Department of Biology, Queen's University, Kingston, Ontario K7L 3N6, Canada
* Author for correspondence (e-mail: chinsang{at}biology.queensu.ca)
Accepted 20 June 2005
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SUMMARY |
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Key words: Eph Receptor Tyrosine Kinase, Robo receptor, Morphogenesis, Cell movements, C. elegans, Synthetic lethal
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Introduction |
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Null mutations in either the vab-1 Eph RTK or efn-1
ephrin result in defective cell movements, and, as a result, the embryos
usually die or have severe morphogenesis defects
(Chin-Sang et al., 1999;
George et al., 1998
). However,
the fact that the penetrance of the lethality is not complete suggests that
there is genetic redundancy in C. elegans Eph RTK signaling, that is,
other signaling pathways may work together or in parallel with the VAB-1 Eph
RTK. Indeed a divergent ephrin, EFN-4, and a leukocyte common antigen-related
(LAR) receptor protein tyrosine phosphatase (RPTP), PTP-3, work redundantly,
or in parallel, with the VAB-1 Eph RTK
(Chin-Sang et al., 2002
;
Harrington et al., 2002
).
We report that the VAB-1 Eph RTK shows a dosage-dependent genetic
interaction with the conserved axon guidance receptor, SAX-3/Robo. The
Roundabout (Robo) family of receptors are evolutionary conserved and have been
implicated in mediating axon guidance events, specifically axon repulsion upon
binding its ligand Slit (Brose et al.,
1999; Challa et al.,
2001
; Kidd et al.,
1998
; Lee et al.,
2001
; Seeger et al.,
1993
; Zallen et al.,
1998
) (for a review, see Wong
et al., 2002
). Like the vertebrate and Drosophila
counterparts, the C. elegans Robo homolog SAX-3 is required for
multiple axon guidance events, including midline crossing, ventral guidance
and nerve ring positioning (Zallen et al.,
1999
; Zallen et al.,
1998
). However, the role of SAX-3 in early embryonic cell
movements has not been described. Four-dimensional video microscopy revealed
that sax-3 embryos display defects in neuroblast gastrulation cleft
closure, as well as ventral movements of the epidermal cells, during ventral
closure, similar to those observed in the vab-1 (Eph RTK) and
efn-1 (Ephrin) mutants. sax-3 mutants also display epidermal
phenotypes that are not observed in vab-1 mutants, and we provide
evidence for a role of SAX-3 in the morphogenesis of epidermal cells
independently of VAB-1. We report that double-mutant combinations in
sax-3 and vab-1 display a completely penetrant embryonic
lethal phenotype. Furthermore, double-mutant combinations between
vab-1 and slt-1, the ligand for SAX-3, show significantly
enhanced cell migration defects in comparison with vab-1 single
mutants, revealing a role for SLT-1 in embryogenesis. We also show that the
VAB-1 tyrosine kinase binds to the intracellular portion of SAX-3,
specifically at the juxtamembrane and CC1 (conserved cytoplasmic region 1)
region. The SAX-3 receptor and VAB-1 Eph RTK are co-expressed on a subset of
neuroblasts, which is consistent with these two receptors forming a complex.
Our results suggest that SAX-3/Robo and VAB-1 can act together to regulate
early neuroblast movements and axon guidance.
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Materials and methods |
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Chromosome II: vab-1 (e2, e699, dx31)
(George et al., 1998);
ptp-3 (op147) (Harrington et al.,
2002
); mec-7::gfp (muIs32) a GFP marker for
mechanosensory neurons (gift from Dr C. Kenyon's lab).
Chromosome IV: efn-4(bx80)
(Chin-Sang et al., 2002);
ajm-1::gfp (jcIs1) (Mohler et
al., 1998
).
Chromosome X: sax-3(ky123), the ky123 allele deletes the
signal sequence and the first exon and is likely to be a null allele
(Zallen et al., 1998);
slt-1(eh15), the eh15 allele is a duplication of the
slt-1 locus with an out-of-frame deletion in both copies
(Hao et al., 2001
).
