1 Umeå Centre for Molecular Pathogenesis, Umeå University, S-901 87
Umeå, Sweden
2 Department of Developmental Biology, Wenner-Gren Institute, Stockholm
University, S-106 91 Stockholm, Sweden
* Author for correspondence (e-mail: christos{at}devbio.su.se)
Accepted 5 August 2002
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SUMMARY |
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Key words: Tracheal branching, Cell migration, Robo, Slit, Drosophila
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INTRODUCTION |
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The tracheal system develops from 20 clusters of ectodermal cells, each
containing about 80 cells. After invagination and without further cell
division, each epithelial cluster extends sequentially primary, secondary,
fusion and terminal branches to generate the tubular network that facilitates
larval respiration (Manning and Krasnow,
1993; Samakovlis et al.,
1996a
). The regular outgrowth pattern of the primary branches is
determined by the localized expression of signaling factors in the surrounding
tissues. Among these signals, Branchless (Bnl), a member of the Fibroblast
Growth Factor family, first directs the outgrowth of multicellular branches to
its site of expression, and it then induces the activation of a set of
terminal branching genes in the leading cells of the primary branches
(Sutherland et al., 1996
).
Single terminal cells then form a unicellular branch, migrate over substantial
distances and finally stretch and bind to distinct parts of the target tissue
to facilitate respiration. A single terminal cell of each ganglionic branch
(GB), for example, targets each hemisegment of the embryonic ventral nerve
cord (VNC). A cluster of bnl-expressing cells just outside the CNS
attracts the GB toward the CNS. The GB cells migrate ventrally along the
intersegmental nerve (ISN), but just before reaching the entry point into the
CNS, they break their contact with ISN and turn posteriorly to associate with
the segmental nerve (SN) (Englund et al.,
1999
). This substrate switch is promoted by the expression of
adrift (aft), a bnl-induced gene required in the
trachea for efficient entry into the CNS
(Englund et al., 1999
). Inside
the CNS, the GB1 cell extends over a distance of about 50 µm, from the
entry point into the CNS via four different neural and glial substrata to its
target on the dorsal side of the neuropil
(Englund et al., 1999
). During
the first 20 µm of its journey inside the CNS, the GB1 cell moves its cell
body and nucleus along the exit glia, the SN and ventral longitudinal glia
towards the midline (Englund et al.,
1999
) (Fig. 1A).
The rest of the path is covered by a long cytoplasmic projection that turns
dorsally at the midline and reaches the dorsal part of the neuropil by the end
of embryogenesis (Fig. 1B,C).
The signals that guide GB1 migration inside the CNS are not known but the
substrata that the GB contact along its path could potentially provide
important guidance cues.
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We investigate the importance of glial substrata in guiding the GB1 inside
the CNS. By genetic ablation experiments, we show that different glial cells
provide distinct positional cues to the trachea. Longitudinal glia are first
required for GB1 migration towards the midline, whereas midline and channel
glia are necessary for inhibiting it from crossing the midline and to make it
migrate dorsally through the neuropil. We show that Slit signaling plays a
major role in the migration of the GB1 cell. Slit is produced by midline cells
(Rothberg et al., 1988;
Rothberg et al., 1990
) and
prevent GBs from crossing the midline of the VNC. Slit is also required as an
attractant for the outgrowth of the primary, dorsal and visceral branches. The
Slit receptors Roundabout (Robo) (Brose et
al., 1999
; Kidd et al.,
1998a
) and Roundabout 2 (Robo2)
(Rajagopalan et al., 2000a
;
Simpson et al., 2000a
) are
both required in the trachea independently of their function in axonal
migration. The analysis of the tracheal robo and robo2
mutant phenotypes suggests that they may mediate different responses to the
Slit signal. These results provide a first insight into the signaling
mechanisms that guide the GB in the CNS, and identify an in vivo system for
the study of bi-functional role of Slit in epithelial cell guidance at the
level of single cells.
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MATERIALS AND METHODS |
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The following GAL4 and UAS strains were used: w;C321c (on second
chromosome, provided by A. Hidalgo)
(Hidalgo et al., 1995);
Mz520 and Mz820 (on second chromosome, both provided by G.
Technau) (Ito et al., 1995
);
SRF-GAL4 (on third chromosome, K. Guillemin, personal communication);
btl-GAL4 (Shiga et al.,
1996
); elav-GAL4 (#458); twi-GAL4 (#914) and
en-GAL4 (#233) (all three from Bloomington Stock Center);
UAS-lacZ (Brand and Perrimon,
1993
); w;UAS-ricinA/CyOwgen11lacZ
(Hidalgo et al., 1995
);
UAS-robo (Kidd et al.,
1998a
), UAS-slit
(Kidd et al., 1999
) and
UAS-comm (Kidd et al.,
1998b
) (all three provided by C. Goodman); UAS-robo and
UAS-robo2 (HA-tagged, both provided by B. Dickson)
(Rajagopalan et al., 2000b
);
and UAS-EGFPF/CyO, which harbors an enhanced GFP construct that is
targeted to the cellular membrane (provided by R. Palmer)
(Finley et al., 1998
).
The enhancer trap marker 1-eve-1 (Tracheal 1), which was used to
visualize tracheal cells, has been described previously
(Perrimon et al., 1991).
Immunostaining
Embryo fixation, staining, light and confocal fluorescence microscopy were
as described (Samakovlis et al.,
1996b). The tracheal lumen-specific antibody used was mAb2A12
(Developmental Studies Hybridoma Bank, University of Iowa) diluted 1:3. The
anti-DSRF monoclonal antibody was mAb2-161 (1:2000) from M. Gilman (Ariad
Corporation, Boston, MA). Rabbit antiserum against ß-galactosidase
(Cappel) was used at 1:1500. Rabbit antiserum against Robo2 (from B. Dickson)
(Rajagopalan et al., 2000a
)
was used at 1:100 for immunofluorescence. The mouse monoclonal antibodies
against Robo (mAb13C9) (Kidd et al.,
1998a
) and Fasciclin II (mAb1D4)
(Van Vactor et al., 1993
) were
used at 1:10 and 1:50, respectively (both from C. Goodman). Mouse monoclonal
against Slit (C555.6d) was diluted 1:10 (from B. Dickson)
(Rothberg et al., 1990
).
