Department of Biology, McGill University, 1205 Dr Penfield Avenue, Montreal, Quebec H3A 1B1, Canada
* Author for correspondence (e-mail: siegfried.hekimi{at}mcgill.ca)
Accepted 7 September 2004
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SUMMARY |
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Key words: mau-2, Migration, Guidance, slt-1, slit, Caenorhabditis elegans, Maternal effect, Cell autonomous
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Introduction |
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A number of genes that are known to be required for proper migration in
C. elegans were identified through screens for behavioural mutants
(Brenner, 1974), the subsequent
characterization of which revealed specific neuroanatomical defects (e.g.
Hedgecock et al., 1990
;
Hedgecock et al., 1985
;
McIntire et al., 1992
). Other
screens have used surrogate markers such as transparent larvae or withered
tails (Forrester et al., 1998
;
Wightman et al., 1997
). Yet
more genes were identified through screens for mutants that affect the
position of particular neurones and axons, regardless of their behavioural
consequences, such as sax-3, which affects the development of the
nerve ring (Zallen et al.,
1998
), and nid-1, which affects the position of nerve
bundles (Kim and Wadsworth,
2000
).
The molecular characterization of some of these genes has revealed that a
wide range of proteins contribute to the proper development of the nervous
system of the worm (reviewed by Antebi et
al., 1997; Blelloch et al.,
1999
). Some proteins act as guidance cues or as receptors for
these guidance cues, other are components of the extracellular matrix or
regulate cell adhesion, and yet other proteins participate in the cellular
response to guidance cues. A number of proteins required for the guidance of
migrations are novel, such as MIG-13 (Sym
et al., 1999
), CED-12
(Gumienny et al., 2001
),
UNC-53 (Stringham et al.,
2002
), VAB-8 (Wolf et al.,
1998
), and UNC-76 (Bloom and
Horvitz, 1997
), and their biochemical functions remain
unknown.
Most of the genes required for the development of the nervous system
identified so far are zygotic. In an attempt to identify genes involved in the
earliest stages of the patterning of the nervous system, a genetic screen for
maternal-effect behavioural mutants was carried out
(Hekimi et al., 1995). Three
mutations (qm4, qm5 and qm40) identified in this screen
define the gene mau-2 (mau stands for maternal-effect
uncoordinated).
mau-2 mutants are severely uncoordinated and egg-laying defective,
and these phenotypes are completely penetrant
(Hekimi et al., 1995;
Takagi et al., 1997
). In
addition, approximately one-fifth of the larvae fail to complete development
and become filled with fluid (the Lid phenotype). At the anatomical level,
mau-2 mutants display defects in the positions of cell bodies and
processes that belong to the locomotory, egg-laying and osmoregulation
systems. In fact, numerous cells that undergo long-range migrations during the
development of the worm are misplaced in mau-2 mutants, including
neurones (e.g. AVM, PVM, SDQR/L and HSNR/L), axons (e.g. of AVM, PLM, and
CAN), as well as the distal tip cells (DTCs)
(Takagi et al., 1997
).
mau-2 mutations affect migrations that occur embryonically as well as
post-embryonically. For example, the embryonic migration of the cell body of
HSN and the extension of the axons of PLM and CAN, are affected in
mau-2 mutants. In addition, the extension of the axon of AVM, the
projection of motoneurone axons into the dorsal cord, the migration of the Q5
descendants and of the DTCs, all of which occur during larval development, are
abnormal in mau-2 mutants. In addition, migrations that occur along
both the longitudinal and the circumferential body axes of the worm, oriented
anteriorly-, posteriorly-, ventrally- or dorsally-, are all affected in
mau-2 mutants. Thus, rather than being involved in a specific subset
of migrations, mau-2 is broadly required for the proper migration of
cells and axons.
Importantly, migrating cells frequently undergo excessive migrations in
mau-2 mutants, such that their final position is beyond the target
region where the migration should have halted. For example, the SDQR and HSN
neurones, which undergo anteriorly directed migrations during development, are
frequently placed too anteriorly in mau-2 mutants
(Takagi et al., 1997). In
addition, the axon of AVM, which normally projects ventrally, can instead
extend posteriorly for long distances in mau-2 mutants. Thus,
mutations in mau-2 do not impair the motility of the cells per se,
but rather affect the proper guidance of these migrations. Here, we report
that mau-2 encodes a novel protein that functions cell autonomously
to guide migrations during the development of the nervous system.
