1 Department of Developmental Biology, Wenner-Gren Institute, Stockholm
University, S-106 96 Stockholm, Sweden
2 Department of Medical Nutrition, Karolinska Institute, Stockholm, Sweden
3 Department of Natural Sciences, Södertörns Högskola, S-141 04
Huddinge, Sweden
4 Umeå Centre for Molecular Pathogenesis, Umeå University, S-901 87,
Umeå, Sweden
* Author for correspondence (e-mail: christos{at}devbio.su.se)
Accepted 27 April 2004
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SUMMARY |
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Key words: Drosophila, ru, Egfr, Epithelial migration, VNC midline
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Introduction |
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The morphogenesis of the embryonic tracheal (respiratory) network depends
on the charted migration of 2000 epithelial cells deriving from 20
epidermal invaginations. These cells undergo three successive rounds of
branching to generate a tubular network that extends along stereotyped paths
towards specific target tissues. The last branching event produces thin,
unicellular terminal branches that associate with distinct organs
(Samakovlis et al., 1996a
;
Manning and Krasnow, 1993
).
The ventral nerve cord (VNC) is invaded by 20 ganglionic branches (GBs), which
sprout from the lateral trunk of the trachea. GB migration towards and inside
the CNS is highly stereotyped and has been described in detail elsewhere
(Englund et al., 1999
). Each
GB initially tracks along the inter-segmental nerve and towards the CNS. GB1,
the leading cell of the ganglionic branch, enters the nerve cord and changes
substrate to track along the segmental nerve, proceeding ventrally on top of
the longitudinal fascicles and towards the CNS midline. Finally, after
reaching the midline, GB1 takes a sharp turn and migrates dorsally through the
dorsoventral channel and then turns posteriorly on the dorsal side of the VNC.
At the end of embryogenesis, GB1 will have trailed a remarkable 50 µm
inside the CNS. Genetic analysis has uncovered a number of factors that are
necessary for this fixed migratory path: the FGF homolog Branchless is
required to guide the GBs towards the CNS and to induce them to enter it
(Sutherland et al., 1996
), in
part by inducing the expression of the nuclear protein Adrift
(Englund et al., 1999
). Once
inside the CNS, Slit (Rothberg et al.,
1988
), the main repulsive cue for axons at the midline, becomes a
key guiding cue for the migrating GBs. Slit controls several, distinct aspects
of ganglionic branch pathfinding into the CNS: it is first required to attract
GBs towards the CNS, an effect mediated by its receptor Robo2, and then to
prevent GBs from crossing the midline once they reach it, which is mediated by
Robo (Englund et al.,
2002
).
To identify additional signals that steer GB1 migration, we screened a
collection of P-element insertions for GB pathfinding phenotypes. One of the
strains recovered showed a specific GB1 midline-cross phenotype reminiscent of
robo or slit mutants, but unlike mutants in the
slit pathway, had no defects in axonal pathfinding. The mutation was
found to affect roughoid/rhomboid 3 (ru - FlyBase) an
intramembrane protease that activates Egfr ligands
(Wasserman et al., 2000). Our
analysis indicates that Rhomboid 3 defines a new signalling centre for
tracheal repulsion from the midline. Rho3 is expressed by the VUM midline
neurons, where it activates an Egfr ligand. Egfr and Ras but
not Raf, yan or mbc, are required in GB1 for its turn away
from the midline. The analysis of loss-of-function and overactivation
phenotypes suggests that EGF itself is not a chemorepellent for GBs, instead
it appears to provide a necessary activation switch for the interpretation of
a yet unknown, Slit-independent, repellent function of the midline.
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Materials and methods |
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Molecular identification of the inga locus
The inga P-element line contains a single P-element, as analysed
by Southern blot. Genomic DNA flanking the P-element was obtained by plasmid
rescue in E. coli after cleavage of the genomic DNA with
EcoRI or BamHI. This DNA was sequenced and used to search
the databases. The search identified also a cDNA clone that was obtained from
Research Genetics: LP02893. The inga P-element was inserted at
position 137648 of the Celera contig AE003741 and 345 bp upstream of the first
exon of the cDNA LP02893.
Antibodies, embryo staining and whole-mount in situ hybridisation
Embryo fixation, antibody staining, light and confocal fluorescence
microscopy were performed as described previously
(Samakovlis et al., 1996b).
