Department of Zoology, University of British Columbia, Vancouver V6T 1Z4, Canada
* Author for correspondence (e-mail: auld{at}zoology.ubc.ca)
Accepted 23 January 2003
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Rho, Rac, GTPase, Peripheral glia, Drosophila, actin, Microtubule, Cytoskeleton
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The Drosophila embryo is an ideal model for the analysis of glial
cell migration and nerve ensheathement. Mutant phenotypes of genes important
in Drosophila peripheral glial migration can be readily detected,
because the pattern of the peripheral nerves is very simple. In addition to
their extensive migration, peripheral glia project long cytoplasmic sheaths
around the axon tracts. Loss or disruption of the peripheral glial sheath
results in defasciculated axon tracts, that is also an easily detectable
phenotype (Sepp et al., 2001).
As development of the peripheral glia in the Drosophila embryo has
only recently been characterized, our knowledge of genes important in
peripheral glial development is very limited. However, it is known that
peripheral glia have an important role in mediating axon guidance during PNS
development (Sepp et al.,
2001
).
As little is known about how peripheral glial cell migration occurs,
studies on other cell types are useful to develop hypotheses on glial
migration. Dynamic rearrangement of the actin cytoskeleton is crucial for the
migration of multiple cell types (Hall and
Nobes, 2000). In addition, interaction between the migrating cell
and its underlying substrate is an important factor in a the ability of a cell
to migrate (O'Connor and Bentley,
1993
; Lin and Forscher,
1993
). Thus, it is likely that actin rearrangements are involved
in peripheral glial cell migration. As well, it is possible that neurons
express molecules that stimulate the migration of glia along their axons by
directly affecting actin dynamics.
The small Rho GTPases Rho, Rac and Cdc42 are mediators of actin dynamics in
motile cells and during cell morphogenesis
(Hall and Nobes, 2000). Each
GTPase has been implicated in the formation of different Actin-based
structures. In in vitro analysis of fibroblasts, Cdc42 is involved in
extension of filopodia, Rho mediates stress fiber formation, and Rac is
involved in membrane ruffling and the formation of lamellipodia
(Nobes and Hall, 1995
). The
small Rho GTPases also have distinct functions in Drosophila neurons.
Rac1 is involved in axonal outgrowth and steering, Cdc42 is involved in neuron
morphogenesis and cell positioning, and RhoA is involved in neuroblast
proliferation and is essential for dendritic but not axonal growth
(Luo et al., 1994
;
Kaufmann et al., 1998
;
Lee et al., 2000
;
Hakeda-Suzuki et al., 2002
;
Ng et al., 2002
). The current
knowledge of the function of Rho GTPases in glia is limited to in vitro
studies. Cdc42 is found to control cell polarity in astrocyte monolayers and
affects cell motility in Schwann cell cultures
(Cheng et al., 2000
;
Etienne-Manneville and Hall,
2001
). Rac1 is also involved in cell motility
(Cheng et al., 2000
), while
RhoA activity affects cell morphology in cultured Schwann cells
(Brancolini et al., 1999
).
As Rho GTPases are widely expressed during embryogenesis, mutations in
these genes affect many different tissue types. Therefore, in determining the
effects of the GTPases in a specific tissue, standard mutant analysis is not
always appropriate. We have chosen to analyze the function of Rho GTPases in
peripheral glia during embryonic nervous system development of
Drosophila. For these experiments, we ectopically expressed mutant
and wild-type constructs specifically in the glia using the GAL4/UAS gene
expression system (Brand and Perrimon,
1993). Distribution of actin-GFP was characterized in the
wild-type and mutant backgrounds. The studies have shown that Rho and Rac have
distinct activities in peripheral glial cell migration and nerve
ensheathement, while Cdc42 did not appear to have a major role in peripheral
glial cell development.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
For ectopic Rho GTPase expression, the repo::actin-GFP line was crossed to
each of the following lines: UAS-RhoAV14
(Fanto et al., 2000),
UAS-RhoA (wild type) (Harden et
al., 1999
), UAS-RhoAN19
(Strutt et al., 1997
),
UAS-DRac1wt, UAS-DRac1V12, UAS-Drac1N17, UAS-Drac1L89, UAS-Dcdc42V12
and UAS-Dcdc42N17 (Luo et al.,
1994
). For constructs where multiple P-element transformants were
available, the repo:actin-GFP line was crossed to the alternate
transformant lines and phenotypes of the progeny were analyzed to verify that
mutant phenotypes were due to ectopic expression of the UAS constructs rather
than their genomic insertion sites.
