Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294, USA
Author for correspondence (e-mail:
jamila.horabin{at}med.fsu.edu)
Accepted 23 August 2005
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SUMMARY |
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Key words: Hedgehog, Sex-lethal, Decapentaplegic, Body size, Drosophila
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Introduction |
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Hedgehog (Hh) acts as a morphogen and specifies cell fate during
development, patterning several different tissues (reviewed by
Ingham and McMahon, 2001;
Hooper and Scott, 2005
). Its
receptor Patched (Ptc), inhibits a second transmembrane protein, Smoothened
(Smo). On Hh binding, the inhibition of Smo by Ptc is relieved, enabling Smo
to activate the transcription factor Cubitus interruptus (Ci). Cells not
exposed to Hh express the 75 kDa isoform of Ci
(Aza-Blanc et al., 1997
), a
proteolyzed form of Ci which acts as a transcriptional repressor. Hh signaling
generates full length Ci, a 155 kDa isoform, which activates transcription of
target genes including wingless (wg),
decapentaplegic (dpp) and ptc.
The regulated processing of Ci is realized through a complex of Ci with the
cytoplasmic components of the Hh pathway (reviewed by
Hooper and Scott, 2005). The
complex is tethered to Smo (Jia et al.,
2003
; Lum et al.,
2003
; Ogden et al.,
2003
; Ruel et al.,
2003
) and microtubules by Costal 2 (Cos2), a protein with sequence
similarity to the motor domain of kinesin. On Hh signaling, the complex is
released to result in full length Ci in the nucleus
(Robbins et al., 1997
;
Sisson et al., 1997
;
Zhang et al., 2005
).
Depending on the degree of Ci activation, different downstream Hh targets are activated. In the wing disc, graded Ci activation is accomplished by the expression of Hh in the posterior compartment; Hh diffuses into the anterior compartment and differentially activates Ci. At the highest level of activation, Ci activates engrailed (en) and ptc in the cells closest to the anteroposterior (AP) boundary. Slightly lower levels of Ci activation drive dpp expression, a few cells away from the AP boundary; still lower levels drive the expression of genes from the Iroquois complex further from the AP boundary.
Previously, we showed that Hh promotes the nuclear entry of Sxl in both
germ cells and somatic cells (Vied and
Horabin, 2001; Horabin et al.,
2003
). In the anterior compartment of the wing disc, Ptc appears
to be a positive effector of this Hh promoted nuclear entry, while Smo has no
role (Horabin et al., 2003
).
Here, it is shown that promotion of Sxl nuclear entry by Hh requires the
cholesterol but not palmitoyl modification on Hh. The cholesterol modification
also allows Sxl to enhance the levels of full-length Ci. Signaling by Hh is
thus augmented, resulting in an increase in activation of Ci targets. I
propose that this augmentation is how Sxl increases female size to produce
sexually dimorphic animals.
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Materials and methods |
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Immunofluorescence, LMB incubations and staining of discs
Anti-BrdU monoclonal antibodies were from Zymed and used at 1:40.
Anti-Phospho Histone3 antibodies were from Upstate Biotechnology, used at
1:1000. Anti-pMad antibodies were used at 1:150. Anti-Sal were used at 1:400
and anti-Ptc at 1:5. The rest of the antibodies and staining procedures are
described by Horabin et al. (Horabin et
al., 2003).
Western blot analysis
These were performed as previously described
(Vied and Horabin, 2001). To
obtain loading matches of either Ci or ß-tubulin, different amounts of
adult male and female extracts were loaded on the same gel and probed with
anti-Ci and anti-ß-tubulin.
BrdU incorporation
Dissected and inverted larval heads from UAS-Sxl mated to
ap-GAL4/CyO flies raised at 25°C were incubated in 20 µM BrdU
(Sigma) in Schneider's Drosophila medium for 35 minutes at room temperature
before fixing and staining as in Johnston and Shubiger (Johnston and
Shubinger, 1996).
