1 Samuel Lunenfeld Research Institute of Mount Sinai Hospital, 600 University
Avenue, Toronto M5G 1X5, Canada
2 Department of Molecular and Medical Genetics, University of Toronto, Toronto
M5S 1A8, Canada
* Author for correspondence (e-mail: culotti{at}mshri.on.ca)
Accepted 7 January 2004
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SUMMARY |
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Key words: Plexins, Migration, RHO-GTPases, C. elegans
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Introduction |
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Good candidates for downstream effectors of guidance cues and their
receptors are the small GTPases of the RHO family, RAC, RHO and CDC-42. These
GTPases regulate actin cytoskeleton dynamics in neurons
(Luo, 2000) and in
non-neuronal cells (Hall,
1998
), and they act as molecular switches cycling between a
GTP-bound `on' state and a GDP-bound `off' state
(Hall, 1998
). Positive and
negative regulators of RHO GTPases include guanine exchange factors (GEFs) and
GTPase-activating proteins (GAPs), respectively
(Dickson, 2001
).
RHO-family GTPases have been implicated in axon pathfinding and cell
migration through the analysis of constitutively active or dominant-negative
forms of these proteins and their effectors in cultured migrating cells
(Eickholt et al., 1999;
Jin and Strittmatter, 1997
;
Kuhn et al., 1999
). However,
the involvement of RAC GTPases in regulating cell movements and morphogenesis
in vivo has been best demonstrated through genetic analyses of model
organisms. For example, the three known RAC genes in C. elegans, ced-10,
mig-2 and rac-2, have been thoroughly examined for their effects
on CAN cell, gonadal leader cell (e.g. distal tip cells of the hermaphrodite
gonad), P cell and axon growth cone migrations, plus apoptotic cell
phagocytosis (which involves aspects of cell migration), using both genetic
and RNAi-induced loss-of-function approaches, as well as genetic
gain-of-function approaches (Kishore and
Sundaram, 2002
; Lundquist et
al., 2001
; Spencer et al.,
2001
; Zipkin et al.,
1997
). C. elegans RAC GTPases clearly have shared
(redundant or same pathway) and distinct (parallel pathways) functions,
sometimes dependent on cell type and in other cases dependent on the aspect of
migration being examined in a particular cell type. For example ced-10,
mig-2 and rac-2 have largely redundant functions in CAN and
GABAergic axon guidance, and in CAN cell migration, but mig-2 and
ced-10 have distinct functions in determining the direction of the
third phase of DTC migration (Lundquist et
al., 2001
), with double-mutant analysis suggesting that these two
genes act in the same pathway to regulate this migration. The C.
elegans RAC GEF activity of UNC-73, previously shown to be involved in
axon guidance and cell migration (Steven
et al., 1998
), behaves genetically as though it activates CED-10,
MIG-2 and RAC-2 in vivo (Lundquist et al.,
2001
), and is therefore another important component of axon
guidance and cell migration signaling mechanisms. The Drosophila and
vertebrate homologs of C. elegans UNC-73 appear to have
evolutionarily conserved functions in related signaling pathways
(Awasaki et al., 2000
;
Bateman et al., 2000
;
Liebl et al., 2000
;
Newsome et al., 2000
).
In the literature it is unclear whether specific RHO-family GTPases have
the same function in different cell types or in different situations. Several
studies have shown that attractive guidance cues activate RAC or CDC-42 to
promote cell or growth cone advance (Luo,
2000; Mueller,
1999
; Suter and Forscher,
1998
), whereas repulsive cues activate RHO to inhibit cell or
growth cone advance, or to induce retraction
(Dickson, 2001
;
Jalink et al., 1994
;
Luo, 2000
;
Yuan et al., 2003
). However,
the axon guidance receptor most directly implicated in regulating RHO and RAC
activities for its output is the Semaphorin receptor Plexin. Both
Drosophila and C. elegans lack the other major class of
semaphorin receptors, the neuropilins, but Drosophila Sema-1a binds
Plexin A and C. elegans SMP-1 (Ce-sema-1a) binds Plexin-1 (PLX-1)
(Fujii et al., 2002
;
Winberg et al., 1998
).
Drosophila PlexB and mammalian PlexB1 directly bind the activated
GTP-bound form of RAC but not its inactive GDP-bound form
(Driessens et al., 2001
;
Hu et al., 2001
;
Vikis et al., 2000
).
Drosophila PlexB also binds GDP and GTP forms of RhoA (Rho1
FlyBase) and has been proposed to stimulate RhoA
(Hu et al., 2001
).
In Drosophila, it has been proposed that semaphorin-activated
Plexin B (PlexB) sequesters RACGTP and thereby downregulates its
downstream serine/threonine kinase effector PAK while stimulating the RHO
pathway (Hu et al., 2001;
Vikis et al., 2002
). According
to this view, inactivation of the RACGTP-dependent growth cone
spreading mechanism is a pre-requisite step for RHO-induced collapse
stimulated by Plexin B (Hu et al.,
2001
; Vikis et al.,
2002
). This model is based on in vivo gain-of-function studies and
has not yet been validated by loss-of-function studies. Nonetheless, these
Drosophila studies indicate that semaphorin signaling through plexins
is an excellent starting point for understanding how the activation of
particular guidance receptors affect signaling through RHO family GTPases to
influence cell movements and morphogenesis.