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Materials and methods |
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and see below.
Hermaphrodites were injected with cosmids, deletion constructs and pRF4
(Mello and Fire, 1995) at
concentrations of 5, 50 and 100 ng/µl, respectively. Insert orientation in
cosmid C09H6 was determined by restriction mapping.
Neuroanatomical observations
The position of neurones, axons and fascicles was examined using the
reporter unc-31::gfp, which is expressed in virtually all neurones
and in vulval muscles (Takagi et al.,
1997); the axon of HSN, using tph-1::gfp
[mgIs71; (Sze et al.,
2000
)]; AVM, using the mec-4::gfp reporter. In
tissue-specific rescue assays, plasmids for mec-4::gfp or
unc-31::gfp was injected at 100 ng/µl. Examination of maternally
rescued animals was carried out using an integrated unc-31::gfp. For
the analysis of genetic interactions with unc-40::myr and slt-1,
zdIs4 was used. The morphology of the axon of AVM was scored only when
the position of the AVM cell body was normal.
Molecular analysis
General molecular manipulations were carried out as described
(Sambrook et al., 1989).
Pfu polymerase was used for PCR (Stratagene). All cloned inserts were
sequenced.
mau-2 transcript
SL1- or SL2-specific primers, combined with primers specific for predicted
exons, were used on two independent first-strand mixed-stages cDNA libraries.
RT-PCR products and yk462a (gift of Dr Kohara) were sequenced. mau-2
encodes a single message of 2010 bp (not including SL1, nor the poly A tail).
The 5'UTR is SL1 transpliced at the acceptor site CAG, 6 bp upstream of
the initiating ATG. The polyadenylation signal is probably AAAAAA, located 211
bp downstream of the TAA Stop codon. The polyadenylation cleavage site is 8 bp
downstream of the polyadenylation signal, resulting in a 222 bp long
3'UTR. yk462a lacks the 5'UTR and the first 9 coding base pairs,
and part of the 3'UTR.
mau-2 mutant alleles
A library of UV-TMP-mutagenized worms was screened by nested PCR with
primers specific for the gene mau-2 and qm160 was isolated
(Edgley et al., 2002). The
primer sequences correspond to the following bases on cosmid C09H6:
27984-28007, 32138-32161, 27507-27530 and 31892-31915. qm160 fails to
genetically complement qm5.
The region of mau-2 was sequenced on two independent PCR products amplified from genomic DNA of N2, qm4, qm5, qm40 and qm160.
Northern blot analysis
Worm populations were synchronized at different developmental stages as
described (Wood, 1988). Total
RNA was isolated using Trizol (Gibco). A mau-2-specific probe made by
32P-radiolabelling (Ready-to-go kit, Pharmacia) using the entire
insert of yk462a was used for northern blotting.
Anti-MAU-2 polyclonal antibodies and western analyses
A BSA-conjugated peptide located (ASRRMLSVENLTPL-VANMPASK, residues 552-573
near the C terminus of MAU-2) was synthesized (Sheldon Center, McGill
University) and injected into two rabbits (Animal Resources Center, McGill
University). Polyclonal antibodies were affinity purified from crude serum on
an Affigel column (BioRad) (Harlow and
Lane, 1999). The purified antibodies detect the conjugated antigen
of an expected size of 70 kDa, free peptide at
2.5 kDa, a band of the
expected size of MAU-2 (
68 kDa) in N2 but not in mau-2(qm160)
extracts, and a band at
68 kDa in mau-2(qm160) mutants carrying
wild-type copies of mau-2 on an extrachromosomal array. Few other
non-specific bands were detected.
Total protein extracts were prepared by grinding. Affinity purified anti-MAU-2 antibody (see below) was used at a 1:5 dilution, and donkey anti-rabbit IgG antibody (Jackson Laboratories) at 1:1000, followed by detection with ECL (Amersham). Analysis MAU-2 in maternally rescued animals: 400 Dpy worms for each genotype and stage were picked, and denatured in sample buffer.
Reporter constructs for mau-2
For translational fusions between mau-2 and gfp,
synthetic PstI and BamHI sites on primers allowed
directional cloning of bases 27054-32154, 24119-32154, or 22063-32154 of C09H6
into pPD95.77.