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Results |
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To test whether either of the lipid modifications plays a role in Hh
promoted Sxl nuclear entry, female wing discs expressing Hh with only a single
modification were examined. HhN encodes the N-terminal region of Hh that is
palmitoylated but, because it does not undergo autoproteolytic processing,
does not contain the cholesterol moiety
(Porter et al., 1996). As
previously reported, this form of Hh is functional for Ci activation and
full-length Ci is detected distantly anterior of the AP boundary. Where HhN
levels are maximal, there is a reduction of full-length Ci
(Fig. 1A,D), most likely from
the activation of en, which inhibits Ci transcription
(Schwartz et al., 1995
). HhN
does not increase Sxl nuclear levels, however
(Fig. 1B,C,E). The normal high
nuclear levels in the posterior compartment and graded nuclear localization in
the anterior compartment (Horabin et al.,
2003
) are detected, with no change in the cells expressing
HhN.
The alternative single modification [cholesterol without palmitoyl
(C84S-Hh)], by contrast, was active with respect to Sxl
(Fig. 2E; see Fig. S1B in the
supplementary material). In Fig.
2, the dppGAL4 driver was used to drive expression of
C84S-Hh. Relative to endogenous Hh, about threefold less of the nuclear export
inhibitor Leptomycin B (LMB) (Nishi et
al., 1994) was required to detect nuclear Sxl in these discs,
suggesting that the nuclear Sxl is effected primarily by the ectopic Hh.
Sxl stabilizes Ci on Hh signaling
C84S-Hh has been shown to dominantly destabilize Ci, decreasing the
expression of Hh target genes. Patterning of the wing is compromised and the
size of the region between veins L3 and L4 is reduced. C84S-Hh is also unable
to rescue the embryonic segmentation phenotype caused by loss of Hh
(Lee et al., 2001).
We also found that C84S-Hh destabilizes Ci, but only in males. Females show the opposite effect, increasing the levels of full-length Ci (compare Fig. 2A with D; see Fig. S1 in the supplementary material). The nuclear Ci detected is in a broad band reflective of the dpp expression zone (note that C84S-Hh is expected to diffuse from its source of expression), and requires several fold less of the nuclear export inhibitor LMB for detection. Under the same conditions, male discs have a weaker signal for Ci and the protein is not nuclear.
|
Curiously, the presence of Sxl does not temper the wing patterning defect
caused by the ectopic expression of C84S-Hh; the reported narrowing between
wing veins L3 and L4 (Lee et al.,
2001) is the same in the two sexes. The form of Ci that Sxl
stabilizes through C84S-Hh must not be the form responsible for Hh
patterning.
Sxl enhances the rate of Ci nuclear entry as well as its levels
The results above were obtained with variant Hh. If Sxl alters the rate of
full-length Ci production and/or its nuclear entry, one might predict that the
endogenous protein should show a difference between the sexes. To test this,
wild-type male and female discs were treated with relatively high levels of
LMB and then stained for Ci and Sxl in the same dish. Examining the discs
shows that it is indeed possible to sex the discs without probing for Sxl.
Male discs consistently show a more diffuse Ci signal, indicative of the
protein being distributed in the cytoplasm. By contrast, female discs show a
more punctate signal and Ci protein appears more distinctly nuclear
(Fig. 3A-F).
If Sxl is responsible for this difference, expression of ectopic Sxl in male discs should alter the behavior of full-length Ci. So as to have an internal control, Sxl was expressed in only the dorsal compartment of the disc with the apterous GAL4 (ap-GAL4) driver, leaving the ventral half of the disc in the wild-type condition. Fig. 3G-I shows that, in the compartment where Sxl is expressed, higher levels and more nuclear full-length Ci are detected in cells near the AP boundary of male discs. Ectopic Sxl was also able to enhance full-length Ci production in females, although less dramatically (Fig. 3J-L). This is not surprising given that they already express Sxl. Under the same conditions, Tra, the immediate target of Sxl in sex differentiation, does not have an effect (data not shown) suggesting that Sxl itself, or another target regulated by Sxl, is responsible.
To further test the idea that females stabilize more Ci than males, the amount of endogenous full-length Ci in males versus females was compared using Western blot analysis. As can be seen in Fig. 4, which uses ß-tubulin as a loading control, when the loading level of Ci is the same in the two sexes, there is more protein loaded in the male lane. Conversely, for the same loading level of protein, females have higher amounts of full-length Ci. These results are consistent with the tissue staining results that suggest that, in addition to increasing the rate of nuclear entry, Sxl enhances the production of full-length Ci.
Sxl boosts the Hh signal
To determine whether the effects of Sxl on full-length Ci production alters
the expression of its downstream targets, the expression of ptc, en
and dpp was analyzed in male and female wing discs expressing ectopic
Sxl. In all cases, Sxl was expressed in the dorsal compartment using the
ap-GAL4 driver, so that the ventral half of the disc could be used as
an internal control.
