Department of Molecular Genetics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA
Author for correspondence (e-mail:
nbaker{at}aecom.yu.edu)
Accepted 30 July 2003
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SUMMARY |
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Key words: Drosophila eye, Morphogenetic furrow, Hedgehog, Decapentaplegic, Notch, Delta
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Introduction |
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Progression of the morphogenetic furrow depends primarily on Hedgehog,
which is secreted by differentiating photoreceptor neurons. Hedgehog is
necessary for furrow progression (Ma et
al., 1993). Ectopic Hh expression is sufficient to initiate
ectopic furrows in the anterior undifferentiated region
(Heberlein et al., 1995
).
Despite the primary role of Hh, cells unable to receive Hh signals are still
able to differentiate because Hh triggers the secretion of secondary signals,
and cells unable to respond to Hh still respond to these other signals
(Strutt and Mlodzik, 1997
).
Dpp is one important secondary signal, and acts redundantly with Hh. Cells
must be able to respond to either Hh or Dpp to begin differentiating
(Heberlein et al., 1993
;
Greenwood and Struhl, 1999
;
Curtiss and Mlodzik, 2000
). It
has been proposed that Notch signaling also contributes
(Baker and Yu, 1997
;
Li and Baker, 2001
). Ectopic
Notch and Dpp together can initiate differentiation as effectively as ectopic
Hh does (Baonza and Freeman,
2001
).
In this paper we sought to define the individual roles and interactions of each signal through the study of loss-of-function mutations affecting response to Hh, Dpp or N signals, both alone and in combination. We also sought to determine the basis of the redundancy between Hh and Dpp, and how the two signals replace each other, and we investigated whether there is redundancy between Hh and N, and for what events, if any, each signal is individually sufficient in the absence of the others.
Although redundant functions can be studied through ectopic expression
experiments, a loss-of-function approach that removes components from the
redundant pathways has the advantage of addressing gene function at the normal
time and place, and at normal expression levels. As each of Hh, Dpp and N
signals are important many times during Drosophila development from
embryogenesis onwards, it was necessary to use a conditional genetic approach.
We employed mosaic analysis using the FLP/FRT system to obtain clones of
retinal cells lacking function of Hh, Dpp and N pathway components. Each of
these genes also plays roles in the initiation of the morphogenetic furrow at
the posterior margin of the eye field, so that furrow initiation fails when
eye margin cells are mutated, leaving the region to the anterior
undifferentiated (Lee and Treisman,
2002). The present study is therefore restricted to the
progressive onset of differentiation by cells within the retinal field.
Initially, we found that removing Smo did not prevent accumulation of the Ci activator Ci155. However, we found that differentiation of Smo-mutant cells depended not on Ci, but on Dpp and N signaling. Perhaps because Dpp and Hh would otherwise act over different ranges, the pace of furrow progression is constrained by inhibitors, such as Hairy, which are themselves regulated by Dpp, Hh and N.
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Materials and methods |
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smo
Clones were obtained in: eyF; smo3
FRT40/[armlacZ] FRT40, hsF; smo3
FRT40/[armlacZ] FRT40, hsF; smoD16
FRT40/[armlacZ] FRT40, and y hsF;
smo1 FRT42 en
/smo3 FRT42 [smo+ hs:Gfp]
[ci+]; ci94
/Dp(1;4)y+ spa larvae with equivalent results.
smo3 and smoD16 are
both null alleles (Chen and Struhl,
1998).
smo tkv
Clones were obtained in: hsF; smo3
tkvstrII FRT40/M [armlacZ] FRT40 larvae.
tkvstrII is a null allele
(Greenwood and Struhl,
1999).
tkv
Clones were obtained in: hsF; tkva12 FRT40/M
[armlacZ] FRT40 larvae as described
(Burke and Basler, 1996).
tkva12 is a null allele
(Penton et al., 1994
).