Constructs for tissue-specific expression of mau-2
Extrachromosomal arrays carrying mau-2(+) do not give maternal
rescue (non-transgenic worms derived from a transgenic mau-2
hermaphrodite are invariably Mau). In addition, a promoterless transgene
containing the entire mau-2 cDNA (pCB42) cannot rescue the
mau-2 mutants.
pCB42 contains the entire mau-2 cDNA from the ATG to the last codon, excluding the Stop, fused in frame to gfp. yk462a was the template, and the missing 5'UTR, along with a PstI site was added on the 5' primer. The 3' primer contained a synthetic BamHI site for cloning into pPD95.77.
Plasmids were obtained by cloning promoters in front of mau-2 in
pCB42. Pdpy-7::mau-2::gfp contains a 0.9 kb fragment
corresponding to the promoter of the gene dpy-7
(Gilleard et al., 1997);
Pmyo-3::mau-2::gfp, a 2.5 kb fragment for the
myo-3 promoter (Okkema et al.,
1993
); Punc-119::mau-2::gfp, a 1.2 kb region
for the unc-119 promoter (Maduro
and Pilgrim, 1995
); Pmec-7::mau-2::gfp, a
contains a 0.8 kb fragment for the mec-7 promoter
(Hamelin et al., 1992
).
These constructs were injected into N2 and qm160 at 10 ng/µl, along with pRF4 (100 ng/µl) and mec-4::gfp or unc-31::gfp at 100 ng/µl.
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Results |
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To gain insight into the function of mau-2, we determined to what extent the development of m+z animals proceeds normally. We examined mau-2(qm5) maternally rescued animals at the anatomical level using the reporter unc-31::gfp, which allows the visualization of the nervous system and the vulval muscles. In contrast to mau-2(qm5) mutants, the nervous system of m+z is virtually indistinguishable from that of the wild type: the ventral nerve cord, the commissures and laterals are normally organized, the dorsal cord is always fasciculated, and the cell body of migratory neurones such as AVM, SDQR and HSN, as well as of the DTCs, are normally positioned (Fig. 1). The defect of the axon of AVM is also completely maternally rescued. Thus, the parts of the nervous system that underlie locomotion develop normally in maternally rescued animals. As the excretory and locomotory systems are laid down largely during embryogenesis or shortly after hatching (L1 and early L2 larval stages), it appears that sufficient wild-type mau-2 product can be supplied by the mother for the early development of its progeny to occur normally. However, maternally rescued mau-2 animals display anatomical defects in their egg-laying system, consistent with their abnormal egg laying. In particular, at least one out of the four vm1 vulval muscles is abnormally placed in 24% of the maternally rescued animals (Fig. 1). In addition, the axon of one of the two HSN neurones projects abnormally in 21% of maternally rescued animals, often extending laterally from the cell body first, and projecting ventrally at a position anterior to the vulva, instead of posterior to it. Other aspects of the egg-laying system, such as the placement of the vm2 vulval muscles and uterine muscles, which are more difficult to examine, might also be affected in mau-2 m+z animals, possibly accounting for the completely penetrant egg-laying defect.
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The mutant phenotype of all four mau-2 alleles is identical for
phenotype penetrance and expressivity, at both the dissecting microscope and
neuroanatomical levels. We have previously described the phenotype of qm4,
qm5 and qm40 (Takagi et al.,
1997), and now describe qm160. Like the other alleles,
all mau-2(qm160) animals are uncoordinated and egg-laying defective
(41% of the Egl animals actually become bags of worms, n=359), and
17% of the larvae acquire a straight and translucent appearance and die
the Lid phenotype [n=200 larvae
(Takagi et al., 1997
)]. In
addition, the dorsal cord defasciculation defect is similarly affected in
qm160 compared with the other mau-2 alleles and the
penetrance of the defect of the guidance of the AVM axon is very similar to
that of qm5 and qm4 [see below
(Takagi et al., 1997
)]. As all
four mau-2 alleles have identical phenotypes, it appears that they
all result in a complete loss of the gene function.
We examined protein levels in mau-2 mutants compared to the wild
type (Fig. 3A). We developed
affinity-purified polyclonal antibodies directed against MAU-2 and carried out
immunoblot analysis on worm extracts. We believe that a band of 68 kDa,
the expected molecular weight for MAU-2, corresponds to the full-length MAU-2
protein as it is detected in wild-type worms, absent in the deletion allele
mau-2(qm160), and detected in mau-2 mutants that carry
wild-type copies of mau-2. MAU-2 is undetectable in the nonsense
alleles mau-2(qm5) and mau-2(qm40), indicating that they are
likely molecular nulls, although we cannot exclude that undetected truncated
proteins may be produced in these two nonsense mutants. Finally, the level of
MAU-2 is strongly reduced in the mis-sense allele mau-2(qm4),
indicating that the qm4 mutation severely affects the stability of
MAU-2, and probably also the function of the remaining mutant protein, as this
allele is as severe as the other mau-2 mutants. Interestingly, the
Gly residue that is mutated in qm4 is conserved through evolution in
all animals.
