µm on an
Oxford vibratome. Sections were collected in series on glass slides
(Fisher), allowed to dry until the edges adhered, and then placed in
0.1 M PO4 buffer. The slides were
dehydrated through a graded ethanol series followed by two butanol
rinses, were cleared in xylenes, and were mounted in Permount (Fisher).
Immunocytochemistry and histology for cryostat-sectioned embryos.
Stage 40-41 embryos were fixed in 4% paraformaldehyde, rinsed in
PBS, saturated in 30% sucrose, transferred to OCT embedding medium
(Tissue Tek, Miles, Elkhart, IN), and sectioned at 15 µm on a
cryostat (Zeiss, Oberkochen, Germany). Immunocytochemistry to visualize
myc-fusion proteins was performed on sectioned tissues as described
above. Sections also were stained for condensed nuclei by being soaked
in a 1:10,000 solution of Hoechst nuclear stain for 10 min, followed by
PBS rinses. Apoptotic nuclei were visualized with an Apoptag kit
(Oncor, Gaithersburg, MD). Briefly, sections were incubated in a
terminal deoxynucleotidyl transferase solution along with
digoxigenin-labeled nucleotides, rinsed with PBS, incubated with a
fluorescein-conjugated anti-digoxigenin antibody, and viewed with epifluorescence.
Imaging and analysis. Immunopositive RGCs were drawn at
100× magnification with Nomarski optics, using a camera lucida
attachment on a Nikon Optiphot-2; the drawings were scanned on a Color
Onescanner (Apple), and parameters of cell morphology were measured by
using National Institutes of Health Image software. RGCs were
identified by their location in the ganglion cell layer directly
abutting the lens. RGC processes were identified as axons if they grew along the vitreal surface toward the optic nerve head and as dendrites if they were oriented toward the inner plexiform layer. Cell body area
was measured by tracing the main body of the outline of a cell, not
including fine processes that could not be distinguished or drawn
individually. Dendrite length was measured by tracing the length of the
longest primary dendrite, and dendrite number was determined by
counting the number of dendrite tips. Only cells that did not overlap
significantly with other transfected cells and that were located in the
flat, middle sections of the retina (generally including the lens) were
used, because their laminar position could be classified with
certainty. To avoid the inclusion of immature cells in the ciliary
marginal zone, a zone of mitotically active cells that remains on the
edges of the Xenopus retina into adulthood, we
analyzed only cells in the inner two-thirds of the retinal
semicircle. Growth cone area was measured from the widening of the axon
at the base of the growth cone to the edges of the lamellipodia.
Filopodial number and length were determined by counting the number of
filopodia and measuring the longest filopodia. Axonal backbranches were
counted two growth-cone lengths behind the base of the growth cone. All
errors were calculated as SEM, and p values were
determined by using Mann-Whitney nonparametric statistical analysis.
Color slides were taken on a Zeiss Axiophot with Nomarski optics,
scanned with a Nikon L5-1000 film scanner, and processed with Adobe
Photoshop software.
Whole-mount embryo processing, imaging, and analysis.
Embryos were transfected at stage 18 and allowed to develop to
stage 41, at which point they were fixed and processed
immunocytochemically as described above. Both HRP-conjugated and
CY-3-conjugated secondary antibodies (Jackson Laboratories) were used.
The brains were dissected out and mounted on slides; the transfected
axons were viewed on a Nikon Optiphot-2. Axons visualized by using
HRP-conjugated secondary antibodies were drawn at 20× magnification,
using Nomarski illumination and a camera lucida; those visualized with
cy-3 secary antibodies were drawn from a fluorescence image captured
with a CCD camera (SpectraSource Instruments, Westlake Village, CA).
The percentage of labeled axons in each brain that were in the
diencephalon and in the tectum, that grew toward the basal optic
nucleus (BON), or that made targeting errors was calculated. Targeting
error percentages do not include axons that grew only as far as the diencephalon.
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RESULTS |
Overexpression of Rho-family GTPases in Xenopus
retinal cells
Xenopus cells begin to express protein ~8 hr after
transfection, at stage 24 (Holt et al., 1990), and RGCs extend their
first axons 6 hr after this, at stage 27 (Holt, 1989). Dendritogenesis usually begins 5 hr after axon initiation (Sakaguchi et al., 1984; Holt, 1989). Thus, the expressed proteins should be present at the time
of process initiation and elaboration. Transfected cells were labeled
immunocytochemically with an anti-myc primary and an HRP-conjugated
secondary. Protein levels in expressing cells were not quantified;
however, only cells that were stained darkly, and therefore most likely
were expressing high levels of protein, were analyzed. A brown DAB
reaction product was present throughout the cells from dendrite to
growth cone tip. Potentially, the anti-myc staining may not be a
precise reflection of cell morphology because of uneven protein
distribution. However, the fact that the mutant proteins differ from
wild type by a single amino acid substitution on the N terminus of the
protein, which is not predicted to have an effect on intracellular
localization (Adamson et al., 1992) and yet differential effects on
cell morphology and process extension were observed, argues against
this possibility. Labeled cells were present in all retinal laminae for
all constructs, suggesting that neuronal migration within the retina
was not perturbed (data not shown). Although a careful analysis of cell
fate was not performed, all transfected cells were in distinct retinal
laminae. Moreover, appropriate differentiated cell morphologies were
observed for most constructs. In addition, transfected cells in stage
40 retina did not appear to be undergoing cell death, as revealed by
Hoechst and Apoptag stain of their nuclei (data not shown), although we did not examine the possibility of cell death occurring at earlier stages.