Rearrangement: mIn1[mIs14 dpy-10 (e128)] II
(Edgley and Riddle, 2001);
mIn1 mIs14 (a.k.a. mIn1GFP throughout this paper) is a
dominant green fluorescent protein (GFP) balancer for chromosome II, including
the region of vab-1 and ptp-3.
Mutations not referenced here can be found elsewhere
(Riddle et al., 1997). All
C. elegans strains were obtained from the C. elegans
Genetics Stock Center, care of T. Stiernagle (University of Minnesota).
Double-mutant constructs
vab-1(weak); sax-3 double mutants were completely inviable and
were maintained as balanced strains of the genotype vab-1(e2 or e699)/mIn
GFP; sax-3(ky123). These strains segregate only viable GFP animals, as
non-GFP animals (vab-1; sax-3) are dead. Balanced strains with the
vab-1(dx31) null could not be maintained because of the
vab-1 dosage effect (see below). efn-4; sax-3 double
homozygotes were maintained as homozygous lines balanced by an
extrachromosomal array (juEx350) carrying wild-type copies of
vab-1 and efn-4
(Chin-Sang et al., 2002).
ptp-3; sax-3 double mutants were analyzed from balanced strains of
genotype ptp-3/mIn1GFP; sax-3. The penetrance of lethality and
morphogenetic defects were quantified as described previously
(George et al., 1998
).
Dosages studies between the vab-1 and sax-3 genes
vab-1 males were crossed to mIn1GFP/+;
sax-3(ky123) animals to obtain vab-1/mIn1GFP; +/sax-3 GFP
cross progeny. GFP-positive sax-3 animals were picked based on the
Sax-3 notched-head phenotype (vab-1/mIn1GFP; sax-3). We were able to
isolate vab-1(dx31)/mIn1GFP; sax-3(ky123) from vab-1/mIn1GFP(+);
+/sax-3 mothers because sax-3 animals exhibited maternal rescue.
In the subsequent generations, only mIn1GFP; sax-3 animals were
observed, which is consistent with sax-3 animals requiring two copies
of the vab-1 gene to survive. Of 35 putative vab-1/mIn GFP;
sax-3(ky123) GFP notched-head animals, four were of the genotype
vab-1/mIn1GFP;+/sax-3(ky123) double heterozygous and therefore
displayed non-allelic non-complementation for the notched-head phenotype. Note
that vab-1 does not show dominant phenotypes and is completely
recessive (i.e. +/vab-1(dx31), 100% of embryos viable,
n>500). To confirm that even in the presence of one copy of the
wild-type vab-1 gene the sax-3 animals are inviable, we
crossed vab-1(dx31)/mIn1GFP males to sax-3(ky123) animals
and scored for the male cross progeny. Of >100 male cross progeny scored,
only GFP animals (+/mIn1GFP; 0/sax-3) were observed, suggesting that
the non-GFP vab-1(dx31)/+; 0/sax-3 males were dead. Other crosses
using GFP-marked vab-1 males confirmed this result (see below).
Double heterozygous +/vab-1; +/sax-3 combinations
Double heterozygous +/vab-1; +/sax-3 animals were constructed by
crossing GFP-marked vab-1 chromosome (vab-1 (dx31) GFP (mec-7:gfp
muIs32)) males to sax-3 females (animals feminized by feeding
RNAi with fem-1) and +/vab-1(dx31) GFP; + or 0 /sax-3(ky123)
cross progeny was identified as GFP-positive animals. All cross progeny (GFP
positive) that survived developed as hermaphrodites, suggesting that the males
were dead. 57% of the GFP embryos were dead (assume that 50% should be XO male
embryos), suggesting that about 7% of the XX double heterozygous animals were
dead (t-test compared with control cross, P<0.001). A
control cross of mec-7::GFP males crossed to sax-3 females
produced male and hermaphrodite survivors, and 27% of the GFP cross-progeny
embryos were dead. To score the doubly heterozygous +/vab-1; +/sax-3
for amphid neuron defects, we crossed vab-1(dx31) males to
mIn1GFP; sax-3(ky123) hermaphrodites to isolate vab-1/mIn1GFP;
+/sax-3. These animals were stained with the fluorescent dye DiI
(Molecular Probes/Invitrogen) and scored for axon guidance defects (lack of
ventral guidance or anterior positioning, 25%, n=87), as described
previously (Zallen et al.,
1999).