smo Mad
Clones were obtained in: hsF; smo3
Mad1-2 FRT40/[armlacZ] FRT40 larvae as
described (Curtiss and Mlodzik,
2000). Mad1-2 is an insertion in
Mad regulatory sequences that prevents most Dpp signaling with little
effect on growth (Wiersdorff et al.,
1996
).
ci
Clones were obtained in: y hsF; FRT42
[ci+]/FRT42; ci94 larvae as
described (Methot and Basler,
1999). ci94 is a null allele from
which the promoter region and first exon have been deleted, including start
sites for transcription and translation
(Slusarski et al., 1995
;
Methot and Basler, 1999
).
smo ci
Clones were obtained in: y hsF; smo1
FRT42 en /smo3 FRT42
[smo+ hs:Gfp] [ci+];
ci94 larvae as described
(Methot and Basler, 1999).
Control smo clones were obtained in parallel from phenotypically
y+ larvae: y hsF;
smo1 FRT42 en
/smo3 FRT42 [smo+
hs:Gfp] [ci+]; ci94
/Dp(1;4)y+ spa.
Mad ci
Clones were obtained in: y hsF;
Mad1-2 FRT40/[ci+] FRT40;
ci94 larvae. This [ci+]
transgene was provided by R. Holmgren.
tkv ci
Clones were obtained in: y hsF;
tkva12 FRT40/[ci+] FRT40;
ci94, and y hsF;
tkva12 FRT40/M21 [ci+] FRT40;
ci94 larvae.
Su(H) ci
Clones were obtained in: y hsF;
Su(H)47 FRT40 [w+
l(2)35Bg+]/[ci+] FRT40;
ci94 larvae.
Su(H)
47 is a 1.9 kb deletion that removes
the Su(H) l(2)35Bg intergenic region, including the transcriptional
start site and ATG codon of both genes, and is a null allele
(Morel and Schweisguth,
2000
).
Su(H)
Clones were obtained in: y hsF;
Su(H)47 FRT40 [w+
l(2)35Bg+]/[armlacZ] FRT40 larvae.
Mad Su(H) ci
Clones were obtained in: y hsF; Mad1-2
Su(H)47 FRT40 [w+
l(2)35Bg+]/[ci+] FRT40;
ci94 larvae.
Ci misexpression
Fng:Gal4 (gift of M. Mlodzik) was used to misexpress ci derivatives from
UAS transgenes. We also used the Gal4 line
hairyH10 to misexpress UAS:ciCell
anterior to the morphogenetic furrow
(Ellis et al., 1994).
Antibody labelling
Eye discs were labelled for Atonal as described
(Lee et al., 1996). Other
labelling was performed either in 0.1 M sodium phosphate (pH 7.2), 1% normal
goat serum, 0.1% saponin, following a 45 minute fixation in cold PLP
(Tomlinson and Ready, 1987
),
or in 0.1 M sodium phosphate (pH 7.2), 0.3% sodium deoxycholate, 0.3% Triton
X-100, after fixation at room temperature in 3.7% formaldehyde, 100 mM PIPES
(pH 6.95), 1 mM EGTA, 2 mM MgSO4. Preparations were examined using
BioRad MRC600 and Radiance 2000 confocal microscopes, and digital images were
manipulated using Adobe PhotoShop 4.0 and NIH Image 1.62 software. Antibodies
against ß-galactosidase were obtained from Cappel and from the
Developmental Studies Hybridoma Bank (mAb40-1), polyclonal rabbit anti-GFP was
obtained from Molecular Probes, and other antibodies were obtained from their
developers: anti-ato (Jarman et al.,
1994
); anti-Ci155 (mAb2A1)
(Motzny and Holmgren, 1995
);
anti-senseless (Nolo et al.,
2000
); and anti-hairy antibodies
(Brown et al., 1995
).
Preparations were obtained over several years. Consequently, variations in
procedures, antibody batches, and confocal hardware and software make
comparing signal intensity between preparations unreliable, except where
specifically noted in the text.
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Results |
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R8 specification in the morphogenetic furrow is illustrated in
Fig. 1. senseless and
other target genes are expressed in response to rising Atonal activity
(Baker, 2002). Atonal
expression is transient, whereas Senseless is maintained in differentiating R8
cells throughout the eye disc (Nolo et
al., 2000
) (Fig.