We examine genetically the function of C. elegans Semaphorin 1
proteins and Plexin 1 in the positioning of sensory ray 1 cells during male
tail development. C. elegans has two plexin-related genes,
plx-1 and plx-2, encoding Plexin 1 (most closely related to
Drosophila and human Plexin A) and Plexin 2, respectively. C.
elegans also has three semaphorin genes, smp-1, smp-2 and
smp-3, encoding Sema 1A, Sema 1B and Sema 2A/MAB-20, respectively. We
find that in mutants lacking semaphorin 1 genes (i.e. smp-1
and smp-2) or plexin 1 (i.e. plx-1), ray 1 cells
are positioned anterior to their normal position. smp-1 and
smp-2 were shown previously to be required redundantly to prevent
this anterior displacement of the ray 1 cells
(Fujii et al., 2002;
Ginzburg et al., 2002
). We now
show that smp-1 and smp-2 largely require plx-1 for
this function. We further show that prevention of the anterior displacement of
the ray 1 cells also depends on RAC and RHO GTPases independent of smp-1,
smp-2 and plx-1; however, the relative levels of active RAC are
deciphered when Semaphorin 1 activates Plexin 1 signaling. Lowered doses of
specific wild-type RAC-encoding genes can cause a polarity switch in the
Plexin 1-dependent positioning of ray 1 cells.
Based on expression patterns for plx-1, smp-1 and smp-2,
and on the genetic analysis of mutants in these genes in C. elegans,
we propose that at normal cycling RHOGTP and RACGTP
levels, PLX-1 induces an apparent attraction to sources of SMP-1 and SMP-2, by
using the known cell spreading and adhesion functions of RHO-family GTPases.
By contrast, at low RACGTP levels, PLX-1 induces an apparent
repulsion from the same sources of SMP-1 and SMP-2. The anterior displacement
of ray 1 cells caused by plx-1 mutations is suppressed by mutations
in unc-33/CRMP, a known mediator of semaphorin-induced axon growth
cone collapse in other animals (Goshima et
al., 1995). The spatiotemporal expression patterns of plx-1,
smp-1 and smp-2 reporters suggest a cell-based model for the
control of anterior ray 1 displacements, which we have further examined by
cell ablation and ectopic expression studies.
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Materials and methods |
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Linkage Group X (LGX): mig-2(mu28)
(Zipkin et al., 1997) and
mig-2(gm103gf) (Forrester and
Garriga, 1997
).
LGI: smp-1(ev715) (Ginzburg et
al., 2002), unc-73(ev509)
(Steven et al., 1998
),
unc-73(e936) (Desai et al.,
1988
), unc-73(rh40)
(Steven et al., 1998
),
smp-2(ev709) (Ginzburg et al.,
2002
) and mab-20(bx61)
(Baird et al., 1991
).
LGIV: unc-33(e1261) (Li et
al., 1992), ced-10(n1993)
(Ellis et al., 1991
),
lin-1(e1275) (Kimble et al.,
1979
), plx-1(ev724) (this study) and plx-1(nc37)
(Fujii et al., 2002
).
LGV: him-5(e1490) (Hodgkin et
al., 1979).
Strains not isolated in our laboratory were obtained from the C. elegans Genetics Center, care of T. Stiernagle (The University of Minnesota), or from G. Garriga (U. C. Berkeley).
Reverse genetics
A frozen reverse genetics library, which represents 1.7 million mutagenized
haploid genomes, was screened for deletions in the Ceplexin-1 gene
using nested PCR methods (Roy et al.,
2000; Zwaal et al.,
1993
). Once a deletion sample was identified, sib selections were
performed to isolate the homozygous deletion strain NW1391
plx-1(ev724). AmpliTaq GOLDTM (Perkin-Elmer) was used in all PCR
reactions. The isolated deletion allele plx-1(ev724) was outcrossed
with N2 Bristol strain at least five times before further analysis. Defining
the first nucleotide of the initiation codon as the first nucleotide in a DNA
sequence, the plx-1(ev724) allele was deleted for nucleotide pairs
16135 to 18134.
Molecular biology
Standard molecular biology methods
(Sambrook et al., 1989) were
used unless otherwise noted. The
ZAPII (Stratagene) cDNA clone
yk535f1 was provided by Y. Kohara and excised in vivo.
Gene specific cDNA analysis and genotyping
Total RNA was isolated using the standard Trizol (GIBCO-BRL) protocol. A
standard reverse transcription (RT) protocol
(Moon and Krause, 1991) was
used to amplify gene specific products either using oligo dT or random primers
to identify all RNA populations. RT-PCR products comprising wild-type or
mutant cDNAs were cloned into pBSK+ or pGEMT-easy vectors
and sequenced to confirm the ORFs. Ce-plx-1 specific primers flanking
the genomic DNA deletion of plx-1(ev724) were used to follow the
mutation during outcrossing and multiple mutant strain constructions. Primer
sequences are available upon request.
Transgene constructs
A plx-1 transcriptional gfp reporter was constructed by
cloning the 2621 bp sequence immediately 5' to the initiation codon into
the multiple cloning site of pPD95_77 to generate plasmid pPD95_77cplx. A
plx-1(+) rescuing construct was assembled from multiple PCR fragments
encompassing the entire coding sequence of Ce-PLX-1. The 3' portion of
the construct comes from the cDNA yk535f1 and contains 739 bp of the
3'UTR. This plx-1(+) cDNA minigene was cloned downstream of the
promoter sequence of the pPD95_77cplx transcriptional reporter to obtain the
plasmid pZH127. The gfp coding sequence is out of frame in pZH127.
The construct contains the full-length plx-1(+) minigene with 2621 bp
of sequence immediately 5' to the initiation codon and 739 bp of the
3'UTR sequence.
The GFP-encoding portion of pZH127 was put in frame with the PLX-1(+) sequence by fusing it after the PmlI site located four amino acids before the stop codon. For this, a SphI-KpnI fragment was deleted from pZH127, cut with PmlI and re-ligated in combination with a linker sequence into the SphI-KpnI cut pZH127 to obtain the new plx-1 translational reporter plasmid pZH157.