Rabbit antiserum against GFP (Molecular Probes) was diluted 1:1000. Anti-HA
mAb16B12 (Berkeley Antibody Company) was used at 1:1000 or 1:400. Biotin-,
Cy2- and Cy3-conjugated (Jackson Laboratories) and Alexa Fluor-568- and
-488-conjugated (Molecular Probes) secondary antibodies were used at 1:300,
1:500 and 1:400, respectively. Embryo staging was according to Campos-Ortega
and Hartenstein (Campos-Ortega and
Hartenstein, 1985
).
Ablations, rescue and gain-of-function experiments
The ablation, rescue and gain-of-function experiments were carried out
using the UAS-GAL4 system (Brand and
Perrimon, 1993). For all the UAS-GAL4 crosses mentioned below,
embryos were collected for 6 hours at 20°C and then transferred to
29°C for 10 hours to maximize GAL4 activity and they were analyzed with
markers accordingly.
To ablate glial cells w;UAS-ricinA/CyOwgen11lacZ flies were crossed to C321c driver line, which expresses GAL4 in longitudinal glia, or to Mz520 and Mz820, express GAL4 in midline glia and channel glia in the CNS, respectively. Embryos were stained with mAb2A12, anti-ß-galactosidase and mAb1D4 (which stains Fasciclin II-positive axons in the longitudinal connectives). To rescue the tracheal phenotypes of roboz570 and robo21,4,8 mutants, we used the SRF-GAL4 driver, which expresses GAL4 in all tracheal terminal cells from stage 13, to drive expression of UAS-robo and UAS-robo2. In addition, the reciprocal tracheal rescue and the UAS-robo and UAS-robo2 tracheal gain-of-function experiments were made using these strains and embryos were analyzed with mAb2A12, mAb2-161 (marks tracheal terminal cells), mAb1D4, mAb16B12 and mAb13C9. To rescue the robo mutant CNS phenotype UAS-robo was driven by elav-GAL4, which is expressed in all postmitotic neurons in the CNS. For the comm gain-of-function experiment, we used the UAS-comm and the btl-GAL4 driver, which expresses GAL4 in all tracheal cells from stage 11. For the slit gain-of-function experiment in the CNS, we used C321c-GAL4 and elav-GAL4 strains to drive expression of UAS-slit, and to misexpress UAS-slit in the gut epithelium and in the epidermis, we used the twi-GAL4 and en-GAL4 drivers, respectively.
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RESULTS |
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When we ablated longitudinal glia with the C321c-GAL4 line
(Hidalgo et al., 1995), 31% of
GBs (n=112) stalled or turned to migrate posteriorly before reaching
the longitudinal connectives (Fig.
1H and data not shown). The formation of longitudinal axon tracts
as revealed by mAb1D4 staining was not detectably affected in these embryos,
suggesting that longitudinal glia might have a function in guiding the GB to
the ventral side of the neuropil and across the longitudinal tracts. Ablation
of midline glia using the Mz520-GAL4 line
(Ito et al., 1995
) caused a
different phenotype. Nine percent of the GBs (n=112) crossed the
midline, and 5% lingered along the midline or turned anteriorly
(Fig. 1I). Staining with mAb1D4
showed that some axons of the longitudinal tracts also were crossing the
midline (Fig. 1I). These
results suggest that midline glia influence GB1 turning either through direct
signaling or indirectly by affecting the structure of surrounding axon
trajectories. We have also expressed Ricin A in subperineurial and channel
glia using the Mz820-GAL4 line
(Ito et al., 1995
). In these
embryos, 6% of GBs (n=126) turned to migrate posteriorly before
crossing the longitudinal connectives and 8% lingered around the midline (data
not shown). We did not observe any defects in the Fasciclin II-positive axons,
suggesting that dorsoventral channel glia may provide an instructive landmark
for the extension of GB1 from the ventral to the dorsal side of the
neuropil.
In summary, the above results suggest that the different types of glia that become contacted by GB1 inside the VNC provide distinct guidance landmarks for its migration. As the ablation of longitudinal glia results in stalls or misroutings before the longitudinal tracts, these cells are likely to have an attractive function for GB1. The ablation of midline and channel glia leads to midline crossings and lingering, suggesting that these cells may provide both direct or indirect attractive and repulsive landmarks.
Expression of Slit and its receptors during tracheal development
A major determinant of axonal pathways inside the CNS is the repellent
signal Slit. Midline cells express Slit, a large extracellular matrix protein
(Rothberg et al., 1988;
Rothberg et al., 1990
) that
functions both as a short- and long-range repellent, controlling axon crossing
at the midline and mesodermal cell migration away from the midline
(Battye et al., 1999
;
Kidd et al., 1999
). In axon
guidance, the Slit repulsive signal is mediated by the Roundabout (Robo)
receptors (Brose et al., 1999
;
Kidd et al., 1999
;
Kidd et al., 1998b
;
Rajagopalan et al., 2000a
;
Simpson et al., 2000a
).
Different axons express different combinations of the three receptors, which
determine the distance of their projections from the midline along the
longitudinal fascicles (Rajagopalan et
al., 2000b
; Simpson et al.,
2000b
). The midline crossing phenotypes of GBs in embryos
expressing Ricin A in the midline glia suggests that Slit signaling may also
guide GB1 in its turn away from the midline. We double stained embryos
expressing GFP under the control of the pan-tracheal btl-GAL4 driver
(Shiga et al., 1996
), which
drives expression of GAL4 in all tracheal cells from stage11, with antibodies
against GFP and Slit or its receptors, and analyzed their expression by
confocal microscopy. The GB1 cell comes close to the midline source of Slit at
early stage 16 but it then turns dorsally and posteriorly at the midline
(Fig. 2A, top). Slit is also
expressed in several other tissues close to the migrating tracheal branches.
