1 Department of Biochemistry and Molecular Genetics, University of Alabama at
Birmingham, 1918 University Boulevard, Birmingham, Alabama 35294, USA
2 Department of Biological Sciences, Columbia University, New York, New York
10027, USA
* Author for correspondence (e-mail: jhorabin{at}uab.edu)
Accepted 11 September 2003
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SUMMARY |
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Key words: Hedgehog, Sex-lethal, Patched, Signaling, Drosophila melanogaster
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Introduction |
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Hedgehog (Hh) is a secreted protein that patterns and specifies cell fate
during the development of several different tissues. Binding to its receptor
Patched (Ptc), relieves Smoothened (Smo) from the inhibition of Ptc and
enables Smo to activate the transcription factor Cubitus interruptus (Ci). In
cells that have not been exposed to Hh, the predominant form of Ci is the 75
kDa isoform (Aza-Blanc et al.,
1997), which acts as a transcriptional repressor. Phosphorylation
of Ci by protein kinase A (PKA) promotes processing of Ci to the 75 kDa
isoform. This processing is dependent on Supernumerary limbs (Slmb) protein
(Jiang and Struhl, 1998
). Hh
reduces the phosphorylation of Ci and generates the full-length 155 kDa Ci
isoform (Chen et al., 1999
)
that activates transcription of wingless (wg),
decapentaplegic (dpp) and ptc (reviewed by
Ingham, 1998
).
This regulated processing and nuclear import of Ci is achieved through a
complex of Ci with the cytoplasmic members of the Hh signaling pathway, the
known members of which are Costal 2 [Cos2; also known as Costa (Cos)], Fused
(Fu) and Suppressor of Fused [Su(fu)]
(Robbins et al., 1997;
Sisson et al., 1997
;
Stegman et al., 2000
). Fu
appears to be a serine threonine kinase
(Therond et al., 1996
;
Nybakken et al., 2002
); Cos2
has sequence similarity to the motor domain of kinesin
(Sisson et al., 1997
), while
Su(fu) shows no homology to any known protein
(Preat et al., 1993
). The
complex is tethered to microtubules by Cos2, and on Hh signaling is released
from microtubules resulting in full-length Ci in the nucleus
(Robbins et al., 1997
;
Sisson et al., 1997
).
Previously, we showed that the stem cells and early cystoblasts of
Drosophila ovaries use the Hh signaling pathway to regulate the
degradation and trafficking of Sxl into the nucleus
(Vied and Horabin, 2001;
Vied et al., 2003
). As Ci is
not expressed in germ cells, the suggestion was raised that Sxl may replace Ci
in the Hh cytoplasmic complex. We show here that in somatic cells, Sxl is in a
complex that contains all of the known Hh cytoplasmic components, including
Ci. Hh promotes the nuclear entry of Sxl in the wing disc and, surprisingly,
in the anterior compartment Ptc appears to be a positive effector of this Hh
promoted nuclear entry. Our data show a cross talk between the sex
determination and the Hh signaling pathways suggestive of a common functional
origin of some of the Hh cytoplasmic components.
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Materials and methods |
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Immunofluorescence, immunoprecipitations and western blots
Antibodies used have been described by Vied and Horabin
(Vied and Horabin, 2001)
except for anti-Su(fu) (1:100; D. Robbins), anti-Msl-2 (1:250; B. Baker),
anti-Ci (1:3; R. Holmgren), anti-En (1:1; Developmental Studies Hybridoma
Bank) and anti-ß-gal (1:1000; Promega). Biotinylated donkey anti-rat
antibody followed by CyTM3-conjugated streptavidin (Jackson
ImmunoResearch Laboratories, Inc), goat anti-rabbit AlexaTM488, goat
anti-mouse AlexaTM594, goat anti-mouse AlexaTM647 (Molecular Probes)
and propidium iodide or Hoechst were the fluorescent probes. Embryos and discs
were mounted in aquaPolyMount (Polysciences, Inc.). Stainings,
immunoprecipitations from 50 µl of 0- to 8-hour OreR embryo extracts, and
western blots were performed as described by Vied and Horabin
(Vied and Horabin, 2001
). For
the Ci immunoprecipitations from Su(fu)LP embryos, 100
µl of whole embryo extracts were used, as Ci levels are reduced in this
background. Each antibody used in the immunoprecipitation was crosslinked to
the protein A beads to minimize the signal from the heavy and light chains in
the western blot.
