Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3204, USA
Author for correspondence (e-mail:
garriga{at}berkeley.edu)
Accepted 1 August 2005
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SUMMARY |
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Key words: Caenorhabditis elegans, Cell migration, FGF, FGFR, Repellent
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Introduction |
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In vertebrates, homologs of UNC-6 and SLT-1 proteins collaborate in
different ways to coordinate growth cone migrations along the neuroaxis. As
with C. elegans UNC-6, vertebrate Netrins are expressed ventrally and
attract commissural axons to the vertebrate floorplate
(Kennedy et al., 1994;
Serafini et al., 1994
). Sonic
hedgehog, which is expressed in the floorplate and functions as a morphogen to
pattern the ventral spinal cord, also attracts commissural axons ventrally
(Charron et al., 2003
). Slit
coordinates commissural axon guidance at the floorplate, but it does not
collaborate with Netrin and Sonic Hedgehog to guide migrations ventrally.
Instead, Slit expression in the floorplate inhibits commissural axons
expressing its receptor Robo from recrossing the midline
(Brose et al., 1999
;
Long et al., 2004
;
Yuan et al., 1999
;
Zou et al., 2000
). Robo also
forms a complex with the Netrin receptor DCC, inhibiting the ability of DCC to
respond to Netrin and facilitating the extension of these growth cones across
the floorplate (Stein and Tessier-Lavigne,
2001
).
Although molecules that guide migrations along the DV axis have been
identified, the mechanisms that guide migrations along the AP axis are poorly
understood. Wnt proteins, which are conserved secreted glycoproteins that act
as graded morphogens, have recently been shown to guide commissural axons
along the AP axis of the mammalian spinal cord. After crossing the midline,
commissural axons turn anteriorly and extend towards the brain. Several Wnt
proteins have been shown to stimulate commissural growth cones after they have
crossed the midline. These Wnt proteins are expressed in the developing spinal
cord. In particular, Wnt4 RNA is present in a gradient decreasing from
anterior to posterior, placing Wnt4 in a key position to guide commissural
axons anteriorly (Lyuksyutova et al.,
2003).
In C. elegans, the gene vab-8 is both necessary and
sufficient for posteriorly directed migrations of cells and growth cones. Most
posterior migrations require vab-8
(Wightman et al., 1996), and
ectopic expression of vab-8 can reroute anteriorly projected axons
towards the posterior (Wolf et al.,
1998
) (N. Watari-Goshima and G.G., unpublished). The
vab-8 locus encodes at least two novel intracellular proteins that
act in the cells to promote their migration
(Wolf et al., 1998
). How these
proteins regulate these migrations, however, remains unknown.
Unlike VAB-8, the C. elegans transmembrane protein MIG-13 plays a
nonautonomous role in guiding cell migrations along the AP axis
(Sym et al., 1999).
mig-13 loss-of-function alleles display more specific defects than
vab-8 mutants, disrupting the anterior directed migrations of only
the BDU neurons and descendants of the right Q neuroblast. Ectopic expression
of mig-13 from a heat shock promoter, however, induces an anterior
shift in the final positions of neurons that migrate in either direction along
the AP axis, indicating that MIG-13 plays a broader role than was suggested by
the effects of mig-13 mutants. As with VAB-8, the role of MIG-13 in
these migrations remains unclear (Sym et
al., 1999
).
In C. elegans, the fibroblast growth factor (FGF) homolog EGL-17
functions as an attractant for the precise positioning of the anteriorly
directed migrations of the sex myoblasts (SMs)
(Burdine et al., 1998;
Burdine et al., 1997
). In early
larval stages, the SMs migrate from the posterior midbody to positions
flanking the center of the gonad (Sulston
and Horvitz, 1977
). During SM migration, EGL-17 is expressed in
the primary vulval precursor cells (VPCs) and the dorsal uterine (DU) cells of
the somatic gonad, which define the final destination of the SMs
(Branda and Stern, 2000
;
Burdine et al., 1998
). EGL-17
signals through the FGF receptor (FGFR) EGL-15 to attract SMs
(Burdine et al., 1998
;
DeVore et al., 1995
).
In an effort to understand AP guidance in C. elegans, we have
focused on the posterior migrations of the CANs, a pair of bilaterally
symmetric neurons that are born in the head and migrate to the middle of the
embryo (Sulston et al., 1983).
Although a previous screen for CAN migration mutants identified a number of
genes, none of them encoded guidance cues
(Forrester and Garriga, 1997
;
Forrester et al., 1998
). One
explanation for this outcome is that multiple cues contribute to CAN
migration, and therefore removing one might result in only subtle CAN
migration defects. To test this hypothesis, we used sensitized genetic
backgrounds to re-evaluate the potential role of secreted molecules in CAN
migration. The use of these sensitized backgrounds revealed a role for FGF in
CAN migration.
Although mutations in genes encoding EGL-17/FGF and EGL-15/FGFR had
negligible effects on CAN migration, they enhanced the CAN migration defects
caused by other mutations. Further supporting a role for FGF signaling in CAN
migration, the tyrosine phosphatase receptor CLR-1, which inhibits EGL-15/FGFR
signaling to regulate fluid homeostasis
(Huang and Stern, 2004;
Kokel et al., 1998
), also
inhibited EGL-15/FGFR activity in CAN migration. We also show that EGL-17/FGF
functions as a guidance cue for CAN migration. Two cells at the anterior end
of the embryo express EGL-17/FGF when the CANs migrate, suggesting that
EGL-17/FGF may function as a repellent. Consistent with this hypothesis,
ectopic expression of EGL-17/FGF could repel the CANs from the new sites of
expression. Finally, we provide evidence that EGL-15/FGFR acts in the CANs to
promote their posteriorly directed migrations. Our results raise the
possibility that multiple cues might promote guidance of cells and growth
cones along the C. elegans AP axis.
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Materials and methods |
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LGI: unc-40(e1430)
(Hedgecock et al., 1990).
LGII: clr-1(e1745ts)
(Kokel et al., 1998).
LGIII: let-756(s2887) (Roubin
et al., 1999), unc-32(e189
(Brenner, 1974
).