At early stage 14 in the dorsal side of the embryo, two rows of migrating
mesodermal cells that will form the larval heart express Slit. These
cardioblasts are in close proximity to the two leading cells of the tracheal
dorsal branches (DBs), which also migrate towards the dorsal midline and give
rise to the dorsal anastomosis (DB2) and the dorsal terminal branch (DB1)
(Fig. 2A, bottom). Slit
expression is also detected from stage 13 on the surface of the midgut, at the
sites of contact of the growing tracheal visceral branches (VBs)
(Fig. 2B). Finally we detected
Slit in lateral stripes of epidermal cells adjacent to the growing dorsal
trunk (DT) and dorsal branches from stage 13 (data not shown). Are then the
Slit receptors expressed in the trachea? Robo staining can be detected in all
tracheal cells as they invaginate from the epidermis already at stage 11
(Fig. 2C, top). Its tracheal
expression is decreased by stage 13, when it is only weakly expressed in the
dorsal trunk (Fig. 2C, bottom).
We were not able to detect any convincing expression of Robo in the trachea
after stage 14, even when we analyzed serial optical sections of the GB1 cell
along its path in the CNS (Fig.
1 and data not shown). Robo2 is also expressed in all tracheal
cells from stage 11 (Fig. 2D,
top) and it then becomes restricted to the dorsal trunk and dorsal and
visceral branches by stage 13 (Fig.
2D, bottom; Fig.
2E) (Rajagopalan et al.,
2000a
). In contrast to Robo, which becomes undetectable in the
trachea by stage 14, Robo2 expression is stronger and is maintained as late as
at stage 16 in the DB1 and DB2 cells at the dorsal midline (data not shown).
Robo3 expression could not be detected in the trachea
(Rajagopalan et al., 2000a
).
The expression of Slit in tissues surrounding the developing trachea and the
dynamic expression of its two receptors in different tracheal branches
suggested a role for Slit signaling in tracheal branch outgrowth towards their
target tissues.
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Slit signaling is required for ganglionic branch turning at the
midline
Although we could not detect the expression of any of the known Slit
receptors in the GBs, the abrupt turn of GB1 when it comes close to the
midline and the results from the midline cell ablation experiments prompted us
to examine the potential role of Slit in the GB1 pathfinding. We first
analyzed, robo, robo2 and slit mutant embryos double stained
with antibodies against the tracheal lumen and a tracheal nuclear marker. We
also studied embryos of these genotypes, double stained for the tracheal lumen
and Fasciclin II to correlate the tracheal and axonal phenotypes in the same
embryos. In robo mutants, all GBs migrated into the VNC and the
position of GB1 nucleus was not significantly affected
(Fig. 3B). At the midline, 29%
of GBs (n=140) crossed and 26% migrated unusually close to the
midline, where they stalled or turned to migrate dorsally
(Fig. 3B). The characteristic
structure of the three longitudinal connectives in the same embryos was also
severely disrupted by loops of axons crossing the midline several times
(Kidd et al., 1998a)
(Fig. 3G). In contrast to
axons, GB1 crossed the midline only once and migrated along the longitudinal
tracts of the contralateral hemisegment. This phenotype suggested that GB1
might not just passively follow the misrouted axons and argued that Robo might
function as a repellent receptor in GB1 independently of its role in the
neurons. It also suggests that robo is only required to prevent GB1
from crossing the midline and not to repel it once it has entered. If the GB1
phenotype in robo mutants is a primary effect caused by loss of Robo
in the trachea, it should be possible to rescue this phenotype by selectively
expressing robo in GB1. We crossed an UAS-robo transgene and
a SRF-GAL4 driver construct, which expresses GAL4 in all tracheal
terminal cells, into the robo mutant strain. The anti-Robo antibody
was used to detect Robo protein deriving from the transgene. When Robo was
expressed in the GB1 of robo mutants
(robo/robo;UAS-robo/SRF-GAL4), none of the GBs crossed the
midline (Table 1;
Fig. 3L). Instead, 91% of the
GBs that expressed Robo in GB1 (n=118) turned to migrate posteriorly
prematurely (Fig. 3L). In these
tracheal rescued robo mutants, 12% of the GB1 cells did not express
any detectable Robo and 18% of these cells crossed the midline
(Fig. 3L). Thus, Robo
expression in the GB1 cells of robo mutants fully rescues the
tracheal crossing of the midline phenotype. In the reciprocal experiment,
where expression of UAS-robo in homozygous robo mutant
embryos was driven in all neurons by the elav-GAL4 strain
(elav-GAL4/+;robo/robo;UAS-robo/+), the crossing of
Fasciclin II-positive axons was significantly rescued
(Kidd et al., 1998a
)
(Fig. 3K), but 21% of the GBs
(n=168) still crossed the midline
(Table 1;
Fig. 3K). We conclude, that
Robo is required in the GB1 tracheal cell to prevent it from crossing the CNS
midline.
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Robo2 acts as a long distance Slit receptor in the axons of the lateral
longitudinal fascicles (Rajagopalan et
al., 2000a; Simpson et al.,
2000a
). In robo2 mutants, the more distant lateral
fascicle is disrupted and its axons come closer to the midline and
occasionally cross it (Rajagopalan et al.,
2000a
; Simpson et al.,
2000a
) (Fig. 3H).
The analysis of the tracheal phenotypes of two robo2 mutant alleles,
robo24 and robo28, produced similar
results and revealed that the GB defects of robo and robo2
mutants were different. Only 6% (n=160) of the branches crossed the
midline in robo24 embryos and, in contrast to the
robo mutant phenotype, these branches did not extend to the
contralateral longitudinal connectives, they lingered around the midline
instead. The most striking defect of robo2 mutants was that 46% of
the branches were stalled before reaching the midline turning point, and an
additional 11% of them did not even enter the CNS
(Fig. 3C) suggesting that
robo2 may be required earlier than robo for the migration of
the GB1 cell towards the midline glia. We expressed a HA-tagged version of
Robo2 in the GB1 cell under the control of the SRF-GAL4 driver in
robo24 embryos to assess whether we could rescue the
mutant phenotypes. The embryos were double stained with antibodies against the
tracheal lumen and HA to identify branches expressing Robo2. The
number of GBs stalled outside the CNS was markedly reduced in these embryos
(Fig. 3M), 1% (n=220)
compared with 11% (n=160) in robo24 mutants,
suggesting that robo2 is required in the GB1 for its entry into the
CNS. In the same experiment we did not observe any reduction of the number of
branches that cross the midline, suggesting that this phenotype may be
indirect or that the expression levels of transgenic Robo2 protein in the
robo2 mutants where not optimal for rescuing this phenotype. In fact,
17% of the GB1s in these embryos turned prematurely before reaching the
midline (Fig. 3M).