Primary antisera were anti-ß-galactosidase (diluted 1:1500, Cappel),
mAb1D4 against Fasciclin 2 [1:10 (Van
Vactor et al., 1993
)], mAb BP102 that labels all CNS axons (1:50),
mAb2A12 against tracheal lumen (1:5), mAb22C10 labelling a subset of CNS and
PNS axons [1:20 (Zipursky et al.,
1984
)], anti-Repo (1:5), mouse anti-Wrapper [1:10
(Noordermeer et al., 1998
)],
mouse anti-Robo [1:10 (Kidd et al.,
1998
)] and mouse anti-Slit [1:10
(Rothberg et al., 1990
)], each
obtained from the Developmental Studies Hybridoma Bank at The University of
Iowa. Secondary antibodies included biotin (1:300), Cy2 (1:200), Cy3 (1:300)
and Cy5 (1:200) conjugates (Jackson Laboratories), and Alexa Fluor-594 (1:400)
and -488 (1:200) conjugates (Molecular Probes). When necessary, the signal was
developed using Vectastain Elite ABC Kit (Vector Laboratories). Embryos were
visualised with a Zeiss Axioplan2 microscope under Nomarski optics or a Zeiss
confocal microscope. Confocal stacks were processed using the Volocity
software (Improvision) to obtain three dimensional reconstructions.
Whole-mount in situ hybridisation was performed using random-primed,
digoxigenin-labelled roughoid/rho3 cDNA (LP02893, Research Genetics)
as a probe; embryo staging was according to
(Campos-Ortega and Hartenstein,
1985
).
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Results |
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Egfr signalling is required in GBs for midline repulsion
Egfr signalling is known to be required at several steps in the development
of the CNS, including the specification and survival of the midline glia
(Klambt et al., 1991), which
then provide a source of signals that guide neuronal and tracheal migration
inside the VNC. The early functions of EGF signalling make the analysis of
mutations in components of the pathway difficult to interpret in the context
of the migration of GB1 at late stage 16. As an example, a hypomorphic
mutation in the main Egfr ligand, spitz
(Rutledge et al., 1992
)
resulted in severe GB midline crossing defects
(Fig. 4A), but also caused
major defects in axonal migration (Fig.
4C), as Spitz is essential for both midline glia differentiation
and survival (Klambt et al.,
1991
). To overcome the problem of secondary effects, we made use
of a Gal4 strain that exclusively activates gene expression in the terminal
cells of all tracheal branches (SRF>Gal4)
(Jarecki et al., 1999
)
(Fig. 4E), to express a series
of dominant-negative and activated constructs of Egfr pathway components.
Expression of SRF (pruned, blistered)
(Affolter et al., 1994
;
Guillemin et al., 1996
) is
activated at stage 14 and is a marker for GB1 terminal differentiation;
therefore, the expression of transgenes under the control of its promoter
should not interfere with earlier cell specification events in GB1.
|
|
To explore whether part of the rho3 mutant phenotypes are due to its expression in the tracheal cells, we attempted to ablate the GB1s by the expression of Ricin A. If signalling deriving from GB1s was important for their own migration then the ablation of some or most GB1s might result in misrouting of the few remaining ones that escape ablation. However, if GB1 signals were important for the VNC cells, then the ablation of GB1 might cause abnormalities in the pattern and migration of the glia and neurons.
Embryos expressing UAS-RicinA (Hidalgo
et al., 1995) under the control of SRF-Gal4 showed a normal
appearance of glial populations and axonal tracts (as detected by Repo,
Wrapper and Fasciclin 2, respectively Fig.
5). Thus, if any signalling occurs from GBs to CNS cell
populations, it appears unnecessary for the patterning of the latter.
Moreover, the few GB1s that escaped ablation, presumably owing to mosaic Gal4
expression, migrated normally (Fig.
5, arrows), implying that GBs are unlikely to signal to each other
during their migration in the CNS.
To address the potential tissue-specific requirement of rho3
during GB migration, we expressed the close relative Rho1 in different
populations of CNS cells and in GB1s, and assayed the extent of rescue of the
rho3 tracheal phenotype. SRF-directed expression of Rho1 was not
sufficient to significantly rescue the midline cross phenotype of
rho3 ganglionic branches (Fig.