1). hairy encodes a negative regulator of Atonal
function, and is expressed ahead of the morphogenetic furrow and downregulated
as differentiation begins (Fig.
1) (Brown et al.,
1995
). Notch activation downregulates Hairy
(Baonza and Freeman, 2001
).
These events coincide with peak Hh signal at the anterior of the morphogenetic
furrow, as revealed by accumulation of the Ci155 protein
(Motzny and Holmgren, 1995
;
Strutt and Mlodzik, 1996
)
(Fig. 1).
|
|
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Since smo-mutant cells differentiate in response to Dpp
(Greenwood and Struhl, 1999;
Curtiss and Mlodzik, 2000
), we
tested whether Ci155 was being stabilized by Dpp signaling. Ci155 levels were
examined in clones of cells mutant for both smo and tkv, or
for both smo and Mad. tkv encodes the Type I Dpp receptor,
Mad encodes the essential transcription factor target. Ci155 did not
accumulate when both Hh and Dpp signal reception was inactivated, indicating
that Dpp signal reception was required to accumulate Ci155 proteins in the
absence of Hh signal reception (Fig.
2C,D). A modest but reproducible reduction in Ci155 levels was
seen in cells mutant for tkv alone, indicating that Dpp signaling
through Tkv contributes to the level of Ci155, even in the presence of the Hh
pathway (Fig. 2E).
Ci is dispensable
Dpp signaling might promote furrow progression and differentiation by
regulating Ci155 processing to Ci75, as Hh does. If so, ci-mutant
cells should resemble cells that are unable to respond to either Hh or Dpp,
and thus would be unable to differentiate. An alternative model was that Dpp
acted independently of its effects on Ci. If so, ci-mutant clones
should exhibit delayed differentiation as do clones mutant for smo.
Cells homozygous for a deletion of the ci gene were examined to
distinguish between these two models.
Unexpectedly, clones of ci-mutant cells differentiated normally in
all respects (Fig. 3A-C). The
temporal and spatial pattern of ato expression was completely normal
in ci-mutant cells, unlike in cells unable to respond to Hh or in
cells unable to respond to Hh or Dpp (Fig.
3A). Normal neural differentiation occurred in ci-mutant
cells without any delay (Fig.
3B and data not shown). Cells lacking ci were
morphologically normal in adults, both externally and on sectioning to reveal
differentiated cellular morphology (Fig.
3C). There were no defects in ommatidial chirality or planar
polarity. The genotype of the ci-mutant cells was unequivocally
confirmed using antibodies against Ci to detect ci-mutant cells
directly (Fig. 3A). While this
paper was under review, Pappu et al. also reported normal eye development by
large clones lacking ci (Pappu et
al., 2003).
|
If smo signals independently of ci in the eye, smo ci clones should show delayed differentiation like smo clones (e.g. Fig. 3D). If smo is only essential to eliminate Ci75, smo ci clones should develop normally without any delay, like ci clones. Fig. 3E shows that smo ci clones developed normally like ci, and were not delayed like smo clones of the same size (Fig. 3D). Therefore, smo appeared to be essential only to prevent processing of Ci to the Ci75 repressor protein.
We targeted ectopic expression of different forms of Ci protein to the
developing eye. The Gal4 line hairyH10 was used
to target UAS:ciCell expression anterior to the morphogenetic
furrow (Ellis et al., 1994).
ciCell encodes a Ci protein truncated at amino acid 975 and mimics
Ci75 (Methot and Basler,
1999
). Atonal expression was reduced, although eye patterning
occurred normally (data not shown). Fng:Gal4 (a gift of M. Mlodzik) was used
to misexpress UAS:ciCell ventrally
(Fig. 3F). When expressed under
the control of Fng:Gal4, ciCell prevented furrow progression and
differentiation in ventral cells, but permitted furrow progression across the
dorsal region of the eye disc (Fig.