In slit mutants, the CNS axons enter the midline and remain there
forming one large axon fascicle (Kidd et
al., 1999; Rothberg et al.,
1990
) (Fig. 3I).
When we looked at GB pathfinding in slit mutant embryos, we found
defects both outside and inside the CNS. Practically all GBs were misrouted,
and branches did not migrate in the same dorsoventral plane. We found that 17%
of GBs (n=154) stalled outside or inside the CNS and that 37% crossed
the midline (Fig. 3D). The
tracheal phenotypes in robo,robo2 double mutants were similar to the
defects of slit embryos, 21% of the branches (n=140) stalled
outside the CNS, 31% crossed the midline and 45% were misrouted
(Fig. 3E).
This analysis indicates that Slit is an important regulator of GB1 pathfinding towards and inside the CNS. Slit function is mediated in the trachea by the Robo and Robo2 receptors. The differences in tracheal phenotypes in robo and robo2 mutants, suggest that Robo2 functions as a long distance attractant receptor for Slit during GB1 migration towards the CNS, whereas Robo mediates the repellent function of Slit inside the CNS, at the ventral midline.
Ectopic activation of Slit signaling redirects GB1 away from the
midline
To further dissect the function of Slit and its receptors in GB1 migration
we studied the effects of over activation of the pathway by either driving
slit expression in longitudinal glia or manipulating the expression
levels of robo and robo2.
Ectopic expression of slit in the longitudinal glia (C321c-GAL4/+;UAS-slit/+) causes stalls and premature turns in 37% of the GBs (Fig. 4E) suggesting that elevated levels of slit are sufficient to inhibit and repel GB1 migration inside the CNS. The longitudinal tracts in these embryos are formed normally as judged by mAb1D4 staining (Fig. 4E).
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Robo and Robo2 expression can be post-translationally downregulated by
Commissureless (Comm), a transmembrane protein that is expressed on the CNS
midline cells during the formation of axon commissures
(Tear et al., 1996). Comm is
necessary to decrease Robo expression on commissural axons thereby allowing
them to cross the midline (Kidd et al.,
1998b
; Seeger et al.,
1993
). In comm mutants, Robo fails to be downregulated,
no axons cross the midline and as a result no commissures are formed
(Fig. 4B). In comm
mutants, 97% of GBs (n=140) stalled or turned to migrate dorsally and
posteriorly prematurely, before reaching the midline
(Fig. 4B), suggesting that Comm
is also modulating the levels of Robo in the trachea. We hypothesized that if
Robo is downregulated by Comm, then transgenic expression of comm in
the trachea might lead to downregulation of Robo in GB1 and a comm
`gain-of-function' phenotype where GB cross the midline. To test this, we
crossed UAS-comm flies to the btl-GAL4 line
(Shiga et al., 1996
). In these
embryos (UAS-comm/+;btl-GAL4/+) the longitudinal axon tracts
looked normal but 17% of the GBs (n=168) crossed the midline, 14%
turned anteriorly at the midline and 15% stalled prematurely
(Fig. 4F). These tracheal
phenotypes are similar to the defects seen in slit mutant embryos and
further argue for the role of Robo receptors in GB migration.
To overexpress robo and robo2 in GB1 cells, we used the SRF-GAL4 driver and epitope tagged forms of receptor transgenes, UAS-roboHA and UAS-robo2HA, which express similar levels of protein, as judged by immunohistochemical staining. These constructs were chosen to allow comparisons of the different phenotypes caused by the overexpression of the robo or robo2 in the trachea. In UAS-roboHA/SRF-GAL4 embryos, 55% of the HA-expressing GBs (n=104) turned prematurely to migrate posteriorly before coming close to the midline, and 16% stalled at the level of the longitudinal tracts (Fig. 4C). The premature turning phenotype resembles the tracheal defects in comm mutants and argues for a midline repulsive function for robo in GB1. The same phenotype was evident in embryos expressing an untagged version of UAS-robo under the control of the same driver, but there 94% of the Robo-expressing branches (n=104) were turning prematurely. In UAS-robo2HA/SRF-GAL4 embryos, only 12% of the GBs expressing HA (n=105) turn to migrate posteriorly prematurely (Fig. 4D). The quantitative differences in the turning phenotype of the overexpression experiments, taken together with the qualitative differences in the loss-of-function phenotypes for both receptors, suggest that robo2 plays a minor role in GB1 pathfinding inside the CNS and that robo is the major repellent receptor at the midline.
Robo can rescue robo2 but Robo2 can not rescue robo
phenotypes
To further separate the potentially different functions of robo
and robo2, we attempted to rescue the GB1 phenotype of one mutant by
ectopically expressing a transgene encoding the tagged form of the other
receptor. Overexpression of Robo in the GB1 cell of robo2 mutants
(detected by staining against HA) can partially rescue the entry into the CNS
phenotype from 15% (n=238) in
robo28/robo24 embryos to 3.5% (n=123)
in rescue embryos (robo24/robo28;
UAS-roboHA/SRF-GAL4) (Fig.