Rho-family GTPases affect dendritogenesis
To determine whether Rho-family GTPases are involved in the
formation of dendrites in Xenopus neurons in
vivo, we introduced wt, ca, and dn versions of RhoA, Rac1, and
Cdc42 or the control genes luciferase and GFP-myc via transient
transfection into dividing primordial eye cells. Although all cell
types in the retina were transfected, only RGCs were analyzed, because
their identity can be determined by their laminar location and they
allow for analysis of both axonal and dendritic structures. Normally,
RGCs have an extensive dendritic tree that forms in the inner plexiform
layer (Fig. 1A,B). The
effect of expression of Rho-family GTPases on RGC morphology is
illustrated in Figure 1C-K. Qualitatively, cells expressing
activated RhoA and Rac1 displayed the most dramatic and consistent
phenotypes. ca-RhoA expression resulted in the absence of nearly all
dendrites (Fig. 1D), whereas ca-Rac1 caused a
proliferation of processes, giving 70% of the cells expressing this
construct a hairy appearance (Fig. 1G). The expression of other mutant and wt-GTPase proteins usually resulted in a general decrease in the number and length of dendrites.
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Figure 1.
Expression of Rho-family GTPase mutants
affects dendritogenesis. Shown are RGCs in sections of
Xenopus eye immunolabeled with anti-luciferase
(A, B) or anti-myc antibodies
(C-K). All cells are visualized with
HRP-conjugated secondary antibodies. Photomicrographs are oriented with
the PE (pigment epithelium) at the top and the lens at
the bottom. A, A low-power view of a
retina with a luciferase-transfected RGC and an amacrine cell
(AC). Arrowheads indicate RGC and AC
dendrites; arrows indicate two RGC axons exiting the
eye. B, A higher power view of a luciferase-expressing
RGC. Arrowheads illustrate the extensive dendritic tree.
The axon of this RGC is in the next section. C-K, RGCs
expressing wt, ca, and dn RhoA, Rac1, and Cdc42.
Arrowheads indicate dendrites; arrows
indicate axons. Scale bar, 15 µm.
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The effect of different Rho-family GTPase transgenes was quantified by
counting the number of dendrite tips and measuring the longest
dendrite. Dendrites were defined as processes oriented toward the inner
plexiform layer in the retina. Dendrite length and number could not be
measured accurately for cells expressing ca-Rac1, because the processes
could not be distinguished individually. Therefore, a third parameter,
termed "extended cell area," was examined also, in which the
outline of the cell body, including processes that were not separable,
was traced and measured. Most of the GTPase transgenes significantly
decreased the average length of the longest dendrite, ranging from an
~60% decrease caused by dn-Rac1 to an ~90% decrease caused by
ca-RhoA (Fig. 2A). Many constructs also significantly altered the number of dendrite tips (Fig.
2B). Cells expressing wt- and ca-RhoA displayed the
strongest phenotype, with an ~70-90% decrease, and all forms of
Cdc42 caused a significant decline in dendrite tip number. The dramatic
effect of expressing ca-Rac1 on extended cell body area is illustrated in Figure 2C, in which the area is nearly double that of
controls. A smaller, although significant, increase in extended cell
area also was observed for wt-Rac1 and ca-Cdc42, which is probably attributable to some of these cells displaying a "hairy" phenotype. Thus, a loss of both Rac1 and Cdc42 activity negatively impacts dendrite formation in vivo, although an increase in Rac1
activity alone is enough to promote dendritogenesis.
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Figure 2.
Most wt and mutant GTPases cause dendritic
reduction, whereas ca-Rac causes process proliferation.
A, Average length of the longest dendrite measured for
RGCs expressing control proteins or ca-, wt-, or dn-GTPases.
B, Average number of dendritic tips per RGC.
C, Extended cell area of transfected RGCs (includes cell
body plus closely growing, indistinguishable processes). The
numbers in the bars indicate the RGCs
that were analyzed. Error bars are SEM, and p values
(*p < 0.05, **p < 0.01, ***p < 0.0001) indicate differences from
control.
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Rac1 is required for axon initiation
The deregulation of Rho-family GTPases had a dramatic inhibitory
effect on axon initiation. The expression of ca forms of RhoA, Rac1,
and Cdc42 nearly eliminated RGC axons. Similarly, blocking Rac1
function with the dn version severely inhibited axonogenesis. Rarely,
very short axons that failed to exit the eye were seen
(arrows; see Fig. 1G,J), but labeled axons
were never observed in the brain in vibratome sections for these
constructs (data not shown). Several possibilities could explain the
lack of axons. (1) Axons were never initiated. (2) Axons were formed initially but were retracted. (3) Axon initiation was delayed. To
distinguish among these possibilities, we performed an in
vivo time course. At no time were axons detected extending from
cells expressing ca-GTPase mutants or dn-Rac1 (data not shown). Thus, an unregulated increase in the activity of these GTPases, or a loss of
Rac1 activity, severely disrupts axon initiation. Cells expressing
wt-RhoA, Rac1, and Cdc42 initiated and extended axons, indicating that
the cells are able to compensate for the overexpression of wild-type
proteins. Expression of dn-RhoA and Cdc42 did not block axon
initiation, suggesting that these GTPases are not required for axon initiation.
dn-Cdk5 coexpression with ca-Rac1 partially
rescues axonogenesis
Cyclin-dependent kinase 5 (Cdk5) forms a complex with p35 and
regulates neurite outgrowth in culture (Nikolic et al., 1996). A recent
report shows that this complex colocalizes with Rac1 in neuronal growth
cones and is a specific effector of Rac1 (Nikolic et al., 1998). We
wondered whether the inhibition of axonogenesis observed after ca-Rac1
expression could be rescued by the coexpression of a dn version of
Cdk5. Embryos were transfected with ca-Rac1 alone or along with wt- or
dn-Cdk5. The coexpression rate for most constructs is generally >90%
when the DNAs are mixed in the lipofection cocktail (Holt et al., 1990;
Riehl et al., 1996). The number of transfected RGCs with axons in the
retina was determined (Fig.