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|
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A change in en expression in the anterior compartment was less clear cut. As only a few anterior cells at the AP boundary are normally induced to express en by highly activated Ci, and discs expressing Sxl in the dorsal half are frequently distorted owing to an increase in growth (see below), it was hard to gauge whether the narrow row of cells in the anterior compartment expressing En had expanded.
|
To test whether the enhancement of dpp expression by Sxl activates
the downstream targets of the Dpp signaling pathway, the levels of Spalt (Sal)
and phosphorylated Mothers against Dpp (pMad) were examined. Sal is a
transcription factor induced by Dpp signaling. Mad is a downstream
transcriptional target of the Dpp signal that is phosphorylated on activation
of the pathway (Tanimoto et al.,
2000). Sxl increases the intensity of both Dpp responses (Sal in
Fig. 6E,H,K,N; pMad in
Fig. 7E,H) in males and
females; compare the levels of the ventral half with the dorsal half of the
disc in Fig. 6.
If Sxl boosts the Hh signal, removal of Sxl in female discs should reduce the relative strength of the signal. To test this prediction, Sxl-null clones were generated in female discs and the levels of expression of Ci, Sal and dpp-lacZ examined. Very few large clones were recovered in the wing pouch, but the small clones recovered near the AP border all showed a reduction in their levels of full-length Ci (Fig. 8A-C). Larger clones were found in the notum area of the wing disc and these also showed a reduction in Ci (Fig. 8D-G), as well as Sal and dpp-lacZ expression (Fig. 8H-K). The reduction is frequently modest. However, as males have a fully functional Hh signal, elimination of Sxl in females is only expected to reduce not eliminate the signal.
Sxl induces growth in the wing disc
Hh only patterns the region immediately anterior to the AP boundary, which
gives rise to the middle of the wing between veins L3 and L4.
Patterning of the rest of the disc is accomplished by the gradient of Dpp that
is elicited by Hh (Lecuit et al.,
1996; Nellen et al.,
1996
). Dpp also regulates growth of the disc
(Capdevila and Guerrero, 1994
;
Burke and Basler, 1996
;
Lecuit et al., 1996
;
Nellen et al., 1996
),
promoting the cell cycle while maintaining cell size
(Martin-Castellanos and Edgar,
2002
).
To test whether Sxl affects the cell division rate, male and female wing discs expressing ectopic Sxl in the dorsal compartment were analyzed for BrdU incorporation. Fig. 9A-I shows that the rate of BrdU incorporation in the dorsal half of the wing pouch is increased by ectopic Sxl. As BrdU reports DNA synthesis, this suggests that the mitotic index is elevated by Sxl. Under the same conditions, Tra did not produce this effect (data not shown). The number of cells positive for Phospho histone 3 (PH3), a modification that is also indicative of mitosis, also shows an increase when Sxl is ectopically expressed (Fig. 9J-R).
If Sxl induces disc growth, as suggested by the data above, and the augmentation of Hh signaling by Sxl requires the Hh cholesterol moiety, then signaling through just C84S-Hh might be predicted to induce growth. Such an effect would have to overcome the dominant-negative effect of C84S-Hh on endogenous Hh signaling (which normally also functions to grow the disc). Despite these opposing effects, male and female wing discs expressing C84S-Hh and ectopic Sxl by the dpp-GAL4 driver, show a change in their normal proportions that is suggestive of enlargement. All male discs show the posterior En signal with an anterior extension (Fig. 2H), with the rest of the anterior compartment relatively normal in size. In females, the anterior compartment is larger than the posterior, frequently by almost twofold (Fig. 2L).
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Growth induced by Sxl is not cell autonomous
If the growth effects by Sxl are caused through altering the Hh signal then
expressing Sxl in the anterior compartment, in only the cells that respond to
the Hh signal, should be sufficient to induce growth across the entire disc.
To test this prediction, Sxl was expressed in the anterior compartment using
the dpp-GAL4 driver, and effects on disc growth examined. As seen in
Fig. 10E,F, both males and
females show overgrowth not only in the anterior compartment where it is
expected, but also in the posterior compartment. These results clearly
demonstrate that Sxl produces growth effects in a non-autonomous manner.
