1 Skirball Institute of Biomolecular Medicine and Department of Cell Biology,
NYU School of Medicine, 540 First Avenue, New York, NY 10016, USA
2 Department of Biology, Johns Hopkins University, 3400 North Charles Street,
Baltimore, MD 21218, USA
* Present address: Center for Developmental Biology and Department of
Pharmacology, UT Southwestern Medical Center, 6000 Harry Hines Boulevard,
Dallas, TX 75390, USA
Author for correspondence (e-mail:
treisman{at}saturn.med.nyu.edu)
Accepted 16 September 2002
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SUMMARY |
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Key words: Ubiquitin, Morphogenetic furrow, hedgehog, Cubitus interruptus, slmb, Drosophila melanogaster
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INTRODUCTION |
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A small number of signaling pathways appear to direct most developmental
processes. In Drosophila, the BMP family member Decapentaplegic (Dpp)
and the founding member of the Hedgehog family (Hh) are used repeatedly
throughout development. One function of Hh and Dpp is to direct the
progressive differentiation of the eye imaginal disc (reviewed in
Lee and Treisman, 2002). In
the second instar eye disc, hh is expressed in a complex pattern in
both the disc proper and the peripodial membrane, before being refined to a
small domain centered on the dorsoventral midline of the disc's posterior
margin (Cho et al., 2000
). Hh
signals more anterior cells to express dpp and atonal (ato),
which encodes the bHLH transcription factor required for the formation of the
R8 `founder' photoreceptor in each cluster
(Jarman et al., 1994
). These
cells then differentiate as photoreceptors and themselves express hh,
allowing the cycle to propagate toward the anterior of the disc. dpp
is expressed in the morphogenetic furrow, an indentation at the front of
differentiation, where it is responsible for coordinating the timing of
differentiation through synchronization of the cell cycle
(Penton et al., 1997
). Loss of
either Hh or Dpp blocks the initiation of differentiation, while loss of both
blocks progression (Curtiss and Mlodzik,
2000
; Greenwood and Struhl,
1999
). While it is known that Hh activates dpp expression
in the furrow (Heberlein et al.,
1995
; Heberlein et al.,
1993
; Ma et al.,
1993
), it is not known how hh expression is controlled,
nor how dpp is turned off in cells leaving the furrow.
Much has been learned about the regulation of hh expression in
tissues other than the eye. During embryonic development, maintenance of
hh expression in the posterior of each segment requires the homeobox
gene engrailed (en)
(Tabata et al., 1992). In the
head segments, however, hh expression is not controlled by En, but by
several different homeobox and segment polarity genes
(Crozatier et al., 1999
;
Gallitano-Mendel and Finkelstein,
1997
). hh is also regulated by En later in development,
in the posterior compartments of the leg and wing imaginal discs
(Tabata et al., 1995
;
Zecca et al., 1995
). En
regulates hh expression indirectly, by repressing the expression of
cubitus interruptus (ci) in the posterior compartment
(Dominguez et al., 1996
;
Eaton and Kornberg, 1990
;
Schwartz et al., 1995
). In the
absence of Hh signal, the full-length Ci protein (Ci155) is
proteolytically processed to a 75 kDa transcriptional repressor
(Ci75); when the Hh signal is received this processing event is
inhibited, allowing the accumulation of Ci155
(Aza-Blanc et al., 1997
). In
the anterior compartment, Ci75 represses hh expression
(Dominguez et al., 1996
;
Methot and Basler, 1999
). The
Hh signal is transmitted to Ci through two transmembrane proteins, Patched
(Ptc) and Smoothened (Smo) (reviewed by
Ingham and McMahon, 2001
). Hh
binds to Ptc and inhibits its activity; in the absence of Hh, Ptc promotes
internalization, dephosphorylation and degradation of Smo
(Alcedo et al., 2000
;
Denef et al., 2000
;
Ingham et al., 2000
).