3A). Nearly 85% of control
GFP-expressing RGCs had axons. Using highly sensitive CY-3 secary
antibodies on thin cryostat sections, we saw faintly labeled axons that
were not visible with HRP-conjugated secondary antibodies on 19% of
myc-tagged ca-Rac1-expressing cells. Cotransfection with wt-Cdk5 did
not alter the number of ca-Rac1-expressing RGCs that formed axons, as
observed by staining the myc-tag on the ca-Rac1 protein. However,
cotransfection with dn-Cdk5 increased the percentage of
ca-Rac1-expressing RGCs with axons to 48% (Fig. 3A).
Because the cdk5 constructs were not myc-tagged, the rescue of
axonogenesis in the case of dn-cdk5 must be attributable to
coexpression with myc-tagged ca-Rac1. These axons projected normally to
the optic tectum (Fig. 3B). This implies that, for normal
axonogenesis to occur, the activity of Rac1 and its downstream effectors, such as p35/Cdk5, must be regulated tightly. The
cotransfection of wt-Cdk5 in dn-Rac1-expressing RGCs was not able to
rescue axonogenesis (data not shown). dn-Cdk5 cotransfection with GFP
had no obvious effect on RGC axons as determined by GFP fluorescence;
they projected normally to the tectum (data not shown). This finding
contrasts with the results of Nikolic et al. (1996), who observed an
inhibition of axon formation with the expression of dn-Cdk5 in neurons
in culture. This difference may be attributable to compensatory
mechanisms in vivo, or perhaps dn-Cdk5 was not expressed at
high enough levels in our experiments to inhibit axon formation.
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Figure 3.
dn-Cdk5 rescues inhibition of axonogenesis caused
by ca-Rac1. A, Percentage of transfected RGCs with axons
exiting the eye in cryostat sections of stage 41 embryos. The
numbers of axons that were examined are in
parentheses. B, RGC axons coexpressing
ca-Rac1 and dn-Cdk5 project dorsally up through the diencephalon
(Di) to the optic tectum (Te) in a
whole-mount brain. Black dots approximate the tectal
borders. Scale bar, 150 µm.
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Axon extension, target selection, and target recognition
compromised by wt-Cdc42 and dn-RhoA
To determine whether Rho-family GTPases are important in
pathfinding or target recognition, as has been shown in invertebrates (Zipkin et al., 1997; Kaufmann et al., 1998; Steven et al., 1998), we
followed the trajectories of GTPase transgene-expressing axons in
whole-mount brains. Although some axons expressing ca-Rac1 were
observed by using CY-3 secary antibodies in whole-mount, this analysis
could not be performed for this construct because there were too few
axons observed and the staining was faint. Normally, the majority of
RGC axons at stage 41 has grown dorsally through the
contralateral diencephalon to innervate the optic tectum (Fig.
4A). A small fraction
of RGCs also normally innervates the basal optic nucleus (BON), located
in the hindbrain. Axons were classified according to whether they were
within the diencephalon (parentheses; Fig.
4A,B), had innervated the optic tectum
(dots; Fig. 4A,B), or were growing toward
the basal optic nucleus (BON). Greater than 80% of control axons
innervated the tectum, whereas ~10% were still in the diencephalon,
and <5% grew toward the BON (Fig. 4C). Just over 50% of
wt-Cdc42-expressing cells had extended an axon to the tectum at the
same stage, whereas 40% still had axons within the diencephalon. The
growth cone of an axon can been seen just exiting the optic chiasm and
entering the diencephalon in Figure 4B (white
arrowhead). Thus, the overexpression of wt-Cdc42 seems either to
delay axonogenesis developmentally or to cause growth cones to advance
more slowly. A decrease in axons within the tectum also was observed
for dn-RhoA-expressing cells. In this case, there was a fivefold
increase in the percentage of cells that grew toward the BON as
compared with the controls, indicating that the loss of RhoA function
may impair the ability of RGC growth cones to make appropriate target
choices.
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Figure 4.
Axon extension, target selection, and target
recognition defects caused by Rho-family GTPases. A,
GFP-transfected axons of the optic projection (OP)
course dorsally through the diencephalon (Di) toward
their target, the tectum (Te), in a control stage 41 brain. Black dots approximate the tectal borders. A
single faintly labeled axon has left the main tract
(arrows) to project toward the basal optic nucleus
(BON). B, wt-Cdc42-expressing
axons immunostained with an anti-myc antibody. Indicated are an axon
just leaving the optic chiasm and entering the optic pathway in the
diencephalon (white arrowhead), an out-of-focus axon
that has terminated correctly within the tectum
(asterisk), an axon on the ventral border of the tectum
(black arrowhead), and an axon that has
extended past both the optic tectum and the BON (arrow).