For analysis of hypomorphic Rac and RhoA alleles, embryos were collected
from the following stocks: RhoAE3.10/CyO
(Halsell et al., 2000),
RhoAk02107a/CyO (Magie et al.,
1999
), Rac1J10/TM6 B, Tb, Rac1J11/TM6 B, Tb,
Rac2
and Mtl
/TM3, Sb
(Hakeda-Suzuki et al., 2002
;
Ng et al., 2002
). All embryos
and larvae were raised at 21°C.
Embryo and larval staining
Embryos and larvae were stained and mounted for microscopy as reported
previously (Halter et al.,
1995; Sepp et al.,
2000
). The rabbit anti-GFP primary was used at 1:200 (AbCAM,
Cambridge, UK), mouse anti-ß-galactosidase was used at 1:300 (Sigma, St
Louis, MO), mouse anti-Neuroglian (mAb 1B7)
(Hall and Bieber, 1997
) was
used at 1:500, rabbit anti-HRP was used at 1:100 (Jackson Immunoresearch, West
Grove, PA), mAb 1D4 was used at 1:2, and mAb 22C10 (Developmental Studies
Hybridoma Bank, University of Iowa) was used at 1:2. Fluorescent secondaries,
goat anti-rabbit Alexa 488 and goat anti-mouse Alexa 568 were used at 1:200
(Molecular Probes, Eugene, OR). Images were taken on a BioRad MRC 600 confocal
microscope for Fig. 1 and
Fig. 3B, on a
Perkin-Elmer/Yokogawa disk scanning confocal for
Fig. 3A, and all others were
obtained on a BioRad Radiance Plus confocal microscope. Confocal stacks were
processed with ImageJ 1.24 and maximum projections were assembled with Adobe
Photoshop 5.5.
|
|
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Peripheral glia arise during early neurodevelopment at the lateral edge of
the CNS as a compact cone-shaped mass of cells
(Fig. 1A, arrows). Shortly
after the pioneer motorneurons from the CNS project axons into the periphery,
the peripheral glia, which have proliferated into groups of 6 to 8 cells per
hemisegment, begin to extend processes along the newly established axon
tracts. The peripheral glia migrate peripherally as a continuous chain of
cells (Sepp et al., 2000).
With the actin-GFP marker, protrusions were observed which emanated largely
from the leading glial cells in each chain
(Fig. 1B,C concave arrows). At
stage 14, the position of the leading glial cell bodies is variable. In some
segments, the cell body, which does not stain darkly with actin-GFP is
observed at the leading edge of the glial process
(Fig. 1C, solid arrow). In
other segments, the cell body is still located among the cone-shaped mass of
glia closer to the CNS/PNS transition zone, and in this case, the leading edge
of the glial process is often a lamellar-like structure
(Fig. 1C, arrowhead). The
leading glial cell was identified through comparison of Actin-GFP labeling to
previous analyses using glial nuclear and cytoplasmic enhancer traps
(Sepp et al., 2000
) as well as
dye-injection studies (Schmidt et al.,
1997
). The peripheral glia extend filopodia-like extensions not
only along in the correct migratory direction, but also laterally towards the
ventral somatic muscle domain (Fig.