Unlike these other tissues, in the eye the source of hh expression
changes over time; as cells are recruited to differentiate as photoreceptors,
they begin expressing hh. Reflecting this difference in hh
control, en function is not required in the eye disc for hh
expression (Strutt and Mlodzik,
1996). The relationship between hh and dpp
expression is conserved in some tissues, but not in others. In the wing disc,
as in the eye, dpp is transcribed in response to Hh
(Basler and Struhl, 1994
;
Zecca et al., 1995
); in the
leg disc this response is limited, through the activity of Wg, to a dorsal
domain in the anterior compartment (Basler
and Struhl, 1994
; Brook and
Cohen, 1996
; Jiang and Struhl,
1996
). In other tissues, such as the dorsal domain of the early
embryo, dpp expression appears to be independent of Hh signaling
(St Johnston and Gelbart,
1987
). Thus the mechanisms controlling the expression of these
signals are likely to be at least partially context-dependent. In tissues
where Hh regulates dpp expression, dpp is both activated by
Ci155 and repressed by Ci75, allowing robust control of
dpp by Hh (Methot and Basler,
1999
).
To gain further insight into the control of photoreceptor development, we
have conducted mosaic screens for mutations that perturb differentiation
(Benlali et al., 2000;
Lee and Treisman, 2001a
;
Lee and Treisman, 2001b
;
Treisman, 2001
). Many
components of the hh and dpp pathways were recovered in
these screens. We report here our characterization of mutations in the
hyperplastic discs (hyd) locus; hyd encodes a HECT
domain E3 ubiquitin ligase (Callaghan et
al., 1998
; Mansfield et al.,
1994
). Mutations in hyd cause ectopic non autonomous
differentiation in the eye. This effect is due to the independent activation
of hh and dpp expression in hyd mutant tissue,
reflecting independent effects of Hyd on hh expression and
function.
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MATERIALS AND METHODS |
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Immunohistochemistry
Imaginal discs were stained as described previously
(Hazelett et al., 1998).
Primary antibodies used were rat
-Elav (1:1)
(Robinow and White, 1991
),
rabbit
-Atonal (1:10,000) (Jarman
et al., 1994
), rat
-Ci (1:1)
(Motzny and Holmgren, 1995
),
rat anti-Smo (1:500) (Denef et al.,
2000
), mouse
-ß-galactosidase (Promega; 1:200), rabbit
-ß-galactosidase (Cappel; 1:5000), mouse anti-Neuroglian (1:1)
(Hortsch et al., 1990
), mouse
anti-Ptc (1:150) (Capdevila et al.,
1994
) and rabbit
-Hh-N (1:2000)
(Lee and Treisman, 2001b
).
Secondary antibodies were from Jackson Laboratories, conjugated to FITC, Texas
Red or Cy5, used at 1:200. Fluorescent images were obtained using a Leica TCS
NT confocal microscope. In situ hybridization was performed as described
(Maurel-Zaffran and Treisman,
2000
).
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RESULTS |
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While hyd clones in third instar eye discs contained
Elav-expressing photoreceptors, clones in the adult eye appeared to form
either scars in the eye or head cuticle at its margin
(Fig. 1B). We also noticed that
hyd clones in the posterior of the eye disc were generally smaller
than wild-type clones generated in a similar cross and contained reduced
numbers of photoreceptors (Fig.
1D,E). The lack of mutant ommatidia in adult eyes could reflect
either a loss of hyd mutant clones due to competition with the
surrounding tissue, or a later requirement of hyd for viability. To
further examine this question, we made hyd clones in a
Minute genetic background (Morata
and Ripoll, 1975), thereby reversing the growth disadvantage of
hyd mutant tissue (Fig.
1C). These animals survived to pharate adult stages but could not
eclose; they had greatly reduced eyes that nevertheless appeared to contain
properly formed hyd mutant ommatidia
(Fig. 1C). This suggests that
the lack of visible hyd clones in adults is due to their poor
relative growth, leading to their elimination through competition with
wild-type cells. We also examined third instar eye discs containing
hyd/Minute clones, and found greatly reduced eye discs that appeared
to contain full clusters of photoreceptors throughout the eye field
(Fig. 1F). As cells arrest
division when they differentiate, premature differentiation could explain the
small size of hyd/Minute eye discs. Ato is normally expressed in the
morphogenetic furrow and in the youngest R8 photoreceptors
(Fig. 1G); in early third
instar hyd/Minute mutant discs the only remaining Ato staining is in
single (presumably R8) cells (Fig.