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Extensive searches using prediction programs detect no functional domains,
conserved motifs or subcellular localization signals within C.
elegans MAU-2, other than a tetratricopeptide-like domain (IPR008941,
residues 485-495). A tetratricopeptide (TRP) repeat is detected in vertebrate
MAU-2 proteins (IPR001440, residues 443-476 in F. rubripes and
459-492 in H. sapiens). TRP repeats occur in diverse proteins and are
believed to mediate protein-protein interactions and the assembly of
multiprotein complexes. However, common features in the interaction partners
remain undefined (D'Andrea and Regan,
2003; Lamb et al.,
1995
), providing no clue to what the biochemical function of MAU-2
might be. All four mau-2 mutant alleles can be rescued with a
transgene that carries the first five exons of mau-2, which code for
a truncated protein from residues 1 to 371 of MAU-2. Thus, at least when
overexpressed from an extrachromosomal array, this N-terminal region of MAU-2
is sufficient to carry out fully the identified functions of mau-2,
suggesting that it is functionally the most important.
Abundant mau-2 transcripts accumulate in the oocytes
We examined the abundance of the mau-2 transcript throughout
development in wild-type worm populations by northern blot analysis. A single
band of 2 kb, which is the expected size for the mau-2 mRNA, is
detected at all stages (Fig.
3B). The mau-2 transcript is abundant in the embryonic
and young adulthood stages, whereas it is present in low amounts throughout
the larval stages.
Given the maternal contribution of mau-2 to development and the
high transcript level in young adults, we investigated the mau-2 mRNA
levels in the germline by comparing wild-type young adults with adults
carrying mutations that affect the germline
(Fig. 3B). The mau-2
transcript level is extremely reduced in glp-4(bn2ts) mutants, which
fail to develop a germline at 25°C
(Beanan and Strome, 1992),
indicating that most of the mau-2 mRNA in young adults is in the
germline. Furthermore, the level of the mau-2 transcript is very high
in fem-2(b245ts) mutants that make only oocytes at 25°C,
indicating that the oocytes are enriched with mau-2 transcript.
However, the mau-2 transcript is very low in fem-3(q20ts)
adults that make sperm only at 25°C, indicating that the mau-2
transcript is not enriched in sperm. Taken together, these results indicate
that oocytes accumulate high levels of mau-2 mRNA, presumably as a
store to be used by the embryo.
Consistent with our observations, in situ hybridization data reported in
the Nematode Expression Pattern Database
(http://nematode.lab.nig.ac.jp/)
for clone yk462a, which corresponds to mau-2, show an intense signal
in the germline of adults, particularly in the pachytene region, which is a
major site of transcription in the germline
(Lerner and Goldstein, 1988).
In addition, a strong level of mau-2 mRNA is detected at different
embryonic stages, including as early as the two-cell stage.
We further characterized the expression pattern of mau-2 by determining the levels of protein throughout development by immunoblot analysis (Fig. 3C). Overall, the content of MAU-2 protein is similar through all developmental stages, suggesting a requirement for MAU-2 at numerous stages of development, including at times of cellular and axonal migrations. Upon repeated immunoblot analysis, there appeared to be a slight enrichment in the embryos and L1 larvae compared with other larval stages, which could reflect a higher need for MAU-2 during the time when numerous cellular migrations occur.
We examined the level of MAU-2 in the germline mutants glp-4(bn2ts), fem-3(q20ts) and fem-2(b245ts) compared with the wild type (Fig. 3C). The level of MAU-2 in germline mutants is similar to the wild type, indicating that MAU-2 is not particularly enriched in the germline. Thus, the maternal mau-2 product that is provided to the zygote via the oocyte appears to be principally RNA. Maternally contributed mau-2 mRNA is probably responsible for the maternal rescue effect observed with mau-2 mutations.