5E). By contrast, the same transgene expressed under the control
of three CNS-specific Gal4 strains provided a significant rescue of the GB
phenotypes (Fig. 5E).
single minded-Gal4 is initially expressed by all midline cell
precursors, but becomes later restricted to midline glia
(Scholz et al., 1997);
slit-Gal4 is limited to midline glia
(Scholz et al., 1997
). Both
sim- and slit-Gal4 directed expression of Rho1 approximately
halved the occurrence of GB midline crosses in the rho3 mutant
(Fig. 5E). elav-Gal4
is expressed in all post-mitotic neurons but not in midline glia
(Lin and Goodman, 1994
).
Strikingly, elav-Gal4 directed expression of Rho1 suppressed the
rho3 GB midline cross phenotype to 1%
(Fig. 5E). We conclude that
Rhomboid signalling is required in the CNS, rather than in the GB1 itself, to
prevent ganglionic branch midline crossing. The weak rho3 expression
in GB1 might be part of a positive feedback loop, a common feature of Egfr
signalling in flies.
Ras, but not Raf or Yan, mediate GB repulsion from the midline
Egfr signalling is mediated by a number of downstream effectors in
different cell types. In order to determine which one is used in GB1
pathfinding, we analysed a panel of mutants and dominant-negative constructs
of known downstream effectors for their effect on GB migration. myoblast
city (mbc) (Rushton et al.,
1995) is a conserved adaptor necessary for the chemo-attractant
function of Gurken during border cell migration in the ovary
(Duchek et al., 2001
).
mbc alleles had no defects in GB pathfinding (data not shown). As
mbc has negligible maternal contribution and is not readily detected
in tracheal tissues (Erickson et al.,
1997
), we conclude that it is unlikely to have a role in
Egfr-mediated GB repulsion from the midline. We also tested two additional
effectors that have been implicated in Egfr-elicited migratory responses in
other systems, PLC
and PI3K (reviewed by
Schlessinger, 2000
). The fly
PLC
is encoded by the small wing (sl) locus
(Thackeray et al., 1998
).
small wing embryos had extra terminal sprouts emanating from the
primary tracheal branches but showed no specific defects in GB migration
inside the VNC (data not shown).
p60 is a deletion variant of the
adaptor p60, which has dominant-negative effects on PI3K activity in vivo and
in vitro (Rodriguez-Viciana et al.,
1997
; Kodaki et al.,
1994
; Weinkove et al.,
1999
). SRF-Gal4 driven expression of
p60 resulted in a
stalling phenotype of 19% of the GBs (n=280) but not midline crosses
(Fig. 6D). This may reflect a
requirement of PI3-K in the early extension of the GBs towards the midline,
which was also impaired by the expression of the dominant-negative form of
Egfr in GB1 (Fig. 4B).
The activation of Ras is a necessary step in many of the cellular responses
induced by of Egfr signalling in Drosophila
(Rommel and Hafen, 1998). It
leads to the activation of Raf (Li et al.,
1998
), and culminates with activation of the Ets-transcription
factor Pointed and the nuclear export of Yan, another Ets protein, which
antagonises Pnt in the activation of target genes
(O'Neill et al., 1994
;
Tootle et al., 2003
).
SRF-Gal4-directed expression of a dominant-negative form of Ras
(Lee et al., 1996
) resulted in
stalled branches inside or outside the VNC (52%, n=240). Importantly,
a significant number of GBs was grossly misrouted (8%) or crossed the midline
(4%, Fig. 6A, arrow) suggesting
that Ras is required in the GB1cells for their turn away from the midline. The
large proportion of arrests in cell migration observed in these experiments
might reflect a broader requirement for these common effectors in tracheal
cell migration and sprouting.
To analyse whether Egfr mediated repulsion of GB1 from the midline requires
Raf or downstream pathway components, we expressed a dominant-negative form of
Raf (de Celis, 1997) and an
activated form of Yan (Rebay and Rubin,
1995
) under the control of SRF-Gal4
(Fig. 6). These constructs
caused many of the branches to stall or misroute but in neither case could we
find any branches that crossed the VNC midline (n>200). As an
example, expression of the activated Yan construct stalled the migration of
45% of the GBs, and misrouted an additional 7% (n=300), but not a
single midline cross was observed.
In summary, activation of Ras appears to be required for repulsion of GB1 from the midline, whereas the remaining components of the pathway are required for tracheal cell extension inside the VNC but not for the decision to cross the midline barrier.