Yeast two-hybrid assays
Yeast were grown on standard complete and selective media, as appropriate
(Sherman, 1991). Yeast
transformation was performed using a lithium acetate method
(Schiestl and Gietz, 1989
).
For deletion analysis, pGBKT7 and pGADT7 (Clontech) were used as bait and prey
cloning vectors, respectively, and ß-galactosidase activity was measured
qualitatively by X-GAL overlay assays
(Serebriiskii and Golemis,
2000
), or was quantified by liquid ß-galactosidase
(Ausubel et al., 1989
). At
least three independent liquid ß-galactosidase experiments were
performed, and the mean and standard error of the mean (s.e.m.) were
calculated. The VAB-1 kinase domain (669aa-985aa) was cloned into the pGBKT7
GAL-4 DNA-binding domain vector. SAX-3 deletion constructs were made in pGADT7
by cloning cDNA (PCR derived) encoding the various SAX-3 regions, and their
sequence was verified. Amino acid sequences correspond to the SAX-3B isoform
(Wormbase Release WS130). Primer sequences and details of plasmid constructs
are available upon request.
GST pull-down assays
We used a GST `pull-down' assay to confirm the SAX-3/VAB-1 interaction. A
cDNA encoding the vab-1 intracellular region (581aa-1117aa) was
cloned in frame to Glutathione-S-Transferase (pGEX4T-2, Amersham), expressed
in E. coli (BL21 Tuner) and purified on glutathione agarose beads
(GST-Bind, Novagen), according to the manufacturer's protocol. A cDNA encoding
the SAX-3 juxtamembrane to CC1 region (900aa-1030aa) was fused in-frame to
Maltose-Binding Protein (pMALp2x, New England Biolabs) and expressed in E.
coli. Soluble extract containing MBP-SAX-3 (Load) was incubated overnight
at 4°C with either GST (3 mg/ml) or GST-VAB-1 (0.25 mg/ml) bound to 100
µl beads. Unbound fractions were collected, protein bound to beads was
washed three times [25 mM HEPES (pH 7.5) 250 mM NaCl, 5% glycerol, 0.05%
Triton-X-100] and a proportional loading of each sample was analyzed by
SDS-PAGE followed by western blotting. MBP-SAX-3 was detected by using
anti-MBP (New England Biolabs) and secondary HRP-anti-rabbit antibodies
(Upstate) followed by ECL (Pharmacia Biotech).
Immunohistochemistry
Fixation and staining of embryos was performed as described previously
(Chin-Sang et al., 1999;
Finney and Ruvkun, 1990
).
Chicken polyclonal antibodies against GFP (Chemicon) were used at 1:200
dilutions. Rabbit anti-VAB-1 antibodies (antigen: VAB-1-6XHIS intracellular
581aa-1117aa) were used at 1:100 dilutions. MH27 monoclonal antibody
(Francis and Waterston, 1991
)
(anti-AJM-1) was used at a concentration of 1:500. Rhodamine-conjugated goat
anti-mouse (Chemicon), FITC-conjugated goat anti-chicken, and Texas
Red-conjugated goat anti-rabbit secondary antibodies (Jackson ImmunoResearch
Laboratories) were used at 1:500 dilutions.
Transgenic rescue and reporter constructs
To create a rescuing SAX-3::GFP reporter (quEx89), we used a PCR
fusion-based approach (Hobert,
2002) to generate a PCR (Roche Expand Long PCR) product consisting
of 1.2 kb of the sax-3 promoter, the sax-3 genomic region
(exons 1-5) followed by the rest of the sax-3 cDNA fused in frame to
GFP and unc-54 3'UTR sequences derived from pPD95.75 (Dr A.