1C, upper left concave arrow), suggesting that the glia sample
their environment as they travel. The cone-shaped mass of cells begins to
resolve such that the ventral peripheral glial cell (vPG;
Fig. 1D,E, solid arrows), which
will ensheathe the posterior fascicle/segmental nerve (PF/SN), will begin to
separate from the rest of the peripheral glia. The majority of peripheral glia
ensheathe the anterior fascicle/intersegmental nerve (AF/ISN). As the glial
cells advance further along the peripheral nerves, and they expand their
cytoplasmic processes to wrap their associated axons, the overall profile of
actin becomes smoother (compare Fig. 1D
with 1E). Overall, the distribution of actin-GFP is dynamic over
the course of glial migration and nerve ensheathement.
The actin-GFP distribution in peripheral glia was compared with the
microtubule cytoskeleton in embryos expressing both tau-lacZ
(Hidalgo et al., 1995), which
binds microtubules, and actin-GFP using the repo:GAL4 driver. The
actin-GFP profile of mature embryos appears to have more punctate structures
than the long filamentous profile of the microtubule cytoskeleton. The
actin-GFP and tau-lacZ patterns both appear fill the same extent of
the growing cell processes. For example, the vPG glial processes extend
cytoplasmic processes along the SNa motorneuron branch until early larval
stages. At the distal tip of this growing sheath, actin-GFP labeling was at
the leading edge, as was tau-lacZ staining
(Fig. 2A,B, solid arrows). The
observation suggests that both the actin and microtubular cytoskeletons are
used in peripheral glial process extension. However, from inspection of the
overall amount of overlap in staining of actin-GFP and tau-lacZ, the
actin and microtubule profiles do not appear to have a large amount of
similarity inside the cell processes even though the two patterns are found in
all general regions of the cytoplasm (Fig.
2).
|
Rho mediates peripheral glial migration and morphogenesis
To determine whether the RhoA GTPase is involved in peripheral glial cell
migration and axon ensheathement, wild-type and transgenic constructs of Rho
were overexpressed in peripheral glia using the repo:GAL4 line. Glial
phenotypes were observed using the actin-GFP marker and secondary effects on
sensory neurons were observed by double staining embryos with mAb 22C10.
Secondary effects on motorneuron development as detected by mAb 1D4 staining
were subtle, and are not shown here. It is important to note that expression
of the transgenic constructs was generated in a wild-type RhoA background.
Ectopic expression of constitutively active RhoA (RhoAV14) in peripheral glia prevents the cells from migrating peripherally, manifesting as dense clusters of glia arrested at their birthplace at the CNS/PNS transition zone (compare Fig. 1B with Fig. 4D). The phenotype of RhoAV14 overexpression is distinctive when compared with all other transgenic phenotypes generated in this study. Typically, long actin-containing fibers extend out of the glial clusters (Fig. 4D, concave arrows), and there are large expanses of PNS tracts with no glial sheaths whatsoever (compare Fig. 4D with 4E, solid arrows). The aberrant spike structures of the peripheral glia do not always project along sensory axon pathways (Fig. 4F, concave arrow). The lateral line glia fail to extend processes to interconnect between hemisegments (compare Fig. 4A,D, arrowheads) and the lateral chordotonal PNS glial cells appear collapsed and rounded (Fig. 4D, asterisk) although their associated lateral chordotonal neurons appear properly formed (Fig. 4E, asterisk). The sensory axon tracts in these mutants appear defasciculated although their pathfinding to the CNS is generally normal (Fig. 4E, arrow). The glial stalling phenotype is highly penetrant (97%, n=268) compared with repo::actin-GFP wild types (3%, n=185).
|
Similar to overexpression of the wild-type form, the ectopic expression of
dominant-negative RhoAN19 yielded subtle glial phenotypes with sensory axon
defasciculation as a secondary effect. The vPG cell failed to separate from
the main peripheral nerve branch (Fig.
4J, concave arrow) at 26% (n=196) compared with 9%
(n=185) in the repo::actin-GFP wild type. All RhoAN19
embryos died before the first larval stage. It has been previously shown that
even minor disruption of the peripheral glial-based blood-nerve-barrier can
result in embryonic lethality (Auld et al.,
1995).