1I), implying that the morphogenetic furrow has already reached
the anterior margin. From this we infer that differentiation in
hyd/Minute discs still progresses from posterior to anterior, but at
an accelerated rate.
hyd mutant cells ectopically express hedgehog
Growth and differentiation of cells in the eye disc both depend on Hh and
Dpp secreted by more posterior cells. The non autonomous differentiation and
overgrowth caused by hyd clones suggested that the clones might be
producing one or both of these molecules; a similar phenotype is produced by
ectopic expression of hh or activation of the hh pathway
ahead of the furrow (Heberlein et al.,
1995; Pan and Rubin,
1995
; Strutt et al.,
1995
). We therefore looked at hh expression in
hyd clones using both an enhancer trap line
(Lee et al., 1992
) and
antibody staining. hyd mutant clones anterior to the furrow indeed
expressed hh-lacZ and Hh protein earlier than surrounding wild-type
cells (Fig. 2A-C and data not
shown). When we made hyd clones in a Minute background, we
observed hh-lacZ expression throughout the eye disc
(Fig. 2E). The widespread
hh expression in hyd/Minute clones may explain the
accelerated differentiation and small size of these eye discs.
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The ectopic hh expression we observed in hyd mutant
clones could be a consequence, rather than a cause, of their premature
differentiation. To rule out this possibility we generated hyd
ato double mutant clones. Clones mutant for ato cannot form
the R8 photoreceptor (Jarman et al.,
1995), which is itself required for the recruitment of
photoreceptors R1-R7. Thus ato mutant clones do not differentiate,
unless a cell at the margin of a clone is recruited by a neighboring wild-type
R8. We confirmed that ato single mutant clones do not express
hh (data not shown). We found that while hyd ato mutant
clones did not differentiate, they nevertheless expressed hh-lacZ and
were capable of directing ectopic differentiation in surrounding wild-type
tissue (Fig. 2G-I). This
demonstrates that loss of hyd function has a direct effect on
hh expression that is not simply due to differentiation.
As an E3 ubiquitin ligase, Hyd is likely to promote the degradation of one
or more proteins. Based on the ectopic expression of hh in
hyd mutant clones, we hypothesized that Hyd was acting in the
anterior of the third instar eye disc to prevent premature expression of
hh. hyd is expressed in proliferating tissues in the embryo and
larva, but its expression in the eye disc had not been described in detail
(Mansfield et al., 1994).
Using in situ hybridization, we found that hyd RNA was highly
expressed in the anterior of the eye imaginal disc, especially around the
dorsoventral midline (Fig. 2F).
hyd was expressed at lower levels towards the dorsal and ventral
margins but was still restricted to the anterior. This expression pattern is
consistent with a role for hyd in preventing the premature expression
of hh.
Loss of hedgehog function partially suppresses the
hyd phenotype
If the ectopic differentiation and overgrowth associated with hyd
clones is due to ectopic hh expression, it should be possible to
rescue this phenotype by removing hh function from the clones. We
therefore determined the phenotype of hyd hh double mutant clones. We
obtained similar results with three hh alleles,
hhrJ413 (Heberlein et
al., 1993), hhts2
(Ma et al., 1993
) grown at
29°C, and the null allele hhAC
(Lee et al., 1992
). While
hh mutant clones appear wild type unless located on the margin of the
eye disc (Dominguez and Hafen,
1997
) (Fig. 3B),
hyd hh double mutant clones showed a partial suppression of the
hyd phenotype. Ectopic photoreceptors were no longer present in or
around hyd hh clones (Fig.
3C), and hyd hh double mutant clones generated in a
Minute background had only a few photoreceptors associated with the
remaining wild-type tissue (Fig.
3D). Thus hyd mutant tissue requires Hh in order to
differentiate. However, some hyd hh mutant clones were still able to
stimulate proliferation of surrounding tissue, leading to overgrowth of the
adult eye (Fig. 3C,E).
dpp has also been shown to stimulate proliferation in the eye disc
(Pignoni and Zipursky, 1997).