Fire's laboratory). A final 10.4 kb PCR product (10 ng/µl) was co-injected
with pRF4 (30 ng/µl) marker and transgenics were obtained as described
(Mello et al., 1991
). The
SAX-3 minigene (quEx99) consisted of 1.5 kb of the sax-3
promoter fused to the whole 3.8 kb sax-3 cDNA and followed by 0.7 kb
of the sax-3 3'UTR. The final mini-gene PCR product was
co-injected with odr-1::RFP transformation marker (gift from Dr C.
Bargmann's Laboratory). For ajm-1::SAX-3 (quEx100) and
F25B3.3::SAX-3 (quEx102), we used the same strategy as for
the mini-gene, where sax-3 promoter sequence was replaced with 1.6 kb
sequence of ajm-1 promoter and 1.5 kb sequence of F25B3.3
promoter, respectively. The vab-1 rescuing mini-gene (pCZ47) was as
described previously (George et al.,
1998
).
4D video microscopy
4D video microscopy was carried out essentially as described previously
(Chin-Sang et al., 2002).
Recordings were made at room temperature (22°C) using a Zeiss Axioplan 2
microscope with a 63x Plan-neofluar objective lens, Axiocam and
Axiovison software. Fifteen to twenty (0.5 to 1 µm) z sections
were taken at 1-2 minute intervals throughout development. We recorded 20
sax-3(ky123) embryos of which 10 did not complete embryogenesis. For
time-lapsed imaging of GFP-expressing embryos, we replaced the standard UV
(100 W Mercury short arc burner) lamp with a 250 W Halogen fiber-optic cold
light source (Zeiss KL2500 LCD). Images were captured every 5-8 minutes.
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Results |
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In the course of making double-mutant combinations with vab-1 and sax-3/Robo, we identified a gene-dosage dependence of vab-1 in that +/vab-1(dx31); sax-3 heterozygotes were completely inviable (see Materials and methods). We also confirmed the gene dosage requirement for vab-1 by crossing vab-1(dx31) males to sax-3 animals. Because sax-3 is on the X chromosome, half of the cross progeny should be +/vab-1(dx31); 0/sax-3 (males) and the other half +/vab-1(dx31); +/sax-3 (hermaphrodites). From several independent crosses, all cross progeny developed as hermaphrodites, suggesting that heterozygous +/vab-1; 0/sax-3 males die as embryos (Fig. 1). Therefore, removing just one copy of the vab-1 gene reveals an essential role for sax-3 during embryogenesis. The double heterozygous +/vab-1; + or 0/sax-3 embryos (hermaphrodites and males) from vab-1 crossed to sax-3 displayed greater than 50% lethality. Furthermore, +/vab-1(dx31); +/sax-3(ky123) showed weak penetrance notched-head phenotypes, demonstrating non-allelic non-complementation (see Materials and methods).
|
Because it is possible that VAB-1 and SAX-3 may function together during axon guidance, we asked whether these two genes display a gene-dosage interaction, as seen in the embryonic lethality phenotype. We show that the double-heterozygous +/vab-1; +/sax-3 animals display defects in axon guidance in head neurons. Twenty-five percent of the double heterozygous animals displayed an anterior positioning and/or defects in ventral guidance of the amphid neurons similar to the phenotype of sax-3 mutant animals (Fig. 3). Thus, vab-1 and sax-3 show non-allelic non-complementation in axon guidance.
|
In 30% of the sax-3 embryos examined, the gastrulation cleft remained open at the time of epidermal ventral enclosure and the epidermal cells failed to contact each other at the ventral midline. As a result, the embryos ruptured at the ventral side (Fig. 2C, Class I, strongest phenotype). In 20% of the embryos there were no apparent defects in gastrulation cleft closure, and although the epidermal cells met at the ventral side, they failed to form stable contacts (Class II). These animals also ruptured at the ventral midline. Another striking phenotype displayed by vab-1 and efn-1 mutants was an epidermal notched head, which was most likely caused by improper epidermal ventral enclosure in the anterior region. sax-3(ky123) mutant animals also show a `notched' head phenotype (17% strong notches), similar to that seen in vab-1 and efn-1 mutants, which is consistent with sax-3 exhibiting similar embryonic defects as Eph RTK signaling defective animals (Fig. 5A).