LMB incubations and staining of discs
Two to 3 days after heat shock, female wandering larvae were bisected and
the head portion turned inside out in 1x PBS. The tissue was washed in
DS2 medium (Mediatech, Inc.) and then incubated, while gently rocking, for 3
hours in 50-200 ng/ml LMB in DS2 medium. After 3 hours the tissue was fixed in
4% paraformaldehyde and stained as previously described for ovaries
(Vied and Horabin, 2001).
Su(fu)LP control discs were treated identically except
that LMB was left out of the incubation.
Mounting of forelegs and wings
Females in which the forelegs had transformed sex combs were desiccated in
ethanol, the legs were removed and mounted in Permount (Fisher). They were
photographed with an Olympus AX70 using the Zeiss Axiovision program.
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Results |
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Wing discs uncover differential Hh signaling
The differential rate of nuclear entry of Sxl along the wing disc AP axis
is suggestive of an involvement of the Hh patterning system. In the wing disc,
Hh is produced in the posterior compartment cells, but activates Ci in a
graded manner in the cells of the anterior compartment where both its
receptor, Ptc, and Ci are expressed. Ptc is thought to limit the range of the
effects of Hh, binding to and internalizing the ligand. As a consequence, only
a row eight to ten cells deep at the AP border expresses full-length Ci
(Chen and Struhl, 1996).
Full-length Ci activates the expression of the Hh target genes, ptc,
dpp and wg. In the row of three to four cells right at the AP
border where activation of the pathway is at its highest, engrailed
(en) expression is also activated by Hh
(Strigini and Cohen,
1997
).
To test the involvement of Hh in Sxl nuclear entry, female wing discs of
the hhMRT background were stained for both full-length Ci
and Sxl. hhMRT is a cold sensitive, gain-of-function
allele that causes overgrowth and ectopic venation in the anterior distal
portions of the wing disc (Tabata and
Kornberg, 1994). Full-length Ci was used to report the location of
ectopic Hh. As can be seen in Fig.
4A-F, the higher levels of nuclear Sxl (brighter signal) coincide
with the cells that have more Ci. Under these conditions and unlike wild-type
discs, the cells in the anterior compartment frequently show more nuclear Sxl
than the cells in the posterior compartment. Removal of Hh, using
hhts2 animals shifted to the non-permissive temperature of
29°C for 12 or 36 hours, reduces the levels of nuclear Sxl in both the
anterior and posterior compartments of the wing pouch (12 hours shown in
Fig. 4G-I). These results
indicate that, as seen in ovaries (Vied et
al., 2003
), Hh promotes the entry of Sxl into the nucleus in
somatic cells and led us to test whether the other components of the pathway
also have a role.
Surprisingly, given the effect of Hh on Sxl, removal of Smo had no effect (in either the anterior or posterior compartment). This was observed at room temperature (Fig. 4J-L) as well as at 18°C, the latter a harsher condition for the strong loss-of-function smo2 allele (Fig. 4M-Q). Control stains indicated that Ci was affected under these conditions and flies with the reported wing defects were recovered (not shown). Conversely, loss of Ptc was found to reduce the rate of Sxl nuclear entry in the anterior compartment (Fig. 5A-F). This was observed with two ptc loss-of-function alleles, ptcS2 and ptc16. Close examination shows the two ptc alleles are slightly different in their effects. In the case of ptcS2, which binds to and internalizes Hh but fails to relieve Smo inhibition, nuclear Sxl is absent between ptc negative clones that are close together (arrowhead in Fig. 5E). By contrast, in the case of ptc16, a protein null allele, nuclear Sxl can be found in the cells between ptc clones (arrowhead in Fig. 5B). These observations are consistent with the idea that (a) a Hh gradient affects the rate of Sxl nuclear entry, (b) PtcS2 protein can sequester Hh from cells in more anterior regions of the wing pouch, an effect the protein null allele does not have, and (c) Ptc acts positively in the Sxl response to the presence of Hh.
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This result is readily explained by the fact that Ptc D584 is able to sequester Hh but is unable to promote Sxl nuclear entry. Ptc D584 in the dorsal posterior compartment restricts the diffusion of Hh into the anterior compartment, so reducing the nuclear levels of Sxl. Ci, however, responds to the state of Smo. In the presence of the dominant negative Ptc D584N protein, Smo is released from the inhibition of the wild-type Ptc in a Hh-independent manner. Ci is thus activated, resulting in the increase in growth. Sxl, in contrast, is dependent on Ptc activation by Hh to accelerate its nuclear entry.