4H). Overexpression could also rescue the weak midline crossing
phenotype of robo2. None of the GBs crossed the midline compared with
the 6% midline crossing phenotype in
robo28/robo24. In addition, robo
overexpression in robo2 mutants caused a gain-of-function phenotype
with 50% of the branches turning prematurely inside the CNS
(Fig. 4H), similar to the
robo overexpression phenotype in wild-type embryos. Thus,
robo expression in the GB1 cells of robo2 mutant rescues the
CNS entry and midline crossing phenotypes. When Robo2 was ectopically
expressed in GB1 of robo mutant embryos
(robo/robo;UAS-robo2HA/SRF-GAL4), it did not provide substantial
rescue activity. Twenty-two percent of GBs (n=111) still crossed the
midline compared with 29% (n=140) in the robo mutant
(Fig. 4G). In addition,
robo2 overexpression induced slightly fewer gain-of-function
premature turns in robo mutants (7% compared with 11% in the wild
type). These results argue that although robo misexpression can
substitute for the absence of robo2 during the migration of GB1
towards the CNS, overexpression of robo2 can not significantly
substitute for robo in its repellent function at the midline.
Functions of Slit and its receptors in other tracheal branches
The dynamic expression of Slit in several tracheal targets and the
branch-restricted expression of robo and robo2 during
tracheal development suggested an additional role of Slit signaling in
tracheal branches that do not target the CNS. We analyzed slit, robo
and robo2 embryos carrying the 1-eve-1 lacZ marker, in order
to visualize defects in tracheal cell migration and branching. In late stage
16 slit mutant embryos, the migration of the dorsal branches towards
the heart was disrupted. Twenty percent of the branches (n=100) were
either completely missing or stalled at various lengths
(Fig. 5C). In addition, 28% of
the branch fusion events that interconnect the tracheal network of either side
of the embryo over the dorsal midline were also disrupted. Given the
expression of Slit in the developing heart from stage 14, this phenotype
suggests an attractive function for Slit in dorsal branch migration towards
the dorsal midline of the embryo. slit embryos showed also defects in
the migration of the GBs towards the lateral and ventral muscles and the CNS
(Fig. 5F). In slit
mutants, the cells of the ganglionic branches appeared to be extending
projections towards the muscles but their directions were random and they were
falling back towards the lateral trunk
(Fig. 5F). Slit protein was
also detected along the developing midgut at the attachment sites of the
visceral branches (Fig. 2B). In
slit mutants, all primary visceral branches grew towards the gut but
the migration of the secondary branches on the target appeared irregular in
some embryos with occasional projections extending more dorsally or ventrally
along the midgut (data not shown). The tracheal phenotypes of
robo28 mutants were similar to the defects of
slit embryos. Eighteen percent of the dorsal branches
(n=220) were stalled or missing in robo2 embryos and 30% of
the remaining branches that had extended towards the dorsal midline failed to
fuse over the heart (Fig. 5B).
In addition, as in slit embryos, the outgrowth of the GBs from the
lateral trunk (Fig. 5E) was
also sporadically affected. We could not detect any primary branch outgrowth
phenotypes, in robo mutants (Fig.
5A,D). We have carefully examined more than 20 embryos from two
different robo alleles and we conclude that robo is not
required for the migration of tracheal branches towards targets outside the
CNS. Every slit and robo2 mutant embryo analyzed shows
defects in the outgrowth of the dorsal branches and the extension of the
fusion branches over the dorsal midline, the expression pattern and mutant
phenotypes of slit and robo2 mutants suggest that the
expression of Slit outside the CNS provides an attractant signal for the
tracheal cells and that the tracheal receptor for this signal is Robo2.
|
Ectopic Slit attracts tracheal branches to its sites of
expression
If Slit is an attractive signal for the migration of the dorsal and
visceral branches towards their targets, its ectopic expression in other
tissues should attract tracheal branches towards the new expression sites. We
expressed UAS-slit with the en-GAL4 driver in stripes of
epidermal cells along the dorsoventral axis located above the growth tracts of
the dorsal branches (Fig. 6A).
In these embryos, 23% of the dorsal trunk branches (n=340) that
normally extend anteriorly and posteriorly to interconnect the ten tracheal
metameres on either side of the embryo were affected
(Fig. 6B). This defect became
more pronounced when we ectopically expressed UAS-slit in the
engrailed stripes in slit mutant embryos. Fifty-two percent of the
dorsal trunk fusion events were disrupted (n=240); the dorsal trunk
branches, instead of extending anteriorly and posteriorly, seemed to elongate
dorsoventrally along the ectopic source of Slit
(Fig. 6C). These results
suggest that endogenous Slit is required for the migration of the dorsal trunk
branches and that ectopic Slit expressed on epidermal stripes can redirect
this migration to the site of its expression.
|
To analyze the role of Slit and its receptors in the formation of the
visceral branches, we examined the phenotypes in the visceral branches of
embryos expressing UAS-slit in the midgut visceral mesoderm and other
mesodermal tissues under the control of the twi-GAL4 driver
(Fig. 6D,E). In wild-type
embryos, six out of the 10 tracheal metameres send visceral branches towards
the gut. The most anterior (T1) and posterior (T10) metameres as well as trunk
metameres T3 and T9 do not extend any visceral branches
(Manning and Krasnow, 1993)
(Fig. 6H). Overexpression of
UAS-slit in the visceral mesoderm generated new visceral branches
from T3 in 11% of the embryos (Fig.
6I), indicating that overexpression of slit can attract
the migration of tracheal cells to the gut and generate new branches. We then
asked whether Robo2 exclusively mediates the attractant function of Slit. We
directed UAS-slit expression in the visceral mesoderm under the
twi-GAL4 driver in robo28 mutants. We could not
detect any new branches growing from trunk segments T2 to T9 in the 25 embryos
we examined (Fig. 6F),
suggesting that robo2 is necessary to mediate the tracheal attractant
function of Slit in the visceral branches. The analysis of UAS-slit
overexpression in the visceral mesoderm in robo mutant embryos
revealed a surprising phenotype. 34% of the embryos had extra visceral
branches in the trunk segments. These were either new branches deriving either
from T3 and T9 or from bifurcations of the wild-type branches in the rest of
the metameres (Fig. 6G,J). As
the percentage of new branches induced by Slit is threefold higher in
robo mutants than in the wild-type, the results suggest that
robo is an antagonist of Robo2 in the Slit-mediated attraction of the
visceral branches.