The remaining non autonomous overgrowth induced by hyd hh double
mutant clones might therefore be due to ectopic expression of dpp.
Using a dpp-lacZ reporter construct, we observed ectopic dpp
expression in and around hyd mutant clones anterior to the furrow
(Fig. 3F and data not shown).
Ectopic dpp expression appeared more widespread than ectopic
hh expression and occurred in more anterior regions of the disc,
raising the possibility that dpp misexpression is not merely induced
by ectopic Hh in the clones but is an independent consequence of the loss of
hyd. Indeed, we found that dpp was still expressed in some
hyd hh mutant clones (Fig.
3G,H), although its expression was limited to clones close to the
morphogenetic furrow. Thus in the absence of hyd function,
dpp expression is no longer strictly regulated by Hh signaling.
Hyd independently regulates hh and dpp
expression
The persistence of dpp expression in hyd hh double mutant
clones suggested that dpp regulation by Hyd was at least partially
independent of hh (Fig.
3G,H). One molecule that is known to transcriptionally regulate
both hh and dpp is Ci. In the wing imaginal disc it has been
shown that Hh controls dpp expression both by suppressing the
production of Ci75, which inhibits dpp expression, and by
activating Ci155, which activates dpp expression
(Methot and Basler, 1999). In
addition, Ci75 inhibits hh expression in the anterior
compartment of the wing disc; ci transcription is repressed by
en in the posterior compartment, allowing hh to be expressed
there (Dominguez et al., 1996
;
Methot and Basler, 1999
). To
test whether hyd was acting through Ci to affect hh and
dpp expression in the eye disc, we drove the expression of a
truncated constitutive repressor form of Ci (Ci76)
(Aza-Blanc et al., 1997
)
specifically in hyd mutant clones using the MARCM system
(Lee and Treisman, 2001a
;
Lee and Luo, 1999
).
dpp-lacZ was no longer expressed in hyd clones expressing
UAS-ci76 in or anterior to the furrow
(Fig. 4A,B). However,
Ci76 did not prevent the ectopic expression of hh in
hyd mutant clones (Fig.
4D-F). Continued hh expression in these clones sometimes
led to ectopic differentiation in tissue surrounding the clone
(Fig. 4D-F). Thus
Ci76 is sufficient to block dpp but not hh
expression in hyd clones, suggesting that Hyd regulates hh
and dpp expression through at least partially independent
mechanisms.
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If the overgrowth phenotype induced by hyd hh double mutant clones is indeed due to their misexpression of dpp, it should be blocked by introducing Ci76 into the mutant cells. Indeed, when we generated hyd hh mutant clones expressing UAS-ci76, the adult eyes no longer exhibited any overgrowth (Fig. 4C). Thus all the ectopic growth and differentiation caused by loss of hyd in the eye can be attributed to independent effects on hh expression and activation of the Hh pathway.
Loss of hyd causes accumulation of Ci but not
Smoothened
If hyd regulates dpp expression by altering Ci activity,
loss of hyd should lead to upregulation of full-length, active Ci. We
indeed observed increased levels of full-length Ci in hyd mutant
clones in the anterior of the eye disc (data not shown). However, this could
be due to misexpression of hh in the same clones. To determine
whether hyd has a direct effect on Ci, we examined hyd hh
double mutant clones anterior to the morphogenetic furrow. High levels of
full-length Ci accumulated in these clones
(Fig. 5A-C), confirming that
Hyd normally reduces Ci levels independently of Hh activity.
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Hyd might act directly on Ci to promote its proteolytic cleavage or
degradation. An alternative possible target for Hyd activity is Smoothened
(Smo). Smo is a transmembrane protein that acts positively in Hh signaling
(Alcedo et al., 1996). Smo
levels are kept low by the receptor protein Patched (Ptc) in the absence of
Hh, but Smo is stabilized and localized to the membrane when Hh binds to Ptc
(Alcedo et al., 2000
;
Denef et al., 2000
;
Ingham et al., 2000
). To test
whether Hyd normally contributes to Smo degradation, we stained eye discs
containing hyd, hh clones with Smo antibody. No Smo accumulation was
apparent in the clones (Fig.
