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First published online May 16, 2007
doi: 10.1242/10.1242/dev.001164
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.
* Author for correspondence (e-mail: beckendo{at}berkeley.edu)
Accepted 11 March 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Derailed, WNT5, WNT4, RYK, Migration, Salivary gland
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Three
different branches of Wnt signaling lie downstream of Frizzled receptors. In
what is called the canonical Wnt pathway, the presence of a Wnt signal
stabilizes cytoplasmic ß-catenin, leading to its nuclear translocation
and the activation of the transcription factor TCF (Pangolin - FlyBase) or LEF
(Logan and Nusse, 2004). The
other two branches, which do not require ß-catenin, are referred to as
the planar cell polarity (PCP) and calcium pathways
(Cadigan and Liu, 2006;
Logan and Nusse, 2004).
A completely different type of Wnt signaling uses
related-to-tyrosine-kinase (RYK) receptors rather than Frizzled receptors.
Members of the RYK subfamily have unusual, but highly conserved, amino acid
substitutions in their intracellular kinase domains that eliminate kinase
activity (Callahan et al.,
1995; Halford et al.,
1999; Hovens et al.,
1992; Oates et al.,
1998). In addition, RYKs have an extracellular Wnt inhibitory
factor (WIF) domain or Wnt-binding domain that allows RYKs to function as Wnt
receptors. In Drosophila, WNT5 has been shown to bind to the WIF
domain of the RYK receptor Derailed (DRL)
(Yoshikawa et al., 2003). Much
like Frizzled receptors, RYKs are important for a variety of developmental
processes, including axon guidance, organogenesis and craniofacial development
(Bonkowsky et al., 1999;
Fradkin et al., 2004;
Halford et al., 2000;
Inoue et al., 2004;
Lu et al., 2004).
These two different Wnt pathways are both known for their roles in cell
migration and axon guidance (Bovolenta et
al., 2006; Kamitori et al.,
2005; Nelson and Nusse,
2004). In addition, we find that they are required for salivary
gland migration. Much of our knowledge about cell migration in general has
been gained by studying single motile cells in culture. While these studies
have contributed greatly to our understanding of the mechanics of cell
movement, they do not provide us with a very clear picture of cell migration
in the three-dimensional context of a living organism. Furthermore, in many
cases, cells do not migrate alone within the embryo, but rather migrate as
part of a larger tissue (Lecaudey and
Gilmour, 2006). Migration of the salivary glands in
Drosophila embryos offers an opportunity to explore these
processes.
The Drosophila embryonic salivary glands provide a morphologically
simple system in which to study collective cell migration. The salivary gland
anlage is specified by the homeotic gene, Sex combs reduced
(Scr), which activates several factors, including the transcription
factor fork head (fkh) in the salivary placodes
(Panzer et al., 1992). During
early stage 11, the circular salivary placodes form and are visible as two
groups of cells on either side of the ventral midline in parasegment 2
(Fig. 1A,B). The salivary gland
cells invaginate into the interior of the embryo at a 45° angle during
stage 12 until they contact the visceral mesoderm
(Fig. 1C,D). The tubular
salivary glands then turn toward the posterior, continuing their migration
until all of the salivary gland cells have internalized
(Fig. 1E,F). During this phase
of migration, the salivary gland tip cells extend lamellipodial protrusions
and, using integrin-based motility, actively travel along the visceral
mesoderm. The substrate for this movement is the circular visceral mesoderm
(CVM) that will ultimately form the inner layer of the gut muscle
(Bradley et al., 2003;
Kerman et al., 2006;
Vining et al., 2005). During
their migration, the glands are guided by the chemoattractant Netrin and the
chemorepellent Slit to arrive at their correct position within the embryo
(Kolesnikov and Beckendorf,
2005). By stage 14, the longitudinal visceral mesoderm (LVM),
which will form the outer layer of the gut muscle, migrates from the posterior
part of the embryo anteriorly over the CVM and separates the distal tip of
salivary glands from the CVM (Fig.
1J). Separation of proximal portions of the salivary gland from
the CVM occurs as the gut contracts posteriorly. After the migration has
finished, the distal tip of the salivary gland maintains contact with the LVM
(Fig. 1G,H,K)
(Vining et al., 2005).