Abundant MAU-2 protein is detected in maternally rescued animals up to the L3 larval stage
We examined the level of MAU-2 protein in mau-2(qm5) maternally
rescued animals at the L3 and L4 stages compared with the wild type
(Fig. 3D). Given that no MAU-2
protein is detected in mau-2(qm5), any MAU-2 protein detected in
m+z animals derived from a
qm5/+ mother must result from a maternal contribution. MAU-2 protein
is detected in maternally rescued animals, and up to the third larval stage,
the level of MAU-2 in maternally rescued animals is very similar to that of
the wild type. This indicates that maternally contributed mau-2 is
sufficient to sustain a wild-type protein level up to the L3 stage. By the L4
larval stage, the level of MAU-2 is reduced in
m+z animals compared with the wild type:
a low level is present in early- to mid-L4 larvae, and it is no longer
detected in late-L4 larvae, while it is detected in the wild type. Thus, the
maternal contribution of mau-2 product runs out at the L4 stage in
maternally rescued worms. In addition, these results indicate that new MAU-2
protein is translated at the L4 stage in wild-type worms, as the level of
MAU-2 is higher in L4 than L3 in wild-type larvae (400 worms were loaded in
each lane). Thus, mau-2 is re-expressed at the time of the
development of most of the egg-laying system.
A functional mau-2::gfp transgene is expressed ubiquitously by mid-embryogenesis and subsequently becomes restricted to the nervous system
To gain more insight into the expression pattern of mau-2, we
generated transgenes containing the entire genomic region of mau-2,
including a region of upstream sequence, as well as all exons and introns,
fused to the green fluorescent protein (gfp) gene
(Fig. 4A). In three different
constructs, mau-2::gfp was driven by 1 kb, 4 kb and 6 kb of the
putative mau-2 promoter region. All three translational fusions
expressed from extrachromosomal arrays completely rescued the defective
locomotion, egg laying and larval development of all four mau-2
mutant alleles (but did not recapitulate the maternal rescue effect) and
produced an identical expression pattern. The expression level of
mau-2::gfp is 10 fold stronger when driven by 4 or 6 kb of
putative mau-2 promoter, suggesting that regulatory elements for the
transcription of mau-2 reside more than 1 kb upstream of the
gene.
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In addition, the MAU-2::GFP fusion appears to entirely and uniformly fill the cytoplasm of all neurones, including along their axonal projections, and but excluding the nucleus. The human counterpart of MAU-2 (KIAA0892), expressed as a GFP fusion under the control of the Pcmv promoter in transiently transfected COS7 cells, also appeared cytoplasmic, and excluded from the nucleus (data not shown).
Neuronally expressed mau-2 is necessary and sufficient to rescue the mau-2 mutants
mau-2 is required for the guidance of cellular and axonal
migrations during the development of the worm, and rescuing
mau-2::gfp transgenes are expressed, among other cells, in cells that
migrate during development. It is possible therefore that mau-2 could
function inside the migratory cells to participate in their guidance. To
directly determine where mau-2 is required during development, we
expressed mau-2(+) in distinct tissues of the worm and assessed where
the activity of mau-2 is required for normal development. We
generated constructs that drive the expression of mau-2(+) in the
hypodermis, the body wall muscles, the entire nervous system, or in six
mechanosensory neurones, by placing the mau-2 cDNA fused to the
gfp gene under the control of the promoters of the genes dpy-7,
myo-3, unc-119 and mec-7, respectively. We verified the
expression pattern of these constructs carried on extrachromosomal arrays and
found that they were identical in both the wild-type and mau-2(qm160)
backgrounds (Fig. 5A).
Pdpy-7::mau-2::gfp was expressed in hypodermal cells from
the early comma stage in embryogenesis onwards,
Pmyo-3::mau-2::gfp was expressed in the body wall muscles
from the comma stage onwards, Punc-119::mau-2::gfp was
expressed throughout the nervous system from the comma stage onwards (as well
as in vulval muscles in late larvae and adults), and, finally,
Pmec-7::mau-2::gfp was expressed in the mechanosensory
neurones (AVM, ALMs, PVM, PLMs) from late embryogenesis onwards.
