Department of Molecular, Cell, and Developmental Biology, Sinsheimer
Laboratories, University of California, Santa Cruz, CA 95064, USA
* Present address: Department of Biology, Queen's University, Kingston, Ontario
K7L 3N6, Canada
Author for correspondence (e-mail:
chisholm{at}biology.ucsc.edu)
Accepted 4 September 2002
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SUMMARY |
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Key words: Morphogenesis, C. elegans, Ephrin, Semaphorin, EFN-4, VAB-1
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INTRODUCTION |
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We have previously shown that the C. elegans Eph receptor VAB-1
and its ephrin ligand EFN-1 (previously called VAB-2) function in epidermal
enclosure (Chin-Sang et al.,
1999; George et al.,
1998
). During epidermal enclosure, VAB-1 and EFN-1 are
predominantly expressed and required in underlying neurons but not in the
epidermal cells. Time-lapse analysis of vab-1 and efn-1
mutant embryos revealed specific defects in the organization of neural
precursors, manifested as a failure to close the `gastrulation cleft', a
depression in the ventral surface of the embryo left after ingression of
endodermal and mesodermal precursors. Because epidermal cells migrate over the
descendants of these ventral neural precursors, a simple model for the
non-autonomous effects of vab-1 or ephrin mutants on epidermal
enclosure is that they arise as a consequence of an altered neural substrate.
The defects in neural cell organization in C. elegans Eph signaling
mutants may resemble cell sorting defects observed in vertebrates when Eph
signaling is disrupted (Mellitzer et al.,
1999
; Xu et al.,
2000
).
The relative simplicity of the C. elegans Eph signaling network
allows a systematic analysis of its roles in a single morphogenetic process. A
single Eph receptor (VAB-1) and four ephrins (EFN-1 through EFN-4) have been
identified in C. elegans (Wang et
al., 1999). efn-1 mutations, obtained from forward
genetic screens, cause phenotypes weaker than but otherwise indistinguishable
from those of vab-1 mutants; expression, binding and genetic data
indicate that EFN-1 functions as a ligand for VAB-1 in embryonic morphogenesis
(Chin-Sang et al., 1999
).
efn-2 and efn-3 mutants, obtained by reverse genetics,
appear essentially normal during embryogenesis; EFN-2 is expressed
embryonically in a pattern similar to EFN-1. The efn-1 null phenotype
is much weaker than that of vab-1, and an efn-1 efn-2 efn-3
triple mutant resembles a vab-1 knockout, suggesting that
efn-2 and efn-3 play subtle roles in embryogenesis that are
partly redundant with efn-1 (Wang
et al., 1999
) (S. L. M. and A. D. C., unpublished) The similarity
of the efn-1 efn-2 efn-3 triple mutant phenotype to the
vab-1 null phenotype suggests that these three ephrins might account
for all signaling functions of the VAB-1 Eph receptor. VAB-1/EFN-1 signaling
also appears to function partly redundantly with a pathway involving the
LAR-like receptor tyrosine phosphatase PTP-3
(Harrington et al., 2002
).
We report our analysis of a fourth worm ephrin, EFN-4, focusing on its
functions in embryonic morphogenesis. We show that EFN-4 is also required for
movement of embryonic neural precursors and enclosure of the epidermis, but
that its role is distinct from those of VAB-1 or EFN-1. Unexpectedly,
efn-4 mutations show strong synergistic interactions with mutations
in vab-1 and efn-1, suggesting that EFN-4 may function in a
VAB-1-independent pathway. efn-4 mutations also synergize with
ptp-3 mutations, suggesting that EFN-4 does not function solely in
the PTP-3 pathway. Through a comprehensive analysis of vab-1 efn
double mutants we find that only efn-4 mutations show strong
synergism with vab-1 receptor mutants. It had been suggested that the
Semaphorin-2A homolog MAB-20 might function in the EFN-4 pathway
(Roy et al., 2000). We find
that efn-4 and mab-20 mutations show either epistasis or
weak mutual suppression, suggesting that these genes could act in common or
opposing pathways in embryonic morphogenesis.
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MATERIALS AND METHODS |
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Rearrangements used were: mIn1 mIs14
(Edgley and Riddle, 2001); and
mDp1(IV; f).