An unc-73(+) gene driven by the plx-1 5' regulatory
sequence was constructed by sub-cloning a 6340 bp NcoI-SmaI
cDNA fragment of pZH63 (Steven et al.,
1998), encoding the full-length UNC-73, into a modified version of
pPD95_77cplx transcriptional reporter construct. For this sub-cloning, the
PstI site in the multiple cloning site of pPD95_77cplx was mutated to
generate an NcoI site. The resulting plasmid (pZH163) encodes a
full-length unc-73(+) under the control of the plx-1
promoter.
A smp-1 translational GFP reporter gene was obtained by ligating a
GFP cassette, PCR amplified from pPD95_77, into the unique NheI site
(exon 12) of the pVGS1a plasmid containing a 10 kb XbaI genomic
fragment from the smp-1 locus
(Ginzburg et al., 2002). The
resulting plasmid (pVGS1a::GFP) encodes the entire extracellular domain, the
transmembrane domain and an intra-cellular GFP reporter. The original plasmid
pVGS1a has the ability to completely rescue the phenotypes of smp-1
mutant animals (Ginzburg et al.,
2002
).
Generation and analysis of transgenic strains
Transgenic strains were as follows:
Transgenic strains were generated by co-microinjection of the DNA mix into
the distal gonad arms of N2 or him-5(e1490) hermaphrodites
(Mello and Fire, 1995). DNA
mixes consisted of a test construct at a concentration of 50 mg/µl or 30
mg/ml and a co-injection marker to create a final DNA concentration of 100
mg/µl. Transgenic extrachromosomal arrays were integrated using a UV
irradiation-based method (Mitani,
1995
). Integrated alleles were backcrossed five times before
phenotypic analysis.
RNA interference
RNAi constructs were made by sub-cloning a PCR fragment representing a
unique sequence from the targeted gene into the multiple cloning site of L4440
(Timmons et al., 2001). The
targeted genes included a C. elegans homolog of RHO
(Y51H4A.3), two C. elegans homologs of RHO-kinases
(C10H11.9/let-502 and K08B12.5) and exon 8 of
unc-33 (Y37E11C.1). Plasmids were transfected into bacterial strain
HT115. Bacteria were induced with IPTG using a variation of Protocol Number 2
from Kamath (Kamath et al.,
2001
). After induction, bacteria were immediately used to seed NGM
growth plates.
Cos 7 transfection and sub-cellular localization
A DNA construct encoding full-length C. elegans plx-1 MYC-tagged
cDNA under the control of the CMV promoter
(Fujii et al., 2002) was
transfected into Cos7 cells using lipofectamine following the manucfacturer's
protocol (Gibco). Cells were grown at 37°C in 5% CO2 for 24
hours post-transfection in RPMI media supplemented with 10% fetal bovine
serum. Prior to immnunostaining, cells were fixed in 4% paraformaldehyde for
10 minutes, then permeabilized with PBS containing 0.2% Triton X-100. Cells
were immunostained using a mouse monoclonal anti-MYC antibody (9E10, Santa
Cruz) and an Alexa 488-conjugated anti-mouse secondary antibody. Cells were
costained with rhodamine-conjugated phalloidin (Molecular Probes). Cell
morphology was observed using DIC optics of a DMRA2 Leica microscope.
Microscopy
Male tail anterior ray 1 displacement and ray fusion events were scored by
mounting 1 mM levamisole-treated animals on 2% agarose pads for observation
using DIC optics. The ajm-1::GFP translational reporter
(Simske and Hardin, 2001) was
visualized with an Applied Precision Deconvolution microscope or a Leica DMRXA
microscope to assess epidermal cell morphologies.
Standard errors for percentages of the anterior ray 1 phenotypes were
calculated assuming a binomial distribution with the observed percentage value
and the actual sample size. Statistical tests were carried out using a
standard (two-tailed) comparison of two proportions
(Moore and McCabe, 1998). All
P values represent the probability that the measured penetrance of
the phenotype is significantly different between two strains. A P
value less than 0.05 was considered significant. All comparisons described as
significant in the Results were based on this criterion.
Laser ablations
Laser cell ablations were performed using a Leica DMLFS confocal
microscope. Briefly, him-5(e1490) third larval stage males were
anaesthetized using 10 mM sodium azide in M9 solution and mounted on 2%
agarose pads. Developing hook cells were located using the ajm-1::GFP
reporter and ablations were assisted with the Leica confocal software (version
11.04). Worms were recovered after the ablations and allowed to grow 24 hours
before scoring the male tail phenotype. Using this same protocol, two control
ablations of L3 stage ray 3 cells were both successful at specifically
eliminating ray 3 in the adult.
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Results |
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In contrast to the wild type, plx-1 mutant ray 1 cells often fail to detach from the SET during the late fourth larval stage (Fig. 2H, compare with Fig. 2G). This occurs almost invariably with the severe class 1 phenotype, but the milder class 2 phenotype is occasionally observed without this persistent adhesion of ray 1 to the SET (data not shown).
plx-1 normally works in the same genetic pathway as smp-1 and smp-2
Single mutants for smp-1(ev715) and smp-2(ev709) have an
incompletely penetrant anterior ray 1 defect. However, the smp-1(ev715);
smp-2(ev709) double mutant is synergistically enhanced for this phenotype
(Ginzburg et al., 2002). The
severity of the ray 1 phenotype was re-examined in smp-1 and
smp-2 mutants according to criteria defined above (anterior displaced
ray 1 classes 1 and 2), and compared with the ray 1 phenotype observed in
plx-1(ev724). Interestingly, the phenotype is temperature-sensitive
for smp-1(ev715); smp-2(ev709) double mutants and for the
plx-1(ev724) single mutant (Table
1), suggesting the existence of an unknown temperature-sensitive
process involved in preventing anterior displacement of ray 1 that is revealed
when Sema-1/PLX-1 signaling is absent. At the restrictive temperature
(25°C), the penetrance of the anterior class 1 and class 2 ray 1
phenotypes combined is slightly but significantly higher (P<0.005)
in the plx-1(ev724) single mutant compared with in the
smp-1(ev715); smp-2(ev709) double mutant
(Table 1) (e.g. 32% versus 29%,
and 50% versus 35%, for class 1 and 2 defects, respectively). This suggests
that PLX-1 has some minor function in ray 1 positioning that is independent of
Semaphorin 1 signaling.