Rho3 midline crosses are due to decreased Egfr/Ras signalling in GB1, rather than to the lack of a directional cue from the midline
An important issue for cell migration in complex landscapes is the
discrimination between signals that directly provide spatial information and
others that facilitate the interpretation of different instructions. To find
out whether the expression of Rho3 generates a spatial cue for the migrating
GB1, we first increased the amount of Egfr ligand secreted from the midline by
expressing the closely related protease Rho1
(Bier et al., 1990a;
Urban et al., 2001
) in midline
cells, with the sim-GAL4 driver (Scholz et
al., 1997
). Rhomboid 1 is a functionally redundant partner of Rho3
during eye and leg development (Wasserman
et al., 2000
; Campbell,
2002
) and has been shown to have similar biochemical specificity
to Rho3 (Urban et al., 2002
).
In these embryos, 18% of the GB1s turned posteriorly prematurely, before
reaching their characteristic positions close to the midline. Thus, an
increase in the amount of ligand from the midline can repel GB1s from its
source. If the Egfr ligands provided a spatial guiding signal, overactivation
of Egfr signalling in the GB1 either by the expression of activated forms of
the receptor, Ras or the Rho1 activator, should disturb the asymmetric
response to it, and might cause a random misrouting phenotypes, crossings of
the midline or a prominent early arrest of its migration. We expressed two
different forms of activated Egfr (Queenan
et al., 1997
; Lai et al.,
1995
) (see Materials and methods), Rhomboid 1
(Bier et al., 1990a
), Rhomboid
3 (Wasserman et al., 2000
) and
activated Ras in the terminal cells, and analysed the pathfinding phenotypes
in GB1. In all cases, overactivation of Egfr signalling in GB1 resulted in
misroutings but no significant midline crossing. Notably, the overexpression
of all constructs in the terminal cells induced GBs to turn prematurely before
reaching the midline (`early turns'; Fig.
7). This phenotype was generated both by the overexpression of
activated Egfr, activated Ras and Rhomboid 3
(Fig. 7), and became more
prominent in embryos overexpressing Rho1 in their terminal cells, probably
owing to higher levels of activity provided by this transgene. In
Rho1-overexpressing embryos, 60% of the affected branches (26% of the total,
n=300) were turning posteriorly before reaching the midline
(Fig. 7). The GB1 expression of
non-cell autonomous Egfr activators, such as Rhomboid, runs the risk of
affecting the surrounding neurons and glia, as well as the migrating GB.
Nevertheless, the longitudinal fascicles of the CNS appeared unaffected in
these Rho1-overexpressing embryos (Fig.
7), suggesting that the overexpression of Rho1 in the terminal
cells did not substantially influence the patterning and migrations of neurons
and glia. The premature turning phenotypes generated either by increasing the
amounts of active Egfr ligand deriving from the midline, or by raising the
levels of Egfr signalling in GB1 are similar. These results, coupled with the
analysis of dominant-negative constructs in the trachea (see above), suggest
that the midline crossing phenotype in rho3 mutants is due to
decreased levels of Egfr signalling in GB1, rather than to the lack of spatial
information. Instead, the high level of Egfr activation, which is normally
reached at the midline, appears important for the interpretation of a yet
unidentified directional signal.
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Discussion |
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The expression of rho3 in VUMs and its function in GB1 guidance
away from the midline identifies a new role of Egfr signalling in the VNC.
Unlike rho1, rho3 mutants have a normal VNC pattern in which
longitudinal connectives and glial populations appear normal, suggesting that
rho3 is specifically required for GB1 guidance. Expression of
dominant-negative forms of the EGF receptor or Ras in GB1 phenocopied the
rho3 guidance phenotype. In addition, overactivation of Egfr
signalling in the trachea was sufficient to redirect GB1 and induce early turn
phenotypes. Finally, rho3 is required in parallel to slit,
the main repulsive cue deriving from midline glia
(Kidd et al., 1999). Taken
together, these results suggest that rho3 mutant GB1s are misrouted
because of reduced levels of Egfr/Ras signalling in GB1 cells, rather than to
indirect, subtle defects of midline patterning or signalling capacity in
rho3 mutants. This leads us to propose a simple model in which Rho3
activates one or more Egfr ligands secreted by the midline cells. Reception of
this signal by migrating GBs is mediated by Egfr and Ras, and promotes turning
away from the midline.