Scale bar, 150 µm. C, Percentage of RGC axons
transfected with control (GFP), wt-, or dn-GTPase proteins that
terminated within the optic tectum and were located in the diencephalic
portion of the optic pathway or that projected toward the BON in stage
41 whole-mount brains. Axons that made target recognition errors were
not included in this analysis. The numbers in
parentheses indicate the axons that were examined.
D, Percentage of RGC axons transfected with control
(GFP), wt-, or dn-GTPase proteins that made target recognition errors
in stage 41 whole-mount brains. Errors included axons that grew
posteriorly past the tectum and axons that grew ventrally and dorsally
along the tectal border but that did not innervate the tectum. Only
axons that grew past the diencephalon were included in this analysis.
The numbers in parentheses indicate the
axons that were examined.
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Retinal ganglion cells with axons that misexpressed GTPase transgenes
did not display penetrant pathfinding phenotypes, because most of these
exited the eye correctly, crossed the optic chiasm to enter the
contralateral diencephalon, and then grew dorsally toward the tectum or
the BON. However, many more axons expressing wt-Cdc42 and dn-RhoA made
target recognition errors than did those expressing GFP or other GTPase
constructs. A brain with several axons overexpressing wt-Cdc42 is shown
in Figure 4B. One axon has grown past the tectum and
the BON and is extending posteriorly into the hindbrain
(arrow; Fig. 4B). Another axon
(black arrowhead) also has turned at the ventral border of
the tectum. Other errors that were observed include axons that grew
along the border of the tectum dorsally but failed to innervate it. The
percentage of axons making target recognition errors increased
approximately threefold for both wt-Cdc42 and dn-RhoA as compared with
the controls (Fig. 4D). No increased pathfinding or
targeting errors were found with either wt-Rac1 overexpression (Fig.
4D) or in RGCs expressing ca-Rac1 where axonogenesis
was rescued with dn-Cdk5 (data not shown). These observations indicate
a role for RhoA and Cdc42 in the transducing pathway or target
recognition signals in this system.
Growth cone morphology altered by Cdc42 and RhoA
To examine why RhoA and Cdc42 affected target recognition, we
looked at the morphology of growth cones, the structures responsible for sensing and responding to cues in the extracellular environment. Control RGC growth cones exhibited brush-like lamellipodia and finger-like filopodia (arrowheads; Fig.
5A). wt-Cdc42-overexpressing growth cones were larger and more complex and commonly had an increased
number of backbranches along their axons (arrows; Fig. 5E). Of the growth cones expressing dn-RhoA, 58% had
abnormal, thickened filopodia with a balled appearance
(arrowheads; Fig. 5D). In both cases these growth
cones had certain aspects of the transformed appearance of normal
growth cones as they first entered the tectum, such as increased
complexity, backbranching, and altered filopodial morphology (Harris et
al., 1987). It is, therefore, interesting to speculate that these
signaling pathways are involved in target recognition.
View larger version (129K):
[in this window]
[in a new window]
|
Figure 5.
Rho-family GTPase mutants influence
growth cone morphology. Shown are RGC growth cones in sections of
Xenopus brain immunostained with anti-luciferase
(A) or anti-myc antibodies
(B-F). A, A growth cone
transfected with the control protein luciferase;
arrowheads indicate filopodia. B-F,
Growth cones transfected with wt or dn mutant GTPases.
Arrowheads in D indicate the abnormal,
balled filopodia observed on dn-RhoA-expressing growth cones.
Arrows in E indicate backbranches
observed on axons of wt-Cdc42-expressing cells. Notice also the
unusually large growth cone. Scale bar, 15 µm.
|
|
Growth cone morphology was analyzed by measuring the area, the number
of filopodia, and length of the longest filopodium (Fig. 6). For each construct the growth cones
were examined at many different points in the pathway. However, the
differences observed in growth cone morphology were not correlated with
a preponderance of growth cones in a particular brain region. Growth
cones overexpressing wt-Cdc42 had 50% more filopodia, were 40%
larger, and had 80% more axonal backbranches than controls. Expressing
dn-Cdc42 had the opposite effect: decreasing the number of filopodia on
average by ~50%, length of the longest filopodium by ~30%, and
growth cone area by ~25%. Overexpressing wt-RhoA caused a similar
decrease in growth cone area but did not affect significantly the
filopodia number or length of the longest filopodium. Clearly, Cdc42
plays a major role in promoting growth cone structures in
vivo. Interestingly, increasing the activity of this GTPase, and
thus growth cone complexity, seems detrimental to either axon
initiation or extension and to target recognition. Although dn-RhoA did
not change significantly any of the parameters that were measured, it
did alter filopodial morphology in many growth cones. This altered
morphology potentially could explain the differences observed in target
choice and recognition in growth cones in which RhoA function was
inhibited.
View larger version (40K):
[in this window]
[in a new window]
|
Figure 6.
Cdc42 regulates RGC growth cone complexity.
A, Average number of filopodia per growth cone for RGC
growth cones expressing control, wt-, or dn-GTPase proteins.
B, Average length of the longest filopodium on RGC
growth cones. C, Average growth cone area.
D, Average number of backbranches found on the axon two
growth-cone lengths behind the initial swelling of the growth cone. The
numbers in the bars indicate the growth
cones that were examined. Error bars are SEM, and p
values (*p < 0.05, **p < 0.01, ***p < 0.0001) indicate differences from
control.