LGIV: soc-2(n1774)
(Selfors et al., 1998),
let-60(sy101sy127), let-60(n2021), let-60(n1046)
(Beitel et al., 1990
;
Ferguson and Horvitz, 1985
;
Han and Sternberg, 1991
),
mIs11[myo-2::gfp, pes-10::gfp, F22B7.9::gfp].
LGV: unc-34(gm104)
(Forrester and Garriga, 1997),
unc-34(e951) (Brenner,
1974
), vab-8(e1017)
(Manser and Wood, 1990
),
vab-8(gm84) (Wolf et al.,
1998
).
LGX: egl-17(n1377)
(Burdine et al., 1997),
sax-3(ky200ts) (Zallen et al.,
1998
), unc-6(ev400)
(Hedgecock et al., 1990
),
sem-5(n2019) (Clark et al.,
1992
), egl-15(n1477), egl-15(n484), egl-15(n1454)
(DeVore et al., 1995
),
slt-1(eh15) (Hao et al.,
2001
).
The extrachromosomal array otEx1270 with the
dpy-7::egl-15(5A)cDNA transgene, as well as the egl-15
rescuing arrays otEx1254 and otEx1267, and let-765
rescuing arrays otEx1467 and otEx1468, were kindly provided
by O. Hobert (Bulow et al.,
2004). Transgenes generated in this study are described below.
egl-15 and egl-17 constructs and germline transformations
In the egl-15(neu*) construct, the transmembrane domain of EGL-15
was replaced with that of the Neu oncoprotein, which results in constitutive
activity of the EGL-15 receptor (Kokel et
al., 1998). The plasmid NH#420, which contains the
egl-15(neu*) construct, was injected into a soc-2(n1774)
mutant background at a concentration of 50 ng/µl. As co-transformation
markers, the pRF4 plasmid containing the dominant rol-6(su1006)
mutation (Mello et al., 1991
)
and a ttx-3::gfp transgene
(Hobert et al., 1997
) were
co-injected at 50 ng/µl and at 20 ng/µl, respectively. Germline
transformation was conducted using standard procedures described by Mello et
al. (Mello et al., 1991
) to
generate the extrachromosomal array gmEx224. The presence of the
soc-2 mutation was necessary to prevent the lethality caused by
constitutive EGL-15 activity (Kokel et
al., 1998
). As a control, the genomic egl-15 construct
NH#150 (Huang and Stern, 2004
)
was injected into a soc-2(n1774); vab-8(gm84) mutant background at a
concentration of 50 ng/µl with pRF4 at 50 ng/µl and a
ttx-3::gfp transgene at 20 ng/µl.
Global EGL-17 expression was induced using the plasmid NH#402, which
contains hsp::egl-17::gfp (provided by Michael Stern). An
extrachromosomal array was generated by injecting into wild-type
hermaphrodites NH#402 at 50 ng/µl along with pRF4
(Mello et al., 1991) at 20
ng/µl and ttx-3::gfp (Hobert
et al., 1997
) at 20 ng/µl. The resulting extrachromosomal array
gmEx197 was then crossed into a vab-8(gm84) mutant
background. Strains containing the hsp::egl-17::gfp array were scored
at 25°C, a temperature that induced detectable EGL-17::GFP expression.
To express EGL-17 ectopically, we generated a
Pmab-5::egl-17cDNA::gfp translational fusion. The primers
5'-AAGCTTGATTCAGGGTGGTCGACAGT-3' and
5'-AAGCTTAATATTTTAGTTATTAAGATTCCAAGTC-3' were used to amplify by
PCR an 8.8 kb mab-5 promoter fragment with flanking HindIII
sites. The mab-5 promoter was cloned into the HindIII site
of the plasmid pPD95.85 (provided by Andy Fire). The egl-17 cDNA was
amplified by PCR from the plasmid NH#315
(Burdine et al., 1997) using
the primers 5'-TCCCCCCGGGGCTATGCTCAAAGTCCTAC-3', which contains an
XmaI site, and 5'GGCACCGGTGATTTTCTGGATTTCATTGTAC-3',
which contains an AgeI site. The egl-17 cDNA was then
inserted the plasmid pPD95.85 cut with XmaI and AgeI. This
construct was then cut with XmaI and XhoI, and the insert
was cloned into the mab-5/pPD95.85 plasmid cut with the XmaI
and XhoI. The resulting plasmid pTF2, which contains
Pmab-5::egl-17::gfp, was injected into an egl-17(n1377)
mutant background at a concentration of 50 ng/µl with the co-transformation
markers pRF4 (50 ng/µl) and ttx-3::gfp (20 ng/µl). Animals
carrying the resulting extrachromosomal array gmEx256 were then
scored for CAN migration defects.
We also constructed a Plim-4::egl-17::gfp transgene by PCR amplification of a 3.6 kb lim-4 promoter using the primer 5'-AAACTGCAGCGAGTTGAATTAGATGGGC-3', which contains a PstI site, and 5'-TCCCCCCGGGGGTGCAACTTGTGACAGC-3', which contains an XmaI site. The lim-4 promoter was cut with PstI and XmaI, and inserted into pPD95.85 plasmid cut with PstI and XmaI. The egl-17 cDNA in pPD95.85, described previously, was cloned into the lim-4::gfp construct using XmaI and AgeI sites. The extrachromosomal array gmEx244 was generated by injecting egl-17(n1377) mutants with 50 ng/µl of pTF3, which contains Plim-4::egl-17::gfp, and 50 ng/µl of pRF4.
In order to assess whether EGL-15 functions cell autonomously, we used the plasmid NH#1078 (provided by Michael Stern), which contains a 1.2 kb ceh-10 promoter that drives egl-15 genomic DNA. The resulting Pceh-10::egl-15 transgene was injected at 50 ng/µl with the co-transformation markers pRF4 (50 ng/µl) and ttx-3::gfp (20 ng/µl) into a vab-8(gm84); egl-15(n484) mutant background. Animals expressing the extrachromosomal array gmEx280 [ceh-10::egl-15] were then scored for CAN migration defects.