To further establish the requirement of Ptc in transmitting the Hh signal to bring about Sxl nuclear entry, clones mutant for Ptc were made in hhMRT/+ wing discs. If Ptc acts downstream of Hh, its absence should negate the effect of the ectopic Hh on Sxl. This was indeed the case. ptc clones (ptcS2 and ptc16) showed the same effect as in discs that have no ectopic Hh (ptc16 shown in Fig. 5J-L). Full-length Ci and nuclear Sxl were high around the ptc clones because of the ectopic Hh from the MRT allele, but within the ptc clones the levels of nuclear Sxl are reduced. Ci levels were also lower within the clones (induction of en in the clones is antagonistic to high levels of Ci expression, as seen in cells right at the AP border).
Altogether, these data indicate that the presence of the Hh ligand can be signaled in a more complex manner than previously thought. Ptc can communicate the presence of Hh to the Hh cytoplasmic complex and the two Hh `targets' differentially respond to the states of the membrane components.
Some of the Hh pathway cytoplasmic components affect Sxl in the wing
disc
As the Hh membrane components show a differential effect on Sxl and Ci, we
examined the cytoplasmic components for their effect on the nuclear entry rate
of Sxl. In wing discs of females with clones mutant for
PKAH2 or slmb2, Sxl showed no
differences from wild type, suggesting these two genes have little if any
effect (data not shown). Unlike in embryos, we found that mutation of
cos2 had no obvious effects on the levels or nuclear entry rate of
Sxl in either compartment.
Homozygous fu and Su(fu) mutant discs were examined.
Discs mutant for the kinase specific allele, fumH63, were
identified by the broadening of the expression domain of full-length Ci
protein (Ohlmeyer and Kalderon,
1998). Therefore loss of the Fu kinase had no effect on Sxl
(Fig. 6A-C). However, the
strong type I fu allele, fu94, which is truncated
for the Fu regulatory domain (Lefers et
al., 2001
) did have an effect
(Fig. 6D,E). In contrast to the
wild type AP gradient of nuclear Sxl seen in the anterior compartment of
heterozygous discs, Sxl was uniformly nuclear across what we presume are the
homozygous mutant discs. In some cases, a very weak anterior gradient of
nuclear Sxl could be detected (Fig.
6D). These results suggest that the Fu regulatory domain is
normally inhibitory to Sxl nuclear entry in the anterior compartment. The same
result was observed for Su(fu)LP
(Fig. 6F).
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Since Su(fu) has such a strong influence on the nuclear entry rate of Sxl, we determined the distribution of the protein in wild-type discs. An uneven A versus P distribution of Su(fu) would explain the compartmental difference in Sxl localization, and signaling by Hh would account for the nuclear Sxl in the cells at the AP border and its gradation into the anterior compartment. Fig. 6H shows that Su(fu) is uniformly expressed across the wing disc. Its levels, therefore, cannot explain the difference in the rate of nuclear to cytoplasmic shuttling of Sxl along the anterior to posterior axis. Thus, while the effects on Sxl in the anterior compartment show a dependence on the known Hh signaling components, it is not clear what promotes the rapid nuclear entry of Sxl in the posterior compartment. Ptc clones have no effect (and Ptc RNA and protein are not detected in the posterior compartment), but removal of Hh does reduce the nuclear entry rate of Sxl (Fig. 4G-I).
Mutations in Hh signaling genes cause weak sex transformations
Forelegs of females with clones mutant for the various Hh pathway genes
were examined to determine whether the Hh signaling pathway had consequences
on the sex determination process. With the exception of slmb which
had no effect, almost all of the Hh pathway components, including
smo, induced very weak sex transformations represented by a slight
thickening of the bristles on the foreleg
(Fig. 7A-E). In the case of
cos2 clones, occasional females had forelegs with
significantly thickened bristles, almost as thick as those in the male sex
comb.
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Discussion |
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At least three new functional aspects of the Hh pathway are suggested:
A positive role for Ptc, but in this case in conjunction with Smo, in
promoting cell proliferation during head development has recently been
reported (Shyamala and Bhat,
2002). In this situation, however, Hh acts negatively on both Ptc
and Smo in their activation of the Activin type I receptor, suggesting an even
greater variance from the canonical Hh signaling process.