3G). Fng:G4 and hH10 were also used
to drive expression of UAS:ciU, which encodes a Ci protein with a
deletion of amino acids 611-760, which is defective in processing to Ci75 and
which behaves as a Hh-dependent activator protein
(Methot and Basler, 1999
), and
UAS:ZnAD and UAS:ZnRD transgenes, in which the DNA binding domain of Ci was
coupled to transcriptional activator and repressor domains, respectively
(Hepker et al., 1997
). These
were without detectable effect (data not shown). Taken together, these
findings suggested that differentiation might depend on blocking production of
Ci75 in response to Hh or Dpp.
Ci-independent furrow progression in response to Dpp and N
There also had to be a ci-independent signal required to induce
differentiation even in the absence of Ci. If differentiation depended
entirely on eliminating Ci75, deletion of the ci gene would remove
the barrier to differentiation anywhere in the eye field, but instead
ci-mutant cells initiated differentiation in the same temporal
progression as wild-type cells (Fig.
3A). ci-independent differentiation could not depend on
Hh, because smo ci cells also initiated differentiation with
precisely normal timing (Fig.
3E).
The second signal might be Dpp, acting independently of Ci. If this was correct, we would expect that Mad ci mutant cells would fail to differentiate. Mad ci mutant cells were examined and were found to differentiate, but such differentiation was delayed (Fig. 4A,B). This result confirmed that Dpp signaling through Mad contributed to differentiation in the absence of ci, but that some signal from posterior cells was still able to progress slowly through Mad ci mutant cells.
|
Differentiation was not observed in tkv ci-mutant clones. Clones of cells mutant for both tkv and ci grew poorly, and only small clones were recovered (Fig. 4C,D). Such clones were rarely recovered in the posterior region of the eye disc, where they seemed to sort out. Large clones of tkv ci-mutant cells were obtained using the Minute Technique. These large clones always had round shapes, indicating that there was reduced mixing with wild-type cells throughout the eye disc (data not shown). No retinal differentiation was detected. These findings indicate that some Dpp signaling occurred in Mad ci-mutant cells, which contributed to the slow differentiation of Mad ci cells. Clones of Mad Su(H) ci-mutant cells also failed to differentiate, indicating a role for N signaling in the delayed differentiation of Mad ci-mutant cells (Fig. 4E).
N augments Dpp signaling
Two interpretations of the role of N could be considered. One was that N
signaling and Dpp signaling were each required for differentiation in the
absence of ci. The alternative was that N activity enhanced
sensitivity to Dpp so that the limited Dpp signaling that occurs in
Mad-mutant cells became sufficient for delayed differentiation.
If N signaling was required for differentiation in the absence of ci, we would predict that Su(H) ci-mutant cells would be unable to differentiate. However, Su(H) ci-mutant cells did initiate differentiation, but such differentiation was delayed (Fig. 4F). Thus N signaling was not absolutely required for differentiation. Delayed differentiation in the absence of Su(H) and ci must depend on Dpp signaling, as it did not occur in Mad Su(H) ci-mutant cells. Su(H) mutations must reduce the effectiveness of Dpp, as the timing of differentiation was normal in ci-mutant cells but delayed in Su(H) ci-mutant cells (differentiation in ci-mutant clones must be due to Dpp signaling, because tkv ci-mutant cells did not differentiate). These results indicate that N signaling made Dpp signaling more effective, at least in the absence of Ci.
Ci155 augments Dpp signaling
The differentiation of Su(H) ci-mutant cells differed from that of
Su(H)-mutant cells. Su(H)-mutant cells show a profound
neurogenic phenotype in which the majority of mutant cells take an R8 fate
(Ligoxygakis et al., 1998)
(Fig. 4G). Morphogenetic furrow
progression can accelerate so that differentiation begins earlier at the
anterior of large Su(H)-null clones than it does in nearby wild-type
regions (Li and Baker, 2001
)
(Fig. 4G). By contrast,
differentiation of Su(H) ci-mutant cells was delayed, and R8
differentiation was increased only moderately compared with wild type
(Fig. 4F). These data show that
differentiation without Su(H) was more effective in the presence of
ci. This was the first data to indicate a role of Ci155 that could
not be replaced by deleting the ci gene to eliminate Ci75. More
evidence is reported below.