Scoring CAN positions
To determine the extent of CAN migration in wild-type and mutant animals,
the CAN positions were scored relative to the positions of hypodermal nuclei
(V1, P1/2, V2, P3/4, V3, P4/5 and V4) using Normarski optics. Only newly
hatched first larval stage (L1) hermaphrodites were scored. We only scored the
positions of the CANs that had migrated posterior of the V1 cell, because we
could not distinguish CANs located anterior to V1 from other neurons in the
head. When we could not detect a CAN neuron posterior to V1, we scored it as
anterior to V1.
To score the CAN migration defects of strains carrying the larval lethal alleles egl-15(n1456) and let-756(s2887), we observed the progeny of hermaphrodites that were homozygous for these alleles and carried a rescuing egl-15 or let-756 extrachromosomal transgenic array, respectively. The CANs of L1s not expressing ceh-22::gfp, which was used as a co-injection marker for the egl-15 and let-756 rescuing arrays were scored. To ascertain the CAN position of strains with the larval lethal let-60(sy101sy127) allele, we balanced let-60(sy101sy127) with the integrated transgene mIs11 which expresses a gfp cDNA from the myo-2 promoter. We then scored L1s not expressing GFP as let-60(sy101sy127) homozygotes. We mapped mIs11 to within 1 m.u. of let-60.
A two-sample z-test comparing the proportion of CANs in specific positions was used to determine whether two strains showed statistical differences in CAN migration defects. The statistics program StatCrunch was used for the analysis and can be found online at http://www.statcrunch.com/. A P-value of less than 0.01 was considered to be statistically significant. Unless otherwise stated, the P-values shown were determined using a two-sample z-test on the proportion of CANs found in the most anterior position.
Embryonic expression pattern of EGL-17
The transgene ayIs9 contains an integrated array that expresses a
gfp cDNA from a 10.5 kb egl-17 promoter region
(Branda and Stern, 2000). We
also generated a translational fusion that expresses egl-17::gfp from
the 10.5kb egl-17 promoter of ayIs9 (pTF4). This construct
and the co-transformation marker pRF4 were injected at 50 ng/µl each into
wild-type hermaphrodites to create the transgenic array gmEx283. The
embryonic expression pattern of EGL-17 was assessed for both arrays by
visualizing GFP fluorescence on the Zeiss Axioskop2 microscope, and pictures
were obtained using a Hamamatsu ORCA-ER digital camera with Openlab
software.
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Results |
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Although a role for SLT-1 in CAN migration was predicted, EGL-17 had not
been shown previously to play a role in CAN migration. FGF/EGL-17 and its
receptor EGL-15 are essential for the normal migrations of the SMs
(Burdine et al., 1998). Neither
molecule, however, has been shown to function in any additional, long-range
cell migration. Like the egl-17 mutation, mutations in
egl-15 had negligible effects on the CAN migrations. Mutations in
both genes resulted in a similar enhancement of the CAN defects of
vab-8(gm84) mutants (Fig.
1). Enhancement of CAN migration defects by egl-17 and
egl-15 mutations is also observed in the sensitized background of
unc-34(e951) and unc-34(gm104) null alleles
(Fig. 1; data not shown).
unc-34 encodes a member of the Ena/VASP family of proteins and
functions in many cell and growth cone migrations
(Colavita and Culotti, 1998
;
Forrester and Garriga, 1997
;
McIntire et al., 1992
;
Yu et al., 2002
). Enhancement
of unc-34 null alleles indicates that egl-17 and
egl-15 function, at least partially, in parallel to the
unc-34 pathway. Elimination of vab-8 function results in an
almost complete failure of CAN cell migration, precluding us from asking
whether egl-17 or egl-15 mutations enhanced the CAN defects
of mutants lacking all vab-8 activity.
EGL-15/FGFR possesses two genetically distinct functions that are mediated
by different isoforms. EGL-17/FGF attracts the SMs through a specific isoform
of the FGF receptor known as EGL-15(5A), and a second FGF known as LET-756
acts though a different isoform of the receptor known as EGL-15(5B) to
regulate fluid homeostasis and axon outgrowth
(Bulow et al., 2004;
Goodman et al., 2003
;
Huang and Stern, 2004
). The
let-756(s2887) null allele neither displayed CAN migration defects
nor significantly altered the CAN defects of vab-8(gm84) mutants
(Fig. 1). The mutation
egl-15(n484) specifically eliminates the 5A isoform, the
egl-15(n1477ts) mutation reduces the activity of both isoforms, and
the egl-15(n1456) mutation should eliminate all egl-15
function (DeVore et al.,
1995
). All three mutations showed similar enhancement of the CAN
migration defects caused by vab-8(gm84)
(Fig. 1). vab-8(gm84);
egl-17(n1377), egl-15(n484) showed CAN migration defects similar to
vab-8(gm84); egl-17(n1377) and vab-8(gm84); egl-15(n484)
mutants (Fig. 1). Taken
together, these observations indicate that EGL-17/FGF acts through the 5A
isoform of EGL-15/FGFR to promote CAN migration.
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SEM-5/GRB2 is involved in CAN migration
The Src homology 2 (SH2)/SH3 adaptor protein SEM-5/GRB2
(Clark et al., 1992), the
leucine-rich repeat protein SOC-2 (Selfors
et al., 1998
) and the Ras homolog LET-60
(DeVore et al., 1995
) can all
function downstream of EGL-15. Genetic data implicate SEM-5 but not the other
molecules in FGF-mediated SM migration
(DeVore et al., 1995
;
Schutzman et al., 2001
).
sem-5(n2019), soc-2(n1774), let-60(n2021), let-60(sy101sy127) and
let-60(n1046) single mutants showed no CAN defects (Figs
3,
4). We then tested whether the
mutations in sem-5, soc-2 and let-60 could enhance the CAN
defects of vab-8(gm84) mutants. As with the egl-17 and
egl-15 mutations, sem-5(n2019) enhanced the CAN defects of
vab-8(gm84) mutants (Fig.
3). This observation is consistent with but does not prove that
SEM-5 acts as a downstream mediator of EGL-15 in CAN migration.