The RhoA embryonic lethal hypomorphic mutants RhoAE3.10
and RhoAk02107b were analyzed for peripheral glial
phenotypes. The mutants were stained with anti-Neuroglian, which recognizes
peripheral glial membranes and the epidermis
(Fig. 5, green), and the
embryos were counterstained with the anti-HRP neuronal marker
(Fig. 5, red). The embryonic
phenotypes were severe, as RhoA is required for many aspects of embryo
development (Magie et al.,
1999). In the PNS, HRP-positive sensory neurons were largely
absent; however, some motor axon tracts were visible
(Fig. 5B). Peripheral glial
sheaths were not visible on significant regions of the axon tracts
(Fig. 5B, arrows) in all
hemisegments analyzed (n=89). As both the RhoA hypomorphic and
transgene overexpression phenotypes both showed glial disruption and embryonic
lethality, the data suggest that RhoA GTPase is an important mediator of glial
migration and axon wrapping.
|
Expression of constitutively active Rac1 (Rac1V12) disrupted peripheral glial migration along axon tracts, resulting in unensheathed expanses of neurons (Fig. 6D, solid arrows). In addition, the peripheral glial processes appeared abnormally thin along significant sections of the nerves (Fig. 6D, concave arrows). The peripheral nerves of these embryos were defasciculated (Fig. 6E, arrows). The peripheral glial stall phenotype was scored at 91% (n=257).
|
Similar to the ectopic wild type Rac1 mutant, glial expression of the dominant negative Rac1N17 form generated subtle glial wrapping phenotypes, where the overall profile of the glial sheath was abnormal while the sensory axons were defasciculated (Fig. 6M,N). In some hemisegments (17%, n=163), small gaps in the glial sheath were apparent (Fig. 5M).
Glial expression of the Rac1L89 mutant transgene caused the projection of ectopic lamellar-like structures from the glial sheaths (Fig. 6J, concave arrows). The sensory neuron projections in these mutants were largely normal, but in cases where sensory neurons were misplaced, the peripheral glia extended processes to associate with the aberrant projections (Fig. 6J,K, concave arrows). The sensory axon tracts were defasciculated in many hemisegments (69%, n=194). To examine more closely the ectopic lamellar-like projections of the peripheral glia, embryos with Rac1L89 overexpression were co-labeled for either motor- or sensory neurons. The glial ectopic lamellar-like structures, which varied in size, did not reach out over peripheral motor or sensory branches in many cases (Fig. 7). Using higher magnification, small filopodia-like structures were observed extending from the lamellae (Fig. 7C).
|
|
Cdc42 does not affect peripheral glial development
To determine whether the GTPase Cdc42 is involved in aspects of peripheral
glial development, the constitutively active transgenic construct
UAS-Dcdc42V12 and the dominant-negative form UAS-Dcdc42N17
were crossed individually to the glial-specific repo:GAL4 driver with
the UAS-actin-GFP marker in the background. The glial phenotypes in
the resultant progeny were analyzed with live GFP fluorescence and also with
immunofluorescence for confocal microscopy. Overexpression of the activated
form (Cdc42V12) did not affect peripheral glial migration or nerve wrapping
compared with the wild type (data not shown). Furthermore, the embryos were
viable and hatched into first instars, suggesting that the seal of the
blood/nerve barrier was intact. Overexpression of the dominant-negative form
(Cdc42N17) similarly did not lead to any detectable disruptions of the glial
sheath or the underlying neuronal patterns (data not shown). Together with
previous observations showing that the Cdc42 null mutant has normal sensory
neuron patterning (Genova et al.,
2000), the data suggest that Cdc42 does not have a major role in
peripheral glial development.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Distribution of actin-GFP in peripheral glia
For the current study, the actin-GFP was used to image the actin
distributions of peripheral glia in the embryonic and larval stages. The
transgenic method of labeling actin used in this study most probably
represents the endogenous actin distributions in the cell since actin networks
are composed of polymerized monomeric actin, and the actin-GFP is incorporated
into the intrinsic filamentous actin networks of cells
(Verkhusha et al., 1999). This
method is currently the most effective means of visualizing peripheral glia in
vivo. Standard labeling of embryos with fluorophore-conjugated phalloidin to
visualize actin distributions in peripheral glia is not useful, as actin is
strongly expressed throughout the embryo, including the somatic musculature.