![]() |
DISCUSSION |
---|
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---|
Attractive and repulsive functions of Slit
The elegant analysis of axonal guidance at the midline of the fly CNS
established the Slit repellent signal as major determinant of axonal pathways
(Kidd et al., 1999;
Kidd et al., 1998a
;
Rothberg et al., 1990
). A
gradient of Slit emanating from the midline prevents axons from crossing the
midline through the activation of Robo receptors but it also functions as a
long range repellent to position axons in distinct lateral fascicles. This
later function is mediated by the expression of different combinations of
Robo, Robo2 and Robo3 on axons that take distinct positions along the
longitudinal tracts (Rajagopalan et al.,
2000a
; Simpson et al.,
2000b
).
Mammalian Slit can also function as a positive regulator of axonal
elongation and branching of sensory axons from the rat dorsal root ganglia
(Wang et al., 1999) and more
recently Slit was found to play an attractive role for muscles during their
extension to muscle attachment sites on the Drosophila epidermis
(Kramer et al., 2001
). The
molecular mechanism behind the different responses to Slit remains unknown.
Repulsion versus attraction could reflect a difference in receptor subunit
composition or variations in the cytoplasmic signal transduction machinery of
the responsive cells. The complex expression pattern of Slit on several
tissues close to the growing tracheal branches together with the tracheal
migration defects in slit mutants indicate that it plays an important
role in epithelial cell guidance. Lack of Slit affects the oriented outgrowth
of the dorsal, visceral and ganglionic primary branches, the cells of these
branches either stall their migration towards the Slit expressing target or
they become misrouted. Overexpression of Slit with a mesodermal GAL4 driver is
sufficient to attract new branches towards the gut and overexpression of Slit
on epidermal stripes running along the dorsoventral axis of the embryo
redirects the anteroposterior migration of the dorsal trunk branches along the
new sites of Slit expression. This re-orientation phenotype becomes stronger
in slit mutants indicating that endogenous slit provides a
migration cue for these branches. The analysis of loss-of-function and
overexpression phenotypes indicates that Slit is a chemoattractant for the
outgrowth of several primary tracheal branches towards their targets.
The analysis of GB1 phenotypes in slit mutants argues for a repellent function at the midline. In the absence of functional Slit from the CNS midline 37% of the GB1 cells cross the midline barrier and ectopic of slit on the longitudinal glia causes GB1 to stall or turn prematurely when it approaches the longitudinal tracts. Thus, Slit functions as a bi-functional guidance signal in the trachea. The tracheal phenotypes of slit in primary and secondary branches are not fully penetrant, emphasizing the importance of other signals in guiding the tracheal branches to their targets. What is the relationship of Slit to the known guidance cues? As Slit is required for the outgrowth of some primary branches, one might have expected that overexpression of Slit in the epidermis by en-GAL4 would partially rescue the complete absence of tracheal branches in bnl mutants. We did not detect any branch outgrowth in bnl mutant embryos overexpressing slit (data not shown), suggesting that the ability to respond to Slit requires the activity of the Bnl/FGF signaling cascade in the trachea. The most prominent primary branch phenotype of slit mutants is the sporadic lack of outgrowth of the dorsal branches. Dpp/TGFß signaling is required for the outgrowth of these branches, suggesting that the localized strong expression of the Robo and Robo2 receptors might be regulated by Dpp signaling. The abundance Robo and Robo2 was, however, unaffected in null mutants for the Dpp co-receptor, Thickveins (Tkv), or in embryos overexpressing a dominant active from of Tkv in the trachea (data not shown), suggesting that Dpp is not likely to regulate the tracheal responses to Slit.
Different functions for Robo and Robo2 in the trachea
In CNS and muscle development Slit function is mediated by the Robo
receptors (Brose et al., 1999;
Kidd et al., 1999
;
Kidd et al., 1998b
;
Kramer et al., 2001
;
Rajagopalan et al., 2000a
;
Simpson et al., 2000a
).
robo and robo2 are expressed in the trachea and the tracheal
phenotypes of robo, robo2 double mutant embryos were very similar to
the phenotypes of slit mutants, indicating that the tracheal
responses to Slit are mediated by Robo and Robo2. Robo and Robo2 receptors can
form homo- and heterodimers in vitro
(Simpson et al., 2000a
) and
the differences it their expression patterns suggests that they might mediate
different responses to Slit. Indeed, the comparison of the phenotypes between
the mutants for either of the two receptors genes revealed some intriguing
differences. In robo embryos, the GBs erroneously cross the midline,
suggesting that slit signaling via robo mediate repulsion
away from the midline. In robo2 mutants on the other hand, GBs fail
to enter the CNS, suggesting that Robo2 may mediate an attractive response to
Slit. In addition, the stalls in the migration of the dorsal branches detected
in slit embryos were only found in robo2 mutants; no
stalling phenotypes were detected in the tracheal branches that did not target
the CNS in robo mutants. There is also a difference between the
phenotypes generated by overexpression of robo and robo2.
Overexpression of Robo in GB1 causes most of the branches to turn away from
the midline prematurely. This phenotype is much weaker in embryos
overexpressing Robo2, indicating that Robo is a more potent repulsive receptor
in the GB. In addition, tracheal overexpression of Robo2 cannot rescue the
robo mutant GB phenotype, even though tracheal expression of Robo
can. This result further indicates that Robo and Robo2 are not identical in
their output and they cannot simply substitute for one another.
To further investigate whether different receptor complexes may mediate different responses to Slit, we took advantage of the phenotypes caused by overexpression of Slit in the gut. In wild-type embryos, ectopic Slit can attract new visceral branches to its site of expression. This attractive function of Slit requires Robo2, as overexpression of Slit with the same driver did not induce branch outgrowth in robo2 mutants. Robo alone cannot mediate the attractive response to Slit in the visceral branches, instead it appears to function as an antagonist of the attractive signal mediated by Slit and Robo 2 in the visceral branches, because the number of new branches induced by Slit in robo mutants is three times higher than the number of branches induced under the same conditions in wild-type embryos.