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![]() |
Discussion |
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The Hh signal is enhanced in females
The data presented here show that when Sxl is present, the Hh signal is
augmented. This is seen as an increase in full-length Ci in whole-mount tissue
(Fig. 3), and in western blots
which give a more quantitative sense of protein levels
(Fig. 4). In addition to
elevating the levels of full-length Ci, several of the Hh downstream targets,
including ptc, dpp and some of the downstream targets of Dpp, show an
increase in expression. Conversely, removal of Sxl in female cells shows a
reduction in the strength of the Hh signal
(Fig. 8).
|
Hh enhancement of Sxl nuclear entry also depends on the cholesterol and not
the palmitoyl modification. Given that Ci and Sxl are in a complex in the
cytoplasm and both respond to the Hh cholesterol modification, it is tempting
to speculate, although the data presented does not address this issue, that
the two proteins may also enter the nucleus as a complex. This may be the
method by which Sxl stabilizes Ci, diverting it from rapid proteolysis,
particularly the highly activated form that is functionally detectable
(Ohlmeyer and Kalderon, 1998)
but has not been identified biochemically.
Stabilization of full-length Ci by Hh with only the cholesterol
modification in females is in contrast to what occurs in males. We also found,
as previously reported (Lee et al.,
2001), that this form of Hh can destabilize Ci as well as
compromise the Hh signal, but only in males
(Fig. 2A). The effect of the
cholesterol moiety contrasts with the palmitoyl that potentiates Hh in
activating Ci for patterning. This is generally also true in vertebrates,
where the cholesterol modification appears to have less of a role in
patterning and a more significant role in the release and extracellular
transport of the Hh ligand (Feng et al.,
2004
).
Hh signal is not at its maximum
In both sexes, ectopic expression of Sxl shows an increase in intensity of
ptc expression, indicating it is possible to further elevate the Hh
response. Other than en, which was difficult to score in our
experiments, ptc requires the highest levels of Ci activation for its
transcription.
In females, the ectopic Sxl elevates ptc expression in the cells near the AP boundary, but the depth of the cells showing this highest level of Ci activation is reduced. A reduction in the number of cells transcribing ptc, when compared with the wider but less intense width of ptc transcription in the control half of the disc, suggests a restriction in Hh diffusion. Elevated ptc transcription is expected to produce more Ptc at the membrane, which should sequester more Hh close to the AP boundary. This result shows that Sxl can both enhance the Hh response and effectively alter the Hh gradient.
In males, the increase in ptc transcription induced by Sxl both intensifies and widens the ptc expression zone. This suggests that the activation of Ci is at a lower peak in males than in females, and its enhancement by ectopic Sxl does not reach the same maximum that additional Sxl in females produces.
Ectopic expression of Sxl in the dpp expression zone has
previously been shown to adversely affect female wing development, narrowing
the region between veins L3 and L4. This defect was taken to suggest that the
relative concentrations of both Ci and Sxl are important for their normal
function (Horabin et al.,
2003). The data presented here support that conclusion while
providing an explanation for the apparent decrease in effectiveness of the Hh
signal. As outlined above, when additional Sxl is expressed, the slope of the
Hh gradient becomes steeper. As Hh directly patterns the L3 to L4 wing vein
region, a steeper gradient of Hh will reduce the area patterned because the
normal Hh patterning minimum is reached more rapidly. The L3 to L4 intervein
region should correspondingly become narrower. No adult males expressing Sxl
were recovered (presumably because of upsets in dosage compensation) so their
wings could not be scored.
Depending on the expression driver used, ectopic Sxl is not only lethal to
males but also females. This is perhaps not altogether surprising given that
Sxl can modulate the signal strength of a molecule crucial to the development
of numerous tissues. The in vivo concentration of Sxl is, most likely, tightly
controlled. Yanowitz et al. (Yanowitz et
al., 1999) demonstrated that Sxl negatively regulates translation
of its own mRNA. Combined with its positive autoregulatory splicing feedback
loop, which ensures that essentially all of the Sxl mRNA is spliced
in the productive female mode in females, this dual negative and positive
autoregulation implies a homeostasis that keeps the concentration of Sxl in a
predetermined fixed range. The potent effect of Sxl on the Hh signal makes the
requirement for this dual regulation more readily understood.
