1 Department of Oncogene Research, Research Institute for Microbial Diseases,
Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan
2 RIKEN Center for Developmental Biology, Chuo-ku, Kobe 650-0047, Japan
3 Department of Biology, Faculty of Sciences, Kyushu University Graduate School,
Hakozaki, Fukuoka 812-8581, Japan
Author for correspondence (e-mail:
okadam{at}biken.osaka-u.ac.jp)
Accepted 23 September 2005
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SUMMARY |
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Key words: Src family tyrosine kinase, SRC-1, Cell migration, Axon guidance, C. elegans
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Introduction |
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The gonad of a Caenorhabditis elegans hermaphrodite is located in
the anterior-right and posterior-left areas of the body cavity
(Fig. 1A). This bilobed gonad
develops during larval development from a 4-cell primordium positioned in the
ventral midbody. The shape of the two gonad arms is determined by the
migratory path of a distal tip cell (DTC) at the leading edge of each arm
(Kimble and Hirsh, 1979;
Hedgecock et al., 1987
). DTC
migration proceeds through three sequential linear phases
(Fig. 1B). Phase I is the
centrifugal migration along the ventral bands of the body wall muscles away
from the midbody. Phase II begins with a right-angled turn of DTC, after which
it migrates along the inner surface of the epidermis from the ventral to the
dorsal muscle bands. Finally, in phase III, DTC makes another right-angled
turn and then migrates centripetally along the dorsal muscle bands back
towards the midbody (Fig. 1B)
(Kimble and Hirsh, 1979
;
Hedgecock et al., 1987
).
DTC migration is regulated by several extracellular guidance cues
(Hedgecock et al., 1987;
Leung-Hagesteijn et al., 1992
;
Blelloch et al., 1999
;
Montell, 1999
;
Lehmann, 2001
). The
ventral-to-dorsal migration of DTC during phase II is mediated in part by the
netrin family protein UNC-6 and its receptors UNC-5 and UNC-40
(Hedgecock et al., 1990
;
Culotti and Merz, 1998
). UNC-5
and UNC-40 are expressed by DTC, whereas UNC-6 is expressed by the ventral
body wall muscles. These observations suggest that UNC-5 and UNC-40 mediate
the chemorepulsion of DTC away from the ventrally expressed UNC-6
(Wadsworth et al., 1996
;
Su et al., 2000
). It has also
been suggested that UNC-129/TGFß is involved in DTC migration during
phase II (Colavita et al.,
1998
; Nash et al.,
2000
). These guidance cues are transduced into cell signaling
pathways to achieve cell migration. One of these pathways is the small G
protein Rac signaling pathway, which is part of the cellular machinery
involved in remodeling the actin cytoskeleton during DTC migration in response
to these guidance cues. Upstream regulators of CED-10/Rac include CED-2/CrkII,
CED-5/Dock180 and CED-12/Elmo, which control DTC migration by regulating the
actin cytoskeleton via an evolutionally conserved mechanism
(Wu and Horvitz, 1998
;
Reddien and Horvitz, 2000
;
Gumienny et al., 2001
;
Wu et al., 2001
;
Zhou et al., 2001
;
Reddien and Horvitz,
2004
).
|
The extracellular cues directing the migration of neuronal cells and growth
cones also modulate the cytoskeletal organization. It has been shown that the
Rac family proteins CED-10, MIG-2 and RAC-2, and their regulator UNC-73/Trio
influence the actin cytoskeleton and regulate the migration of neuronal cells
and growth cones in C. elegans
(Zipkin et al., 1997;
Steven et al., 1998
;
Lundquist et al., 2001
;
Lundquist, 2003
). Furthermore,
the regulation of the actin cytoskeleton by UNC-34/Enabled and UNC-115/abLIM,
which act downstream of UNC-40/DCC, is implicated in the axon guidance of the
AVM and DA/DB motoneurons and the axon pathfinding of the CAN and PDE neurons
(Yu et al., 2002
;
Gitai et al., 2003
;
Struckhoff and Lundquist,
2003
; Chang et al.,
2004
). Although these molecules, together with small G proteins,
are potential effectors that drive cell migration, the signaling pathways that
directly relay the extracellular guidance cues to these effectors of cell
migration are still unclear.