Three Drosophila Egfr ligands are activated by Rhomboid proteases:
Gurken, which is only present in oocytes, Spitz and Keren
(Ghiglione et al., 2002;
Reich and Shilo, 2002
;
Urban et al., 2002
), the
latter expressed in embryos below the detection level of in situ hybridisation
or antibody staining (Reich and Shilo,
2002
; Urban et al.,
2002
). Thus, the ligand activated by Rho3 to guide GB1 migration
is very likely Spitz, as it is expressed and is functional at the midline
(Golembo et al., 1996
) (data
not shown), but we cannot formally exclude a contribution by Keren.
How does Rho3 guide ganglionic branch migration?
The mammalian EGF receptors regulate migration in a variety of contexts,
but in all known examples they appear to promote responses to
chemoattractants. They do so by directly affecting cytoskeletal organisation,
mainly through the PI3K, PKC or PLC pathways. The proper activation of the fly
Egfr is also necessary for the migration of border cells towards the source of
Gurken in the dorsal part of the oocyte
(Duchek and Rorth, 2001).
During this migration Egfr activation is coordinated with the activation of
the fly PDGF/VEGF receptor homologue and requires the conserved adaptor
protein Mbc (Dock 180/CED-5) (Duchek et
al., 2001
). Mbc provides a link to activated Rac and actin
re-arrangements, which lead to the stereotyped attraction of the border cells
towards the oocyte (Duchek and Rorth,
2001
; Duchek et al.,
2001
). It is, however, unclear whether Egfr provides the necessary
spatial information for border cells during their pathfinding, or if it is
required for the interpretation of positional cues provided by Pdgf/Vegfr or
other receptors (Montell,
2003
).
There are substantial differences in the ways by which Egfr controls migration in GB1 and in border cells. Our analysis indicates that Egfr signalling is not a chemotactic cue for tracheal pathfinding, it rather reveals a surprising role in mediating repulsion from the signalling source. In addition, mbc mutants did not show any midline crossing phenotypes that would resemble the phenotypes of rho3 or the ones generated by inactivation of the receptor. Furthermore, the increase of signalling levels in GB1, either by the expression of Rho1, activated receptor or activated Ras, resulted in a significant phenotype opposite of that of the rho3 mutants: induced GBs to turn early before reaching the midline. This suggests that at the appropriate distance from the midline, Egfr activation becomes a switch to initiate the turn of GB1 away from it. Hence, an experimental increase of signalling levels can shift the crucial switch further away from the midline, while decreased signalling causes midline crosses. In essence, migrating GBs use Egfr activation to efficiently compute their relative distance from the midline, fine-tuning their response to the repulsive and attractive cues originating from it.
Rho3 signalling in GB1 may provide a switch that changes attraction to repulsion
Migration in general, and axonal pathfinding at the midline in particular,
is known to rely on a number of guidance signals, at times redundant ones
(Dickson, 2002;
Montell, 2003
). The major
midline repulsive signal for GB1 is Slit, yet a genetic test showed that
rho3 acts in parallel to Slit. We hypothesize that Egfr works in an
analogous manner by activating a second, yet undiscovered, signalling system
for GB repulsion. Such a guidance cue may be specific for GB1 migration, as
axonal fascicles are not affected in rho3 mutants. Alternatively, the
activation of Egfr in GB1 provides an epithelial specific regulation of a
common repulsive signal used by both axons and GB1.
What could this repulsive signal be? Likely candidates fall in the short
list of conserved signals repelling axons and non-neural cells in different
systems: Netrins, Semaphorins and Ephrins (reviewed by
Dickson, 2002). Netrins are
involved in the repulsion of motor axons in both vertebrates and invertebrates
(Harris et al., 1996
;
Mitchell et al., 1996
;
Keleman and Dickson, 2001
) and
both Drosophila Netrins are expressed at the CNS midline, where they
mediate attraction of commissural axons
(Mitchell et al., 1996
).
Semaphorins and Ephrins are also capable of repelling axons and non-neural
cells in different contexts (Dickson,
2002
; Van Vactor and Lorenz,
1999
; Mellitzer et al.,
2000
), and they therefore represent possible guiding cues for GBs.
Intriguingly, each family uses receptor tyrosine kinases as receptors (in the
case of Ephrin) or co-receptors (in the case of Semaphorins). Most of these
signals are bi-functional, they can elicit both attractive and repulsive
responses on the receiving cells depending on context. Egfr activation in GB1
may lead to the post-translational modifications that activate a repellent
receptor or inactivate an attractant one and may represent a general `switch'
mechanism for changing the orientation of cell migration depending on the
strength of RTK signalling.
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ACKNOWLEDGMENTS |
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