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| |
DISCUSSION |
This study demonstrates that Rho-family GTPases regulate the
initiation, extension, and elaboration of dendrites, axons, and growth
cones in vertebrate neurons in vivo. In addition, by
expressing wt, ca, and dn forms of Rac1, RhoA, and Cdc42 in RGCs and
examining multiple aspects of neuronal process development, we have
illustrated differences in the functional roles for these GTPases in a
single cell type. Several findings have emerged from this study, many of which are consistent with previous studies. (1) Axonogenesis is
inhibited by the unregulated increase in activity of all three Rho-family GTPase members, but only Rac1 activity is required for
axonogenesis. The partial rescue of the ca-Rac1 phenotype with dn-Cdk5
suggests that Rac1 activity must be maintained within certain levels
for normal axonogenesis. (2) Dendritogenesis requires the function of
both Rac1 and Cdc42, whereas the expression of ca-Rac1 results in a
dramatic proliferation of dendrites. (3) Target recognition relies on a
normal balance of Cdc42 and RhoA activity; Cdc42 and RhoA also have
striking effects on growth cone morphology. Table
1 summarizes these results.
Specificity of mutant Rho-family GTPases
The dn-GTPases used in this study function by competitive
inhibition; they bind irreversibly to guanine nucleotide exchange factors (GEFs), molecules that regulate the exchange of GDP for GTP,
thus preventing the activation of endogenous GTPases (Diekmann et al.,
1991). Imperative to the interpretation of results obtained with these
dns is the assumption that each affects only one GTPase and does not
inhibit other family members. Two lines of evidence argue that these
dns act specifically. First, differential effects were observed in this
study after the expression of dn-Rac1, RhoA, or Cdc42. Second, specific
GEFs for RhoA (Gebbink et al., 1997), Rac1 (Michiels et al., 1995), and
Cdc42 (Zheng et al., 1996) have been identified. The ca mutants are
unresponsive to GTPase-activating protein (GAP) stimulation of their
intrinsic GTPase (Ridley et al., 1992) and thus exert their influence
without interacting directly with endogenous regulatory proteins as the
dn mutants do. Although these GTPases have not been demonstrated in the
Xenopus nervous system, it is likely that they are present,
because Rho-family GTPases and their regulatory proteins are highly
conserved and have been localized to nervous tissues in a variety of
vertebrate and invertebrate species (Olofsson et al., 1988; Didsbury et
al., 1989; Luo et al., 1994; Zipkin et al., 1997; Albertinazzi et al., 1998; Kuhn et al., 1998; Steven et al., 1998).
A role for RhoA in regulating target recognition and
target choice
Our results confirm in vitro observations that
increased RhoA activity inhibits process formation (Jalink et al.,
1994; Gebbink et al., 1997; Kozma et al., 1997). The expression of
ca-RhoA in vivo dramatically reduced the average length of
the longest dendrite plus the number of dendrite tips and prevented
axonogenesis. In addition, we have demonstrated novel requirements for
RhoA activity in generating normal filopodial morphology and in target
selection and recognition. It is of interest that growth cones in
culture treated with C3-transferase, an inhibitor of RhoA, also display an abnormal morphology in that they exhibit little or no lamellipodial spreading (Jin and Strittmatter, 1997). The alterations in target selection and recognition observed in this study after dn-RhoA expression could be attributable to several factors. One may be improper myosin function in the growth cones. We have shown that myosin
is required for normal Xenopus RGC growth cone motility in vivo (Ruchhoeft and Harris, 1997), and it has been
demonstrated that RhoA regulates myosin II activity (Amano et al.,
1996; Kimura et al., 1996). It is also possible that these defects
arise via RhoA effectors that interact with actin cytoskeleton, such as p140mDia, a protein that regulates the activity of profilin, an actin
monomer-sequestering protein (Watanabe et al., 1997). Alternatively, other RhoA effectors that have been identified but not yet functionally characterized, such as PKN (Amano et al., 1996; Watanabe et al., 1996)
and rhotekin (Watanabe et al., 1996), may be involved.
Rac1 is required for axon initiation and
promotes dendritogenesis
The absence of axons after dn-Rac1 expression indicates that Rac1
activity is required for Xenopus RGC axon initiation,
although Cdc42 activity is not. The expression of ca-Rac1 also resulted in the absence of axons, suggesting that either an unregulated increase
or decrease in the activity of Rac1 is detrimental. This result agrees
with a study by Luo et al. (1994) in Drosophila, in which
the early expression of either dn- or ca-Rac1 eliminated axons and
later expression caused growth cone stalling, and with a recent study
in chick motor neurons in vitro in which both ca- and
dn-Rac1 drastically reduced neurite length (Kuhn et al., 1998). Visualization of the actin cytoskeleton of growth cones in both of
these systems revealed an increase in F-actin in ca-expressing growth
cones and a decrease in dn-expressing growth cones, suggesting that
Rac1 controls the cycling of actin polymerization and depolymerization. Too much or too little polymerization may upset the delicate balance required to generate an axon. That a dn version of Cdk5, an effector of
Rac1 signaling, can partially rescue the inhibition of axonogenesis caused by ca-Rac1 in Xenopus RGCs supports this argument.
The overexpression of wt-Rac1 did not prevent axonogenesis or affect growth cone morphology in this system. These cells may be able to
compensate via regulatory mechanisms for the increased presence of a wt
protein, whereas the activity of a nonregulatable ca form may be more
difficult to overcome.
The expression of Rac1 mutants in this study also had a dramatic effect
on dendritogenesis. dn-Rac1 caused a decrease in the length of the
longest dendrite and the number of dendritic tips, whereas the
introduction of a ca form resulted in a proliferation of dendrites.