All efn-4 mutations were EMS-induced, with the exception of bx80 (diepoxybutane). S. Brenner isolated the efn-4 mutations e36 and e660; J. Hodgkin isolated e1746; X. Huang isolated ju134. All efn-4 alleles failed to complement bx80. efn-4(ju314), isolated independently from e1746, was found to cause similar phenotypes and to result from the same molecular lesion.
e819 is an X-ray-induced mutation isolated by S. Brenner and
assigned to mab-20 by mapping and complementation tests (data not
shown). mab-20(e819) causes embryonic and larval lethal phenotypes
similar in penetrance to those of mab-20(ev574), suggesting that it
causes a strong loss of function. The weak vab-1 allele
e1029 has been found to result from a mis-sense mutation (C212Y) in
the extracellular domain of VAB-1. Penetrance of lethality and morphogenetic
defects was quantitated as described previously
(George et al., 1998); all
estimates of penetrance were obtained from counts of between 3 and 6 complete
broods.
Double mutant construction and analysis
vab-1(0); efn-4 double mutants were completely inviable and were
maintained as balanced strains. We initially used dpy-9 as a balancer
for efn-4; however, such strains were unstable, owing to
recombination between dpy-9 and efn-4. We subsequently
constructed more stable balanced strains of genotype vab-1/mIn1 mIs14;
efn-4. vab-1 efn-4 double homozygotes were identified as
non-GFP-expressing progeny from such heterozygotes. Some vab-1 efn-4
double mutants constructed using weak vab-1 alleles were subviable;
rare viable and fertile non-GFP-expressing progeny were identified as
segregants from mIn1 mIs14-balanced strains. Such homozygous
vab-1 efn-4 double mutants could be propagated very slowly, and
displayed >99% lethality (see Fig.
5). Some vab-1; efn-4 double mutants were maintained as
homozygous lines balanced by an extrachromosomal array (juEx350)
carrying the wild-type copies of vab-1 and efn-4. juEx350
was formed by co-injection of the vab-1(+) cosmid M03A1 (1
ng/µl), the efn-4(+) cosmid F56A11 (1 ng/µl), the
plasmid pRF4 (30 ng/µl), the SUR-5-GFP marker pTG96 (30 ng/µl) and
pBluescript (40 ng/µl). ptp-3 efn-4 double mutants and vab-1
ptp-3 efn-4 triple mutants were analyzed from balanced strains of
genotype ptp-3 (or vab-1 ptp-3)/mIn1 mIs14;
efn-4.
|
mab-20 efn-4 double mutants were constructed using dpy-9 as a marker in trans to efn-4; all double mutants were confirmed by multiple complementation tests. We used one-way ANOVA (Statview) to compare differences in penetrance of phenotypes.
RNA interference of vab-1
For dsRNA-mediated interference of vab-1 a 1.9 kb
SpeI-PstI fragment of the vab-1 cDNA was subcloned
into the RNAi vector L4440. RNAs were synthesized and annealed as described
(Harrington et al., 2002), and
injected at a concentration of
70 ng/µl. Injected hermaphrodites were
allowed to recover for 12 hours; eggs laid over the ensuing 24 hours were
analyzed. RNAi of vab-1 resulted in phenotypes approximately half as
penetrant as the null mutant (data not shown).
Molecular analysis of efn-4
efn-4 corresponds to the predicted gene F56A11.3. cDNAs from this
gene had been isolated in a genome-wide cDNA sequencing project (Maeda et al.,
2001). The longest cDNA, yk449e2, was sequenced completely; this cDNA lacks
the first 11 bases of the predicted coding sequence. The predicted EFN-4
preprotein consists of 348 amino acid residues; an N-terminal signal sequence
is predicted to be cleaved after residue A20, and a C-terminal GPI addition
signal sequence is predicted to be cleaved near S328. The lesions of the
alleles were determined by sequencing genomic DNA from the mutants, as
described (George et al.,
1998). Sequences of primers are available on request. Using
Southern blotting of genomic DNAs and sequencing, we determined that
bx80 is a deletion of 1838 bp, encompassing exon 2 and parts of
introns 1 and 2. The breakpoints of bx80 are: 5'
gttgttaacaacaaaa[bx80]aaacctaaatttt 3'.
Transgenic rescue and reporter constructs
To generate a genomic clone containing efn-4, we subcloned a 16.1
HindIII-EagI genomic DNA fragment from cosmid F56A11 into
pSL1190, creating pCZ148. This clone contains 5.3 kb genomic sequence 5'
to the EFN-4 ATG. Transgenic lines (juEx150, juEx152, juEx153) were
made by co-injection of pCZ148 and the marker plasmid pRF4 (both at 50
ng/µl) in an efn-4(bx80) background and the penetrance of the
posterior morphology (Vab) phenotypes quantitated in transgenic (Roller)
animals. In a typical transgenic line the penetrance of the Vab phenotype was
0.7% (2/300 Rollers were Vab), compared with 27% in control lines bearing pRF4
alone (n=175). To overexpress EFN-4 we also used the transgene
juEx350 described above.