Hh-dependent differentiation requiring Ci155
One model for the importance of Ci75 was that Ci75 antagonized Dpp and N
function. In this view the only normal role of Hh would be to help
downregulate Ci75 at the furrow so that Dpp and N could act more promptly.
Delayed differentiation in smo clones would be due to ci75
antagonizing the Dpp/N function that we have found is sufficient for normal
furrow progression in the absence of ci. This model does not predict
delayed differentiation in Su(H) ci-mutant clones
(Fig. 4F; see above).
To isolate the role of Hh we examined cells mutant for Mad and
Su(H). If the only role of Hh was to derepress Dpp and N signaling,
then Mad Su(H)-mutant cells would not differentiate because of
missing Dpp and N signals. By contrast, Mad Su(H) cells
differentiated at the normal rate (Fig.
4H). A neurogenic phenotype reflected dependence of lateral
inhibition on Su(H) (Ligoxygakis
et al., 1998). Initiation of Atonal expression has also been
reported in Dl Medea-mutant cells, which should resemble Mad
Su(H) cells in lacking N and Dpp signaling
(Baonza and Freeman, 2001
).
These results show that Dpp and N were dispensable for differentiation if Hh
signaling was intact. As Mad Su(H) ci cells did not differentiate,
differentiation of Mad Su(H) depended on a positive role of Ci that
was not mimicked by deleting the ci gene to remove Ci75. Thus Hh,
through Ci155, could drive differentiation in the absence of Dpp and N
signaling.
N and Hh each turn off Hairy
Notch signaling contributes to differentiation by downregulating Hairy and
Extramacrochaetae expression at the morphogenetic furrow
(Baonza and Freeman, 2001).
Hairy is a transcriptional repressor protein that antagonizes Atonal
(Ohsako et al., 1994
;
Brown et al., 1995
). We
monitored Hairy expression to evaluate the contributions of Hh, Dpp and N
signaling to turning off Hairy, and to correlate this with differentiation.
The results are summarized in Table
1. Hh, Dpp and N signals do not appear essential to turn Hairy on,
although Dpp does contribute because Hairy levels appear lower in clones
mutant for tkv, Mad and their combinations with other mutations
(Greenwood and Struhl, 1999
)
(Fig. 4, and data not
shown).
One, or more, of Dpp, Hh or N signaling is required to turn Hairy off at the morphogenetic furrow, because Hairy expression was maintained cell autonomously in Mad Su(H) ci clones (Fig. 4E). Hairy was turned off in Mad ci clones and tkv ci clones, although downregulation was delayed in the center of the clone (Fig. 4B,D). This implies that N signaling is sufficient to downregulate Hairy in response to a signal from more posterior cells outside the clone.
N signaling was required, and was sufficient, for Hairy downregulation, because Hairy was not shut off in Su(H) ci clones (Fig. 4F). If N signaling was essential to shut off Hairy under all circumstances then we would expect Hairy expression to be maintained autonomously in Su(H) clones. By contrast, there was only a brief delay to shutting off Hairy in Su(H) clones (Fig. 4G). Hairy was also shut off in Mad Su(H) clones, although after a delay (Fig. 4H). These results show that either N or Hh signals from posterior cells is sufficient to shut off Hairy expression, but that Hairy expression is maintained indefinitely in Mad Su(H) ci and Su(H) ci cells unable to respond to either pathway. Downregulation of Hairy in response to Hh as well as N explains why N is not required for differentiation in response to Hh, even though it is required for differentiation in response to Dpp.