To determine whether plx-1(+), smp-1(+) and smp-2(+) work in the same genetic pathway, a triple mutant was constructed. At 25°C, the class 1 and class 2 ray 1 defects combined were slightly but significantly suppressed (P<0.0005) in the plx-1(ev724); smp-1(ev715); smp-2(ev709) triple when compared to plx-1(ev724) (Table 1). Despite the minor differences in penetrance and expressivity between the mutants, these results strongly suggest that plx-1 and smp-1 and smp-2 function largely in the same pathway (see also Fig. 6 and Discussion).
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Of possible relevance, the developing hook is located close to the ray 1
cell cluster during the third larval stage at the time we first observe
abnormal anterior ray 1 positioning in plx-1 and semaphorin
1 mutants (Fig. 2A,E,F,
and above results). As we observed positioning of the ray 1 cell cluster
anterior to the hook in plx-1, smp-1 and smp-2 single
mutants, and in smp-1; smp-2 double mutants, it is possible that
SMP-1 and SMP-2 expression from the hook may normally attract the
PLX-1-expressing ray 1 cells to keep them in their normal, more posterior
position (i.e. closer to the ray 2 cells). To further examine this
possibility, we characterized ray 1 cell positioning in lin-1 mutants
males. In lin-1 mutant males, additional hooks are present anterior
to their normal position because of a ventral epidermal cell lineage defect
(Sulston and Horvitz, 1981)
(Fig. 5H-L), which we have
found does not affect the ray lineages. The smp-2::gfp
transcriptional reporter is expressed in the normally positioned hook and in
the anterior hooks in lin-1(e1275) animals
(Fig. 5H). An anterior ray 1
phenotype essentially identical to the one in plx-1(ev724) mutant
males is observed in lin-1(e1275) mutant males. The penetrance of
anterior ray 1 defects is 3% at both 16°C and 25°C for the less severe
class 2 phenotype, but 14% and 28% at 16°C and 25°C, respectively, for
the more severe class 1 phenotype (n=150). In those
lin-1(e1275) mutant animals that have a nearly normal looking ectopic
hook, we find that ray 1 is often positioned much closer to this ectopic hook
than to the normal hook throughout larval development
(Fig. 5I-L).
The anterior displacement of ray 1 observed in lin-1(e1275) mutant males is considerably suppressed when smp-1(+) function is taken away. For example, in smp-1(ev715); lin-1(e1275) double mutants, the severe ray 1 anterior displacement (class 1) caused by an ectopic hook is 5% (n=58) compared with 28% (n=150) for lin-1(e1275) alone. These results further support the hypothesis that semaphorin 1 expression from the hook attracts ray 1 cells.
To confirm the possible involvement of Semaphorin 1 proteins expressed from the male hook in normally attracting ray 1 cells to the posterior side, we laser ablated hook precursor cells in L3 stage males (see Materials and methods). In hook-ablated animals, anterior displacement of ray 1 cells was observed for four out of seven sides examined. However, the ray 1 cells did not differentiate into a fully developed ray, suggesting that factors expressed by the hook are also required for ray 1 cell differentiation.
mig-2, ced-10 and unc-73 also prevent the anterior displacement of male ray 1 cells
To determine whether RAC GTPases are involved in ray 1 positioning, we made
use of mutations in two existing RAC GTPase genes in C. elegans,
mig-2 and ced-10. Cell migration and phagocytosis of apoptotic
cells are affected by mutations in these genes, although no male tail defects
were reported previously (Kishore and
Sundaram, 2002; Lundquist et
al., 2001
; Zipkin et al.,
1997
).
mig-2(mu28) and ced-10(n1993) single mutants exhibit very
low penetrance anterior ray 1 displacement defects
(Table 2). To test for possible
redundancy between the two RAC-encoding genes for ray 1 positioning, we
attempted to construct a mig-2(mu28); ced-10(n1993) double mutant.
However, double-mutant animals are sterile, and die as embryos and early
larvae as reported previously (Kishore and
Sundaram, 2002; Lundquist et
al., 2001
). Nonetheless, mig-2(mu28); ced10(n1993)/+
males survive and exhibit a severe anterior ray 1 displacement defect compared
with respective single mutants (Table
2). This suggests that mig-2 and ced-10 normally
function redundantly or in series (see Discussion) to prevent anterior
displacement of ray 1 cells. We have not examined the effects of
rac-2 on ray 1 cell positioning, although it also may act redundantly
or in series with mig-2 and ced-10 in this process.
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Like the mig-2(mu28); ced-10(n1993) double mutant, the
unc-73 null allele is lethal
(Steven et al., 1998),
preventing us from determining the ray 1 anterior phenotype in this context.
However, this analysis clearly demonstrates that UNC-73 is necessary for
preventing anterior displacement of ray 1 cells.
unc-73, and by implication ced-10 and mig-2, act in plx-1-expressing cells to prevent anterior displacement of ray 1 cells
Biochemical analyses have shown that UNC-73 activates RAC GTPases, and
genetic analyses show that unc-73 requires ced-10 and
mig-2, which encode RAC GTPases, for its activity in cell migrations
and axon guidance (Kishore and Sundaram,
2002; Lundquist et al.,
2001
; Spencer et al.,
2001
; Steven et al.,
1998
). A simple explanation is that UNC-73 is a GEF activator for
both CED-10 and MIG-2 involved in cell migrations.