Taken together these results suggest that there are qualitative differences between the cellular responses to Robo and Robo2 activation and that each receptor plays a unique role in the control of tracheal cell migration. What is the basis for this difference?
Migrating growth cones encounter many guidance cues along their paths and
these signals can be either attractive, repulsive or bi-functional. The
response to a guidance signal is determined by the intracellular domain of its
receptor. Bashaw and Goodman (Bashaw and
Goodman, 1999) showed that a chimeric receptor with the ectodomain
of Frazzled (the receptor that mediate attractive response to Netrin) and the
cytoplasmic domain of Robo (the receptor that mediate repulsive response to
Slit) mediate a repulsive response to Netrin. The reciprocal Robo-Frazzled
chimeric receptor, however, mediated an attractive response to Slit. Thus, a
major determinant of the cellular response to a signal is the specific signal
transduction machinery that will be activated upon ligand binding. Robo
promotes neuronal repulsion at the midline through its cytoplasmic tail which
binds directly to Abl, Ena/Vasp (Bashaw et
al., 2000
) and srGAPs (Wong et
al., 2001
). These downstream effectors are then thought to
directly modulate actin polymerization and cellular extension during axonal
pathfinding.
A second mechanism for axon repulsion derives from the thorough studies of
Robo and Netrin signaling in vitro with cultured Xenopus neurons.
There, the cytoplasmic domain of Robo was found to mediate axonal repulsion
partly by directly binding to the cytoplasmic domain of the Netrin receptors
and thus silencing the attractive Netrin signal
(Stein and Tessier-Lavigne,
2001).
Several recent studies of neuronal guidance cues have revealed that the
generation of either attractive or repulsive responses by the same ligand also
depends on the type of receptor or receptor complex expressed on the surface
of the growth cone. Netrins, for example, mediate attraction via DCC homodimer
receptor and repulsion via UNC-5 homodimer receptor and DCC/UNC-5 heterodimer
receptor (Colavita and Culotti,
1998; Hong et al.,
1999
; Keleman and Dickson,
2001
; Leonardo et al.,
1997
). Our results suggest a similar scenario for the
interpretation of the Slit signal by tracheal cells. Cells expressing the
Robo2 homodimer perceive Slit as an attractant, whereas cells expressing the
Robo homodimer or the Robo/Robo2 heterodimer perceive it as a repellent. One
possible explanation for the different responses to different receptor
complexes is that they may activate different subsets of signal transduction
pathways inside the tracheal cells. This is not unlikely, as Robo and Robo2
differ in their cytoplasmic domains. Robo2 lacks two of the cytoplasmic motifs
that are found in the Robo receptors in various species
(Rajagopalan et al., 2000a
;
Simpson et al., 2000a
), motifs
that are required in Robo for it to regulate midline crossing
(Kidd et al., 1998a
).
In addition to their function in neuronal guidance, vertebrate Slit
homologs and their receptors are also involved in the migration of leukocytes
(Wu et al., 2001) and lung
development (Xian et al.,
2001
). The dual function of Slit mediated by different receptor
complexes in the trachea may represent a general mechanism for its function
also in other systems. The identification of downstream effectors of the
different receptor complexes will help to elucidate the molecular mechanism
that translates the Slit extracellular signals into attractive or repulsive
responses.
![]() |
ACKNOWLEDGMENTS |
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REFERENCES |
---|
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---|
Bashaw, G. J. and Goodman, C. S. (1999). Chimeric axon guidance receptors: the cytoplasmic domains of Slit and Netrin receptors specify attraction versus repulsion. Cell 97,917 -926.[Medline]
Bashaw, G. J., Kidd, T., Murray, D., Pawson, T. and Goodman, C. S. (2000). Repulsive axon guidance: Abelson and Enabled play opposing roles downstream of the Roundabout receptor. Cell 101,703 -715.[Medline]
Battye, R., Stevens, A. and Jacobs, J. R.
(1999). Axon repulsion from the midline of the
Drosophila CNS requires slit function.
Development 126,2475
-2481.
Brand, A. H. and Perrimon, N. (1993). Targeted
gene expression as a means of altering cell fates and generating dominant
phenotypes. Development
118,401
-415.
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.[Medline]
Campos-Ortega, A. J. and Hartenstein, V. (1985). The Embryonic Development of Drosophila melanogaster. New York: Springer-Verlag.
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]
Englund, C., Uv, A. E., Cantera, R., Mathies, L. D., Krasnow, M.
A. and Samakovlis, C. (1999). adrift, a novel
bnl-induced Drosophila gene, required for tracheal
pathfinding into the CNS. Development
126,1505
-1514.
Finley, K. D., Edeen, P. T., Foss, M., Gross, E., Ghbeish, N., Palmeer, R. H., Taylor, B. J. and McKeown, M. (1998). dissatisfaction encodes a Tailless-like nuclear receptor expressed in a subset of CNS neurons controlling Drosophila sexual behavior. Neuron 21,1363 -1374.[Medline]
Hidalgo, A., Urban, J. and Brand, A. H. (1995).
Targeted ablation of glia disrupts axon tract formation in the
Drosophila CNS. Development
121,3703
-3712.
Hong, K., Hinck, L., Nishiyama, M., Poo, 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.[Medline]
Hummel, T., Schimmelpfeng, K. and Klämbt, C. (1999). Commissure formation in the embryonic CNS of Drosophila. Dev. Biol. 209,381 -398.[CrossRef][Medline]
Ito, K., Urban, J. and Technau, G. M. (1995). Distribution, classification and development of Drosophila glial cells in the late embryonic and early larval ventral nerv cord. Roux's Arch. Dev. Biol. 204,284 -307.
Keleman, K. and Dickson, B. J. (2001). Short- and long-range repulsion by the Drosophila Unc5 Netrin receptor. Neuron 32,605 -617.[Medline]
Kidd, T., Brose, K., Mitchell, K. J., Fetter, R. D., Tessier-Lavigne, M., Goodman, C. S. and Tear, G. (1998a). Roundabout controls axon crossing of the CNS midline and defines a novel subfamily of evolutionarily conserved guidance receptors. Cell 92,205 -215.[Medline]
Kidd, T., Russell, C., Goodman, C. S. and Tear, G. (1998b). Dosage-sensitive and complementary functions of Roundabout and Commissureless controlaxon crossing of the CNS midline. Neuron 20,25 -33.[Medline]
Kidd, T., Bland, K. S. and Goodman, C. S. (1999). Slit is the midline repellent for the Robo receptor in Drosophila. Cell 96,785 -794.[Medline]
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.