|
sax-3/Robo exhibits epidermal defects not observed in vab-1 mutants
Previously (Chin-Sang et al.,
1999; George et al.,
1998
) suggested that the epidermal cell migration defects seen in
ephrin signaling mutants arise from defects in the organization of the
neuronal substrate that they migrate over, and not directly from a lack of
ephrin signaling in epidermal cells. Because the DIC time-lapse showed that
sax-3 mutants have similar defects in epidermal cell migration during
ventral enclosure, we questioned whether there is the same deficient mechanism
that is proposed in ephrin signaling. We analyzed the movement of epithelial
cells in greater detail by 4D time-lapse microscopy of an AJM-1::GFP reporter
construct and MH27 (anti-AJM-1) antibodies, which localize to adherens
junctions of epidermal cells. Wild-type embryos carrying AJM-1::GFP
(jcIs1) illustrate two epidermal morphogenetic steps that occur at
approximately the same time on opposite sides of the embryo: (1) ventral
migration of the contralateral pairs of epidermal cells to the ventral midline
where they contact one another to wrap the embryo in the epidermal monolayer
(Fig. 4A); and (2)
intercalation of the two dorsal-most rows of epidermal cells that later fuse
to each other and form the dorsal syncytium
(Fig. 4C)
(Podbilewicz and White, 1994
;
Williams-Masson et al., 1998
).
In sax-3 embryos, AJM-1::GFP was expressed and localized to the
junctions of the epidermal cells, as in wild-type embryos. We analysed 45
embryos and we observed three kinds of epidermal defects in
sax-3(ky123) embryos. The most dramatic defect was the disruption of
the migration of the ventral pocket cells due to the lack of underlying
substrate cells, caused by the failure of gastrulation cleft closure (13
embryos). In nine of those 13 embryos, the leading cells met at the ventral
midline, and, in the other four embryos, the leading ventral epidermal cells
failed to migrate or migrated abnormally. These 13 embryos ruptured at the
ventral midline and could be classified as Class I, as described for DIC
time-lapse above (Fig. 4D). The
second defect observed was a change in the shape and position of the dorsal
epidermal cells that led to cell intercalation and fusion defects (four
embryos; Fig. 4F). In addition,
three of these embryos also failed to undergo ventral enclosure. The last
defects observed were abnormalities in the lateral epidermis (12 embryos). In
C. elegans embryos, 10 epidermal cells (H0, H1, H2, V1-V6 and T) form
a lateral row of seam cells on each side of the embryo
(Fig. 4B). In sax-3
mutants, one or more seam cells were displaced. For example, V1, V3 and V5
were often shifted ventrally or dorsally, resulting in inappropriate cell
contacts between adjacent cells (Fig.
4E). The remaining 16 embryos enclosed and elongated without
epidermal defects. In summary, the sax-3 gene is necessary for at
least three different processes during epithelial morphogenesis of the embryo:
for ventral epidermal migration, dorsal epidermal cell migration and fusion,
and for the alignment of the lateral seam cells. In addition, sax-3
is required for the neuroblast movements during gastrulation cleft
closure.
|
SAX-3 functions in both the epidermis and nervous system
Because the dorsal epidermal defects observed in sax-3 mutant
embryos cannot be easily reconciled by neuroblast signaling (as is proposed
for the ventral epidermal defects observed in vab-1 and
efn-1), we questioned whether SAX-3 could function cell autonomously
in epidermal cells. We expressed SAX-3 specifically in either epidermal or
neuronal cells using the ajm-1 and F25B3.3 promoters,
respectively. SAX-3 tissue-specific expression was scored for its ability to
rescue the embryonic lethality of sax-3 mutants. The ajm-1
gene is activated only after the epidermal cells are specified and therefore
the ajm-1 promoter is suitable to quantify the activity of SAX-3 in
epidermal cells (Mohler et al.,
1998). F25B3.3 is a RAS1 guanine nucleotide-exchange
factor that is expressed early in neuroblasts and throughout the adult nervous
system (Altun-Gultekin et al.,
2001
). Both ajm-1::SAX-3 and F25B3.3::SAX-3
constructs were able to partially rescue the lethality in
sax-3(ky123) mutant embryos (Fig.