Although unc-73, ced-10 and mig-2 prevent the anterior displacement of ray 1 cells, it is not clear whether they do so by acting in the ray cells, in the hook or, perhaps, even in a third cell type. If unc-73 affects ray cell migrations by acting in the hook or in a cell type other than the ray cells, then we might expect a deficit in unc-73 function to cause abnormal regulation of smp-1 expression. However, smp-1::GFP expression was totally normal in unc-73(e936), suggesting that unc-73 is unlikely to affect ray cell movements by acting in the hook or a cell type other than the ray precursors.
To examine this question more directly, we also expressed unc-73(+) under the control of the plx-1 promoter. This promoter was previously shown to drive expression of a gfp reporter primarily in the ray 1 cells of the male tail and not in the hook. If unc-73 and plx-1 act in the same set of cells, a plx-1::unc-73(+) transgene should rescue unc-73 mutant ray 1 positioning defects, but it should not rescue them if plx-1 and unc-73 act in different cell types. As shown in Table 2, this rescue was nearly complete (1% class 1 and 6% class 2 defects, n=146), showing that UNC-73, and by implication its effectors CED-10 and MIG-2, probably function in the ray 1 cell (or its descendants) to prevent displacement of ray 1 to an abnormal anterior position.
unc-73, mig-10 and ced-2 can position ray 1 cells without plx-1 activity
Each of the RAC signaling genes described above (mig-2, ced-10 and
unc-73) is at least partially required for normal ray 1 positioning
independent of PLX-1 signaling. Mutations in each of these genes are also
enhanced by the plx-1(ev724) null, which on its own has an
incompletely penetrant ray 1 defect even though it is predicted to be totally
non-functional (Table 3). The
mild ray displacement phenotype caused by mig-2(mu28) is enhanced by
reducing plx-1(+) function (Table
3), the greater the reduction in plx-1(+) dose, the
greater the enhancement. An even stronger enhancement phenotype is observed in
plx-1(ev724); ced-10(n1993) double-mutant animals
(Table 3). Thus when there is a
loss of only mig-2 function, or of only ced-10 function,
plx-1(+) is still required to prevent anterior displacement of ray 1
cells.
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RAC family genes switch the `polarity' of Semaphorin 1 signaling that occurs through Plexin 1
The results reported above demonstrate that threshold levels of MIG-2 and
CED-10, and activation of PLX-1 by SMP-1 and SMP-2, are required to prevent
anterior displacement of ray 1 cells in the male tail (see above). To
determine whether the severe anterior ray 1 defect caused by a strong
reduction in RAC function is dependant on plx-1(+), the dose of
plx-1(+) was reduced in a strain in which RAC was already strongly
compromised (as judged by the penetrance of anterior ray 1 defects).
Unexpectedly, in mig-2(mu28lf); ced-10(n1993)/+ males carrying only
one copy of plx-1(+), a significant suppression rather than
enhancement of the severe ray anterior phenotype occurs
(Table 4). This result
demonstrates that at low levels of RAC activity, plx-1(+) is required
for the anterior displacement of ray 1 cells, i.e. the opposite of the
function of plx-1(+) at normal levels of RAC, which is to prevent
anterior displacement of ray 1 cells.
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CRMP/UNC-33 functions in anterior ray 1 positioning and opposes posterior ray 1 positioning mechanisms that function independently of PLX-1
The ray1 anterior displacement phenotype of a strong RAC loss-of-function
[mig-2(mu28); ced-10(n1993)/+ double mutant] depends to some extent
on PLX-1 and its putative ligands SMP-1 and SMP-2. As it is possible that
anterior ray 1 displacement results in part from a Semaphorin 1-induced
repulsion of PLX-1 expressing ray 1 cells (see Discussion), we decided to
examine the effects of mutations in unc-33, which encodes proteins
related to mammalian CRMP proteins known to be required for axon growth cone
repulsions induced by Semaphorins (Goshima
et al., 1995). The anterior ray 1 defect is rarely observed in
unc-33(e1261) mutant males (Table
5) suggesting that unc-33 is not absolutely required for
normal posterior positioning of ray 1. However, unc-33(e1261)
suppresses significantly the severe (class 1) anterior ray 1 phenotype of
plx-1(ev724) mutants (Table
5). unc-33(+) function is therefore at least partially
required for the anterior ray 1 displacement phenotype observed in
plx-1(ev724).
|
Some hints about the mechanism used to regulate the reversal in the ray 1 positioning function of PLX-1 at low RAC levels may also be gleaned from the plx-1(+) overexpressing arrays. For example, the anterior ray 1 phenotype induced by the evIs162 [plx-1(+)] transgene array is also enhanced by mig-2(mu28) (Table 5), but does not rescue the severe ray 1 displacement of mig-2(mu28); ced-10(n1993)/+ (80% class 1 and 9% class 2 defects, n=110). One interpretation of this result is that the PLX-1(+) function provided by evIs162 can mimic the enhancement of mig-2(mu28) by ced-10(n1993)/+, possibly because PLX-1(+) overexpression from the array causes a reversal in the positioning function of PLX-1, just as mig-2(mu28); ced-10(n1993)/+ does. This could happen if, for example, the higher ratio of functional PLX-1 to functional RAC is what determines the reversal in the ray positioning function of PLX-1(+).
We have shown that the ray 1 defects observed in plx-1(ev724) mutants can be suppressed by a mutation in unc-33, demonstrating a requirement for UNC-33/CRMP in anterior ray 1 displacement that is independent of plx-1 function. Consistent with this, the ray 1 anterior displacement observed at 25°C in evIs162[plx-1(+)] (which we argue above is probably caused by overexpression of wild-type PLX-1 protein) is significantly suppressed by performing RNAi on unc-33 (Table 5; 17% versus 0% for the ray 1 class 1 and 39% versus 18% for the ray 1 class 2 defects).