Tyrosine phosphorylation is required for crucial functions in multicellular
animals such as cell differentiation, cell adhesion and migration, axon
guidance and cell-cell communication
(Hunter, 2000). The Src family
of non-receptor protein tyrosine kinases (SFKs) serves as a crucial molecular
switch that transmits extracellular cues into the intracellular tyrosine
phosphorylation events that lead to the cellular responses
(Brown and Cooper, 1996
;
Sicheri and Kuriyan, 1997
;
Thomas and Brugge, 1997
). In
C. elegans, there are two SFK orthologs, src-1 and
src-2/kin-22 (Bei et al.,
2002
; Hirose et al.,
2003
). A deletion allele of src-1, cj293, was originally
isolated by imprecise transposon excision
(Bei et al., 2002
). The
src-1(cj293) allele lacks the SH2 and kinase domains and is
potentially a null allele. Homozygous src-1(cj293) hermaphrodites are
themselves viable but produce inviable embryos, suggesting SRC-1 plays several
essential roles in early development. It has been shown that SRC-1 is required
for the accumulation of tyrosine-phosphorylated proteins at the membrane
boundary between the P2 and EMS cells, and functions in parallel with Wnt/Wg
signaling to specify the endoderm and to orient the division axis of EMS in
the early embryo (Bei et al.,
2002
). However, the functions of SRC-1 in organogenesis and the
development of nervous system, where SRC-1 is abundantly expressed, remain
unknown (Hirose et al.,
2003
).
To address the roles SRC-1 plays in the later stages of C. elegans development, we have characterized homozygous src-1(cj293) hermaphrodites. We found that SRC-1 is essential for directing the migration of DTCs and a subset of neuronal cell bodies, and the growth cone path findings, which are regulated by different guidance cues including UNC-6/netrin. Furthermore, analyses of the genetic interactions between SRC-1 and potential downstream factors revealed that SRC-1 transduces the various extracellular guidance cue that direct cell migration via different pathways that depend on the cell type.
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Materials and methods |
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The hT2 translocation chromosome balances src-1; the
qIs48 insertion onto hT2 allows src-1(cj293)/+
heterozygotes to be distinguished from src-1(cj293) homozygotes by
the presence or absence of the GFP marker inserted onto hT2
(Wang and Kimble, 2001).
Double-mutant strains were constructed by standard methods. The mutations in
the double mutants were confirmed by DNA sequencing or PCR for the
src-1(cj293) mutation.
Phenotypic analysis
Gonad morphology was observed on a 5% agar pad in M9 buffer by Nomarski
differential interference contrast microscopy. The DTC migration pattern was
inferred from the gonadal morphology of young adults at 20°C except as
indicated. At the L1 stage, the positions of the AVM, ALM and PVM neurons were
determined by using mec-7::gfp, muIs32
(Ch'ng et al., 2003), the
position of CAN was determined by using che-23::gfp, kyIs4
(Forrester and Garriga, 1997
),
and the position of HSN was determined by using unc-86::gfp, kyIs179
(Shen and Bargmann, 2003
). The
images of the neuronal cells were captured by a LSM-510 confocal
laser-scanning microscope (Zeiss). The positions of AVM, ALM, CAN, HSN and PVM
were scored on the basis of their positions relative to the V-cell daughters,
as these are stationary landmarks. A defect in the AVM axon morphology was
scored when it failed to extend in the anterior direction after the nerve ring
branch. A defect in the PVM axon morphology was scored when it failed to turn
in the anterior direction after reaching the ventral nerve cord. Defects in
ALM, CAN, HSN and PLM migration were determined by comparisons with their
wild-type morphology. This morphology was defined by electron microscopic
reconstitution of the C. elegans nervous system
(White et al., 1986
).
Feeding RNAi
RNAi was performed essentially as described by Timmons and Fire
(Timmons and Fire, 1998).
After preparing the src-1 RNAi feeding plate, some parental worms
were allowed to lay eggs on the plates for 3-4 hours and were then removed.
The remaining eggs were cultured into young adults and then assessed for gonad
morphology.
|
Transgenic strains
Transgenic lines were generated using standard techniques
(Mello et al., 1991). For
rescue experiments, a lin-44::gfp construct was co-injected as a
marker (50 µg/ml). To rescue the defect in DTC migration, the expression
construct was injected at 1 µg/ml into the src-1/hT2[qIs48]
heterozygote with an injection marker. The defect in DTC migration was then
scored in the src-1(cj293) homozygote carrying the extrachromosomal
array. To rescue the defects in the positioning of AVM cell body,
src-1 or src-1K290M in pPD96.41 was injected at 50 µg/ml
into the src-1/hT2[qIs48] heterozygote with an injection marker. To
rescue the aberrant growth cone migration of PVM, src-1 or
src-1K290M in pPD96.41 was injected at 50 µg/ml into the
src-1/hT2[qIs48] heterozygote with an injection marker. The axon
trajectories of PVM were observed by fluorescence microscopy and were compared
with those in the src-1(cj293) homozygote. For the precocious
expression of unc-5, the plasmid pSU16 (emb-9::unc-5)
construct (a gift from J. G. Culotti and L. Brown) was injected at 1 µg/ml
into the src-1/hT2[qIs48] worms with an injection marker. The gonad
morphology of the resulting heterozygotes (src-1/+) and homozygotes
(src-1/src-1) was then analyzed.