With phalloidin labeling, the actin staining of peripheral glia does not stand
out in comparison with the high intensity labeling of the muscles (K.J.S. and
V.J.A., unpublished).
Our results showed that the distribution of actin over the course of
embryonic development is dynamic, especially during the migratory stages of
peripheral glia. As observed previously, peripheral glia migrate into the
periphery as a continuous chain of cells
(Sepp et al., 2000). With
actin-GFP labeling, we observed spike-shaped filopodial protrusions from the
leading glia, while the follower glia had a smoother actin distribution. It is
possible that the leading glial cell does the majority of pathfinding into the
periphery, while the following glia simply adhere to the leading cell during
migration. Follower glia must have important rearrangements of their actin, as
they all expand their cytoplasmic processes as they migrate peripherally. It
is also interesting to note that both actin and microtubules were observed in
the region of the leading edge of expanding peripheral glial processes. Thus
as for other cells (reviewed by Goode et
al., 2000
), both microtubules and actin could work together in
glia during migration.
The observation that leading glia project spike-shaped protrusions while
followers do not is very similar to what has been observed with GFP labeling
of lateral line PNS glia in the developing zebrafish
(Gilmour et al., 2002), and
also for tracheal cells in Drosophila
(Ribeiro et al., 2002
). It is
possible that migrating chains of many different cell types share similar
mechanisms for movement. As Drosophila peripheral glial migration is
similar to that of zebrafish PNS glia, the data suggest that mechanisms of PNS
glial migration could be conserved through evolution. The in vivo distribution
of actin in migrating PNS glia in vertebrates is not known. The analysis of
actin and microtubules in primary cultures of vertebrate glia is quite
different from what we observed. For example, in migrating astrocyte
monolayers, actin is observed close to the cell body while microtubules are
observed at the extreme leading edge
(Etienne-Manneville and Hall,
2001
). It is likely that the actin distribution of glia varies
depending on whether the glial cells contact neurons or not, as nerve
ensheathement is affected by actin dynamics, as observed in our experiments.
Despite the difficulties in visualizing actin in vivo, it is likely that
research in a genetic vertebrate model amenable to imaging such as zebrafish
should yield high quality images of glial actin in the future.
Function of small GTPases in developing peripheral glia
The small GTPases Rho, Rac and Cdc42 are expressed in the nervous systems
of both Drosophila and vertebrates
(Luo et al., 1994;
Terashima et al., 2001
). The
specific functions of the GTPases in axon outgrowth has been studied
extensively, although little is known about how they are involved in glial
development. In the developing Drosophila embryo, mutations in the
small GTPases disrupt the development of many tissues including neurons. This
complicates the analysis of GTPase function in glia, as peripheral glia use
axonal pathways as a migrational substrate. In Rac GTPase mutations, for
example, axon extension is disrupted (Luo
et al., 1994
; Kaufmann et al.,
1998
; Hakeda-Suzuki et al.,
2002
; Ng et al.,
2002
). Thus, glial stalling phenotypes could not be attributed
specifically to loss of GTPase function in the glia. As well, RhoA null mutant
embryos lacking maternal contributions of RhoA have severely disrupted overall
morphology and segmentation (Magie et al.,
1999
). Thus, phenotypes in the nervous system would be secondary
effects of disrupted embryonic morphogenesis. Complementary analysis of both
transgene overexpression and standard loss-of-function mutant analysis is
therefore required to understand the specific roles of RhoA, Rac1 and Cdc42 in
peripheral glia.
The migration of glia is very different from that of a neuronal growth cone. For glial migration, the cell body containing the nucleus moves and cytoplasmic processes elongate as the glia travel along the axons. When the glia have finished migrating, they must wrap their processes tightly around the axonal bundles to seal the nerves from the surrounding environment. This migration profile is thus unique and is important to understand given that migration and wrapping of glia is implicated in neuropathies and regeneration after nervous system injury.