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Yoshikawa et al.,
2003), Src64PI
(Dodson et al., 1998),
Src42E1 (Tateno et
al., 2000), dnte00722, fz1,
sli2, w1118, Wnt2L and
Wnt4EMS23 (from the Bloomington Stock Center).
The following GAL4 and UAS lines were used: UAS-drl,
UAS-drl
i
(Yoshikawa et al., 2001;
Yoshikawa et al., 2003),
UAS-Wnt5 (Fradkin et al.,
2004), UAS-Wnt4
(Cohen et al., 2002),
UAS-fzN, UAS-fz2N (Zhang and
Carthew, 1998), UAS-dshDIX,
UAS-dshbPDZ (dominant-negative constructs for canonical Wnt
pathway) and UAS-dshDEP+,
UAS-dshDEP+ (dominant-negative construct for PCP pathway)
(Axelrod et al., 1998),
UAS-TCFN (Bloomington Stock Center),
fkh-GAL4 (B. Zhou, PhD thesis, University of California, 1995), and
bap-GAL4 (Weiss et al.,
2001).
Immunocytochemistry and in situ hybridization
Embryo fixation and staining were performed as described
(Chandrasekaran and Beckendorf,
2003). The salivary gland, apical-specific antibody used was mouse
anti-Crumbs (CRB) (Cq4; Developmental Studies Hybridoma Bank, University of
Iowa) at 1:25 and the salivary gland nuclear-specific antibody used was rabbit
anti-FKH (1:1000). In addition, rabbit anti-SG2 (PH4
SG2 - FlyBase) was
used at 1:3000 to visualize the salivary glands
(Abrams et al., 2006). Rat
anti-Titin (Sls - FlyBase) was used at 1:500 to visualize the LVM
(Machado et al., 1998). Rabbit
anti-ß-galactosidase antibody (Roche) was used at 1:1000. The mouse
anti-FASIII (FAS3 - FlyBase) (7G10) antibodies were all obtained from the
Hybridoma Bank and used at 1:10. Alexa Fluor 546 and 488 (Molecular Probes)
secondary antibodies were used at 1:500. Fluorescent in situ hybridization was
performed as described (Tautz and Pfeifle,
1989) with modifications
(Harland, 1991) using
antisense digoxigenin-labeled probes. Mouse anti-DIG (Roche) was used at 1:100
with the Alexa Fluor 546 (Molecular Probes) secondary antibody at 1:250. After
washing, embryos were cleared with 50% glycerol, then 70% glycerol and
visualized on a Zeiss 510 confocal microscope.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). We found that drl RNA expression commenced at
stage 11 in the dorsoposterior portion of the salivary placodes, encompassing
the initial site of invagination and thus the cells that will later form the
distal tip of the salivary gland (Fig.
2A). During stage 12 drl RNA expression decreased, but an
enhancer trap for drl,
drl3.765, showed
ß-galactosidase expression, which suggests that DRL persists at the tip
of the migrating salivary gland through stage 16
(Fig. 2B).
Expression of drl in the salivary placode is dependent on Scr and fkh
Since drl RNA expression in the salivary placodes begins at stage
11, later than primary Scr target genes, drl might be
indirectly activated by Scr through one of these target genes. As
expected, we found that drl expression was absent in Scr
mutant embryos (Fig. 3B,
compare with A). Among the primary Scr targets, we tested three
transcription factors that are required for salivary gland development. In
embryos mutant for huckebein (hkb) or trachealess
(trh), drl expression remained unchanged (data not shown).
In contrast, fkh-mutant embryos lacked drl expression in the
salivary placodes, although its expression in the epidermis and central
nervous system (CNS) was unaffected (Fig.
3C, compare with A).
|
). The conspicuous expression of drl at the tip of
the salivary gland, along with its previously reported role in axon guidance,
suggested that drl might be required for salivary gland guidance. We
found that in 40% of drl-null embryos the salivary glands curved
ventromedially toward the CNS, instead of lying parallel to the midline of the
embryo as in wild type (Fig.
2D,I). Interestingly, this abnormal curvature affected only the
distal tip of the salivary gland, the cells that specifically express
drl, and was first seen in mutant embryos at stage 14. This is the
time that the tip cells normally detach from the CVM and associate with the
LVM. We found that the drl-mutant glands did not maintain contact
with the LVM. Instead they curved ventromedially, and the tip frequently
adhered to the somatic mesoderm (Fig.