While the effects on Sxl in the anterior compartment show a dependence on
the known Hh signaling components, it is not clear what promotes the rapid
nuclear entry of Sxl in the posterior compartment. Su(fu) is expressed
uniformly across the disc so it does not appear to be responsible for the AP
differences, and ptc clones have no effect (and Ptc RNA and protein
are not detected in the posterior compartment). Removal of Hh, however,
reduces the nuclear entry rate of Sxl in both compartments. In this regard,
the parallel between Hh pathway activation and Sxl nuclear entry in the
posterior compartment is worth noting. Ramirez-Weber et al.
(Ramirez-Weber et al., 2000)
demonstrated that Fu is also activated in the posterior compartment in a
Hh-dependent manner, even though Ptc is not present. It is not clear what
mediates between Hh and Fu.
A changing Hh cytoplasmic complex?
The data also suggest that the Hh cytoplasmic complex may have slightly
different compositions in different tissues and/or at developmental stages. In
the female germline (Vied and Horabin,
2001) and in embryos, the absence of Cos2 leads to a severe
reduction in Sxl levels. However, in wing discs when mutant clones are made
using the same cos2 allele, there is no effect on Sxl. We suggest
that between the third instar larval stage and eclosion, the composition of
the Hh cytoplasmic complex may change again to make Sxl more sensitive to
Cos2. This would explain why removal of Cos2 can produce sex transformations
of the foreleg even though mutant clones in wing discs (and also leg discs;
unpublished observations) show no alterations in Sxl levels.
A similar argument might apply to the weak sex transformations of forelegs
produced by PKA clones. Alternatively, PKA may have
a very weak effect but our assay on wing discs is not sufficiently sensitive
to allow detection of small effects; PKA was found to have a modest
effect on Sxl nuclear entry in the germline
(Vied and Horabin, 2001). Sxl
is sufficiently small (38-40 kDa) to freely diffuse into the nucleus, or the
protein may enter the nucleus as a complex with splicing components. This may
account for the limited sex transformations caused by removal of Hh pathway
components.
Removal of several of the Hh pathway components, such as smo,
gives the same weak sex transformation phenotype, even though smo had
no effect on Sxl nuclear entry. Additionally, there is no correlation between
a positive and a negative Hh signaling component and whether there is a
resulting phenotype. Changing the dynamics of the activation state of the Hh
cytoplasmic complex may perturb the normal functioning of Sxl, since Sxl
appears to be in the same complex as Ci. For example, if the Hh pathway is
fully activated because of a mutant condition, the relative amounts of Sxl in
the cytoplasm versus nucleus at any given time, may be different from the
wild-type condition. Perturbing the usual cytoplasmic-nuclear balance could
compromise the various processes that Sxl protein regulates. Sxl acts both
positively and negatively on its own expression through splicing
(Bell et al., 1991) and
translation (Yanowitz et al.,
1999
) control and, additionally, regulates the downstream sex
differentiation targets. The latter could also be responsible for the weak sex
transformations seen, in view of the recent demonstration that
doublesex affects the AP organizer and sex-specific growth in the
genital disc (reviewed by Christiansen et
al., 2002
).
A cytoplasmic to nuclear shuttling complex?
With the exception of Cos2, which can produce relatively substantial
effects on Sxl levels in embryos as well as sex transformations in the
foreleg, the effects of removal of any of the other Hh pathway components are
generally not large. The strong effects of Cos2 on Sxl could be because it
affects the stability of Sxl. However, Sxl depends on an autoregulatory
splicing feedback loop for its maintenance making the protein susceptible to a
variety of regulatory breakdowns. If Cos2 altered the nuclear entry of Sxl,
for example, its removal could compromise the female-specific splicing of
Sxl transcripts by reducing the amounts of nuclear Sxl. Splicing of
Sxl transcripts would progressively fall into the male mode to
eventually result in a loss of Sxl protein.
Cos2 and Fu have been reported to shuttle into and out of the nucleus, and
their rate of nuclear entry is not dependent on the Hh signal
(Méthot and Basler,
2000). That Ci and Sxl are complexed with the same Hh pathway
cytoplasmic components, and share and yet have unique intracellular
trafficking responses to mutations in the pathway, makes it tempting to
speculate that the Hh cytoplasmic components may have had a functional origin
related to intracellular trafficking that preceded the two proteins. Whether
this reflects a more expanded role in regulated nuclear entry remains to be
determined.
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ACKNOWLEDGMENTS |
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