Other studies have shown that ca-Rac1 expression in mouse Purkinje
cells in vivo increases the number of dendritic spines (Luo
et al., 1996), ca-Rac1 expression in rat cortical neurons in culture
increases dendrite number (Threadgill et al., 1997), and the
overexpression of chicken Rac1B enhances neurite number and branching
in retinal cells in culture (Albertinazzi et al., 1998). These results,
as well as the data from this study, all point to a major role for Rac1
in dendrite formation. The influence of Rac1 on dendritic proliferation
could be mediated via downstream effectors of Rac1, such as gelsolin
(Arcaro, 1998; Azuma et al., 1998) and cofilin (Arber et al., 1998;
Yang et al., 1998), which regulate actin polymerization cycling.
Whatever the mechanism of dendritic proliferation, the differential
effects of ca-Rac1 on dendrites versus axons in Xenopus RGCs
suggest intriguing differences in the development of these structures.
Growth cones have been observed on the tips of developing dendrites
(Maslim et al., 1986), and the regulation of actin dynamics could vary from that found in axonal growth cones.
Cdc42 regulates growth cone morphology in vivo
Unlike Rac1, Cdc42 activity was not required for axon initiation,
because dn-Cdc42-expressing RGCs initiated and extended axons normally.
Manipulating Cdc42 function did impact growth cone morphology.
Overexpression of the wt protein increased growth cone size and
filopodia number, whereas dn-Cdc42 expression decreased growth cone
size, filopodial number, and filopodial length. Intriguingly, overexpressing wt-Cdc42 caused both an increase in growth cone complexity and in the percentage of axons that made target recognition errors. Similar to what was observed with dn-Rho, only a small percentage (~10%) of RGCs made target recognition errors. This may
be attributable to variability among the mixed population of RGCs, some
of which may be more sensitive to perturbation. In fly and worm, in
which mutant GTPases have been found to cause pathfinding errors, only
an identified subset of cells exhibits aberrant pathfinding (Zipkin et
al., 1997; Kaufmann et al., 1998). wt-Cdc42-expressing axons also were
significantly shorter than controls, although it is unclear whether
this is attributable to slower axon extension or delayed axon
initiation. Because of their increased size and complexity, these
growth cones may have advanced more slowly or may have been hindered by
abnormal filopodial and lamellipodial motility.
Interestingly, an unregulated increase or decrease of Cdc42 inhibited
dendritogenesis, which is similar to the effect mutant Rac1 expression
had on axon initiation in this system. Potentially, aspects of actin
dynamics are controlled by Cdc42 as well as Rac1 during
dendritogenesis. Cdc42 has been shown to induce actin polymerization in
a cell-free system via a pathway involving p21-activating kinase (Pak1)
(Zigmond et al., 1997).
Previous work on Rho-family GTPases has illustrated that the roles
these molecules play can be extremely context-dependent, which could
make the development of a generalized, comprehensive picture of their
function impossible. In this study we have shown distinct roles for
RhoA, Rac1, and Cdc42 in the formation of neuronal processes in a
single cell type in a developing, in vivo, vertebrate preparation. The thorough nature of the present study may provide insight into the collaborative nature of these three proteins in the
formation of axons, dendrites, and growth cones.
| |
FOOTNOTES |
Received May 7, 1999; revised July 1, 1999; accepted July 15, 1999.
This work was supported by National Institutes of Health and Medical
Research Council grants (to W.A.H. and C.E.H.) and a Lucille P. Markey
Fellowship (to M.L.R.). We thank Alan Hall and Jerold Chun for
providing various GTPase cDNA constructs and Anna Philpott for advice
and for providing Cdk5 cDNA constructs. We also thank Sarah McFarlane,
Barbara Lom, Emilie Marcus, Anne Vincent, and Nick Spitzer for helpful
comments on this manuscript; Anna Marnick for library work; and Darwin
Berg, Jim Posokony, David Rapaport, and Don Cleveland for allowing the
use of their equipment.
Correspondence should be addressed to Dr. Maureen Ruchhoeft, University
of California at San Diego, 3115 Pacific Hall, Mail Code 0357, La
Jolla, CA 92093.