2H, Table 1).
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) to express full-length DRL throughout the salivary
glands of drl mutants. The gland guidance defects of drl
mutants were completely rescued in embryos carrying the fkh-GAL4 and
UAS-drl constructs (Fig.
2E,I). In contrast, we were unable to rescue the drl
mutant phenotype using
UAS-drli, a construct lacking
the intracellular domain, and thus the atypical kinase domain, of drl
(Yoshikawa et al., 2003)
(Fig. 2F,I).
During embryonic development, WNT5 is expressed predominantly in the CNS
(Fradkin et al., 2004;
Yoshikawa et al., 2003).
Wnt5 RNA expression begins during stage 12 and WNT5 protein starts to
accumulate primarily in the posterior commissures during stage 13
(Fradkin et al., 2004). The
CNS, and thus the expression domain for Wnt5, is adjacent to the
salivary glands during their posterior migration, making an interaction
between the ligand in the CNS and its receptor in the salivary gland tip
possible.
In embryos lacking Wnt5, the tips of the salivary glands migrated ventromedially, just as they do in drl-mutant embryos (Fig. 2G). The Wnt5D7; drlR343 double mutant showed the same phenotype with similar penetrance as either of the single mutants alone, suggesting that drl and Wnt5 act in the same pathway (Fig. 2I). Embryos heterozygous for both drl and Wnt5 also showed the ventral curving defect with a slightly lower penetrance (Fig. 2I).
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). Drl-2, like drl, has been implicated in
axon guidance (S. Yoshikawa and J. B. Thomas, personal communication). Since
drl-null mutations caused only a partially penetrant salivary gland
phenotype, we tested whether the other two Drosophila RYKs might be
acting redundantly with drl. By themselves, both dnt and
Drl-2 mutant embryos showed qualitatively similar phenotypes to those
of drl mutants, but with significantly lower penetrance
(Fig. 4B,C,E). We also
discovered dominant genetic interactions between mutations in drl and
Drl-2, suggesting that these two genes might be acting in the same
process (Fig. 4D,E). As embryos
lacking both drl and Drl-2 did not show an enhancement over
either of the single mutants with regard to salivary gland migration, it
remains unclear whether these genes act redundantly in salivary gland
positioning. Taking the results in this section together, we conclude that WNT5, probably emanating from the CNS, acts through the DRL receptor at the tip of migrating salivary gland to keep the gland on track and prevent it from bending toward the CNS. The DNT and DRL-2 receptors may play a minor role in this WNT5 signaling.
WNT4 signaling is also required for salivary gland migration
Since relatively little is known about the interactions between
drl and its ligands, we wanted to test whether additional Wnt ligands
might be needed for salivary gland migration. Of the seven known Wnt homologs
in the Drosophila genome (Adams et
al., 2000; Rubin et al.,
2000), we tested wg, Wnt2, Wnt4 and Wnt5 mutants
for salivary gland defects. We were unable to ascertain whether wg is
needed for salivary gland guidance due to its earlier requirement for
segmentation. Wnt2 mutant embryos did not exhibit salivary gland
defects. However, lack of Wnt4 did disrupt salivary gland guidance.
Embryos mutant for Wnt4 displayed a ventral curving phenotype that is
similar to drl and Wnt5 mutants, except that the entire
gland, rather than just the tip, was curved ventromedially
(Fig. 5B). This positioning
defect occurred earlier than the drl phenotype, disrupting salivary
gland positioning as early as stage 12. At this stage in wild-type embryos,
Wnt4 is expressed ventral to the migrating salivary glands in narrow
ectodermal stripes and in the assembling ventral nerve cord
(Graba et al., 1995).
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Wnt4 is also expressed in the CNS and its mutant phenotype
suggests that it might also be a repellant. Accordingly, Wnt4,
ectopically expressed in the visceral mesoderm, forced the salivary glands
away from the visceral mesoderm (Fig.
6C). Unlike Wnt5, misexpression of Wnt4 rerouted
the entire gland, not just the leading cells. This phenotype suggests that
WNT4 does not act as a ligand for DRL; instead it may act as a ligand for
either FZ or FZ2, both of which are broadly expressed in salivary glands
(Bhanot et al., 1999;
Park et al., 1994). This
possibility is explored further in the next section.