An EFN-4::GFP reporter was constructed by insertion of the GFP intron
cassette from vector pPD121.89 into the Sac II site of the rescuing
genomic clone pCZ148, to create the EFN-4::GFP clone pCZ147. This clone is
predicted to encode EFN-4 with GFP inserted in-frame close to the N terminus
of the mature protein, after residue 33. Three transgenic lines (juEx151,
juEx205 juEx206) were established by injection of pCZ148 (40 ng/µl)
with the co-injection marker plasmid pRF4, and two lines (juEx206,
juEx210) formed by co-injection of pCZ147 and the lin-15(+)
plasmid pLin-15EK into lin-15(n765) animals. All EFN-4::GFP
transgenic lines yielded similar expression patterns. Chromosomal integrants
of juEx210 (juIs109 and juIs110) were made by X-ray
mutagenesis. The lethality of an efn-4(box80) strain is suppressed
from 32% to
5% by the juIs109 transgene. Staining with
anti-EFN-4 antisera was performed on animals overexpressing EFN-4, either from
the EFN-4-GFP array (juIs109) or from EFN-4-overexpressing animals
(juEx350). To examine co-expression of EFN-4-GFP and VAB-1, we
established a VAB-1-overexpressing array (juEx445) by injection of
the vab-1(+) cosmid M03A1 (15 ng/µl) and pRF4 (30 ng/µl) into
juIs109-carrying animals. Co-expression of EFN-1 and EFN-4 was
examined by making animals transgenic for juIs109 and the
efn-1 transgene juIs53.
Anti-EFN-4 antibody production
To express EFN-4 in bacterial cells, we cloned DNA encoding amino acid
residues 26-325 (i.e. EFN-4 lacking secretion and GPI-addition signal
sequences) into pET21a (Novagen), creating the expression clone pICS531. The
resulting fusion protein is tagged with 6xHis at the C terminus. EFN-4-6HIS
was induced with 0.1 mM IPTG in BL21 (DE3) bacteria and purified on Ni-NTA
beads (Qiagen) under denaturing conditions (8 M urea). Purified EFN-4-6HIS was
dialyzed in 1xPBS, lyophilized and injected into rabbits for antibody
production (Animal Pharm). Anti-EFN-4-HIS6 antibodies were preabsorbed to
total protein acetone powders derived from, E. coli, HEK 293T and
efn-4(bx80) protein lysates. Affinity-purified anti-EFN-4 antibodies
were kept at a final concentration of 1-5 mg/ml.
Fixation and staining of C. elegans embryos were as described
(Finney and Ruvkun, 1990).
Chicken anti-GFP antibodies (Chemicon), affinity-purified anti-EFN-4,
anti-EFN-1 (Chin-Sang et al.,
1999
), anti-VAB-1 (S. E. G. and A. D. C., unpublished) and MH27
monoclonals (Francis and Waterston,
1991
) were used at 1:200 to 1:500 dilution. All fluorescently
conjugated secondary antibodies were used at 1:500 dilution in antibody buffer
A. Data were collected on a Leica TCS-NT confocal microscope.
Accession Number
The GenBank Accession Number for the efn-4 cDNA is AF410936.
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RESULTS |
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We reasoned that mutations in efn-4 might cause defects in
epidermal morphogenesis similar to those caused by mutations in the VAB-1 Eph
receptor or the EFN-1 ephrin. The mab-26(bx80) mutation, identified
on the basis of male tail morphogenetic defects
(Chow and Emmons, 1994), causes
variable defects in epidermal morphology throughout the body, and maps to the
same genomic region as efn-4 (Fig.
1A). We found that a 16.1 kb genomic DNA clone (pCZ148) containing
the efn-4 locus completely rescued all mab-26(bx80)
phenotypes in transgenic lines (see Materials and Methods). The bx80
mutation is a 1.8 kb deletion in efn-4, and four additional
mab-26 alleles result from point mutations in this gene, confirming
that efn-4 corresponds to mab-26; we subsequently refer to
this gene as efn-4. All five efn-4 mutations are recessive
and cause similar morphogenetic defects
(Table 1;
Fig. 2). The three strongest
alleles (bx80, e36, and ju134) probably abolish
efn-4 function.
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When expressed in human 293T cells, EFN-4 is localized to the cell surface,
as detected with anti-EFN-4 antibodies (data not shown). In experiments using
VAB-1-AP fusion proteins to determine the affinity of VAB-1/EFN-4
interactions, as previously described
(Chin-Sang et al., 1999), we
were unable to detect binding of VAB-1-AP (10 nM) to EFN-4-expressing cells.