| |
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M. Piper, A. Dwivedy, L. Leung, R. S. Bradley, and C. E. Holt
NF-Protocadherin and TAF1 Regulate Retinal Axon Initiation and Elongation In Vivo
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[Abstract]
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B. K. Garvalov, K. C. Flynn, D. Neukirchen, L. Meyn, N. Teusch, X. Wu, C. Brakebusch, J. R. Bamburg, and F. Bradke
Cdc42 Regulates Cofilin during the Establishment of Neuronal Polarity
J. Neurosci.,
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[Abstract]
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M. K. Sfakianos, A. Eisman, S. L. Gourley, W. D. Bradley, A. J. Scheetz, J. Settleman, J. R. Taylor, C. A. Greer, A. Williamson, and A. J. Koleske
Inhibition of Rho via Arg and p190RhoGAP in the Postnatal Mouse Hippocampus Regulates Dendritic Spine Maturation, Synapse and Dendrite Stability, and Behavior
J. Neurosci.,
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[Abstract]
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H. Chen and B. L. Firestein
RhoA Regulates Dendrite Branching in Hippocampal Neurons by Decreasing Cypin Protein Levels
J. Neurosci.,
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[Abstract]
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T. Hayashi, T. Okabe, Y. Nasu-Nishimura, F. Sakaue, S. Ohwada, K. Matsuura, T. Akiyama, and T. Nakamura
PX-RICS, a novel splicing variant of RICS, is a main isoform expressed during neural development
Genes Cells,
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[Abstract]
[Full Text]
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A. Dwivedy, F. B. Gertler, J. Miller, C. E. Holt, and C. Lebrand
Ena/VASP function in retinal axons is required for terminal arborization but not pathway navigation
Development,
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[Abstract]
[Full Text]
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S. Marshak, A. M. Nikolakopoulou, R. Dirks, G. J. Martens, and S. Cohen-Cory
Cell-Autonomous TrkB Signaling in Presynaptic Retinal Ganglion Cells Mediates Axon Arbor Growth and Synapse Maturation during the Establishment of Retinotectal Synaptic Connectivity
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[Abstract]
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E. I. Charych, B. F. Akum, J. S. Goldberg, R. J. Jornsten, C. Rongo, J. Q. Zheng, and B. L. Firestein
Activity-Independent Regulation of Dendrite Patterning by Postsynaptic Density Protein PSD-95
J. Neurosci.,
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[Abstract]
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H. Liu, T. Nakazawa, T. Tezuka, and T. Yamamoto
Physical and Functional Interaction of Fyn Tyrosine Kinase with a Brain-enriched Rho GTPase-activating Protein TCGAP
J. Biol. Chem.,
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Y. Nasu-Nishimura, T. Hayashi, T. Ohishi, T. Okabe, S. Ohwada, Y. Hasegawa, T. Senda, C. Toyoshima, T. Nakamura, and T. Akiyama
Role of the Rho GTPase-activating protein RICS in neurite outgrowth
Genes Cells,
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[Abstract]
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A. M. Rajnicek, L. E. Foubister, and C. D. McCaig
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E. S. Ruthazer, J. Li, and H. T. Cline
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J. Neurosci.,
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S. Woo and T. M. Gomez
Rac1 and RhoA Promote Neurite Outgrowth through Formation and Stabilization of Growth Cone Point Contacts
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M. Chen, K. G. Lucas, B. F. Akum, G. Balasingam, T. M. Stawicki, J. M. Provost, G. M. Riefler, R. J. Jornsten, and B. L. Firestein
A Novel Role for Snapin in Dendrite Patterning: Interaction with Cypin
Mol. Biol. Cell,
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E. L. Tudor, M. S. Perkinton, A. Schmidt, S. Ackerley, J. Brownlees, N. J. O. Jacobsen, H. L. Byers, M. Ward, A. Hall, P. N. Leigh, et al.
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E. Kvachnina, G. Liu, A. Dityatev, U. Renner, A. Dumuis, D. W. Richter, G. Dityateva, M. Schachner, T. A. Voyno-Yasenetskaya, and E. G. Ponimaskin
5-HT7 Receptor Is Coupled to G{alpha} Subunits of Heterotrimeric G12-Protein to Regulate Gene Transcription and Neuronal Morphology
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E. M. Y. Moresco, S. Donaldson, A. Williamson, and A. J. Koleske
Integrin-Mediated Dendrite Branch Maintenance Requires Abelson (Abl) Family Kinases
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M. Negishi and H. Katoh
Rho Family GTPases and Dendrite Plasticity
Neuroscientist,
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[Abstract]
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E.-E. Govek, S. E. Newey, and L. Van Aelst
The role of the Rho GTPases in neuronal development
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B. Bryan, V. Kumar, L. J. Stafford, Y. Cai, G. Wu, and M. Liu
GEFT, A Rho Family Guanine Nucleotide Exchange Factor, Regulates Neurite Outgrowth and Dendritic Spine Formation
J. Biol. Chem.,
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S. Gehler, G. Gallo, E. Veien, and P. C. Letourneau
p75 Neurotrophin Receptor Signaling Regulates Growth Cone Filopodial Dynamics through Modulating RhoA Activity
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A. Lee, W. Li, K. Xu, B. A. Bogert, K. Su, and F.-B. Gao
Control of dendritic development by the Drosophila fragile X-related gene involves the small GTPase Rac1
Development,
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T. Nakazawa, A. M. Watabe, T. Tezuka, Y. Yoshida, K. Yokoyama, H. Umemori, A. Inoue, S. Okabe, T. Manabe, and T. Yamamoto
p250GAP, a Novel Brain-enriched GTPase-activating Protein for Rho Family GTPases, Is Involved in the N-Methyl-D-aspartate Receptor Signaling
Mol. Biol. Cell,
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[Abstract]
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E. K. Scott, J. E. Reuter, and L. Luo