WNT5 and WNT4 act on discrete pathways to influence different stages of salivary gland placement
There are no data to support WNT4 acting as a ligand for DRL. In fact,
previous studies have shown that, in contrast to Wnt5, Wnt4 does not
interact genetically with drl in the CNS and fails to bind to DRL
(Yoshikawa et al., 2003).
However, WNT4 does bind to two of the Drosophila Frizzled homologs,
FZ and FZ2 (Wu and Nusse,
2002). Hence, WNT4 might work through the canonical Wnt pathway
rather than through the WNT5-DRL pathway during salivary gland development. To
test this, we expressed dominant-negative constructs of fz and
fz2 (Zhang and Carthew,
1998) specifically in the salivary glands. Expression of either
UAS-fzN or UAS-fz2N in the salivary glands resulted in
curved salivary glands similar to those in Wnt4-mutant embryos; a
large portion of the salivary gland curved toward the CNS and this curving
began early, as the gland migrated along the circular visceral mesoderm
(Fig. 5C,D). fz-mutant
embryos show this same ventral curving phenotype, despite the presence of
fz2, which acts redundantly with fz during segmentation of
the embryo (Fig. 5E)
(Bhanot et al., 1999).
Furthermore, Wnt5D7;Wnt4EMS23 double mutants
had a higher penetrance of salivary gland curving than either of the single
mutants alone, emphasizing that two independent Wnt pathways are needed for
proper salivary gland guidance (Fig.
5F,J). Similarly, the penetrance of ventral curving in
drl mutant embryos was enhanced from 40 to 79% in the
drlR343; fz1 double mutant
(Fig. 5G,J). In both
Wnt5D7;Wnt4EMS23 and
drlR343;fz1 double mutants, a combination of
phenotypes was seen: both ventral curving specific to the salivary gland tip
and curving affecting a large portion of the salivary gland. Taken together,
these data demonstrate that there are two Wnt pathways regulating salivary
gland migration: a Wnt4-fz/fz2 signaling pathway that is required
throughout the gland, and a Wnt5-drl signaling pathway that
specifically affects the tip of the migrating salivary gland.
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N) in their salivary
glands resembled Wnt4 mutants, with ventral curving that affected a
large percentage of the salivary gland
(Fig. 5H). We also tested the
effects of several dsh dominant-negative constructs that specifically
disrupt either the canonical Wnt signaling pathway or the PCP pathway
(Axelrod et al., 1998). The
dsh dominant-negative transgenes that affect only PCP signaling
(UAS-dshDEP+,
UAS-dshDEP+) had no affect on salivary gland guidance;
however, transgenes specific to the canonical Wnt pathway
(UAS-dshDIX, UAS-dshbPDZ) caused
ventrally curved salivary glands resembling Wnt4, Tcf, fz and
fz2 mutants (Fig.
5I,J). These results strengthen our conclusion that the canonical
Wnt pathway, activated by the WNT4 ligand, is involved in the early stages of
salivary gland migration.
Src kinases genetically interact with drl in salivary glands
Our analysis indicates that WNT4 is most probably signaling through the
canonical Wnt pathway, but what lies downstream of Wnt5 and
drl is less clear. Recent experiments have suggested that Src kinases
might be involved. Src64 (Src64B - FlyBase) binds to
drl in a yeast two-hybrid screen and genetically interacts with
drl in the developing nervous system (R. Wouda, J. N. Noordermeer and
L. G. Fradkin, personal communication). In addition, Src64 and
drl have been shown to interact, either directly or indirectly, in
Drosophila mushroom body development
(Nicolai et al., 2003). We
found that drl and Src kinase genes also interacted genetically in
the salivary glands. Src64 mutant embryos displayed ventral curving
of salivary gland tips, similar to drl mutant embryos
(Fig. 7B,G), as did drl
Src64 doubly heterozygous embryos
(Fig. 7C). Placement of
Src64 downstream of drl is further supported by our finding
that homozygous drl Src64 double mutants had a similar frequency of
guidance defects as either single mutant
(Fig. 7G).