RNA interference with rho-1-encoded GTPase, or let-502- or K08B12.5-encoded RHO-kinases, enhances anterior displacement defects of a plx-1 null and an unc-73 hypomorph
Recent studies have reported that vertebrate Plexins and
Drosophila Plexin B bind the active GTP-bound RAC GTPase
(RACGTP), and both RHOGDP and RHOGTP
(Driessens et al., 2001;
Hu et al., 2001
;
Rohm et al., 2000
;
Vikis et al., 2000
). To
examine the possibility that RHO GTPases might be involved in preventing the
anterior displacement of ray 1 cells, RNAi experiments were performed on the
RHO GTPase encoded by rho-1, and on the putative RHO-kinase effectors
encoded by let-502 and K08B12.5. All RNAi experiments
involved feeding larvae with bacteria designed to produce specific ds-RNAs
(see Materials and methods). RNA interference with each of these three genes
produced mildly penetrant ray 1 anterior displacement phenotypes
(Table 6). Each of them also
significantly enhanced the anterior ray 1 displacement defects of
unc-73(e936) animals (Table
6). RNAi of rho-1 on plx-1(ev724) animals also
significantly enhanced the class 1 defect
(Table 6). RNAi of
rho-1 at 25°C did not enhance plx-1(ev724) for unknown
reasons (not shown); however, RNAi of RHO-kinase encoding genes marginally
enhanced these defects in plx-1(ev724)
(Table 6). These results
suggest that C. elegans RHO GTPases, like the C. elegans RAC
GTPases MIG-2 and CED-10, are also involved in preventing the anterior
displacement of ray 1 cells in developing males.
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Discussion |
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A second mechanism for ray 1 positioning involves the RAC sub-types of the
RHO family of GTPases, MIG-2, CED-10 and their putative activator UNC-73 (a
RAC GEF). Loss-of-function mutations in mig-10 or ced-10
alone cause few, if any, effects on ray 1 positioning; however, concomitant
reductions in the dosage of wild-type mig-2 and ced-10 genes
[i.e. mig-2(mu28); ced-10(n1993)/+] causes significant anterior ray 1
displacements. This suggests that the RAC GTPase sub-types of the RHO family
of GTPases normally act redundantly to prevent anterior ray 1 displacement;
however, because mig-2(mu28) might not be a null allele, we cannot
rule out the possibility that these RAC GTPase sub-types act in series. In
either case, these results are consistent with the finding that even partial
loss-of-function mutations in unc-73 have significant ray 1 defects,
as unc-73 has been shown to be required for MIG-2, CED-10 and
RAC-2 functions in other types of cell migrations
(Kishore and Sundaram, 2002;
Lundquist et al., 2001
). In
the future, it should prove interesting to examine the effects of
simultaneously reducing all RAC gene functions on ray 1 cell positioning,
including the remaining known rac-2 C. elegans gene
(Lundquist et al., 2001
).
As RAC and RHO GTPases are traditionally thought to act antagonistically in
guiding migrating axon growth cones (and by implication in cell positioning),
we examined the effects of reducing the function of the single known C.
elegans RHO GTPase gene rho-1. Although RNAi of rho-1
in control him-5 animals did not dramatically affect ray 1 cell
positioning, it did dramatically enhance the anterior displacement of ray 1
cells of unc-73(e936) and plx-1(ev724) mutant animals.
Similar results were obtained by RNAi of RHO-kinase genes let-502 and
K08B12.5. These results suggest that RHO-1 and putative RHO effectors
act in the same sense as RAC GTPases and their putative activator UNC-73,
which is to prevent the anterior displacement of ray 1 cells. This is contrary
to the reported antagonistic roles for RHO and RAC functions in axon growth
cone migration (Dickson, 2001;
Jalink et al., 1994
;
Luo, 2000
;
Mueller, 1999
;
Suter and Forscher, 1998
;
Yuan et al., 2003
), but is
certainly not the only exception to this view to be found in the literature
(Dickson et al., 2001; Driessens et al.,
2001
).
The nearly complete penetrance of unc-73; plx-1 double mutants further suggests that UNC-73 and PLX-1 functioning together could account for all of the normal posterior positioning of ray 1. In principle, they could do this by acting in the same or in different cell types. For example, PLX-1 could act in the ray 1 cells, whereas UNC-73 and the RAC GTPases could act in some nearby tissue (e.g. the nearby hook, see below). We examined this possibility by using the plx-1 promoter to drive expression of unc-73(+) in plx-1-expressing cells. The fact that we obtained nearly complete rescue of unc-73(e936) (1% class 1 and 6% class 2 defects) by an extrachromosomal transgene array carrying plx-1::unc-73(+) strongly indicates that UNC-73, and by implication the RAC GTPases it putatively activates, MIG-2 and CED-10, normally function cell-autonomously to position the ray 1 cells.
The fact that unc-73, mig-2 or ced-10 mutations enhance the plx-1 null for anterior ray 1 defects, demonstrates that RAC GTPases and UNC-73 function in parallel to Semaphorin 1 protein signaling through PLX-1. At the very least, the synergistic effects of unc-73 and plx-1 mutations on the penetrance of anterior ray 1 defects indicate that PLX-1 function is partially redundant with the RHO family GTPases. However, it is important to note that these genetic analyses do not rule out the possibility that the RHO family GTPases also act in the same pathway as PLX-1 and may be intracellular effectors of the PLX-1 signal transduction machinery.
A switch in PLX-1 function caused by an alteration in the relative levels of RAC reverses the polarity of ray 1 cell positioning
Whether or not the RHO family of GTPases acts as PLX-1 effectors, one thing
that appears fairly certain is that the RHO family GTPases can affect PLX-1
signal transduction in a profound way. This is demonstrated by our finding
that at high (normal) doses of wild-type RAC genes, plx-1 acts to
prevent anterior displacements of ray 1, but at low doses of wild-type RAC
genes, PLX-1 signaling is switched in the polarity of the response that is
elicited by Semaphorin 1 proteins instead of being required to prevent
anterior displacements of ray 1, it is required to cause them.