Hairy retards differentiation
Downregulating Hairy was neither necessary nor sufficient for
differentiation. Whereas Mad Su(H) ci-mutant cells both failed to
differentiate and maintained Hairy expression
(Fig. 4E), tkv ci
cells did not differentiate even though Hairy expression was shut off
(Fig. 4C,D). By contrast, Hairy
expression was maintained in Su(H) ci-mutant clones, even though
Su(H) ci cells could differentiate. However, Hairy downregulation may
contribute to prompt differentiation because differentiation is delayed in
Su(H) ci compared with ci, and is less neurogenic in
Su(H) ci than in Su(H)
(Fig. 4F,G).
As Hairy was shut off by either Hh or N signaling, Hairy maintenance away
from the boundaries of Mad ci, tkv ci and Mad Su(H) clones
shows that these genotypes were defective for Hh and Dl secretion. Hh is
normally secreted by differentiating photoreceptor cells
(Ma et al., 1993). Atonal and
then Egf receptor activity promotes expression of Dl, the activating ligand
for Notch (Baker and Yu, 1998
;
Tsuda et al., 2002
). Both Hh
and Dl expression should be absent or delayed in Mad ci, tkv ci and
Mad Su(H) clones.
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Discussion |
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The pathways implied by our results shown in Fig. 5. Fig. 5A shows how Hh, Dpp and N signaling pathways act within each cell. Fig. 5B illustrates the spatial and temporal relationships of the extracellular signals during morphogenetic furrow progression.
|
We find that Hairy is downregulated redundantly by Hh and N signaling. Prolonged Hairy expression is not sufficient to block differentiation completely but it does antagonize it (e.g. in Su(H) ci clones). Downregulation of Hairy in response to Hh as well as N explains why both ci and Su(H) mutant clones can differentiate promptly, and why N enhances differentiation in response to Dpp but is not required for differentiation in response to Hh.
Hh or Dpp is sufficient for differentiation
Comparison between Mad Su(H) ci cells that do not differentiate
and Mad Su(H) cells that do shows that Hh signaling is sufficient to
initiate eye differentiation. This is consistent with previous studies of
ectopic Hh activation (Heberlein and
Moses, 1995; Li et al.,
1995
; Ma and Moses,
1995
; Pan and Rubin,
1995
; Strutt et al.,
1995
). Our experiments confirm this conclusion at the normal time
and place of Hh signaling at the anterior of the morphogenetic furrow, and
confirm directly that Dpp and N signaling are not necessary for Hh signaling
to be sufficient.
Comparison between Mad Su(H) ci cells and Su(H) ci cells
shows that Dpp signaling is sufficient to initiate eye differentiation in its
normal location in the absence of Hh or N signals, but such differentiation is
delayed. The normal timing of differentiation is restored by combined Dpp and
N signals (in ci clones). This is the basis for the ectopic
differentiation on co-expression of Dpp and Dl ahead of the furrow
(Baonza and Freeman, 2001).
Superficially, our results differ from previous ectopic expression studies
that concluded that Dpp signaling alone was not sufficient to induce ectopic
differentiation in all locations (Pignoni
and Zipursky, 1997; Greenwood
and Struhl, 1999
; Baonza and
Freeman, 2001
). This discrepancy is probably explained by the
baseline repressor activity of Su(H) protein
(Hsieh and Hayward, 1995
;
Morel and Schweisguth, 2000
).
Our previous work shows that without N signaling, repressor activity of Su(H)
protein retards differentiation (Li and
Baker, 2001
). Dpp signaling is sufficient for differentiation in
our experiments where the Su(H) gene has been deleted. In the
presence of the Su(H) gene, Dpp may be most effective at locations
where there is little Su(H) repressor activity, such as close to the
morphogenetic furrow where N signaling is active.
Comparison between Mad Su(H) ci cells, which do not differentiate,
and Mad ci or tkv ci cells, which differentiate slowly or
not at all, shows that Notch signaling alone is insufficient for
differentiation. Premature differentiation reported when N is activated
ectopically ahead of the furrow must reflect endogenous Dpp signaling at such
locations (Baonza and Freeman,
2001; Li and Baker,
2001
).