We tested whether the other Drosophila Src kinase, SRC42 (SRC42A - FlyBase) also interacts genetically with drl. We did find drl-Src42 interactions, but they were more complex than the drl-Src64 interactions. While Src42 homozygous mutant embryos had salivary gland defects that included ventral curving at the tip of the salivary gland (Fig. 7D), they also displayed more generalized curving defects that occurred earlier than the drl phenotype (Fig. 7E). Similar to the Src64 interaction, drl Src42 doubly heterozygous embryos displayed guidance problems that closely resembled the drl mutant phenotype (Fig. 7F). Thus, it appears that Src42 may be working downstream of drl late in salivary gland development, but may play a role in earlier salivary gland positioning as well.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). In the
first phase, the salivary glands invaginate into the embryo at a 45°
angle, moving dorsally until they reach the visceral mesoderm. fkh,
RhoGEF2 and 18 wheeler have been shown to regulate apical
constriction of the salivary gland cells during this invagination process
(Weigel et al., 1989;
Myat and Andrew, 2000a;
Nikolaidou and Barrett, 2004;
Kolesnikov and Beckendorf,
2007). In addition, hkb and faint sausage are
needed for proper positioning of the site of invagination
(Myat and Andrew, 2000b). No
guidance cues have been identified for this first phase of migration; it may
be that the patterns of constriction and cell movements at the surface of the
embryo are sufficient to direct the invaginating tube.
|
). Netrin, which is expressed in the CNS and the visceral
mesoderm, works to maintain salivary gland positioning on the visceral
mesoderm. At the same time, Slit acts as a repellent from the CNS to keep the
salivary glands parallel to the CNS. Here we have shown that there is a third
guidance signal, WNT4, acting through FZ or FZ2 receptors, that is also
required in the second phase of salivary gland migration
(Fig. 8A). Loss of Wnt4,
fz or fz2 in the embryo resulted in salivary glands that were
curved in a ventromedial direction. This curving affected a large portion of
the salivary gland and may have resulted from the fact that the fz
and fz2 receptors, in contrast to drl, are expressed
throughout the salivary gland. Furthermore, dominant-negative transgenes that
disrupt the function of DSH or TCF caused the same phenotype, suggesting that
transcription induced by the canonical Wnt signaling pathway is needed to
maintain the proper migratory path of the salivary glands on the CVM. The
migration along the CVM takes more than 2 hours for completion, which would
leave adequate time for a transcriptional response. Although Wnt4 and slit are both required for the second phase of migration, and their mutants show similar, though distinguishable, phenotypes, we believe that they act independently. While most slit-mutant embryos have medially curving salivary glands, embryos lacking Wnt4 had salivary glands that curved in a distinctly different, ventromedial, direction. Embryos doubly mutant for Wnt4 and slit showed predominantly one or the other phenotype and neither phenotype increased in severity (data not shown). These results suggest, though they do not prove, that Wnt4 and slit act in distinct pathways.
Atypical Wnt signaling mediates final positioning of the salivary glands
After the entire salivary gland has invaginated, migrated posteriorly
within the embryo and lies parallel to the anteroposterior axis of the embryo,
the distal ends of the salivary glands come into contact with the LVM. We have
shown that drl and Wnt5 are required for this late phase of
salivary gland positioning (Fig.
8B). Loss of either drl in the salivary gland or
Wnt5 in the CNS resulted in the distal tip of the salivary gland
being misguided to a more ventromedial position. This change in the shape of
the salivary gland was seen only after the salivary glands were no longer in
contact with the CVM (after stage 13). Thus we propose that drl is
required during the third phase of salivary gland migration, as the salivary
gland detaches from the CVM and contacts the LVM.
The striking expression of drl at the tip of the salivary gland
makes the leading cells uniquely different from the rest of the salivary gland
cells. These cells project lamellipodia upon reaching the visceral mesoderm
and beginning their posterior migration. They may act to both guide and pull
the rest of the gland during migration
(Bradley et al., 2003). Cells
at the tip of a migrating organ are frequently specialized to guide migration.