By contrast, neither the soc-2 nor the let-60 mutations significantly affected CAN migration in a vab-8(gm84) mutant background, suggesting that neither soc-2 nor let-60 function in CAN migration (Figs 3, 4). However, as soc-2(n1774) is a hypomorphic allele we cannot completely rule out soc-2 involvement in CAN migration. Furthermore, let-60(sy101sy127) null mutants arrest as larvae and were scored as progeny of heterozygous hermaphrodites, raising the possibility that a maternal contribution of let-60 may mask its role in CAN migration. Nevertheless, the lack of an effect for either hypomorphic (n2021) or gain-of-function (n1046) alleles that reduce or increase let-60 activity zygotically and maternally supports the hypothesis that let-60 is not involved in CAN migration (Fig. 3).
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To constitutively activate EGL-15, we used the egl-15(neu)
transgene (Kokel et al.,
1998). Because expression of the egl-15(neu) construct
causes lethality, all strains were scored in a soc-2(n1774) mutant,
which suppresses the lethality caused by the egl-15(neu) transgene.
soc-2(n1774) had no effect on CAN migration either alone or in
combination with a vab-8 mutation
(Fig. 4B). Expression of
egl-15(neu) enhanced the vab-8(gm84) CAN migration defects,
while expression of wild-type egl-15 did not
(Fig. 4B).
Increasing EGL-15 activity in a clr-1 mutant suppressed the CAN defects of the vab-8 mutants, but increasing EGL-15 with an activating mutation enhanced the defects. Although these results may appear inconsistent, they support a model where asymmetric activation of EGL-15 by EGL-17 guides the migrating CANs. In this model, EGL-15 is normally activated asymmetrically in the CAN by an EGL-17 gradient. When CLR-1 function is reduced, we propose that normal asymmetric activity of EGL-15 is increased, resulting in the suppression of CAN migration defects. We propose that the activating mutation increases all EGL-15 activity, masking the asymmetric activation by EGL-15 and leading to an enhancement of CAN migration defects. The effects of the activated EGL-15 receptor and global EGL-17 expression on CAN migration suggest that both the asymmetric activation of EGL-15 and the distribution of EGL-17 are important for CAN guidance.
EGL-17 is a CAN repellent
The distribution of endogenous egl-17 expression during the time
of CAN migration may address whether EGL-17 attracts or repels the CANs. We
analyzed the GFP expression pattern of animals carrying ayIs9, an
egl-17::gfp transcriptional reporter transgene
(Branda and Stern, 2000), and
gmEx283, a functional egl-17::gfp translational reporter
transgene that can rescue the enhancement of vab-8(gm84) CAN
migration defects caused by egl-17(n1377)
(Fig. 5A). At the time when the
CANs are migrating, we detected GFP in only two hyp5 hypodermal cells
at the anterior tip of the embryo (Fig.
5B-D). This expression pattern suggests that EGL-17 repels the
CANs. To test this hypothesis, we expressed egl-17 ectopically both
at the anterior end of the embryo using the lim-4 promoter
(Plim-4::egl-17::gfp) and in the posterior midbody using a
mab-5 promoter (Pmab-5::egl-17::gfp)
(Fig. 6A). The LIM homeodomain
LIM-4 functions in the cell-fate specification of olfactory neurons.
lim-4 is expressed in the AWB neurons as well as a few other head
neurons (Sagasti et al.,
1999
). mab-5 encodes a homolog of Drosophila
Antennapedia, and its expression in the posterior midbody region of the worm
is required for proper epidermal, neuronal and mesodermal cell differentiation
(Costa et al., 1988
;
Wang et al., 1993
). If our
hypothesis that EGL-17 acts as a CAN repellent is correct, then the
Plim-4::egl-17::gfp transgene should promote normal CAN migration,
while the Pmab-5::egl-17::gfp transgene should hinder migration. As
predicted by the repellent model, the Plim-4::egl-17::gfp transgene
partially rescued the CAN migration defects of vab-8(gm84);
egl-17(n1377) double mutants, while the Pmab-5::egl-17::gfp
transgene enhanced the CAN migration defects of vab-8(gm84) mutants
(Fig. 6B). These experiments
suggest that EGL-17 can repel the CANs.
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Discussion |
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FGFs play multiple roles in development
FGFs and their receptors are highly conserved proteins involved in a wide
variety of developmental events that include patterning, differentiation and
morphogenetic movements. In Drosophila, the FGF Branchless and its
receptor Breathless can provide different functions for tracheal cell
migrations. Branchless can function through Breathless as an attractant for
primary branching in tracheal development and then as a permissive cue for
secondary branches. Later, Branchless is an inducer and chemoattractant for
terminal branches (Jarecki et al.,
1999; Lee et al.,
1996
).
Two different FGFs act as guidance cues during chick gastrulation. FGF8
expression along the primitive streak repels cells away from the streak,
whereas FGF4 expression in the head process and notochord attracts cells
emerging from the anterior streak (Yang et
al., 2002). Cells expressing a dominant negative FGFR1 fail to
respond to FGF4 attraction, suggesting that FGFR1 may function as a receptor
for FGF4 (Yang et al.,
2002
).
C. elegans has a single FGFR, EGL-15, and two FGFs, EGL-17 and
LET-756. EGL-15/FGFR has at least five functions: an essential function in
fluid balance regulation revealed by the larval lethality of egl-15
null mutants; a guidance function in attracting the migrating SMs; a role in
axon outgrowth; a role in maintenance of axon bundles; a guidance function in
repelling the migrating CANs (Bulow et al.,
2004; DeVore et al.,
1995
; Stern and Horvitz,
1991
). EGL-15/FGFR acts in the hypodermis, an epithelial cell type
that encases the worm, to regulate fluid homeostasis, axon outgrowth and the
maintenance of axon bundle integrity (Bulow
et al., 2004
; Huang and Stern,
2004
).