5A), reducing the embryonic lethality from 48% to 23% for
ajm-1::SAX-3; sax-3 transgenic animals, and to 17% lethality
for F25B3.3::SAX-3; sax-3 transgenic animals, suggesting
that the sax-3 gene functions both in epidermal and neuroblast cells.
Surprisingly, the epidermal `notched-head' phenotype was rescued only by the
neuronal F25B3.3::SAX-3 construct and not the epidermal
ajm-1::SAX-3 construct (Fig.
5A).
SAX-3/Robo is expressed in the same neuroblasts as the VAB-1 Eph RTK
SAX-3 expression has been reported in the nervous system, particularly
during the stage of axonal outgrowth in the embryo
(Zallen et al., 1998).
However, we re-examined the SAX-3 expression pattern (specifically in the
embryo), and we were able to extend the SAX-3 expression pattern by creating
transgenic animals that carried the entire SAX-3 receptor fused to GFP
expressed from its own promoter (see Material and methods). The SAX-3::GFP
construct was able to partially rescue the sax-3 embryonic lethality
and the notched-head phenotype, suggesting that SAX-3::GFP has functional
activity (Fig. 5A). SAX-3::GFP
is expressed in early neuroblasts during gastrulation cleft closure and in the
underlying neurons during ventral enclosure, similar to VAB-1 and EFN-1.
However, unlike VAB-1 or EFN-1, SAX-3::GFP was also observed in epidermal
cells (Fig. 5B)
(Chin-Sang et al., 1999
;
George et al., 1998
). To
examine SAX-3 and VAB-1 co-localization, we used anti-VAB-1 antibodies and
anti-GFP antibodies on transgenic embryos expressing VAB-1 (pCZ47)
(George et al., 1998
) and the
SAX-3::GFP construct. VAB-1 and SAX-3 co-localized on neuroblasts, consistent
with SAX-3 and VAB-1 functioning together during this stage of development.
However, the co-localization is not exact and VAB-1 and SAX-3 were also
expressed in separate cells. In general, we found that co-localization of
VAB-1 and SAX-3 occurred more frequently in the early neuroblasts, but in
later embryos (post-ventral enclosure), we found that SAX-3 and VAB-1 were
expressed in separate groups of neighboring cells, reminiscent of the
reciprocal expression patterns of ephrin ligands and their receptors
(Fig. 5C).
The juxtamembrane and CC1 region of SAX-3/Robo binds the VAB-1 tyrosine kinase
Non-allelic non-complementation or gene-dosage sensitivity between two
genes can be interpreted genetically as two proteins acting in a complex.
Because VAB-1 exhibits gene-dose sensitivity with SAX-3 in embryogenesis and
in axon guidance, we tested whether these two receptors can physically
interact. We used the VAB-1 tyrosine kinase region fused to the GAL-4-binding
domain, and the full-length intracellular region of SAX-3 fused to the
GAL-4-activation domain, to show that these two proteins can interact in a
yeast two-hybrid assay (Fig.
6A,B).
|
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Discussion |
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SAX-3 has roles in embryonic cell movements
We report that SAX-3 is necessary for cell migration in two phases of
embryonic development, closure of the gastrulation cleft by short-range
movement of neuroblasts, and epidermal cell movement during ventral enclosure;
defects in cell migration during these two phases are also observed in
ephrin-signaling mutants.
|
The lethality in both sax-3 and vab-1 embryos was caused by a defect in cell movements and not as a result of defective cell differentiation. For example, the structure of the pharynx and intestine were formed correctly in Class I terminal-stage vab-1 and sax-3 embryos, and the GABAergic motoneurons and mechanosensory neurons were specified and expressed using the unc-25::GFP and mec-4::GFP markers, respectively (data not shown). The body muscles were also differentiated judging by the presence of muscle twitching in terminal stage mutants.