Mechanisms of redundancy
Our experiments reveal an outline of the mechanisms of Hh, Dpp and N
redundancy (Fig. 5A). First,
our results show that Mad and Ci independently reinforce differentiation,
presumably through the transcription of target genes because Mad is sufficient
for differentiation in the absence of Ci, and vice versa. Our results show
unequivocally that the transcriptional activator Ci155 activates
differentiation in addition to Ci75 antagonizing differentiation.
It was surprising to find that Dpp stabilizes Ci155 in the absence of Smo,
which suggested Dpp input into Hh signal transduction. Although the
requirement for smo-dependent input through fused makes it
unlikely that Ci155 is functional in smo clones
(Ohlmeyer and Kalderon, 1997;
Methot and Basler, 1999
),
Ci155 accumulation might be associated with reduced Ci75 levels. Ci75 is shown
to repress differentiation in smo clones because smo ci
clones differentiation normally. Ci155 stabilization cannot be due to an
indirect effect of Dpp signaling on Hh, Ptc or Smo expression levels because
the effect is detected in the absence of smo, and, therefore,
reflects an effect on Hh signal transduction components downstream of Smo. One
idea is that Dpp signaling (or Dpp-induced differentiation) may replace
SCFSlimb processing of Ci (which cleaves Ci155 to Ci75) with
Cullin3-mediated Ci degradation, just as normally occurs posterior to the
morphogenetic furrow (Ou et al.,
2002
). In a smo clone, Ci155 would accumulate because Smo
is required for Cullin3 to degrade Ci (Ou
et al., 2002
) (N.E.B., unpublished). However, the
SCFSlimb-to-Cullin3 switch may not be the only effect of Dpp on Ci
processing, because Tkv slightly enhanced Ci155 accumulation even when
smo is present (Fig.
2E).
Finally, downregulation of Hairy by N requires the Su(H) gene. N
also overcomes baseline repressor activity of Su(H) protein to promote
progression of differentiation (Li and
Baker, 2001). This role of N must be independent of Hairy.
Signal combinations control the rate of differentiation
Dl, Hh and Dpp are generally thought to signal over very different
distances. How can signals of such different range substitute for one another
to permit normal eye development? Fig.
5B shows signal sources and targets in the eye disc. Dpp is
transcribed in response to Hh signaling and is produced where Ci155 levels are
highest (Heberlein et al.,
1993; Strutt and Mlodzik,
1996
). Dl is regulated by Hh indirectly through Ato and
Ato-dependent Egfr activity in differentiating cells
(Baker and Yu, 1998
;
Tsuda et al., 2002
). Hh is
expressed most posteriorly of the three, in differentiating photoreceptors
(Ma et al., 1993
).
Eye differentiation uses Hh to progress through cells unable to respond to
Dpp (tkv, Mad) or N (Su(H)). The range of Hh diffusion
depends in part on the shape of the morphogenetic furrow cells
(Benlali et al., 2000). The Dpp
that drives differentiation through ci-mutant cells unable to respond
to Hh must diffuse from outside the ci clones because Dpp synthesis
is Hh dependent (Heberlein et al.,
1993
; Methot and Basler,
1999
). Large ci clones develop normally so Dpp diffusion
cannot be limiting (dpp-mutant clones offer no information about the
range of Dpp because they express and differentiate in response to Hh).
Instead the rate of progression in response to Dpp is controlled by Dl. Dl
signals over, at most, one or two cell diameters at the morphogenetic furrow
(Baker and Yu, 1997
).
The previous view of eye patterning was influenced by the morphogen
function of Hh and Dpp in other discs
(Nellen et al., 1996;
Strigini and Cohen, 1997
). It
was thought that domains of Ato and Hairy expression reflected increasing
concentrations of Hh and Dpp (Greenwood
and Struhl, 1999
; Lee and
Treisman, 2002
). Our data shows that, in the eye, the combination
of signals is important. Differentiation is triggered where Dl and/or Hh
synergize with Dpp, regardless of where the source of Dpp is. The additional
requirements limit Dpp to initiating differentiation at the same locations
that Hh does.
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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