FGF signaling in CAN migration
Our results and the studies on SM migrations demonstrate that a single FGF
and a single FGFR can mediate both attraction and repulsion
(Burdine et al., 1998;
DeVore et al., 1995
;
Stern and Horvitz, 1991
). It
is not unusual for the same guidance cue to function both as an attractant and
a repellent. UNC-6/Netrin, for example, functions as a repellent for cells and
growth cones that migrate dorsally and as an attractant for cells and growth
cones that migrate ventrally (Chan et al.,
1996
; Colamarino and
Tessier-Lavigne, 1995
). Even the same cells can respond
differently to the same cue at different times during development. Migrating
mesodermal cells in Drosophila are first repelled by Slit expression
at the midline and later attracted towards Slit expression at muscle
attachment sites (Kramer et al.,
2001
).
Two mechanisms appear to regulate how migrating cells and growth cones
respond to a guidance cue. UNC-6/Netrin can attract or repel migrating cells
and growth cones depending on the types of receptors that are expressed.
Expression of the UNC-40 homolog DCC, for example, results in an attractive
response, while expression of the UNC5 receptor and DCC results in repulsion
(Hong et al., 1999).
Consistent with a conserved role for this mechanism, repulsion can be mediated
in Xenopus spinal axons by Netrin-induced UNC5/DCC heterodimers.
Alternatively, intracellular molecules can alter the response to a guidance
cue. Netrin, for example, can either attract or repel the growth cones of
cultured embryonic Xenopus spinal neurons depending on the levels of
cAMP within the neuron (Ming et al.,
1997
). cGMP levels play a similar role for other guidance cues
(Xiang et al., 2002
).
Alternate splicing produces two distinct EGL-15 isoforms (5A and 5B) that
contain distinct extracellular domains
(Goodman et al., 2003).
LET-756 signals through the EGL-15(5B) to regulate fluid homeostasis and axon
outgrowth, while EGL-17 signals through EGL-15(5A) to attract the SMs
(Bulow et al., 2004
;
Goodman et al., 2003
).
Although each receptor isoform mediates the response to a distinct FGF, the
different isoforms are capable of responding to either FGF. egl-17
driven from the let-756 promoter can rescue the larval lethality of a
let-756 mutant and, conversely, let-756 driven from the
egl-17 promoter can rescue the SM migration defects of an
egl-17 mutant (Goodman et al.,
2003
). We found that the 5A isoform mutant egl-15(n484)
enhanced CAN migration defects of a vab-8 mutant to a similar extent
as egl-15(n1456) null mutants, which affect both 5A and 5B isoforms.
In addition, a let-756 null allele failed to significantly enhance
vab-8(gm84) CAN migration defects. Therefore, EGL-17 appears to
function through the 5A isoform as both an attractant and as a repellent.
However, it is unclear whether different co-receptors, the levels of cyclic
nucleotides or yet another mechanism determines the attractive or repulsive
functions of EGL-17.
We have shown that similarities and differences exist in downstream
components of EGL-15/FGFR signaling when comparing CAN migration and the other
processes mediated by this pathway. In addition to acting downstream of EGL-15
in SM migration and fluid balance regulation
(Clark et al., 1992), the
adaptor protein SEM-5 is also involved in CAN migration. SOC-2, a leucine-rich
repeat protein, and LET-60/Ras both act downstream of EGL-15 in fluid
homeostasis, and LET-60/Ras functions downstream of EGL-15 in axon outgrowth
(DeVore et al., 1995
;
Selfors et al., 1998
).
However, neither SOC-2 nor LET-60/Ras appears to be involved in directing SM
or CAN migration (DeVore et al.,
1995
) (this study).
Defining additional downstream components of EGL-17/FGF will be important
to determine how EGL-17 can act both as an attractant and a repellent.
Phospholipase C (PLC
) and phosphoinositide 3-kinase (PI3-kinase)
could potentially function downstream of EGL-15/FGFR. Coactivation of
PLC
and PI3-kinase are required for the chemoattraction towards nerve
growth factor (NGF) by Xenopus spinal neurons expressing exogenous
rat TrkA, a receptor tyrosine kinase (Ming
et al., 1999
). Perhaps PLC
and PI3-kinase also contribute
to the signaling of EGL-15 to promote attraction of the SMs or repulsion of
the CANs.
The receptor tyrosine phosphatase CLR-1 inhibits EGL-15/FGFR signaling
Although CLR-1 does not have any obvious role in SM migration, we have
found that, as with fluid homeostasis, CLR-1 inhibits EGL-15/FGFR signaling in
CAN migration (Kokel et al.,
1998). CLR-1 also inhibits UNC-6/Netrin signaling of the C.
elegans AVM growth cones (Chang et
al., 2004
). UNC-34/ENA and the Rac CED-10 function in parallel
downstream of the UNC-40/DCC receptor to mediate UNC-6/Netrin attraction
(Gitai et al., 2003
).
Additionally, CLR-1 mediated inhibition of UNC-40/DDC signaling requires
UNC-34/Ena (Chang et al.,
2004
). This observation differs from our finding that in CAN
migration CLR-1 and UNC-34/Ena can act independently.
EGL-17 may be one of several guidance cues for CAN migration
Although our results show that EGL-17 and its receptor EGL-15 function in
CAN migration, the role of each gene is only revealed in sensitized genetic
backgrounds. Why does FGF signaling play such a subtle role in CAN migration?
One observation that may be relevant to this question is that no other
guidance cue or receptor has been shown to be necessary for CAN cell
migration. Although two receptors, the INA-1/PAT-3 integrin and the RTK CAM-1,
function in the CAN to promote its migration, these molecules are unlikely to
act as guidance receptors. The ligand for INA-1/PAT-3 appears to be the
permissive substrate laminin (Baum and
Garriga, 1997), and the kinase domain of CAM-1 is not involved in
its migration functions (Forrester et al.,
1999
). In fact, the extracellular CRD domain of CAM-1 is both
necessary and sufficient for its role in CAN migration, and genetic
experiments suggest that CAM-1 functions in cell migration by inhibiting Wnt
function (Forrester et al.,
2005
; Kim and Forrester,
2003
).