Significance of the interaction between VAB-1 Eph Tyrosine Kinase and the juxtamembrane CC1 region of SAX-3/Robo
The Robo receptor family is characterized by an extracellular domain
consisting of two to five immunoglobulin domains and two to three fibronectin
type III repeats, and a cytoplasmic domain containing various combinations of
four short Conserved Cytoplasmic motifs called CC0, CC1, CC2 and
CC3 (Bashaw et al., 2000;
Kidd et al., 1998
). These
motifs are thought to serve as binding sites for various intracellular
signaling molecules, including Enabled (Ena) and the Ableson (Abl) tyrosine
kinase (Bashaw et al., 2000
).
We have shown an interaction between the VAB-1 tyrosine kinase domain and the
juxtamembrane and CC1 region of SAX-3/Robo. SAX-3 does not have a CC0
consensus, which is located in the juxtamembrane region of Robo receptors;
however, there are seven tyrosine residues in the SAX-3 juxtamembrane region
that VAB-1 could potentially phosphorylate. Interestingly, the human and mouse
Robo3/RIG-1 lacks the CC1 domain and differs from the other Robo homologs
because it is required for midline crossing, which is opposite to the role of
Robo receptors. It appears to accomplish this by repressing Robo1/2 signaling
(Sabatier et al., 2004
). Thus,
the CC1 region may be important in regulating a repulsive signaling downstream
of Robo. It will be of interest to determine whether VAB-1 phosphorylates
SAX-3 to alter its function, or whether the SAX-3/VAB-1 interaction affects
the UNC-40/SAX-3 interaction or the ability of Abl to regulate SAX-3/Robo
function.
SAX-3/Robo functions independently of VAB-1 and has the potential to form neuronal receptor complexes with the VAB-1 Eph RTK
We propose a model in which SAX-3 has two cooperative roles. The first is
to function together with VAB-1 in AB neuroectoblast movement during late
gastrulation and ventral enclosure to provide a substrate or a signal for
proper epidermal cell movements specifically on the ventral side of the
embryo. A second role for SAX-3 may be to function independently of VAB-1 in
the epidermis for epidermal movements. For example, on the dorsal side,
because sax-3 animals display dorsal epidermal defects that are not
shared by the vab-1 null (Fig.
7). Similarly, sax-3 animals display axon guidance
defects not seen in vab-1 mutants, suggesting that SAX-3 has
VAB-1-independent roles during axon development
(Zallen et al., 1999). This is
also consistent with their expression patterns, where SAX-3 and VAB-1 are
co-expressed early in development but later are expressed in separate cells.
George et al. showed by genetic mosaic analysis that loss of vab-1 in
AB lineages caused strong morphogenetic defects, including the `notched-head'
epidermal defect (George et al.,
1998
). Similarly, the sax-3 `notched-head' defects could
arise as a result of a requirement for SAX-3 in the AB lineages, as
F25B3.3::SAX-3 expression in the AB neuroectoblast specifically
rescues this defect; by contrast, the ajm-1::SAX-3 expression in
epidermal cells did not rescue the epidermal notched-head defect. Furthermore,
the double vab-1; sax-3 mutants do not exhibit any new phenotype over
those observed in single mutants, suggesting that SAX-3 and VAB-1 have common
and partially redundant functions during morphogenetic movements.
|
The vertebrate Robo receptors and Eph RTKs have been shown to mediate
axonal repulsion, and in some cases to work in the same neurons. It has even
been suggested that the downstream signaling between these two neuronal
receptors converge on phosphatidylinositol-3-kinase (PI3K) signaling
(Wong et al., 2004). The
vertebrate Eph RTKs have also been shown to function during angiogenesis, and,
curiously, Magic Roundabout (Robo4) is expressed specifically in the
vasculature and is essential for angiogenesis
(Bedell et al., 2005
;
Suchting et al., 2005
). It
will be interesting to know whether in other organisms the Eph RTKs and Robo
receptors work together in a similar fashion to that which we propose for the
early morphogenetic movements in C. elegans.
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ACKNOWLEDGMENTS |
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REFERENCES |
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