5D-F). Thus loss of hyd leads to accumulation of
full-length Ci without altering the level of Smo.
hyd regulates Ci and hh in the wing disc
Since hyd is expressed in the wing disc and is required for its
normal growth (Mansfield et al.,
1994), we tested whether its effects on wing development might
also be mediated by alterations in hh expression and Ci levels. In
wild-type wing discs, hh is expressed uniformly throughout the
posterior compartment of the wing pouch, while dpp is expressed in
the anterior compartment in a stripe along the AP border
(Basler and Struhl, 1994
).
Ci155 is present at high levels in a similar stripe at the AP
border and at lower levels elsewhere in the anterior compartment
(Motzny and Holmgren, 1995
).
Expression of hh, dpp and Ci155 in hyd clones
remained restricted to the correct compartment. However, some hyd
mutant clones in the posterior compartment expressed elevated levels of
hh-lacZ (Fig. 6A,B).
This misexpression of hh was correlated with a rounded shape and
apparent overgrowth of the clones. The only known regulator of hh
expression in the wing disc is Ci, which is restricted to the anterior
compartment by En-mediated repression; Ci76 represses hh
there (Dominguez et al., 1996
;
Methot and Basler, 1999
;
Schwartz et al., 1995
). These
results suggest that a Ci-independent activator of hh expression must
be present in the posterior compartment and kept in check by Hyd activity.
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In addition, Ci155 was upregulated in anterior hyd mutant clones (Fig. 6E,F). In contrast to the eye disc, we did not observe any hh misexpression in anterior hyd clones (Fig. 6A,B); thus Ci upregulation in hyd clones must be independent of hh. This is consistent with our findings that hyd regulates Ci and hh independently in the eye disc. Smo levels were not significantly increased in hyd mutant clones in the anterior compartment of the wing disc (Fig. 6C,D), suggesting that as in the eye disc, hyd affects Ci independently of Smo.
The F-box protein Slmb has been shown to promote processing of Ci to
Ci75 as a component of an SCF ubiquitin ligase complex
(Jiang and Struhl, 1998;
Miletich and Limbourg-Bouchon,
2000
; Noureddine et al.,
2002
; Theodosiou et al.,
1998
). We therefore compared the effects of slmb and
hyd mutations on Ci levels in the wing disc. Ci155 was
much more dramatically increased in slmb clones than in hyd
clones (Fig. 6I,J). We also
observed an interesting difference between hyd and slmb in
the regulation of dpp. dpp expression was increased in hyd
mutant clones close to the AP border, but was very rarely activated outside
this domain (Fig. 6G,H). In
contrast, slmb mutant clones activated dpp expression only
when they lay outside the wing pouch (Fig.
6K,L) (Miletich and
Limbourg-Bouchon, 2000
), perhaps because of activation of Wg
signaling, which represses dpp expression, within the wing pouch
(Jiang and Struhl, 1998
).
Consistent with these third instar phenotypes, we have not observed anterior
duplications like those resulting from loss of slmb in adult wings
containing hyd mutant clones, although outgrowths did arise from
internal regions of the wing (data not shown). Such duplications would require
dpp to be misexpressed at a distance from its normal domain of
expression (Zecca et al.,
1995
). Ptc expression, which requires activation of the
full-length form of Ci (Methot and Basler,
1999
), was not altered in either hyd or slmb
mutant clones (Wang et al.,
1999
) (data not shown). Slmb and Hyd thus appear to have distinct
effects on Ci protein accumulation and activity, suggesting that they have
either different substrates or different effects on the same substrate.
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DISCUSSION |
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Little is known about the control of hh expression in the eye
disc. In the embryo and other imaginal discs hh expression is
controlled by en, which defines the posterior compartment in a
lineage-dependent manner. The eye disc has no anterior-posterior compartment
boundary, and loss of en function in the eye has no effect
(Strutt and Mlodzik, 1996).