The CANs migrate normally in C. elegans that are mutant for the
homologs of the guidance cues Netrin and Slit, and in worms that are mutant
for the guidance receptor UNC-40. SLT-1/Slit and its receptor SAX-3/Robo, by
contrast, play a subtle role in CAN migration. Like egl-17 and
egl-15 mutations, slt-1 and sax-3 mutations have
little effect on CAN migration in an otherwise wild-type background, but can
alter CAN migration in a sensitized background that contains a
ceh-23::gfp transgene or a vab-8 mutation
(Hao et al., 2001) (this
study). At the time when the CANs migrate, SLT-1 is expressed at the anterior
end of the embryo, consistent with it acting as a repellent. Taken together,
the FGF and SLT-1 results are consistent with a model in which several
different cues guide the CANs to their final destinations. Removing any
specific cue has little effect on CAN migration because the remaining cues are
able to provide adequate guidance information. This model predicts that
genetically sensitized backgrounds are needed to identify the molecules that
guide the CANs and perhaps other neurons that migrate along the
anteroposterior axis.
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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---|
Baum, P. D. and Garriga, G. (1997). Neuronal migrations and axon fasiculation are disrupted in ina-1 integrin mutants. Neuron 19,51 -62.[CrossRef][Medline]
Beitel, G. J., Clark, S. G. and Horvitz, H. R. (1990). Caenorhabditis elegans ras gene let-60 acts as a switch in the pathway of vulval induction. Nature 348,503 -509.[CrossRef][Medline]
Branda, C. S. and Stern, M. J. (2000). Mechanisms controlling sex myoblast migration in Caenorhabditis elegans hermaphrodites. Dev. Biol. 226,137 -151.[CrossRef][Medline]
Brenner, S. (1974). The genetics of
Caenorhabditis elegans. Genetics
77, 71-94.
Brose, K., Bland, K. S., Wang, K. H., Arnott, D., Henzel, W., Goodman, C. S., Tessier-Lavigne, M. and Kidd, T. (1999). Slit proteins bind Robo receptors and have an evolutionarily conserved role in repulsive axon guidance. Cell 96,795 -806.[CrossRef][Medline]
Bulow, H. E., Boulin, T. and Hobert, O. (2004). Differential functions of the C. elegans FGF receptor in axon outgrowth and maintenance of axon position. Neuron 42,367 -374.[CrossRef][Medline]
Burdine, R. D., Chen, E. B., Kwok, S. F. and Stern, M. J.
(1997). egl-17 encodes an invertebrate fibroblast growth factor
family member required specifically for sex myoblast migration in
Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA
94,2433
-2437.
Burdine, R. D., Branda, C. S. and Stern, M. J.
(1998). EGL-17(FGF) expression coordinates the attraction of the
migrating sex myoblasts with vulval induction in C. elegans.
Development 125,1083
-1093.
Chan, S. S., Zheng, H., Su, M. W., Wilk, R., Killeen, M. T., Hedgecock, E. M. and Culotti, J. G. (1996). UNC-40, a C. elegans homolog of DCC (Deleted in Colorectal Cancer), is required in motile cells responding to UNC-6 netrin cues. Cell 87,187 -195.[CrossRef][Medline]
Chang, C., Yu, T. W., Bargmann, C. I. and Tessier-Lavigne,
M. (2004). Inhibition of netrin-mediated axon attraction by a
receptor protein tyrosine phosphatase. Science
305,103
-106.
Charron, F., Stein, E., Jeong, J., McMahon, A. P. and Tessier-Lavigne, M. (2003). The morphogen sonic hedgehog is an axonal chemoattractant that collaborates with netrin-1 in midline axon guidance. Cell 113,11 -23.[CrossRef][Medline]
Clark, S. G., Stern, M. J. and Horvitz, H. R. (1992). C. elegans cell-signalling gene sem-5 encodes a protein with SH2 and SH3 domains. Nature 356,340 -344.[CrossRef][Medline]
Colamarino, S. A. and Tessier-Lavigne, M. (1995). The axonal chemoattractant netrin-1 is also a chemorepellent for trochlear motor axons. Cell 81,621 -629.[CrossRef][Medline]
Colavita, A. and Culotti, J. G. (1998). Suppressors of ectopic UNC-5 growth cone steering identify eight genes involved in axon guidance in Caenorhabditis elegans. Dev. Biol. 194,72 -85.[CrossRef][Medline]
Costa, M., Weir, M., Coulson, A., Sulston, J. and Kenyon, C. (1988). Posterior pattern formation in C. elegans involves position-specific expression of a gene containing a homeobox. Cell 55,747 -756.[CrossRef][Medline]
DeVore, D. L., Horvitz, H. R. and Stern, M. J. (1995). An FGF receptor signaling pathway is required for the normal cell migrations of the sex myoblasts in C. elegans hermaphrodites. Cell 83,611 -620.[CrossRef][Medline]
Ferguson, E. L. and Horvitz, H. R. (1985).
Identification and characterization of 22 genes that affect the vulval cell
lineages of the nematode Caenorhabditis elegans.
Genetics 110,17
-72.
Forrester, W. C. and Garriga, G. (1997). Genes
necessary for C. elegans cell and growth cone migrations.
Development 124,1831
-1843.
Forrester, W. C., Perens, E., Zallen, J. A. and Garriga, G.
(1998). Identification of Caenorhabditis elegans genes required
for neuronal differentiation and migration. Genetics
148,151
-165.
Forrester, W. C., Dell, M., Perens, E. and Garriga, G. (1999). A C. elegans Ror receptor tyrosine kinase regulates cell motility and asymmetric cell division. Nature 400,881 -885.[CrossRef][Medline]
Forrester, W. C., Kim, C. and Garriga, G. (2005). The C. elegans Ror RTK CAM-1 inhibits EGL-20/Wnt signalling in cell migration. Genetics 168,1951 -1962.[CrossRef]
Gilleard, J. S., Barry, J. D. and Johnstone, I. L. (1997). cis regulatory requirements for hypodermal cell-specific expression of the Caenorhabditis elegans cuticle collagen gene dpy-7. Mol. Cell. Biol. 17,2301 -2311.[Abstract]
Gitai, Z., Yu, T. W., Lundquist, E. A., Tessier-Lavigne, M. and Bargmann, C. I. (2003). The netrin receptor UNC-40/DCC stimulates axon attraction and outgrowth through enabled and, in parallel, Rac and UNC-115/AbLIM. Neuron 37, 53-65.[CrossRef][Medline]
Goodman, S. J., Branda, C. S., Robinson, M. K., Burdine, R. D.
and Stern, M. J. (2003). Alternative splicing
affecting a novel domain in the C. elegans EGL-15 FGF receptor confers
functional specificity. Development
130,3757
-3766.