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Expression of mau-2 in AVM can rescue the ventral guidance of the AVM axon
To distinguish between a non-autonomous role of mau-2 between
cells within the nervous system and an autonomous role of mau-2
within the migrating cell themselves, we assessed whether expression of
mau-2(+) in the neurone AVM could rescue the axonal guidance defect
that is displayed by mau-2 mutants. The neurone AVM is born and its
axon migrates during the L1 larval stage
(Sulston and Horvitz, 1977;
Hedgecock et al., 1987
). In
the wild-type L1 larva, the axon of AVM pioneers its ventral migration along
the body wall, from the lateral aspect of the body towards the ventral nerve
cord. The axon then turns anteriorly towards the nerve ring
(Hedgecock et al., 1987
;
White et al., 1986
)
(Fig. 6A). In
30% of the
mau-2 mutants, this axon fails to migrate ventrally and instead runs
anteriorly or posteriorly along the body wall
(Takagi et al., 1997
)
(Fig. 6A). We expressed
mau-2(+) exclusively in AVM and five other mechanosensory neurones
under the control of the promoter of the gene mec-7
(Pmec-7::mau-2::gfp). This expression of mau-2(+)
strongly rescued the projection of the AVM axon, of both posteriorly and
anteriorly misguided AVM axons in three independent transgenic lines
(Fig. 5B). Thus, mau-2
functions within the AVM neurone to guide its axon ventrally. As expected, the
narrow expression of Pmec-7::mau-2::gfp did not rescue the
locomotion, egg laying and larval development of the mau-2 mutants.
Expression of mau-2(+) under the mec-7 promoter also rescued
the abnormal projection of the PLM axons (data not shown), which instead of
projecting laterally, as in the wild type, run ventrally in 40% of the
mau-2(qm4) mutants (Takagi et
al., 1997
). This suggests that mau-2 functions cell
autonomously in other cells as well. In addition, consistent with a
cell-autonomous role for mau-2, overexpression of mau-2(+)
in muscles, hypodermis or the nervous system in a wild-type mau-2(+)
background leaves the guidance of AVM entirely unaffected.
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We first asked whether mau-2 is required for signalling through
the netrin receptor UNC-40. Expression of a hyperactive form of the netrin
receptor UNC-40::MYR in mechanosensory neurones leads to profound defects in
AVM guidance, and also results in highly abnormal cell-body shapes and
branching patterns of AVM and PLM (Gitai
et al., 2003). We found that complete loss of mau-2
function does not suppress the effects caused by expression of UNC-40::MYR (in
a strain carrying the extrachromosomal array kyEx456). In
mau-2(qm160); kyEx456, the severity and penetrance of the
defects was similar to those observed in a wild-type background [83% abnormal
in 108 mau-2(qm160) young adults, and 88% abnormal in 92
mau-2(+) young adults]. Thus, the function of mau-2 is not
absolutely required for the signalling downstream of the unc-40
receptor. Consistent with this, we found that a mau-2(qm4);
unc-6(e78) double mutant, where e78 is a results in a partial
loss of function of unc-6 (Lim
and Wadsworth, 2002
), displays migration defects that are more
severe than either of the single mutants (data not shown), indicating that
mau-2 probably acts in parallel to unc-6 in the guidance of
at least some cells and axons during their migration. We could not, however,
determine the phenotypic consequences of the complete loss of function for
both genes [ev400 is a null allele of unc-6
(Wadsworth et al., 1996
)], as
the mau-2(qm160);unc-6(ev400) double mutant is unviable.
Next, we asked whether mau-2 is required for the signalling of
slt-1. SLT-1 is secreted from the dorsal muscles and functions to
repel the AVM axon, thus contributing to its ventral migration
(Hao et al., 2001).
Misexpression of slt-1 in all of the body wall muscles of the worm,
i.e. in ventral as well as dorsal muscles, results in the lateral projection
of the AVM axon in about 30% of the animals
(Hao et al., 2001
)
(Fig. 6). We asked whether the
complete loss of function of mau-2 could suppress the effect of
misexpressing slt-1. We found that mau-2(qm160) does not
suppress the defects caused by misexpression of slt-1, indicating
that mau-2 is not absolutely required for the signalling of
slt-1. By contrast, mau-2(qm160) enhances the AVM axon
guidance defects generated by slt-1 misexpression
(Fig. 6A,B). In
mau-2(qm160) that misexpress slt-1, the defect of the AVM
axon is profoundly enhanced (
80% abnormal) compared with the
mau-2 mutants (
30%) or to transgenic animals misexpressing
slt-1 (
30%). In addition to this quantitative difference, the
phenotype of mau-2(qm160); myo-3::slt-1 is qualitatively different
from that of the mau-2 mutants or of the animals misexpressing
slt-1: in 7% of the animals, the axon of AVM actually projects
dorsally. These dorsally projecting axons of AVM reach the level of the axon
of ALMR or the dorsal sublateral. They then turn anteriorly, with the axon
frequently changing its dorsoventral position, so that they do not extend in a
straight line towards the head of the worm. This dorsal projection of AVM is a
novel mutant phenotype, specific to the interaction of these two genes. These
results indicate that mau-2(+) could act to reduce the signalling of
slt-1 or in a pathway parallel to slt-1.