These data are consistent with previous evidence that the VAB-1/EFN-4
interaction may be weak (Wang et al.,
1999
) relative to other VAB-1/EFN interactions.
EFN-4 regulates embryonic neuroblast movements and epidermal
morphogenesis
efn-4(bx80) mutants display defects in the morphogenesis of
post-embryonic male tail sensilla (Chow and
Emmons, 1994; Hahn and Emmons,
2002
). efn-4 mutants also have widespread defects in
embryonic morphogenesis of the posterior body: about 20-30% of efn-4
larvae and adults have blunted or mis-shapen posteriors
(Fig. 2E,F), although the head
morphology defects (`Notched head'; Fig.
2G) of vab-1 or efn-1 mutants
(George et al., 1998
;
Chin-Sang et al., 1999
) are
rarely seen in efn-4 mutants. In general the post-embryonic
phenotypes of efn-4 mutants partly overlap those of vab-1 or
efn-1 mutants.
The posterior morphology defects of efn-4 mutants arise during
embryogenesis. We analyzed the development of efn-4 embryos using
four-dimensional (4D) time-lapse microscopy and found that efn-4
mutants displayed defects in the same embryonic morphogenetic processes as are
affected in vab-1: neuroblast movements during closure of the ventral
gastrulation cleft, and enclosure of the epidermis
(George et al., 1998;
Chin-Sang et al., 1999
).
Although some embryonic defects of some efn-4 mutants could be
classified according to the criteria used previously in our analyses of
vab-1 and efn-1, we also observed additional phenotypic
classes, detailed below.
Almost all efn-4 mutant embryos displayed an enlarged and
persistent gastrulation cleft (Fig.
2B,C, 230 and 280 minutes); by contrast, vab-1 or
vab-2 mutants display a much lower penetrance of enlarged clefts. In
wild-type development, the gastrulation cleft is 1-2 µm deep and remains
visible as a gap in the ventral neuroblast sheet for 30 minutes to 1 hour
(Fig. 2A, 230 min). By
contrast, the gastrulation cleft in efn-4 mutants varies between 2
µm and 10 µm in depth (n=94), and persists for longer than in
the wild type. About 20% of efn-4 embryos displayed an enlarged
gastrulation cleft that remained open until the time of epidermal leading cell
migration, resulting in a failure of epidermal enclosure and rupture at the
ventral midline, as shown in Fig.
2B; this phenotype resembles the Class I phenotype of
vab-1 or efn-1 mutants. In contrast to vab-1 or
efn-1 mutants, which frequently rupture during epidermal elongation
(vab-1 class IV), less than 1% of efn-4 embryos arrested in
later embryogenesis. Most efn-4 embryos (65%) displayed a novel
phenotype in which the gastrulation cleft was enlarged and delayed in closing,
yet epidermal enclosure and elongation proceeded normally; such animals hatch
with normal morphology or defective posterior morphology
(Fig. 2C). Six percent of
efn-1 animals display similar transient abnormalities in the
gastrulation cleft that do not result in later epidermal rupture
(Chin-Sang et al., 1999
). In
summary, although there is some overlap, the spectrum of embryonic defects in
efn-4 mutants is different from that seen in vab-1 or
efn-1 mutants.
EFN-4 is expressed in the developing nervous system
To determine the expression pattern of EFN-4 we generated anti-EFN-4
antisera; these antisera were able to detect EFN-4 in animals overexpressing
EFN-4 although not in wild-type animals. We also examined the expression of a
functional EFN-4::GFP reporter construct (see Materials and Methods). We
observed indistinguishable expression patterns using both methods.
We first detected EFN-4 at about the 100-cell stage in a large number of ventral surface cells, identified as neural or epidermal precursors (Fig. 3E). During and after enclosure of the epidermis, EFN-4 was expressed in many neurons and not in epidermal cells (Fig. 3A-C). In later embryos, larvae and adults, EFN-4-GFP was expressed in several head neurons, pharyngeal cells and a small number of lateral and tail neurons, within which EFN-4 was localized throughout the neuronal cell body and in axonal processes (Fig. 3M-O). When compared with the expression pattern of EFN-1, EFN-4 expression is relatively widespread (Fig. 3D-I) and only overlapping in part.