Since hh expression is repressed by Ci76 in the anterior
wing disc (Dominguez et al.,
1996
; Methot and Basler,
1999
), it seemed possible that this was also the case in the eye
disc; Hh could then activate its own expression in more anterior cells by
blocking Ci cleavage. However, we were not able to prevent hh
expression by providing Ci76 to hyd mutant cells in the
eye disc, although this did suffice to repress hh target genes such
as dpp. In agreement with this result, ci mutant clones
anterior to the furrow do not induce ectopic differentiation (N. Baker,
personal communication), indicating that loss of the repressor form of Ci is
not sufficient to allow hh transcription in the eye disc.
hyd must therefore be a component of the Ci-independent mechanism
that restricts hh expression.
Regulation of hh by hyd is also clearly independent of Ci
in the wing disc, since loss of hyd leads to hh upregulation
in the posterior compartment, where Ci is not present, and not in the anterior
compartment. The Groucho (Gro) corepressor has been proposed to contribute to
a Ci-independent mechanism of hh repression in cells close to the
compartment boundary (Apidianakis et al.,
2001). However, the effects of loss of hyd differ from
those of loss of gro, which affects only the anterior compartment of
the wing disc (Apidianakis et al.,
2001
) and promotes excessive photoreceptor differentiation only
posterior to the furrow in the eye disc
(Chanut et al., 2000
),
suggesting that a third mechanism of hh regulation may exist.
hh expression may not be merely a default state resulting from the
absence of the Ci repressor and Gro, but may require another activator, the
levels of which are normally kept in check by Hyd.
Control of dpp expression by hyd, in contrast, appears to be mediated by Ci. Ci155 is upregulated in hyd mutant cells in the eye disc in a hh-independent manner, and ectopic dpp expression in these cells can be blocked by Ci76. In the wing disc, dpp misexpression is limited to the ci-expressing anterior compartment, and is again associated with upregulation of Ci155. Thus hyd acts on Hh signaling as well as hh expression, preventing full activation of the Hh pathway in anterior cells (Fig. 7).
|
Targets of hyd function
Hyd is likely to act as an E3 ubiquitin ligase; its human homologue has
been shown to ubiquitinate at least one substrate in vitro
(Honda et al., 2002). The
substrate specificity of HECT domain E3 ubiquitin ligases appears to reside
within their unique N-terminal domains
(Ciechanover et al., 2000
),
making it difficult to predict the sequence or structure recognized by Hyd.
The only potential clue is that Hyd contains a peptide-binding domain
homologous to the C-terminus of poly(A)-binding protein (PABP)
(Callaghan et al., 1998
;
Kozlov et al., 2001
); the
human HYD protein can interact with Paip1, a binding partner of PABP
(Craig et al., 1998
;
Deo et al., 2001
).
Hyd independently regulates hh and dpp expression,
suggesting either that Hyd has multiple substrates or that its substrate has
multiple functions. dpp expression in hyd clones is blocked
by Ci76, placing the effect of Hyd on dpp upstream or at
the level of Ci activity. Ci155 but not Smo accumulates to high
levels in hyd hh mutant cells in the eye disc and in anterior cells
in the wing disc; thus Hyd may act on Ci itself, on a component of the Hh
pathway between Smo and Ci, or on a nuclear cofactor that stabilizes Ci.
Consistent with an effect on Ci or a cofactor, Hyd protein has been detected
in both the cytoplasmic and nuclear compartments
(Mansfield et al., 1994). The
human Hyd homolog EDD appears to be predominantly localized in the nucleus,
where it interacts with the progesterone receptor and DNA topoisomerase
II-binding protein (Henderson et al.,
2002
; Honda et al.,
2002
). Ubiquitination can function to enhance the potency of
transcriptional activation domains
(Salghetti et al., 2001
);
however, the ectopic gene expression observed in hyd mutant clones
would be difficult to explain by this mechanism.