MIG-2 GTPase cycling may be also required to prevent anterior ray 1 cell
displacement as evidenced by the finding of anterior ray 1 positioning defects
in mig-2(gm103gf), which encodes a mutant form of the RAC-like MIG-2
that is constitutively stuck in a GTP-bound active state by being unable to
exchange GDP for GTP (Zipkin et al.,
1997). Although mig-2(gm103gf) appears to be a
gain-of-function mutation (Zipkin et al.,
1997
), it mimics a loss of function for RAC activity both
phenotypically (i.e. it causes significant anterior displacement defects), and
with respect to its genetic interactions with mutations in genes encoding
other components of the ray 1 positioning mechanism. Of most relevance,
mig-2(gm103gf) anterior ray 1 defects are partially suppressed by
loss of plx-1(+) dosage just as mig-2(mu28); ced-10(n1993)/+
anterior ray 1 defects are. This is consistent with the proposed switch in
PLX-1 activity observed when RAC GTPase levels are low. That
mig-2(gm103gf) mimics the effect of low RAC activity on PLX-1
function allows an examination of the role that Semaphorin 1 proteins might
play in a situation that mimics low RAC activity. In this situation, it
appears that the Semaphorin 1 proteins are also required for manifestation of
the proposed switch in PLX-1 function. The apparent defect in preventing
anterior displacement of ray 1 in mig-2(gm103gf) could result partly
from a requirement for GTPase cycling, from low levels of RACGDP,
or from the proposed ability of constitutively GTP-loaded MIG-2 to bind and
inactivate RAC GEFs [for possible functions of the gm103gf allele see
Lundquist et al. (Lundquist et al.,
2001
)]. The ability of mig-2(gm103gf) to switch the
polarity of ray cell positioning caused by Semaphorin 1 signaling
distinguishes it from unc-73 mutations. The latter presumably have
increased levels of RACGDP, which in principle could account for
the difference.
An intriguing corollary to the molecular mechanisms that underlie
attraction versus repulsion is that the intracellular levels of small
molecules such as cGMP and cAMP can determine whether an axon guidance
receptor mediates an attraction to its ligand or a repulsion away from it
(Song et al., 1998). The
molecular mechanisms by which cGMP and cAMP switch the polarity of
receptor-mediated responses are being elucidated but are still incompletely
understood (Song et al.,
1998
). If switches in guidance receptor activity occur in response
to levels of RHO family GTPases, the study of semaphorin/plexin signal
transduction mechanisms would be an excellent system with which to reveal the
detailed molecular mechanisms underlying these switches, because plexins
interact with both RAC and RHO, and the activation of RHO is reportedly
dependent on plexin receptor stimulation by its semaphorin ligand(s)
(Hu et al., 2001
).
Is active migration involved in anterior ray 1 displacement?
It is distinctly possible that the switch in response to Semaphorin 1/PLX-1
signaling we have observed represents a switch from attraction to repulsion,
similar to the switch from repulsion to attraction of growth cones caused by
cGMP or cAMP (Song et al.,
1998). Anterior ray 1 displacements could be caused by a reversal
in the orientation of migration of the ray 1 cells, or it could simply
represent a passive movement that results from an abnormal adherence of the
ray 1 cell cluster to the lineally related elongating SET cell that it
contacts. There are reasons to imagine a purely adhesive function for PLX-1
signaling in C. elegans (Ginzburg
et al., 2002
), but we favor the migration model for several
reasons. First, we find normally positioned ray 1s that sometimes exhibit an
abnormally persistent SET contact, suggesting that there is no causal
connection between the persistent adhesion per se and anterior displacement of
ray 1 in the mutants. More enlightening is the discovery by Fitch and Emmons
(Fitch and Emmons, 1995
) who
found striking similarities of early larval ray lineages and cell-cell
contacts in the developing male tail of several species of the Rhabditidae
family of nematodes, which includes C. elegans
(Fitch and Emmons, 1995
).
However, in spite of the developmentally early similarities, significant
differences in adult ray position were observed between C. elegans
and other Rhabditidae (Fitch and Emmons,
1995
). Furthermore, species-specific ray position changes occur
that are not in any obvious way correlated with a change in shape of an
associated SET cell. When considered together with our results for C.
elegans male ray 1 cells, the Semaphorin 1 and Plexin 1 guidance system
is involved in what appears to be a migration of the ray 1 cell cluster on the
anteroposterior axis while they contact their clonally related R1.p cell.
The finding that the male hook expresses transcriptional and translational reporters for smp-1 and smp-2 at the same time in development, and that ray 1 cells express plx-1 reporters, suggests a straightforward model for how ray 1 positioning is accomplished (see Fig. 6). At normal RAC levels, the Semaphorin 1 proteins in the hook act as attractants to the PLX-1-expressing ray 1 cells, helping to keep them in a posterior position near the ray 2 cells. At low RAC activity or in the presence of non-cycling RACGTP [i.e. in mig-2(gm103gf)] the semaphorins can no longer act as attractants, but instead are actually actively involved with PLX-1 as repellants to the ray 1 cells, effectively pushing them to the anterior.
Consistent with the idea that semaphorins emitted by the hook attract the ray 1 cells is that, in lin-1 mutant males harboring an ectopic anterior hook, we find anterior ray 1 cells in close proximity to the smp-1- and smp-2-expressing ectopic hook (Fig. 5H-L). Semaphorin 1 downregulation in a lin-1 mutant background [i.e. smp-1(ev715);lin-1(e1275)] significantly suppresses the severe ray 1 anterior displacement toward the anterior ectopic hook. This strongly suggests that the ectopic anterior hook in lin-1 mutants attracts ray 1 cells in a Semaphorin 1-dependant manner. These findings are also most consistent with a role for these molecules in the active migration of ray 1 cells.