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If EFN-4 is a cell-surface ligand for VAB-1 then it should be expressed at least in part in cells that contact VAB-1-expressing cells. Our data show that this is the case. During epidermal enclosure, EFN-4- and VAB-1-expressing cells are widely distributed in the ectoderm, allowing many possible points of contact (Fig. 3J-L). We conclude that EFN-4 expression in embryos is consistent with the possibility that it interacts with VAB-1; interestingly, some cells appear to express both VAB-1 and EFN-4 (e.g. circled cell in Fig. 3J-L).
efn-4 mutations enhance vab-1 (Eph receptor) null
phenotypes and are synthetic-lethal with weak vab-1 mutations
We reasoned that if efn-4 functions only in the same pathway as
the VAB-1 Eph receptor, then the phenotype of a VAB-1 receptor null mutant
[vab-1(0)] should not be enhanced by loss of EFN-4. Alternatively, if
EFN-4 has functions that are independent of the VAB-1 receptor, then a
vab-1(0); efn-4 double mutant might be enhanced relative to a
vab-1(0) mutant. We constructed strains doubly mutant for
vab-1 and efn-4, and found that efn-4 mutations
enhanced vab-1 null alleles. All vab-1; efn-4 double mutants
displayed dramatically enhanced lethality relative to the single mutants, and
were maintained as balanced strains for phenotypic analysis (see Materials and
Methods). Most vab-1(0); efn-4(0) double mutant animals showed the
embryonic-lethal phenotype shown in
Fig.4A, which is more severe
than observed in either vab-1 or efn-4 single mutants, in
that the gastrulation cleft is deeper (average 10 µm, n=19)
and the entire embryo appears mildly disorganized.
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VAB-1 has both kinase-dependent and kinase-independent functions
(George et al., 1998). To
determine if efn-4 mutations synergized with either or both
functions, we compared the phenotypes of vab-1(0) efn-4 double
mutants with those of vab-1(kinase) efn-4 double mutants. We found
that vab-1(kinase); efn-4(0) double mutants displayed strong
synergistic phenotypes that were nevertheless weaker than those of
vab-1(0); efn-4(0) double mutants (compare
Fig. 4A with 4B) suggesting
that EFN-4 acts redundantly with both kinase-dependent and kinase-independent
functions of vab-1. In addition, we constructed strains containing
vab-1 extracellular domain mis-sense mutations (e699, e856,
e1029 or ju8) with either of the hypomorphic efn-4
mis-sense alleles (e660 or e1746), and found that all eight
such double mutant combinations showed synergistic lethality (data not shown).
Because the synthetic-lethal interaction of vab-1 and efn-4
is allele nonspecific we conclude that vab-1 and efn-4
mutations affect parallel, partly redundant pathways.
The synergistic interaction of efn-4 mutations with mutations in the VAB-1 Eph receptor stands in contrast to efn-1 mutations, which do not enhance vab-1(0) embryonic phenotypes (Chin-Sang et al., 1998) (data shown for comparison in Fig. 5). We therefore asked if other C. elegans ephrins showed similar interactions with vab-1 that might be suggestive of vab-1-independent functions. We found that loss of function in efn-2, but not in efn-3, slightly but significantly enhanced the embryonic and larval lethal phenotypes of a vab-1 null mutant (Fig. 5). efn-2 mutants and vab-1 efn-2 double mutants also displayed a reduced brood size (average=158 progeny) compared with vab-1 null mutants [average=281 for vab-1(dx14)] or other efn mutants (data not shown); we have not investigated the basis for the reduced fertility of efn-2 strains. We further tested whether efn-1, efn-2 and efn-3 might have redundant functions in VAB-1-independent pathways using vab-1 dsRNAi (see Materials and Methods) in an efn-1 efn-2 efn-3 triple mutant background. Such vab-1(RNAi) efn-1,2,3 quadruple-mutant embryos displayed morphogenetic defects similar in range and penetrance to the efn-1,2,3 triple mutant (data not shown). Although this experiment is subject to the caveat that RNAi does not fully abolish vab-1 function, we provisionally conclude that efn-1, efn-2 and efn-3 are unlikely to function redundantly in VAB-1-independent processes in morphogenesis.
efn-4 mutations are synthetic-lethal with ptp-3
mutations
Mutations in the LAR-like receptor tyrosine phosphatase ptp-3
cause low-penetrance defects in neural and epidermal morphogenesis and also
show synergistic interactions with vab-1 and efn-1 mutations
(Harrington et al., 2002) that
are, in general, weaker than those of efn-4 mutations. The similarity
of the ptp-3 and efn-4 phenotypes and genetic interactions
led us to test whether ptp-3 might function in the same pathway as
efn-4. We found that ptp-3; efn-4 double mutants displayed a
fully penetrant synergistic embryonic lethality, inconsistent with PTP-3
functioning in a linear pathway with EFN-4
(Fig. 4C). vab-1(0) ptp-3;
efn-4 triple mutant embryos displayed abnormal gastrulation clefts that
were larger than in vab-1(0) efn-4 or ptp-3 efn-4 double
mutants (compare outlines in Fig. 4D with
4A and 4C; see legend to Fig.