For example, the coordinated migration of the tracheal branches in
Drosophila is achieved by induction of distinct tracheal cell fates
within the migrating tips. This is illustrated by the fact that FGF (BNL -
FlyBase) signaling becomes restricted to the tips of the tracheal branches
soon after they begin to extend (Gabay et
al., 1997; Sutherland et al.,
1996). The migration and growth of Drosophila Malpighian
tubules provide another clear example of specialized cells needed at the tip
of a migrating tissue. One cell is singled out to become the tip cell, which
directs the growth of the Malpighian tubules as well as organizes the mitotic
response and migration of the other cells forming each tubule
(Hoch et al., 1994). In other
systems, such as Dictyostelium slugs, cells at the tip of a migrating
group are required and solely able to guide migration
(Dormann and Weijer, 2001).
Our results establish that the leading cells of the migrating salivary glands
have a specialized role to play in proper salivary gland positioning. First
they are required to initiate invagination within the embryo, then they
actively participate in migration along the CVM, and finally they ensure that
the distal tip of the gland will remain associated with the LVM at the end of
the migratory phase.
Despite the fact that we have firmly established Wnt5 and
drl as important for the final placement of salivary glands, the
signaling pathways downstream are not well defined. Because salivary-gland
expression of full-length drl can rescue the drl-mutant
phenotype, but drl lacking the intracellular domain cannot, we are
confident that the intracellular domain of DRL is important for signaling.
Similarly, misexpression of full-length drl can misguide axons in the
ventral nerve cord, but misexpression of drl lacking its
intracellular domain cannot (Yoshikawa et
al., 2003). The genetic interactions found in this study between
drl and Src64 support recent findings suggesting that
Src64 acts downstream of drl in the ventral nerve cord (R.
Wouda, J. N. Noordermeer and L. G. Fradkin, personal communication). In
addition, we have shown that the other Drosophila Src kinase,
Src42, may be required at two stages, during salivary gland migration
along the CVM and downstream of WNT5-DRL signaling as the gland moves onto the
LVM.
Another intriguing finding of this study is the involvement of the two remaining Drosophila RYKs, Drl-2 and dnt, in salivary gland development. The phenotypes of Drl-2 and dnt mutants are less penetrant than drl mutants, but they are qualitatively very similar. Furthermore, embryos doubly heterozygous for drl and Drl-2 have salivary glands that resemble those seen in drl mutant embryos. These three RYKs appear to act in a partially redundant fashion in the salivary glands, as none of the single gene mutations leads to completely penetrant phenotypes. However, we did not see an increase in penetrance of the drl phenotype in embryos lacking both drl and Drl-2. In addition, we were unable to detect transcripts for either Drl-2 or dnt in the salivary gland. While it is possible that dnt and Drl-2 are expressed at very low levels in the salivary gland, they might be acting non-autonomously.
WNT5 and WNT4 signaling pathways operate independently of each other
An interesting dilemma in understanding RYK signaling is how inactive
kinases propagate a signal into the cell. Recent mammalian studies have
suggested that RYKs may associate with another catalytically active receptor,
such as FZ or EPH, at the membrane
(Halford et al., 2000;
Lu et al., 2004;
Trivier and Ganesan, 2002). In
the mouse, the extracellular WIF domain of RYK interacts with FZD8, and it has
been proposed that the two proteins may form a ternary complex with WNT1 to
initiate signaling (Lu et al.,
2004). However, data from flies and nematodes support the argument
that DRL and its Caenorhabditis elegans homolog LIN-18 act
independently of FZ. Genetic studies of cell specification in the nematode
vulva suggest that LIN-18 acts in a parallel and separate pathway from the
LIN-17/FZ receptor (Inoue et al.,
2004). Similarly, reduction of fz and fz2 gene
activity in flies has no effect on a DRL misexpression phenotype in the
ventral nerve cord (Yoshikawa et al.,
2003). Here we have shown that double mutants for the
Wnt4 and Wnt5 ligands and for the fz and
drl receptors both show strong enhancements in comparison to the
single mutants, reinforcing the conclusion that these two ligands are
activating different pathways. In addition, we can separate the functions of
these two pathways by phenotype. The Wnt4-fz/fz2 phenotype becomes
evident earlier and affects a larger portion of the salivary gland than the
Wnt5-drl phenotype. Taken together, these results demonstrate that
there are two independent Wnt pathways regulating salivary gland positioning.
The early WNT4 signal appears to activate the canonical Wnt pathway, whereas
there is a later requirement for WNT5 signaling through DRL and the Src
kinases.
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ACKNOWLEDGMENTS |
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