Involvement of Hh in generating sexually dimorphic body size
Mutations in Sxl that produce sex transformed females generally
result in animals that are small and male-like in size. Females transformed by
mutations in tra appear as males but maintain the female size,
indicating that sexual dimorphic body size is controlled by Sxl.
The enhanced levels of full-length Ci and the data of Figs 5,6,7,8,9,10 suggest that Sxl promotes disc growth. Indeed, when ectopic Sxl is being expressed in the dorsal half, many of the discs, both male and female, show an overgrowth phenotype with the dorsal half of the wing pouch frequently expanded and distorted. This growth effect is non autonomous (Fig. 10), indicating that it is effected by a system that signals beyond the cells expressing Sxl. This is consistent with the idea that Hh signaling is augmented to result in the overgrowth. The experiments described here do not rule out the possibility that Sxl may additionally regulate growth autonomously.
Hh with only the cholesterol modification has the greater impact on Sxl and its stabilization of full-length Ci. However, the Ci that is stabilized does not appear to accomplish Hh patterning. This raises the mechanistic question of how Sxl achieves growth of the entire disc.
Simply reducing the levels of the repressor form of Ci (which is accomplished by increasing the levels of full-length Ci) should increase the expression of the growth factor dpp. This is because dpp is affected by Ci at two levels: absence of the Ci repressor ameliorates repression to give low levels of dpp expression, while activated full-length Ci further elevates dpp transcription. Indeed, while the wing patterning defect caused by the ectopic expression of C84S-Hh narrows the region between wing veins L3 and L4 equally in the two sexes (due to its dominant-negative effect on endogenous Hh), the overall sexual dimorphic size difference is maintained. Consistent with this idea, co-expressing Sxl and Hh with only the cholesterol modification produces an overgrowth phenotype in discs (Fig. 2G-L), indicating Sxl can promote disc growth through this form of Hh.
The growth induced by Dpp has been described as `balanced', involving both
mass accumulation as well as cell cycle progression. The net effect is that
cell size does not change, nor does the ploidy. This is in contrast to growth
induced by hyperactivation of Ras, Myc or Phosphoinositide 3 kinase, which
increase growth but do not induce a progression through the G2/M phase of the
cell cycle and, as a result, increase cell size
(Martin-Castellanos and Edgar,
2002).
We propose that in the wild-type gradient of Hh with both its lipid
modifications, Sxl augments the overall Hh signal to increase both full-length
as well as activated full-length Ci. The two Hh targets (Ci and Sxl) respond
differentially to the various components of the pathway
(Horabin et al., 2003). As Sxl
is able to alter signal strength, the final outcome of the Hh signal must
reflect the balance in activities of the components, modulated by the lipid
moieties recognized, the membrane proteins used (Ptc versus Smo) and the
proteins present in the Hh cytoplasmic complex. The studies reported here
provide a strong rationale for why Sxl resides within the Hh cytoplasmic
complex.
Sxl not only elevates expression of dpp and its downstream targets to induce growth, but is able to elevate ptc expression. Enhancing ptc suggests that the Hh signal is `corrected' for the enlarged patterning field, as short-range patterning has to be controlled by Hh. By enhancing dpp, Sxl indirectly also enhances the long-range patterning system of the disc. Augmenting the Hh signal would thus appear an elegant solution for increasing overall size without changing the basic body plan or pattern. As Sxl is expressed in all female tissues from very early in development and this expression is maintained for the rest of the life cycle, Sxl is constantly available to upregulate the Hh signal. This augmentation must be kept within check, however, because, as argued above, too high an increase can change the overall slope of the Hh gradient, effectively changing the final patterning of the tissue.
The Hh pathway can also control body size in mammals. ptc1
mutations in mice provide an overgrowth phenotype with large body size
(Goodrich et al., 1997;
Hahn et al., 1998
), while
increasing ptc1 expression decreases body size
(Milenkovic et al., 1999
).
Humans with basal cell nevus syndrome, an autosomal-dominant condition caused
by the inheritance of a mutant ptc allele, have been reported to have
multiple developmental abnormalities and, relevant to this study, larger body
size (Gorlin, 1995
). Whether
the mechanism described here is global to sexually dimorphic organisms that
use Hh for patterning remains to be seen.
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ACKNOWLEDGMENTS |
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![]() |
Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/21/4801/DC1
* Present address: Room 3300-G, Department of Biomedical Sciences, 1115 West
Call Street, College of Medicine, Florida State University, Tallahassee, FL
32306, USA
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