Our data suggest that RhoA and Rac1 are both involved in peripheral glial cell migration and nerve ensheathement, and have distinct effects on Actin rearrangement. For example, constitutively active Rac1 (V12) and RhoA (V14) expression resulted in halted migration of cell bodies as well as disrupted cytoplasmic process extension. The phenotypes of the two mutants were very different from one another. Rac1 (V12) mutants showed ball-shaped collapsed glia, while RhoA (V14) mutants had very long, spike-shaped actin processes emanating from the cell bodies. The distinct and extreme phenotypes from these mutants suggest that there is a balance of RhoA and Rac1 activity in wild-type peripheral glia to generate normal migration and cytoplasmic process extension. The concept of a balance of GTPase function being necessary for glial cell migration is also supported by our observations that glial cell migration is stalled in both the gain-of-function and loss-of-function mutations. We interpret these observations as suggesting that there is a balance of GTPase activities that is necessary for glial cell migration. In other words, anything that affects this balance either through a loss of function or gain of function, affects the ability of glial cells to migrate.
The well-characterized cultured fibroblast model has shown that Rac is
involved in lamellipodia formation, while Rho mediates stress fiber
polymerization and Cdc42 is involved in the extension of filopodia
(Nobes and Hall, 1995). It is
possible that Rac1 and RhoA mediate the assembly of similar structures in
peripheral glia. The long, straight actin fibers seen in constitutively active
RhoA (V14) mutants could represent overextended stress fibers. Furthermore,
the massive glial lamellar-like structures that are stimulated by Rac1L89
expression appear very similar to the lamellipodia of cultured fibroblasts
(Nobes and Hall, 1995
). The
biochemical activity of the Rac1L89 mutation is not known, and can act as
either a dominant-negative or constitutively active form, depending on the
cell type (Luo et al., 1994
;
Kaufmann et al., 1998
). The
Rac1L89 phenotype in peripheral glia was most similar to overexpression of
wild-type Rac1 (compare Fig. 5G with
5J), suggesting that the ectopic lamellar structures were a result
of moderate increase in Rac1 activity. Thus, it is possible that the Rac1L89
mutation caused Rac1 to be overactive but not as much as in the Rac1V12
mutation.
It was interesting to note that the ectopic actin-containing projections of
RhoAV14 and Rac1L89 mutants did not always reach over axon tracts, which are
the normal peripheral glial migrational substrates in the wild type. For the
steering of a migrating cell, large amounts of actin polymerization occur at
the contact between the leading edge of the cell and the attractive
migrational substrate (O'Connor and
Bentley, 1993; Lin and
Forscher, 1993
). Perhaps the hyperactivity of the mutant GTPases
enabled the peripheral glia to extend processes out on less adhesive
substrates compared with axons. It was also interesting to note that ectopic
projections of peripheral glia (in the RhoAV14 and Rac1L89 mutants) did not
interfere with axon pathfinding in the periphery. The ectopic glial
projections could be a result of failed glial pathfinding instead.
Interestingly, axons were capable of correctly migrating in the absence of
glial sheaths (in the RhoAV14 and Rac1V12 mutants). It has been noted
previously that peripheral glia mediate sensory axon guidance to the CNS
(Sepp et al., 2001
). Thus,
peripheral glia most probably mediate sensory axon migration to the CNS using
secreted cues.
In conclusion, we have shown that the rearrangement of actin by the small GTPases RhoA and Rac1 is an integral part of peripheral glial migration and nerve ensheathement. This model can be used in the future to dissect GTPase signaling pathways. For example, it could be possible to determine whether the GTPases share common activators or effectors given that the cellular phenotype for mutants of each GTPase is so distinctive. In addition, we have found further characteristics of Drosophila peripheral glial development that are similar to the vertebrate zebrafish model. Thus, future genetic research in Drosophila to understand PNS glial migration will probably help advance the knowledge of the vertebrate field as well.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Auld, V. J., Fetter, R. D., Broadie, K. and Goodman, C. S. (1995). Gliotactin, a novel transmembrane protein on peripheral glia, is required to form the blood-nerve barrier in Drosophila.Cell 81,757 -767.[Medline]
Brancolini, C., Marzinotto, S., Edomi, P., Agostoni, E.,
Fiorentini, C., Müller, H. W. and Schneider, C. (1999).