4 for quantitation of gastrulation cleft morphology). Such triple
mutant embryos appeared qualitatively more abnormal than the double mutants,
in that cells were more mobile and less tightly packed in the embryo. Because
vab-1 ptp-3 efn-4 triple mutants appeared more severely affected than
vab-1 ptp-3, vab-1 efn-4 or ptp-3 efn-4 double mutants,
these data suggest that VAB-1, PTP-3 and EFN-4 may function in distinct yet
partly redundant pathways in embryonic morphogenesis.
EFN-4 and MAB-20 Semaphorin-2A may function in common or opposing
pathways in early embryonic morphogenesis
Loss of function of the C. elegans secreted Semaphorin-2A homolog
MAB-20 causes defects in male tail morphogenesis
(Baird et al., 1991) similar to
those of efn-4 mutants. It has been suggested that these two genes
might affect a common pathway in male tail development
(Chow and Emmons, 1994
;
Roy et al., 2000
). To ask
whether mab-20 and efn-4 might function in the same pathway
in embryogenesis, we examined the embryonic morphogenetic defects of
mab-20 mutants using 4D analysis. We found that mab-20
mutants were frequently defective in closure of the gastrulation cleft
(Fig. 6A, arrow at 230
minutes); in general, the defects resembled those seen in efn-4
mutants. Thus, in addition to its role in enclosure of the posterior
epidermis, semaphorin signaling is also involved in the earlier process of
neuroblast migration.
|
We examined the consequences of reducing both MAB-20 signaling and ephrin signaling on embryonic morphogenesis. We made several mab-20 efn-4 double mutant strains using either null or weak alleles of either gene. In contrast to vab-1, efn-1 or ptp-3 mutations, mab-20 mutations did not show synthetic-lethal interactions with efn-4 mutations. In all mab-20 efn-4 double mutant strains the penetrance of embryonic lethality was not significantly different from that of the corresponding mab-20 single mutants (Fig. 7A). Using 4D microscopy, we confirmed that most mab-20 efn-4 double mutant embryos showed morphogenetic defects that were less severe than expected from additivity of the single mutants. In particular, the gastrulation cleft defects of mab-20 efn-4 double mutants were sometimes less severe than in either single mutant (Fig. 6C,D). The less than additive effect of mab-20 and efn-4 in embryonic morphogenesis suggests that these two genes might either have antagonistic roles or that they might operate in a common pathway.
|
We analyzed later embryonic and larval phenotypes of mab-20 and
mab-20 efn-4 double mutants to explore further whether
mab-20 and efn-4 might function in common or opposing
pathways later in development. mab-20 mutants display defects in
cell-cell associations in the ventral epidermis, manifested as ectopic
contacts between P cells (Roy et al.,
2000). efn-4 mutants displayed normal contacts between
ventral P cells during embryogenesis; furthermore, efn-4 mutations
did not suppress the ectopic P cell contacts due to mab-20 mutations
(62/78 mab-20(ev574) efn-4(e660) embryos displayed ectopic P cell
contacts, compared with 53/75 mab-20(ev574) embryos; P=0.7,
Fisher's exact test). Overall, mab-20 efn-4 double mutants showed an
increased penetrance of larval lethality consistent with additivity of mutant
phenotypes (Fig. 7B). We
provisionally conclude that mab-20 and efn-4 act
independently in later development.
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DISCUSSION |
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By reference to the structures of mouse ephrin B2
(Toth et al., 2001) and of the
ephrin-B2/EphB2 receptor co-crystal
(Himanen et al., 2001
), the
21-residue insertion in EFN-4 appears to affect the loop between
ß-strands F and G. In ephrin B2 this `F-G loop' contributes to the
ligand/receptor binding interface. The F-G loop comprises eight residues in
vertebrate ephrin A proteins and five residues in vertebrate ephrin B proteins
and other C. elegans ephrins, suggesting that an enlarged F-G loop
could affect the binding specificity of EFN-4. Residues in ß-strand G
have also been implicated in binding of vertebrate ephrin-A2 to ADAM
metalloproteases (Hattori et al.,
2000
), suggesting that the EFN-4 insert could affect such an
interaction; however, the sequence of the metalloprotease binding site itself
is poorly conserved in all worm ephrins. Finally, the adjacent G-H domain
[Area II by Toth et al. (Toth et al.,
2001
)] forms the interface between ephrin monomers in the dimeric
crystal structure. An intriguing possibility is that a longer F-G loop might
affect ephrin homo- or heterodimerization and thus could allow EFN-4 to act as
an inhibitory ephrin. Genetic analysis of ephrin double and triple mutants has
not uncovered any evidence for such an antagonistic interaction between C.
elegans ephrins (S. L. M and A. D. C., unpublished). An alternative
possibility, suggested by the apparent receptor-independent nature of EFN-4
function (see below), is that the insert sequence in EFN-4 promotes
dimerization in the absence of receptor.