Leonardo, E. D., Hinck, L., Masu, M., Keino-Masu, K., Ackerman, S. L. and Tessier-Lavigne, M. (1997). Vertebrate homologues of C. elegans UNC-5 are candidate netrin receptors. Nature 386,833 -838.[CrossRef][Medline]
Manning, G. and Krasnow, M. A. (1993). Development of the Drosophila tracheal system. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.
Moffat, K. G., Gould, J. H., Smith, K. and O'Kane, C. J. (1992). Inducible cell ablation in Drosophila by cold-sensitive ricin A chain. Development 114,681 -687.[Abstract]
Perrimon, N., Noll, E., McCall, K. and Brand, A. (1991). Generating lineage-specific markers to study Drosophila development. Dev. Genet. 12,238 -252.[Medline]
Rajagopalan, S., Nicolas, E., Vivancos, V., Berger, J. and Dickson, B. J. (2000b). Crossing the midline: roles and regulation of Robo receptors. Neuron 28,767 -777.[Medline]
Rajagopalan, S., Vivancos, V., Nicolas, E. and Dickson, B. J. (2000a). Selecting a longitudinal pathway: Robo receptors specify the lateral position of axons in the Drosophila CNS. Cell 103,1033 -1045.[Medline]
Rothberg, J. M., Hartley, D. A., Walther, Z. and Artavanis-Tsakonas, S. (1988). slit: An EGF-homologous locus of D. melanogaster involved in the development of the embryonic central nervous system. Cell 55,1047 -1059.[Medline]
Rothberg, J. M., Jacobs, J. R., Goodman, C. S. and Artavanis-Tsakonas, S. (1990). slit: An extracellular protein necessary for development of midline glia and commissural axon pathways contains both EGF and LRR domains. Genes Dev. 4,2169 -2187.[Abstract]
Samakovlis, C., Hacohen, N., Manning, G., Sutherland, D.,
Guillemin, K. and Krasnow, M. A. (1996a). Development of the
Drosophila tracheal system occurs by a series of morphologically
distinct but genetically coupled branching events.
Development 122,1395
-1407.
Samakovlis, C., Manning, G., Steneberg, P., Hacohen, N.,
Cantera, R. and Krasnow, M. A. (1996b). Genetic control of
epithelial tube fusion during Drosophila tracheal development.
Development 122,3531
-3536.
Seeger, M., Tear, G., Ferres-Marco, D. and Goodman, C. S. (1993). Mutations affecting growth cone guidance in Drosophila: Genes necessary for guidance toward or away from the midline. Neuron 10,409 -426.[Medline]
Shiga, Y., Tanaka-Matakatsu, M. and Hayashi, S. (1996). A nuclear GFP/b-galactosidase fusion protein as a marker for morphogenesis in living Drosophila. Dev. Growth Diff. 38,99 -106.
Simpson, J. H., Kidd, T., Bland, K. S. and Goodman, C. S. (2000a). Short-range and long-range guidance by Slit and its Robo receptors: Robo and Robo2 play distinct roles in midline guidance. Neuron 28,753 -766.[Medline]
Simpson, J. H., Bland, K. S., Fetter, R. D. and Goodman, C. S. (2000b). Short-range and long-range guidance by Slit and its Robo receptors: A combinatiorial code of Robo receptors controls lateral position. Cell 103,1019 -1032.[Medline]
Stein, E. and Tessier-Lavigne, M. (2001).
Hierarchial organization of guidance receptors: Silencing of Netrin attraction
by Slit through a Robo/DCC receptor complex. Science
291,1928
-1938.
Sutherland, D., Samakovlis, C. and Krasnow, M. A. (1996). branchless encodes a Drosophila FGF homolog that controls tracheal cell migration and the pattern of branching. Cell 87,1091 -1101.[Medline]
Tear, G., Harris, R., Sutaria, S., Kilomanski, K., Goodman, C. S. and Seeger, M. A. (1996). commissureless controls growth cone guidance across the CNS midline in Drosophila and encodes a novel membrane protein. Neuron 16,501 -514.[Medline]
Van Vactor, D., Sink, H., Fambrough, D., Tsoo, R. and Goodman, C. S. (1993). Genes that control neuromuscular specificity in Drosophila. Cell 73,1137 -1153.[Medline]
Wang, K. H., Brose, K., Arnott, D., Kidd, T., Goodman, C. S., Henzel, W. and Tessier-Lavigne, M. (1999). Biochemical purification of a mammalian slit protein as a positive regulator of sensory axon elongation and branching. Cell 96,771 -784.[Medline]
Wong, K., Ren, X.-R., Huang, Y.-Z., Xie, Y., Liu, G., Saito, H., Tang, H., Wen, L., Brady-Kalnay, S. M., Mei, L. et al. (2001). Signal tranduction in neuronal migration: Roles of GTPase activating proteins and the small GTPase Cdc42 in the Slit-Robo pathway. Cell 107,209 -221.[CrossRef][Medline]
Wu, J. Y., Feng, L., Park, H.-T., Havlioglu, N., Wen, L., Tang, H., Bacon, K. B., Jiang, Z.-H., Zhang, X.-C. and Rao, Y. (2001). The neuronal repellent Slit inhibits leukocyte chemotaxis induced by chemotactic factors. Nature 410,948 -952.[CrossRef][Medline]
Xian, J., Clark, K. J., Fordham, R., Pannell, R., Rabbitts, T.
H. and Rabbitts, P. H. (2001). Inadequate lung development
and bronchial hyperplasia in mice with a targeted deletion in the
Dutt1/Robo1 gene. Proc. Natl. Acad. Sci. USA
98,15062
-15066.