Rho-dependent regulation of cell spreading by the tetraspan membrane protein
Gas3/PMP22. Mol. Biol. Cell
10,2441
-2459.
Brand, A. H. and Perrimon, N. (1993). Targeted
gene expression as a means of altering cell fates and generating dominant
phenotypes. Development
118,401
-415.
Carpenter, E. M. and Hollyday, M. (1992). The location and distribution of neural crest-derived Schwann cells in developing peripheral nerves in the chick forelimb. Dev. Biol. 150,144 -159.[Medline]
Cheng, H.-L., Steinway, M. L., Russell, J. W. and Feldman, E.
L. (2000). GTPases and phosphatidylinositol 3-kinase are
critical for Insulin-like Growth Factor-1-mediated Schwann cell motility.
J. Biol. Chem. 275,27197
-27204.
Etienne-Manneville, S. and Hall, A. (2001).
Integrin-mediated activation of Cdc42 controls cell polarity in migrating
astrocytes through PKC. Cell
106,489
-498.[Medline]
Fanto, M., Weber, U., Strutt, D. I. and Mlodzik, M. (2000). Nuclear signaling by Rac and Rho GTPases is required in the establishment of epithelial planar polarity in the Drosophila eye. Curr. Biol. 10,979 -988.[CrossRef][Medline]
Genova, J. L., Jong, S., Camp, T. and Fehon, R. G. (2000). Functional analysis of Cdc42 in Actin filament assembly, epithelial morphogenesis, and cell signaling during Drosophila development. Dev. Biol. 221,181 -194.[CrossRef][Medline]
Gilmour, D. T., Maishchein, H.-M. and Nüsslein-Volhard, C. (2002). Migration and function of a glial subtype in the vertebrate peripheral nervous system. Neuron 34,577 -588.[Medline]
Goode, B. L., Drubin, D. G. and Barnes, G. (2000). Functional cooperation between the microtubule and actin cytoskeletons. Curr. Opin. Cell Biol. 12, 63-71.[CrossRef][Medline]
Hakeda-Suzuki, S., Ng, J., Tzu, J., Dietzl, G., Sun, Y., Harms, M., Nardine, T., Luo, L. and Dickson, B. J. (2002). Rac function and regulation during Drosophila development. Nature 416,438 -442.[CrossRef][Medline]
Hall, S. G. and Bieber, A. J. (1997). Mutations in the Drosophila Neuroglian cell adhesion molecule affect motor neuron pathfinding and peripheral nervous system pathfinding. J. Neurobiol. 32,325 -340.[CrossRef][Medline]
Hall, A. and Nobes, C. D. (2000). Rho GTPases: molecular switches that control the organization and dynamics of the actin cytoskeleton. Philos. Trans. R. Soc. Lond. B Biol. Sci. 355,965 -970.[CrossRef][Medline]
Halsell, S. R., Chu, B. I. and Kiehart, D. P.
(2000). Genetic analysis demonstrates a direct link between rho
signaling and non-muscle myosin function during Drosophila morphogenesis.
Genetics 155,1253
-1265.
Halter, D. A., Urban, J., Rickert, C., Ner, S. S., Ito, K.,
Travers, A. A. and Technau, G. M. (1995). The homeobox gene
repo is required for the differentiation and maintenance of glia in
the embryonic nervous system of Drosophila melanogaster.
Development 121,317
-332.
Harden, N., Ricos, M., Ong, Y. M., Chia, W. and Lim, L. (1999). Participation of small GTPases in dorsal closure of the Drosophila embryo: distinct roles for Rho subfamily proteins in epithelial morphogenesis. J. Cell Sci. 11,273 -284.