Tissue staining
Adult worms were dissected and fixed essentially as described by Francis et
al. (Francis et al., 1995).
Briefly, the dissected gonads were fixed with 3.7% formaldehyde for 1 hour and
postfixed with 100% methanol for 5 minutes. The specimens were then blocked
with 3% BSA in 0.1% Tween TBS, and stained with anti-phosphotyrosine
monoclonal antibody, 4G10 (Upstate), followed by detection with secondary
antibodies conjugated with Texas Red. The images were captured by confocal
laser-scanning microscopy on a Fluoview FV1000 (Olympus). For the
unc-5 reporter assay, staining for ß-galactosidase activity was
carried out as described previously (Fire
et al., 1990
).
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Results |
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We also analyzed worms that had developed on src-1 RNAi feeding plates. The src-1(RNAi) worms had a similar phenotype to the src-1(cj293) mutants. Observed DTC migrations were classified into three categories (Table 1). The first is the normal migration path [100% in N2, 19% in src-1(cj293), 28% in src-1(RNAi)]. The second type represents the migration path that turns in the opposite direction during the third phase [0% in N2, 16% in src-1(cj293), 8% in src-1(RNAi)]. The third type is the no-turn phenotype, which was the most frequently exhibited phenotype for the src-1(cj293) mutant and the src-1(RNAi) worms [0% in N2, 65% in src-1(cj293), 64% in src-1(RNAi)]. These results raise the possibility that DTCs in the src-1(cj293) mutants are not being controlled by the guidance cues that direct their turning at the appropriate time and position.
|
|
To further examine the contribution of SRC-1 to DTC migration, the
expression of SRC-1 was determined by detecting the expression of a reporter
gene (GFP) expressed under the src-1 promoter. The expression of
src-1 gene was clearly detected in DTCs
(Fig. 5A,B) as described
previously (Hirose et al.,
2003). Furthermore, immunostaining with an anti-phosphotyrosine
antibody (4G10), which specifically recognizes phosphorylated tyrosine
residues of various proteins, revealed high levels of tyrosine phosphorylated
proteins in DTCs (Fig. 5C). By
contrast, DTCs in the src-1(cj293) mutant gave only faint signals
(Fig. 5D), showing that SRC-1
activity is a major source of tyrosine phosphorylation in DTCs.
Genetic interaction of the src-1 gene with other genes involved in DTC migration
To position SRC-1 in a cell signaling pathway involved in DTC migration, we
first analyzed the genetic interaction of src-1 with components of
the Rac pathway, including ced-2/CrkII, ced-5/Dock180,
ced-12/Elmo and ced-10/Rac. Mutations in these genes induce
unregulated turns of DTC migration (Wu and
Horvitz, 1998; Reddien and
Horvitz, 2000
; Gumienny et
al., 2001
; Wu et al.,
2001
; Zhou et al.,
2001
; Reddien and Horvitz,
2004
). We analyzed DTC migration in the following double mutants:
src-1;ced-2, src-1;ced-5, src-1;ced-10 and src-1;ced-12. In
these experiments, the phenotypes of these mutants were evaluated by assessing
the occurrence of typical no-turn phenotype of the src-1(cj293)
mutant (Table 2). The
ced mutations alone did not induce noturn phenotype. However, the
no-turn phenotype of src-1(cj293) mutant was drastically suppressed
in the src-1;ced-2, src-1;ced-5 and src-1 ced-12 double
mutants. The effects were evident in both gonad lobes with the frequency being
decreased from 13%/70% (anterior/posterior lobes) to 2%/13%, 2%/5% and 2%/7%
in the src-1;ced-2, src-1;ced-5 and src-1;ced-12 double
mutants, respectively. These results suggest that SRC-1 potentially acts as a
suppressor of the Rac signaling pathway. Furthermore, in these double mutants,
DTCs turned twice as in wild-type worm, raising the possibility that an
alternative pathway exists that functions in parallel to the
src-1/ced pathway. In src-1;ced-10 double mutant, the
no-turn phenotype was only partially suppressed, with the frequency in the
anteroposterior lobes being 20%/45%, suggesting that other genes function
redundantly with ced-10 in this signaling pathway.