Guan, K. L. and Rao, Y. (2003). Signalling mechanisms mediating neuronal responses to guidance cues. Nat. Rev. Neurosci. 4,941 -956.[Medline]
Hamelin, M., Zhou, Y., Su, M. W., Scott, I. M. and Culotti, J. G. (1993). Expression of the UNC-5 guidance receptor in the touch neurons of C. elegans steers their axons dorsally. Nature 364,327 -330.[CrossRef][Medline]
Han, M. and Sternberg, P. W. (1991). Analysis of dominant-negative mutations of the Caenorhabditis elegans let-60 ras gene. Genes Dev. 5,2188 -2198.[Abstract]
Hao, J. C., Yu, T. W., Fujisawa, K., Culotti, J. G., Gengyo-Ando, K., Mitani, S., Moulder, G., Barstead, R., Tessier-Lavigne, M. and Bargmann, C. I. (2001). C. elegans slit acts in midline, dorsal-ventral, and anterior-posterior guidance via the SAX-3/Robo receptor. Neuron 32, 25-38.[CrossRef][Medline]
Hawkins, N. C. and McGhee, J. D. (1990). Homeobox containing genes in the nematode Caenorhabditis elegans. Nucleic Acids Res. 18,6101 -6106.[Abstract]
Hedgecock, E. M., Culotti, J. G. and Hall, D. H. (1990). The unc-5, unc-6, and unc-40 genes guide circumferential migrations of pioneer axons and mesodermal cells on the epidermis in C. elegans. Neuron 4,61 -85.[CrossRef][Medline]
Hobert, O., Mori, I., Yamashita, Y., Honda, H., Ohshima, Y., Liu, Y. and Ruvkun, G. (1997). Regulation of interneuron function in the C. elegans thermoregulatory pathway by the ttx-3 LIM homeobox gene. Neuron 19,345 -357.[CrossRef][Medline]
Hong, K., Hinck, L., Nishiyama, M., Poo, M. M., Tessier-Lavigne, M. and Stein, E. (1999). A ligand-gated association between cytoplasmic domains of UNC5 and DCC family receptors converts netrin-induced growth cone attraction to repulsion. Cell 97,927 -941.[CrossRef][Medline]
Huang, P. and Stern, M. J. (2004). FGF
signaling functions in the hypodermis to regulate fluid balance in C. elegans.
Development 131,2595
-2604.
Ishii, N., Wadsworth, W. G., Stern, B. D., Culotti, J. G. and Hedgecock, E. M. (1992). UNC-6, a laminin-related protein, guides cell and pioneer axon migrations in C. elegans. Neuron 9,873 -881.[CrossRef][Medline]
Jarecki, J., Johnson, E. and Krasnow, M. A. (1999). Oxygen regulation of airway branching in Drosophila is mediated by branchless FGF. Cell 99,211 -220.[CrossRef][Medline]
Kennedy, T. E., Serafini, T., de la Torre, J. R. and Tessier-Lavigne, M. (1994). Netrins are diffusible chemotropic factors for commissural axons in the embryonic spinal cord. Cell 78,425 -435.[CrossRef][Medline]
Kim, C. and Forrester, W. C. (2003). Functional analysis of the domains of the C elegans Ror receptor tyrosine kinase CAM-1. Dev. Biol. 264,376 -390.[CrossRef][Medline]
Kokel, M., Borland, C. Z., DeLong, L., Horvitz, H. R. and Stern,
M. J. (1998). clr-1 encodes a receptor tyrosine phosphatase
that negatively regulates an FGF receptor signaling pathway in Caenorhabditis
elegans. Genes Dev. 12,1425
-1437.
Kramer, S. G., Kidd, T., Simpson, J. H. and Goodman, C. S.
(2001). Switching repulsion to attraction: changing responses to
slit during transition in mesoderm migration. Science
292,737
-740.
Lee, T., Hacohen, N., Krasnow, M. and Montell, D. J. (1996). Regulated Breathless receptor tyrosine kinase activity required to pattern cell migration and branching in the Drosophila tracheal system. Genes Dev. 10,2912 -2921.[Abstract]
Long, H., Sabatier, C., Le, M., Plump, A., Yuan, W., Ornitz, D. M., Tamada, A., Murakami, F., Goodman, C. S. and Tessier-Lavigne, M. (2004). Conserved roles for slit and robo proteins in midline commissural axon guidance. Neuron 42,213 -223.[CrossRef][Medline]
Lyuksyutova, A. I., Lu, C. C., Milanesio, N., King, L. A., Guo,
N., Wang, Y., Nathans, J., Tessier-Lavigne, M. and Zou, Y.
(2003). Anterior-posterior guidance of commissural axons by
Wnt-frizzled signaling. Science
302,1984
-1988.
Manser, J. and Wood, W. B. (1990). Mutations affecting embryonic cell migrations in Caenorhabditis elegans. Dev. Genet. 11,49 -64.[CrossRef][Medline]
McIntire, S. L., Garriga, G., White, J., Jacobson, D. and Horvitz, H. R. (1992). Genes necessary for directed axonal elongation or fasciculation in C. elegans. Neuron 8, 307-322.[CrossRef][Medline]
Mello, C. C., Kramer, J. M., Stinchcomb, D. and Ambros, V. (1991). Efficient gene transfer in C.elegans: extrachromosomal maintenance and integration of transforming sequences. EMBO J. 10,3959 -3970.[Abstract]
Ming, G. L., Song, H. J., Berninger, B., Holt, C. E., Tessier-Lavigne, M. and Poo, M. M. (1997). cAMP-dependent growth cone guidance by netrin-1. Neuron 19,1225 -1235.[CrossRef][Medline]
Ming, G., Song, H., Berninger, B., Inagaki, N., Tessier-Lavigne, M. and Poo, M. (1999). Phospholipase C-gamma and phosphoinositide 3-kinase mediate cytoplasmic signaling in nerve growth cone guidance. Neuron 23,139 -148.[CrossRef][Medline]
Roubin, R., Naert, K., Popovici, C., Vatcher, G., Coulier, F., Thierry-Mieg, J., Pontarotti, P., Birnbaum, D., Baillie, D. and Thierry-Mieg, D. (1999). let-756, a C. elegans fgf essential for worm development. Oncogene 18,6741 -6747.[CrossRef][Medline]
Sagasti, A., Hobert, O., Troemel, E. R., Ruvkun, G. and
Bargmann, C. I. (1999). Alternative olfactory neuron fates
are specified by the LIM homeobox gene lim-4. Genes
Dev. 13,1794
-1806.