Ablation of the hook precursors caused anterior displacements of ray 1 in four out of seven ray 1s that could be examined, therefore the anterior displacement defects are not fully penetrant in hook-ablated animals. This could mean that hook-independent mechanisms exist for keeping ray 1 in its normal posterior position and is consistent with the finding that even plx-1 null mutations are not fully penetrant for this defect.
Molecular model for ray 1 positioning
Implicit in our results, which clearly show that one function of the RHO
family members (MIG-2, CED-10 and RHO-1), the putative RHO effectors
(RHO-kinases LET-502 and K08B12.5), the two Semaphorin 1 family members (SMP-1
and SMP-2) and their putative receptor (PLX-1) in C. elegans is to
prevent the anterior displacement of ray 1 cells in the male tail, is the
understanding that there must exist an anterior positioning mechanism for ray
1 that counteracts or antagonizes normal Semaphorin 1, PLX-1 and RHO family
GTPase functions (Fig. 6). In
situations where anterior ray 1 displacement occurs, such as when
plx-1(+) levels are low (PLX-1 signaling assumed to be low) or when
plx-1(+) is putatively overexpressed (by evIs162), anterior
displacement appears to require UNC-33/CRMP. This is consistent with the idea
that unc-33 is required in a mechanism that normally opposes
Semaphorin 1/Plexin 1- and/or RHO-family GTPase-mediated attractive
signaling.
How might UNC-33/CRMP mediate what appears to be a repulsion of the ray 1
cells from the hook (see Fig.
6)? UNC-33 could oppose PLX-1 signaling by antagonizing some
component of the PLX-1 signaling pathway, or it may simply be part of a
mechanism providing force to counteract the force of PLX-1 signaling used to
determine ray 1 positioning. One possible molecular mode of UNC-33 activity is
suggested by the finding that CRMP-1 binds and inhibits mammalian RHO-kinase
(Leung et al., 2002), a
probable RHO effector. Therefore UNC-33/CRMP could cause anterior displacement
of ray 1 cells by simply antagonizing RHO GTPase-mediated mechanisms that
prevent anterior displacement (Fig.
6). Another possibility is that UNC-33 could be part of an
independent, parallel acting pathway that causes repulsion. This would be the
classical view of UNC-33/CRMP activity because its function appears to be
directly required for growth cone collapse and repulsion in several systems
(Goshima et al., 1995
;
Hall et al., 2001
). Evidence
for a direct requirement of UNC-33/CRMP in growth cone repulsion comes from
the finding that the Fes/Fps tyrosine kinases upon binding of Sema3A to PlxA1,
are recruited to phosphorylate the cytoplasmic portion of PlexA1 and an
associated complex of the proteins CRAM and CRMP-2 (the latter is a splice
variant of UNC-33/CRMP) (Mitsui et al.,
2002
). Sema3A-induced growth cone collapse of dorsal root ganglion
neurons is suppressed in Fes kinase negative mutants
(Mitsui et al., 2002
), which
shows the requirement of the kinase for collapse and indicates a possible
involvement of Fes/Fps-mediated phosphorylation of CRMP in the plexin
collapsing pathway.
A non-exclusive possibility is that UNC-33/CRMP, by promoting tubulin
hetero-dimer assembly (Fukata et al.,
2002), might disturb the microtubule network and in this way cause
a localized collapse. Recent evidence suggests that growth cone attraction and
repulsion can be completely blocked by specifically inhibiting the dynamics of
microtubule ends in the growth cone (Buck
and Zheng, 2002
).
Based on biochemical interactions described previously and our genetic data
we propose a model (Fig. 6) in
which Semaphorin 1 proteins emitted by the hook primordium bind to PLX-1 on
the ray 1 cell(s) causing the conserved intracellular domains of PLX-1 to bind
cycling RACGTP thereby mediating an attractive response to a
Semaphorin 1 ligand. It remains unclear whether C. elegans PLX-1 can
bind RHO because of sequence divergence in this region between the
Drosophila PlxB and C. elegans PLX-1. However RHO and
RHO-kinase signaling is required in parallel to or in series with MIG-2 and
CED-10 for attraction. This could override a default ray 1 anterior
positioning function, part of which requires CRMP/UNC-33. By inference, at
relatively low levels of rac(+), the ray 1 repulsion mediated by
Semaphorin 1-induced activation of PLX-1 may be dependent on the recruitment
of Fes/Fps tyrosine kinases and phosphorylation of the CRAM
(Mitsui et al., 2002) and
UNC-33/CRMP protein complex, causing cytoskeletal changes leading to ray cell
repulsion (e.g. away from the hook). Another possibility is that RHO-kinase
may normally be antagonized by UNC-33, reducing attraction of ray 1 to the
hook and allowing an unidentified default repulsion mechanism to increase its
activity. These two possibilities are not mutually exclusive, nor do they
exclude other possible molecular mechanisms for UNC-33/CRMP activity, some of
which were discussed above.
Implications of model
The ability of C. elegans PLX-1 to serve as a sensor of levels of
RHO-family GTPases, and to switch its activity accordingly, indicates that
PLX-1 stimulation by Semaphorin 1 proteins is instructive for cell
positioning. This together with the incomplete penetrance of a null mutation
in plx-1 on ray 1 cell migration suggests that there may exist other
membrane-associated receptors with similar functions in regulating ray 1 or
other cell migrations. These receptors, like PLX-1, might sense relative
levels of active RAC and RHO in the cell, perhaps in the same way (e.g. by
direct or indirect binding of these GTPases), and thereby regulate the actin
cytoskeleton associated with the cell membrane at the leading edge of
migration, causing attraction (cytoskeletal growth) toward the ligand(s) of
the receptor when functional cellular RAC levels are high, but causing
repulsion (cytoskeletal collapse) when RAC levels are low.
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ACKNOWLEDGMENTS |
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