|
|
|
It is well established that the chemorepulsive mechanism mediated by
UNC-6/netrin and its receptors UNC-5 and UNC-40 guide DTC migration,
particularly during phase II (Hedgecock et
al., 1990). The programmed expression of unc-5 in DTCs is
shown to time the turning of DTC migration
(Su et al., 2000
). Mutations
in unc-5(e53) and unc-6(ev400), which are putative null
alleles, cause specific defects in the ventral-to-dorsal migration of DTCs. In
either src-1(cj293);unc-5(e53) mutants or
src-1(cj293);unc-6(ev400) mutants, the no-turn defect characteristic
for src-1(cj293) mutant was not significantly affected
(Table 2). These observations
demonstrate that src-1(cj293) mutation is epistatic to
unc-5(e53) or unc-6(ev400) mutations.
As the timing of DTC turning is affected by regulation of unc-5
expression (Su et al., 2000),
we examined the effect of the src-1(cj293) mutation on the expression
of unc-5 by detecting the unc-5 promoter activity
(unc-5B::lacZ). As shown in Fig.
6B, the src-1(cj293) mutant at the L3 stage expressed
unc-5 at the appropriate time, while still showing the no-turn
phenotype. Furthermore, the precocious turning of DTCs that is induced by the
precocious expression of unc-5 under the emb-9 promoter
(emb-9::unc-5) was substantially decreased from 30%/70%
(anteroposterior lobes) in the src-1(cj293)/+ heterozygotes to 7%/14%
in the src-1(cj293) homozygotes
(Fig. 6C, D;
Table 3). These results suggest
that the src-1 mutant phenotype is epistatic to not only precocious
but also normal DTC turning induced by UNC-5, and that SRC-1 is required for
UNC-5 mediated signaling pathway that directs DTC migration.
|
|
|
Defects in growth cone migration due to the src-1(cj293) mutation
The migration and direction of growth cones are also regulated by specific
extracellular guidance cues. To examine whether SRC-1 is involved in these
processes, we observed the effect of the src-1(cj293) mutation on the
axon trajectories of the AVM, ALM, CAN, HSN, PVM and PLM neurons. In the
src-1(cj293) mutant, apparent defects in the axon trajectory
directions were observed for AVM, ALM, CAN and PVM, with a penetrance of 60%,
21%, 38% and 46%, respectively (Fig.
8). AVM axons failed to extend anteriorly after the nerve ring
branch, while ALM axons were slightly bent, probably because of mislocation of
the ALM cell body to ventral side. In CAN neurons, axons terminated
prematurely and often branched inappropriately. Normally, the growth cones of
PVM neurons make a right-angled turn anteriorly after reaching the ventral
nerve cord (Fig. 8H). In the
src-1(cj293) mutant, however, about half of the PVM growth cones
(46%) turn in the opposite direction (Fig.
8I-K). During this inappropriate migration, a substantial number
of the PVM axons (26%) make a reverse turn in the anterior direction, thus
allowing the growth cones to reach the anterior body
(Fig. 8J). These observations
suggest that the PVM growth cones in the src-1(cj293) mutant fail to
respond to the guidance cues that direct their migration along the
anteroposterior axis. This defect was completely rescued by the expression of
SRC-1 but not the kinase-defective form of SRC-1 under the mec-7
promoter (Table 5), indicating
again the cell-autonomous and kinase-dependent role of SRC-1 in cell
migration. However, as in the case of AVM cell body migration, the
ced-5/-12 mutations did not significantly alter the normal PVM growth
cone migration, nor did they modify the src-1(cj293) phenotype
(Table 5). These observations
suggest that SRC-1 uses different downstream pathway in PVM from the one it
uses in DTCs.
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Discussion |
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In C. elegans, defects in DTC migration have been observed in the
ced-2/CrkII, ced-5/Dock180, ced-10/Rac and
ced-12/Elmo mutants (Wu and
Horvitz, 1998; Reddien and
Horvitz, 2000
; Gumienny et
al., 2001
; Wu et al.,
2001
; Zhou et al.,
2001
; Reddien and Horvitz,
2004
). CED-2, CED-5 and CED-12 form a ternary complex that can
trigger the localized remodeling of the actin cytoskeleton through CED-10. It
has also been shown that the Dock180-Elmo complex functions as a guanine
nucleotide-exchanging factor for Rac, and that CrkII binding enhances the
functions of this complex (Brugnera et al.,
2002
). Src may act upstream of this pathway to regulate cell
migration through the actin cytoskeleton as the binding of CrkII to Dock180 is
enhanced in v-Src transformed 3Y1 cells
(Kiyokawa et al., 1998
).