We also examined the mau-2(qm160);slt-1(ev15) double mutants
[ev15 is a null allele of slt-1
(Hao et al., 2001)]. This
double mutant displays an enhanced phenotype for the guidance of AVM. The
overall penetrance of the AVM axon defect appears to be the sum of the effect
of the two single mutations; however, the phenotype of the double mutant is
qualitatively different from that of either of the single mutants. In
mau-2 mutants, one-third of the abnormal axons project posteriorly.
This appears to be suppressed in the double mutant with slt-1.
Moreover, 7% of the AVM axons project dorsally in the double mutant. Again,
this phenotype has not previously been observed to be a consequence of the
loss of function of genes implicated in the guidance of AVM. Together, our
results indicate that mau-2 and slt-1 act independently to
guide the AVM axon ventrally. Although slt-1, unc-6 and
mau-2 are all necessary to ensure that AVM migrates ventrally, the
combined action of mau-2 and slt-1 is required to prevent
AVM from projecting dorsally (Fig.
6C).
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Discussion |
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Our results demonstrate that mau-2 functions cell-autonomously to guide the axon of the AVM neurone. First, mau-2 mutations impair the proper axonal guidance of AVM. Second, a functional mau-2::gfp transgene is expressed in the AVM neurone, including at the time of its axonal migration. Third, expression of mau-2(+) in AVM, in otherwise mau-2 null mutant animals, is sufficient to rescue the axonal guidance defect of AVM. Similar observations were made for the axon of the PLM neurone.
Several lines of evidence suggest that mau-2 may function within other migrating cells to participate in their guidance. mau-2 mutants display defects in the guidance of a number of cellular migrations, including numerous neurones and their axons, and mau-2 is expressed in the affected cells at the time of their migration. Neuronal migration and axon outgrowth start from around 370 minutes and the major nerve tracts form from around 450 minutes. Expression from the functional mau-2::gfp transgene is visible in the embryo, including in neuroblasts and neurones, starting about 300 minutes after the first cleavage. Functional mau-2::gfp keeps being expressed in the nervous system, from the threefold embryonic stage (after 500 minutes) onwards, coincident with the occurrence of additional neural migrations, including the integration of new axons in the dorsal cord at the L1 stage. For example, the positions of the embryonically migrating cell body of HSN and the embryonically extending PLM and CAN axons, is abnormal in mau-2 mutants, and mau-2::gfp is expressed in these cells, including at the time when their migration is occurring in the embryo. Importantly, pan-neuronal expression of mau-2(+) under the unc-119 promoter rescues the mau-2 mutant defects (e.g. locomotion, egg-laying, Lid larval lethality, nervous system organization), indicating that mau-2 functions in other neurones as well. Taken together, these observations suggest that mau-2 may function cell autonomously in other migrating cells as well. Consistent with this, nonautonomous activity of mau-2 is unlikely, as the mau-2 mutant defects are not rescued by expression of mau-2(+) in the mechanosensory neurones, in the hypodermis or in the body wall muscles.
Furthermore, mau-2 may function cell autonomously in cells that develop later, such as those constituting the egg-laying system. In mau-2 mutants, the vulva is frequently asymmetrical and the vulval muscles are often misplaced. Interestingly, our analysis of wild-type and maternally rescued animals revealed that new MAU-2 protein is also produced at the L4 larval stage, and de novo mau-2::gfp expression was observed in the muscle cells, and occasionally in hypodermal cells, of the developing vulva in L3 and L4 larvae. Expression of mau-2 at the L4 stage appears to be necessary for the normal development of the vulva, as vulval muscles attach abnormally in maternally rescued animals.
mau-2 acts in parallel to slt-1 to prevent AVM from projecting dorsally
Simultaneous perturbation of the activities of mau-2 and
slt-1 profoundly affects the guidance of the axon of AVM. As many as
80% of the double null mutants display guidance defects, which is twice
as many as in each of the single mutants, indicating that mau-2 and
slt-1 function in parallel pathways. Importantly, the AVM axon
projects dorsally in a significant number of animals in which both
mau-2 and slt-1 activities are perturbed. This was observed
in the mau-2; slt-1 double mutants, as well as in mau-2
mutants misexpressing slt-1 in all body wall muscles [slt-1
is normally secreted only from dorsal muscles to repel the AVM axon
(Hao et al., 2001
)]. This
indicates that slt-1 and mau-2 act redundantly to prevent
the dorsal projection of AVM.