Evidence that EFN-4 function is not VAB-1-dependent
Several lines of evidence suggest that EFN-4 can function independently of
the VAB-1 Eph receptor. First, the efn-4 mutant phenotype is only
partly overlapping with the vab-1/EphR null phenotype: in particular,
the penetrance of gastrulation cleft defects is significantly higher than seen
in vab-1 strains. Thus, unlike the phenotypes of efn-1
mutants, which are weaker but otherwise indistinguishable from those of
vab-1, the efn-4 phenotypes are distinctly different from
those caused by loss of receptor function.
A second line of evidence is the strong synergistic interaction between
vab-1 and efn-4 mutations. Again, this result contrasts with
the other three ephrins: loss of function in efn-1, efn-2, or
efn-3 does not exacerbate the phenotypes of vab-1 null
mutants, with one exception. The fully penetrant lethality of a vab-1(0);
efn-4(0) double mutant could reflect additivity (80% lethality of a
vab-1 null combined with
30% lethality of an efn-4
null), and thus does not by itself imply redundancy of vab-1 and
efn-4. However, double mutants made with the weakest alleles of each
gene (each resulting in <10% lethality alone) were >99% inviable,
suggesting that loss of efn-4 function strongly sensitizes the animal
to loss of vab-1 function, and vice versa. We propose that
efn-4 and vab-1 function in parallel pathways that perform
related and partly redundant functions in morphogenesis. In support of this, a
clear synergistic interaction was also seen in double mutants between
efn-4 and efn-1; null mutations in either gene cause
30% lethality, whereas a double mutant using null alleles was 100%
inviable (S. L. M. and A. D. C., unpublished).
A third line of evidence that EFN-4 may not solely signal via VAB-1 is the
apparently low affinity of EFN-4/VAB-1 interactions
(Wang et al., 1999). The
affinity of the VAB-1-EFN-4 interaction appears to be much lower than that of
the VAB-1-EFN-1 interaction (Kd
5 nM)
(Chin-Sang et al., 1999
). Even
if EFN-4 does signal partly via VAB-1, this would not invalidate the
conclusion from genetic analysis that EFN-4 also functions in a
VAB-1-independent pathway. Genetic and phenotypic analysis suggests that EFN-2
also may have functions independent of VAB-1. Thus, at least two of the four
C. elegans ephrins appear to have functions that do not require the
only known C. elegans Eph receptor.
How might EFN-4 signal in morphogenesis?
We envisage several possibilities for how EFN-4 (and possibly EFN-2) might
signal in pathways that do not require the VAB-1 receptor. First, a trivial
possibility is that a second Eph-like receptor may exist in the <0.5% of
the C. elegans genome as yet unsequenced, or that such a receptor may
be sufficiently diverged that it is not recognizable using current algorithms.
Both these possibilities seem unlikely given the extensive efforts to identify
worm kinases (Plowman et al.,
1999) and RTKs (Popovici et
al., 1999
).
Second, EFN-4 may signal via a non-Eph-like receptor. The apparent
epistasis of efn-4 and mab-20 mutant phenotypes suggests
that EFN-4 could signal via a component of the MAB-20 pathway (see below).
Vertebrate ephrins can bind ADAM family metalloproteases
(Hattori et al., 2000); EFN-4
could signal via one of the C. elegans ADAM metalloproteases.
Mutations in one such metalloprotease, the kuzbanian ortholog sup-17
cause complex phenotypes that reflect reduction of lin-12 and
glp-1 function (Wen et al.,
1997
); we are currently examining whether sup-17 plays
additional roles in C. elegans ephrin signaling. The receptor
tyrosine phosphatase PTP-3 also functions in parallel to VAB-1 in
morphogenesis (Harrington et al.,
2002
). However, the Ptp-3 and Efn-4 phenotypes are distinct, and
ptp-3 mutations display strong synergistic interactions with
efn-4 mutations, inconsistent with PTP-3 being the sole EFN-4
receptor. Because vab-1 ptp-3 efn-4 triple mutant embryos appear only
slightly more defective than any corresponding double mutants, our data do not
rule out a model in which EFN-4 signals redundantly via VAB-1 or PTP-3.