Furthermore, Hck, a Src family member, binds to Elmo1 through its SH3 domain
in vitro and phosphorylates Elmo1 in cultured cells
(Scott et al., 2002
). These
lines of evidence strongly support our proposal that SRC-1 functionally acts
in the Rac pathway in DTCs. Of the three rac-related genes in C.
elegans (ced-10, mig-2 and rac-2), CED-10 and MIG-2
have been shown to redundantly control the migration of some cells, including
DTCs (Lundquist et al., 2001
;
Wu et al., 2002
). Consistent
with this is our observation that a mutation in either mig-2 or
ced-10 only partially suppressed the no-turn phenotype of DTC
migration in the src-1(cj293) mutant, whereas mutations in ced-2,
ced-5 or ced-12 robustly suppressed this phenotype. These
results suggest that CED-10 and MIG-2 function redundantly downstream of the
CED-2 complex under the control of SRC-1. By contrast, a mutation in the
rac-2 gene enhanced the DTC migration defect in the
src-1(cj293) phenotype, suggesting that RAC-2 is involved in DTC
migration but acts in an alternative pathway independently of SRC-1.
|
Neuronal cell and growth cone migration in the src-1(cj293) mutants
In the src-1(cj293) mutant, we also observed apparent defects in
the migration of neuronal cells. In particular, the positioning of AVM, a
descendant of the QR neuroblast, is strongly affected by the
src-1(cj293) mutation. As with DTC migration, the kinase activity of
SRC-1 is required for its role in the directional migration of QR and its
descendants. Although the cues that guide Q cell migration along the
anteroposterior axis are still unknown, it has been reported that the
transmembrane protein MIG-13 is a key determinant in the final positioning of
AVM (Sym et al., 1999). MIG-13
expression is restricted to the anterior and central body regions and
functions in a non-cell-autonomous manner to promote migration in the anterior
direction. Like the src-1(cj293) mutant, a mig-13 mutant
shows defects in the migration of QR and its descendants, but not in the
migration of QL and its descendants (ALM, CAN and HSN). Furthermore, the final
position of AVM in the mig-13 mutant is very similar to that in the
src-1(cj293) mutant. These observations raise the interesting
possibility that SRC-1 mediates the cell signaling induced by extracellular
MIG-13 cues. Mutations in ced-5/-12 genes that affected DTC turning
did not alter the migration defects of QR and its descendants in the
src-1(cj293) mutants. This shows that the signaling pathways that are
dependent on SRC-1 vary depending on cell type.
The src-1(cj293) mutant also showed defects in the migration of the growth cones of some neuronal cells, namely, AVM, ALM, CAN and PVM. For example, whereas the growth cone of PVM normally makes a right-angled turn in the anterior direction after reaching the ventral nerve cord, in the src-1(cj293) mutant it frequently makes a turn in the opposite direction (Fig. 8). It appears that the axon of PVM in the src-1(cj293) mutant may randomly determine the direction in which it turns. SRC-1 may be involved in the regulation of cell signaling evoked by the guidance cues that direct the migration of a growth cone, suggesting that the migration of a neural growth cone uses the same mechanisms employed by migrating cell bodies like DTCs. However, identification of the guidance cues involved in the attraction or repulsion of the PVM growth cone within the ventral nerve cord will be required to confirm the role of SRC-1 in this process.
In this study, we showed initially that SRC-1 plays a potential role in transducing the netrin signal to the Rac pathway at a particular time of DTC migration. However, our subsequent observations of neuronal cells and growth cones suggest that SRC-1 plays a more general role in directing cell migration, and that it does so via different pathways depending on the cell types. Thus, in response to various extracellular guidance cues, activated SRC-1 may transduce the signals into an appropriate intracellular pathway that probably regulates the cytoskeletal remodeling required for providing polarity information to the cells. In vertebrates, Src family kinases have been shown to respond to a wide variety of extracellular cues, including guidance cues, extracellular matrices and growth factors, and to play roles in regulating a wide variety of cellular functions, including cell adhesion, migration, secretion, endocytosis, proliferation and differentiation. These multifunctional aspects of Src family kinases have hampered the unraveling of their most crucial function(s). Further analysis of the functions of SRC-1, particularly by focusing on the process of cell migration, may help elucidate its basal role, which may be conserved during animal evolution.
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ACKNOWLEDGMENTS |
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Footnotes |
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