Schutzman, J. L., Borland, C. Z., Newman, J. C., Robinson, M.
K., Kokel, M. and Stern, M. J. (2001). The
Caenorhabditis elegans EGL-15 signaling pathway implicates a DOS-like
multisubstrate adaptor protein in fibroblast growth factor signal
transduction. Mol. Cell. Biol.
21,8104
-8116.
Selfors, L. M., Schutzman, J. L., Borland, C. Z. and Stern, M.
J. (1998). soc-2 encodes a leucine-rich repeat protein
implicated in fibroblast growth factor receptor signaling. Proc.
Natl. Acad. Sci. USA 95,6903
-6908.
Serafini, T., Kennedy, T. E., Galko, M. J., Mirzayan, C., Jessell, T. M. and Tessier-Lavigne, M. (1994). The netrins define a family of axon outgrowth-promoting proteins homologous to C. elegans UNC-6. Cell 78,409 -424.[CrossRef][Medline]
Stein, E. and Tessier-Lavigne, M. (2001).
Hierarchical organization of guidance receptors: silencing of netrin
attraction by slit through a Robo/DCC receptor complex.
Science 291,1928
-1938.
Stern, M. J. and Horvitz, H. R. (1991). A normally attractive cell interaction is repulsive in two C. elegans mesodermal cell migration mutants. Development 113,797 -803.[Abstract]
Sulston, J. E. and Horvitz, H. R. (1977). Post-embryonic cell lineages of the nematode, Caenorhabditis elegans. Dev. Biol. 56,110 -156.[CrossRef][Medline]
Sulston, J. E., Schierenberg, E., White, J. G. and Thomson, J. N. (1983). The embryonic cell lineage of the nematode Caenorhabditis elegans. Dev. Biol. 100,64 -119.[CrossRef][Medline]
Svendsen, P. C. and McGhee, J. D. (1995). The
C. elegans neuronally expressed homeobox gene ceh-10 is closely related to
genes expressed in the vertebrate eye. Development
121,1253
-1262.
Sym, M., Robinson, N. and Kenyon, C. (1999). MIG-13 positions migrating cells along the anteroposterior body axis of C. elegans. Cell 98,25 -36.[CrossRef][Medline]
Tessier-Lavigne, M. and Goodman, C. S. (1996).
The molecular biology of axon guidance. Science
274,1123
-1133.
Wadsworth, W. G., Bhatt, H. and Hedgecock, E. M. (1996). Neuroglia and pioneer neurons express UNC-6 to provide global and local netrin cues for guiding migrations in C. elegans. Neuron 16,35 -46.[CrossRef][Medline]
Wang, B. B., Muller-Immergluck, M. M., Austin, J., Robinson, N. T., Chisholm, A. and Kenyon, C. (1993). A homeotic gene cluster patterns the anteroposterior body axis of C. elegans. Cell 74,29 -42.[CrossRef][Medline]
Wightman, B., Clark, S. G., Taskar, A. M., Forrester, W. C.,
Maricq, A. V., Bargmann, C. I. and Garriga, G. (1996).
The C. elegans gene vab-8 guides posteriorly directed axon outgrowth and cell
migration. Development
122,671
-682.
Wolf, F. W., Hung, M. S., Wightman, B., Way, J. and Garriga, G. (1998). vab-8 is a key regulator of posteriorly directed migrations in C. elegans and encodes a novel protein with kinesin motor similarity. Neuron 20,655 -666.[CrossRef][Medline]
Xiang, Y., Li, Y., Zhang, Z., Cui, K., Wang, S., Yuan, X. B., Wu, C. P., Poo, M. M. and Duan, S. (2002). Nerve growth cone guidance mediated by G protein-coupled receptors. Nat. Neurosci. 5,843 -848.[CrossRef][Medline]
Yang, X., Dormann, D., Munsterberg, A. E. and Weijer, C. J. (2002). Cell movement patterns during gastrulation in the chick are controlled by positive and negative chemotaxis mediated by FGF4 and FGF8. Dev. Cell 3,425 -437.[CrossRef][Medline]
Yu, T. W., Hao, J. C., Lim, W., Tessier-Lavigne, M. and Bargmann, C. I. (2002). Shared receptors in axon guidance: SAX-3/Robo signals via UNC-34/Enabled and a Netrin-independent UNC-40/DCC function. Nat. Neurosci. 5,1147 -1154.[CrossRef][Medline]
Yuan, W., Zhou, L., Chen, J. H., Wu, J. Y., Rao, Y. and Ornitz, D. M. (1999). The mouse SLIT family: secreted ligands for ROBO expressed in patterns that suggest a role in morphogenesis and axon guidance. Dev. Biol. 212,290 -306.[CrossRef][Medline]
Zallen, J. A., Yi, B. A. and Bargmann, C. I. (1998). The conserved immunoglobulin superfamily member SAX-3/Robo directs multiple aspects of axon guidance in C. elegans. Cell 92,217 -227.[CrossRef][Medline]
Zou, Y., Stoeckli, E., Chen, H. and Tessier-Lavigne, M. (2000). Squeezing axons out of the gray matter: a role for slit and semaphorin proteins from midline and ventral spinal cord. Cell 102,363 -375.[CrossRef][Medline]
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