The dorsal projection of AVM is a guidance defect that has not previously
been reported for any single or double mutant that affects the guidance of the
AVM axon. In fact, mutations in the genes unc-6, unc-40, slt-1, sax-3,
unc-34 and mau-2 that affect the guidance of AVM, result in the
lateral projection of AVM in 25-40% of the cases
(Hedgecock et al., 1990
;
Takagi et al., 1997
;
Yu et al., 2002
). In these
mutants, when the axon of AVM fails to project ventrally, it instead extends
anteriorly. The AVM axon can project posteriorly as well in the single mutants
mau-2 and unc-6 [in
7-10% of the animals
(Takagi et al., 1997
;
Yu et al., 2002
)]. In
addition, the axon of AVM can extend posteriorly in animals that misexpress
slt-1 in all body wall muscles
(Hao et al., 2001
). The axon
of AVM has been observed to migrate dorsally and reach the dorsal cord, and
then turn anteriorly along the dorsal cord in animals that misexpress UNC-5 in
AVM under the control of a heterologous promoter
(Hamelin et al., 1993
). UNC-5
is a netrin receptor that mediates the repulsion of a growth cone of migrating
motor axons away from the netrin source, but is not expressed in AVM
(Killeen et al., 2002
;
Su et al., 2000
) and does not
appear to normally participate in the guidance of the AVM axon
(Leung-Hagesteijn et al.,
1992
). Thus, our analysis of the interactions between
mau-2 and slt-1 reveals that the AVM axon is capable of
migrating dorsally as a result of the loss of function of genes implicated in
its guidance. Our results also indicate that the ventral guidance of AVM does
not only rely on unc-6 and slt-1, as previously reported
(Yu et al., 2002
).
Our results furthermore indicate the existence of a new mechanism, involving mau-2, that functions in parallel to slt-1 to repel AVM from the dorsal area of the body wall (Fig. 6C). The effects of perturbing both mau-2 and slt-1 gene activities are observed even in the presence of unc-6(+) activity, indicating that the unc-6 mediated attraction of AVM is insufficient to prevent dorsal migration by itself. Although we could not fully characterize the interactions between mau-2 and unc-6, it appears that, unc-6-mediated attraction, slt-1-mediated repulsion and a mechanism involving mau-2, are all partially redundant in the ventral guidance of AVM. It is also possible that the mechanism involving mau-2 may impinge upon both the unc-6 pathway (for example, upstream of unc-40) and the slt-1 pathways, rather than being a fully independent pathway.
It will be interesting to find out how mau-2 participates in the
dorsoventral guidance of the AVM axon and, specifically, to determine if it is
involved in a guidance mechanism entirely distinct from those of netrin and
slit. For example, one question is whether mau-2 might interact with
clr-1, a receptor tyrosine phosphatase that has been recently
implicated in the regulation of netrin signalling
(Chang et al., 2004).
Maternal expression of mau-2
The zygotic activity of mau-2 is sufficient for the normal
development of the worm. Indeed, heterozygous animals produced by a homozygous
mutant mother are completely wild type. In addition, functional
mau-2::gfp transgene expression is first detected as late as by
mid-embryogenesis, which is well passed the time at which the zygotic genome
is turned on [at the onset of gastrulation
(Edgar et al., 1994;
Powell-Coffman et al., 1996
;
Kaltenbach et al., 2000
)].
However, mau-2 mutants can be profoundly maternally rescued, and high
levels of mau-2 transcript are loaded into the oocytes and still
detectable in the very early embryo. Why is mau-2 maternally
contributed at all?
It is possible that mau-2 might play role in very early development that may be masked by the function of redundant, yet non-homologous, genes. It is also conceivable that the maternal contribution of mau-2 serves to ensure a sufficient level of mau-2 expression at the time of the start of mau-2 expression in the zygote. The effect of such a stabilization might not easily be detected in the experimental setting, but might be of sufficient magnitude to have been evolutionarily favoured.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/131/23/5947/DC1
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