However, in the absence of evidence that EFN-4 directly interacts with either
receptor, it is equally possible that VAB-1, EFN-4 and PTP-3 define three
partly redundant pathways in morphogenesis.
Third, ephrins form dimers and higher-order multimers
(Davis et al., 1994;
Toth et al., 2001
). Cell
culture experiments suggest that co-expression of EFN-4 with EFN-1 does not
modulate the ability of EFN-1 to bind VAB-1 (I. D. C.-S. and A. D. C.,
unpublished), although it could alter the specificity of other ephrins so that
a heterodimer could interact with an unknown receptor. Additionally, if VAB-1
does bind EFN-4, the co-expression of VAB-1 and EFN-4 in some cells could
alter the responsiveness of VAB-1 to other ligands, as demonstrated for
co-expressed ephrins and Eph receptors in vertebrate retinal neurons
(Dutting et al., 1999
;
McLaughlin and O'Leary,
1999
).
Finally, EFN-4 might not interact directly with any receptor. Recent data
from several groups have suggested that GPI-linked ephrins, like their
transmembrane counterparts, can also function as bidirectional signaling
proteins (Knoll and Drescher,
2002). Vertebrate Ephrin A proteins may function in reverse
signaling as a consequence of their localization to membrane microdomains
(Bruckner et al., 1999
;
Davy et al., 1999
;
Davy and Robbins, 2000
), and
C. elegans EFN-1 has functions independent of the VAB-1 kinase
(Chin-Sang et al., 1999
). We
speculate that EFN-4 might have evolved to be able to trigger `reverse
signaling' pathways in the absence of receptor binding. For example, the F-G
loop insert in EFN-4 might allow it to dimerize more easily than other
ephrins, leading to activation of pathways in the absence of the VAB-1
receptor. Further analysis of EFN-4 could provide insights into how GPI-linked
ephrins function in bidirectional signaling.
Eph signaling, semaphorin signaling and cell adhesion in the early
C. elegans embryo
mab-26 (efn-4) was proposed to function in the same
genetic pathway as the Semaphorin 2A MAB-20, based on the similarity and
non-additivity of mab-20 and mab-26 mutant phenotypes in
male tail morphogenesis (Roy et al.,
2000). Consistent with the previous analysis of male tail
phenotypes we find that the efn-4 and mab-20 embryonic
phenotypes are overlapping and show less than additive effects in double
mutants. However, our analysis has not conclusively established whether
efn-4 and mab-20 act in a common pathway in embryogenesis.
The apparent epistasis of mab-20 efn-4 double mutants is also
compatible with a model in which mab-20 and efn-4 play
opposing roles. MAB-20 is ubiquitously expressed in embryos during
morphogenesis stages (Roy et al.,
2000
), rendering possible several kinds of interaction with EFN-4
pathways. Although ephrin and semaphorin pathways both can function in
contact-dependent repulsion of axonal growth cones, the consequence of
reducing both pathways simultaneously has not been explicitly addressed, and
many other combinatorial interactions have been observed between axon guidance
pathways (Yu and Bargmann,
2001
). Further analysis of genetic interactions between Eph
signaling and semaphorin signaling will be necessary to elucidate their in
vivo functional relationships.
Most functions of Eph signaling involve modulation of cell-cell adhesion.
Indeed, ephrins were first identified as contact-dependent axon repellents in
the vertebrate central nervous system
(Cheng et al., 1995;
Drescher et al., 1995
;
Nakamoto et al., 1996
).
Numerous studies have confirmed and extended this anti-adhesive role of ephrin
signaling, both in the nervous system and in non-neuronal cells
(Wilkinson, 2001
). However, it
is clear that in some situations ephrin/Eph signaling can promote cell
adhesion (Klein, 2001
). The
cellular basis for the early embryonic defects of C. elegans Eph
signaling mutants remains poorly understood, but probably reflects alterations
in cell adhesion among neural precursors. The phenotype of vab-1 ptp-3
efn-4 triple mutants, in which cells appear more mobile than in the wild
type, hints that in the C. elegans embryo these proteins may function
as parts of a redundant cell-adhesion network. However, it remains unresolved
whether ephrin signaling directly mediates cell adhesion in the C.
elegans embryo, or whether it modulates the function of other cell
adhesion molecules, such as LAD-1 (Chen et
al., 2001
).
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ACKNOWLEDGMENTS |
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Footnotes |
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