Hidalgo, A., Urban, J. and Brand, A. H. (1995). Glia dictate pioneer axon trajectories in the Drosophila embryonic CNS. Development 127,393 -402.
Jessen, K. R. and Mirsky, R. (1999). Schwann cells and their precursors emerge as major regulators of nerve development. Trends Neurosci. 22,402 -410.[CrossRef][Medline]
Kaufmann, N., Wills, Z. P. and van Vactor, D.
(1998). Drosophila Rac1 controls motor axon guidance.
Development 125,453
-461.
Lee, T., Winter, C., Marticke, S. S., Lee, A. and Luo, L. (2000). Essential roles of Drosophila RhoA in the regulation of neuroblast proliferation and dendritic but not axonal morphogenesis. Neuron 25,307 -316.[Medline]
Lin, C. and Forscher, P. (1993). Cytoskeletal remodeling during growth cone-target interactions. J. Cell Biol. 121,1369 -1383.[Abstract]
Luo, L., Liao, Y. J., Jan, L. Y. and Jan, Y. N. (1994). Distinct morphogenetic functions of similar small GTPases: Drosophila Drac1 is involved in axonal outgrowth and myoblast fusion. Genes Dev. 8,1787 -1802.[Abstract]
Magie, C. R., Meyer, M. R., Gorsuch, M. S. and Parkhurst, S.
M. (1999). Mutations in the Rho1 small GTPase disrupt
morphogenesis and segmentation during early Drosophila development.
Development 126,5353
-5364.
Ng, J., Nardine, T., Harms, M., Tzu, J., Goldstein, A., Sun, Y., Dietzl, G., Dickson, B. J. and Luo, L. (2002). Rac GTPases control axon growth, guidance and branching. Nature 416,442 -447.[CrossRef][Medline]
Nobes, C. D. and Hall, A. (1995). Rho, Rac, and Cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell 81,53 -62.[Medline]
O'Connor, T. P. and Bentley, D. (1993). Accumulation of actin in subsets of pioneer growth cone filopodia in response to neural and epithelial guidance cues in situ. J. Cell Biol. 123,935 -948.[Abstract]
Ribeiro, C., Ebner, A. and Affolter, M. (2002). In vivo imaging reveals different cellular functions for FGF and Dpp signaling in tracheal branching morphogenesis. Dev. Cell 2, 677-683.[Medline]
Schmidt, H., Rickert, C., Bossing, T., Vef, O., Urban, J. and Technau, G. M. (1997). The embryonic central nervous system lineages of Drosophila melanogaster II. Neuroblast lineages derived from the dorsal part of the neuroectoderm. Dev. Biol 189,186 -204.[CrossRef][Medline]
Sepp, K. J. and Auld, V. J. (1999). Conversion
of lacZ enhancer trap lines to GAL4 lines using targeted
transposition in Drosophila melanogaster. Genetics
151,1093
-1101.
Sepp, K. J., Schulte, J. and Auld, V. J. (2000). Developmental dynamics of peripheral glia in Drosophila melanogaster. Glia 30,122 -133.[CrossRef][Medline]
Sepp, K. J., Schulte, J. and Auld, V. J. (2001). Peripheral glia direct axon guidance across the CNS/PNS transition zone. Dev. Biol. 238, 47-63.[CrossRef][Medline]
Strutt, K. L., Weber, U. and Mlodzik, M. (1997). The role of RhoA in tissue polarity and Frizzled signaling. Nature 387,292 -295.[CrossRef][Medline]
Terashima, T., Yasuda, H., Terada, M., Kogawa, S., Maeda, K., Haneda, M., Kashiwagi, A. and Kikkawa, R. (2001). Expression of Rho-family GTPases (Rac, cdc42, RhoA) and their association with p-21 activated kinase in adult rat peripheral nerve. J. Neurochem. 77,986 -993.[CrossRef][Medline]
Verkhusha, V. V., Tsukita, S. and Oda, H. (1999). Actin dynamics of lamellipodia in the Drosophila ovary revealed by a GFP-actin fusion protein. FEBS Lett. 445,395 -401.[CrossRef][Medline]