Small GTPase Cdc42 Is Required for Multiple Aspects of Dendritic Morphogenesis
J. Neurosci.,
April 15, 2003;
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G. P. Demyanenko and P. F. Maness
The L1 Cell Adhesion Molecule Is Essential for Topographic Mapping of Retinal Axons
J. Neurosci.,
January 15, 2003;
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H. Fujita, H. Katoh, Y. Ishikawa, K. Mori, and M. Negishi
Rapostlin Is a Novel Effector of Rnd2 GTPase Inducing Neurite Branching
J. Biol. Chem.,
November 15, 2002;
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T. Kubo, T. Yamashita, A. Yamaguchi, H. Sumimoto, K. Hosokawa, and M. Tohyama
A Novel FERM Domain Including Guanine Nucleotide Exchange Factor Is Involved in Rac Signaling and Regulates Neurite Remodeling
J. Neurosci.,
October 1, 2002;
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P. Dergham, B. Ellezam, C. Essagian, H. Avedissian, W. D. Lubell, and L. McKerracher
Rho Signaling Pathway Targeted to Promote Spinal Cord Repair
J. Neurosci.,
August 1, 2002;
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W. M. Jurney, G. Gallo, P. C. Letourneau, and S. C. McLoon
Rac1-Mediated Endocytosis during Ephrin-A2- and Semaphorin 3A-Induced Growth Cone Collapse
J. Neurosci.,
July 15, 2002;
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[Abstract]
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P. S. McQuillen, M. F. DeFreitas, G. Zada, and C. J. Shatz
A Novel Role for p75NTR in Subplate Growth Cone Complexity and Visual Thalamocortical Innervation
J. Neurosci.,
May 1, 2002;
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[Abstract]
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M. D. Kim, P. Kolodziej, and A. Chiba
Growth Cone Pathfinding and Filopodial Dynamics Are Mediated Separately by Cdc42 Activation
J. Neurosci.,
March 1, 2002;
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[Abstract]
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C.-S. Uhm, B. Neuhuber, B. Lowe, V. Crocker, and M. P. Daniels
Synapse-Forming Axons and Recombinant Agrin Induce Microprocess Formation on Myotubes
J. Neurosci.,
December 15, 2001;
21(24):
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[Abstract]
[Full Text]
[PDF]
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P. Penzes, R. C. Johnson, V. Kambampati, R. E. Mains, and B. A. Eipper
Distinct Roles for the Two Rho GDP/GTP Exchange Factor Domains of Kalirin in Regulation of Neurite Growth and Neuronal Morphology
J. Neurosci.,
November 1, 2001;
21(21):
8426 - 8434.
[Abstract]
[Full Text]
[PDF]
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H. Katoh, H. Yasui, Y. Yamaguchi, J. Aoki, H. Fujita, K. Mori, and M. Negishi
Small GTPase RhoG Is a Key Regulator for Neurite Outgrowth in PC12 Cells
Mol. Cell. Biol.,
October 1, 2000;
20(19):
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[Abstract]
[Full Text]
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A. Tashiro, A. Minden, and R. Yuste
Regulation of Dendritic Spine Morphology by the Rho Family of Small GTPases: Antagonistic Roles of Rac and Rho
Cereb Cortex,
October 1, 2000;
10(10):
927 - 938.
[Abstract]
[Full Text]
[PDF]
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A. K. McAllister
Cellular and Molecular Mechanisms of Dendrite Growth
Cereb Cortex,
October 1, 2000;
10(10):
963 - 973.
[Abstract]
[Full Text]
[PDF]
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S. U. Walkley, M. Zervas, and S. Wiseman
Gangliosides as Modulators of Dendritogenesis in Normal and Storage Disease-affected Pyramidal Neurons
Cereb Cortex,
October 1, 2000;
10(10):
1028 - 1037.
[Abstract]
[Full Text]
[PDF]
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A. Y. Nakayama, M. B. Harms, and L. Luo
Small GTPases Rac and Rho in the Maintenance of Dendritic Spines and Branches in Hippocampal Pyramidal Neurons
J. Neurosci.,
July 15, 2000;
20(14):
5329 - 5338.
[Abstract]
[Full Text]
[PDF]
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W. T. Wong, B. E. Faulkner-Jones, J. R. Sanes, and R. O. L. Wong
Rapid Dendritic Remodeling in the Developing Retina: Dependence on Neurotransmission and Reciprocal Regulation by Rac and Rho
J. Neurosci.,
July 1, 2000;
20(13):
5024 - 5036.
[Abstract]
[Full Text]
[PDF]
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N. Arimura, N. Inagaki, K. Chihara, C. Menager, N. Nakamura, M. Amano, A. Iwamatsu, Y. Goshima, and K. Kaibuchi
Phosphorylation of Collapsin Response Mediator Protein-2 by Rho-kinase. EVIDENCE FOR TWO SEPARATE SIGNALING PATHWAYS FOR GROWTH CONE COLLAPSE
J. Biol. Chem.,
July 28, 2000;
275(31):
23973 - 23980.
[Abstract]
[Full Text]
[PDF]
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S. R. Floyd, E. B. Porro, V. I. Slepnev, G.-C. Ochoa, L.-H. Tsai, and P. De Camilli
Amphiphysin 1 Binds the Cyclin-dependent Kinase (cdk) 5 Regulatory Subunit p35 and Is Phosphorylated by cdk5 and cdc2
J. Biol. Chem.,
March 9, 2001;
276(11):
8104 - 8110.
[Abstract]
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[PDF]
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Y. Yamaguchi, H. Katoh, H. Yasui, K. Mori, and M. Negishi
RhoA Inhibits the Nerve Growth Factor-induced Rac1 Activation through Rho-associated Kinase-dependent Pathway
J. Biol. Chem.,
May 25, 2001;
276(22):
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[Abstract]
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T. Rashid, M. Banerjee, and M. Nikolic
Phosphorylation of Pak1 by the p35/Cdk5 Kinase Affects Neuronal Morphology
J. Biol. Chem.,
December 21, 2001;
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[Abstract]
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N. Matsuo, M. Hoshino, M. Yoshizawa, and Y.-i. Nabeshima
Characterization of STEF, a Guanine Nucleotide Exchange Factor for Rac1, Required for Neurite Growth
J. Biol. Chem.,
January 18, 2002;
277(4):
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[Abstract]
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[PDF]
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TOP
ABSTRACT
INTRODUCTION