The SCF ubiquitin ligase complex containing the F-box protein Slmb and the
RING finger protein Roc1 has been implicated in the ubiquitination of Ci that
mediates its processing to Ci75
(Jiang and Struhl, 1998;
Noureddine et al., 2002
;
Theodosiou et al., 1998
;
Wang et al., 1999
). There are
several possible explanations for the apparent overlap between Slmb and Hyd
functions. Slmb may directly ubiquitinate Ci, while Hyd acts on another
substrate; the more dramatic effect of slmb than hyd clones
on Ci accumulation argues for this possibility. However, it is also possible
that Hyd, rather than Slmb, is the direct ubiquitin ligase for Ci. The
consensus sequence for Slmb is not present in Ci, although it has been
suggested that several weakly matching sequences might suffice for its
recognition (Price and Kalderon,
2002
; Winston et al.,
1999
). In addition, two groups have obtained inconsistent genetic
evidence as to whether slmb acts upstream or downstream of
smo and protein kinase A (PKA)
(Jiang and Struhl, 1998
;
Theodosiou et al., 1998
;
Wang et al., 1999
). It is
unlikely that Hyd and Slmb carry out the same process in different cells, as
hyd is expressed throughout the wing pouch
(Mansfield et al., 1994
), and
slmb is clearly active in the same region
(Fig. 6I) (Jiang and Struhl, 1998
),
although its expression has not been examined. Finally, Hyd and Slmb could
both act on Ci, either additively or with different outcomes. For example,
ubiquitination by Slmb promotes processing of Ci to Ci75
(Jiang and Struhl, 1998
),
while ubiquitination by Hyd might promote complete degradation of the Ci
protein. Unfortunately, we were not able to obtain large enough quantities of
hyd hh mutant tissue to test this possibility directly by western
blotting. The lack of hh misexpression in hyd clones in the
anterior compartment of the wing disc suggests that these cells still contain
Ci75 as well as Ci155
(Methot and Basler, 1999
);
loss of hyd may therefore stabilize both forms of the Ci protein
rather than altering their ratio. However, this is not a definitive test of
hyd function, as loss of PKA does not lead to ectopic
hh-lacZ expression despite its effect on Ci processing
(Jiang and Struhl, 1995
;
Jiang and Struhl, 1998
;
Li et al., 1995
;
Pan and Rubin, 1995
).
Hydmediated degradation of both forms of Ci, or its redundancy with Slmb for
Ci cleavage, would explain the limited effect on dpp expression in
hyd hh double mutant clones in the eye disc and in hyd
mutant clones in the wing disc; dpp misexpression in these cases is
restricted to a region in which endogenous Hh may contribute to altering the
ratio of the two forms of Ci.
Ubiquitination is a mechanism commonly used to regulate protein activity by
targeting proteins for degradation or processing, during the cell cycle and in
a number of signaling pathways (reviewed in
Ciechanover et al., 2000). The
Slmb-containing SCF complex is also required for the degradation of Armadillo
(Arm)/ß-catenin, allowing Wnt signaling
(Hart et al., 1999
;
Jiang and Struhl, 1998
;
Miletich and Limbourg-Bouchon,
2000
; Winston et al.,
1999
), as well as for the degradation of IkappaB
(Spencer et al., 1999
;
Yaron et al., 1998
). Hyd is
unlikely to act on Arm in the eye disc, as Arm accumulation would prevent the
ectopic photoreceptor differentiation seen in hyd mutant clones
(Lee and Treisman, 2001a
;
Treisman and Rubin, 1995
);
this may indicate another difference in the substrate specificity of Hyd and
Slmb. However, it is possible that hyd affects Wg signaling in the
wing disc, as hyd mutant clones can induce ectopic expression of the
Wg target gene scute (K. A., unpublished data). The HECT domain
ligases Smurf1 and Smurf2 are important antagonists of BMP signaling,
promoting downregulation of both Smads and receptors
(Kavsak et al., 2000
;
Podos et al., 2001
;
Zhu et al., 1999
).
Itch/Suppressor of deltex, another HECT domain ligase, ubiquitinates Notch
(Cornell et al., 1999
;
Fostier et al., 1998
;
Qiu et al., 2000
). In
addition, nuclear Notch is degraded by Sel-10, while the ligand Delta is the
target of ubiquitination by Neuralized (reviewed by
Lai, 2002
). Our placement of
hyd within the Hh pathway and upstream of hh expression
expands this growing list of cases in which signaling pathways are regulated
by ubiquitination.
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ACKNOWLEDGMENTS |
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