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First published online 15 November 2006
doi: 10.1242/dev.02674
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1 University of Delaware, Department of Biological Sciences, Newark DE,
USA.
2 Duke University, DCMB Group, Biology Department Durham, NC, USA.
* Author for correspondence (e-mail: selva{at}udel.edu)
Accepted 3 October 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Wingless signaling, Wingless, Drosophila, Chaperone, Secretion
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Only recently has it become clear that specialized
mechanisms are required for the processing and packaging of the Wg ligand, the
dissemination of the packaged ligand and its targeting to receiving cells for
efficient Wg signal transduction.
A number of factors have been identified that are essential for efficient
Wg signal transmission, but do not participate directly in the intracellular
signaling pathway in cells receiving the signal. In porcupine
(porc)-mutant embryos, Wg fails to be secreted, but instead
accumulates inside Wg-producing cells. The failure to secrete Wg leads to a
`lawn of denticles' - an embryonic phenotype typical of Wg loss-of-function
mutants (Kadowaki et al.,
1996; Manoukian et al.,
1995). Wnt proteins have recently been shown to be palmitoylated,
and porc probably encodes a palmitoyltransferase necessary for this
post-translational lipid modification
(Willert et al., 2003;
Zhai et al., 2004). In the
porc mutant larvae, Wg is not lipid-modified and fails to be targeted
to plasma-membrane lipid rafts (Zhai et
al., 2004). The importance of rafts in cellular signaling is
inferred from the observation that they are fortified with membrane molecules
responsible for signal transduction (reviewed in
Kurzchalia and Parton, 1999).
Indeed, rafts might serve as an ideal platform for the accumulation and
release of hydrophobic ligands, such as Wg and Hedgehog (Hh, both
palmitoylated and cholesterol-modified), from the plasma membrane
(Chamoun et al., 2001;
Lee and Treisman, 2001;
Micchelli et al., 2002;
Porter et al., 1996). Recent
studies in Drosophila demonstrate that this is clearly the case, as
active forms of secreted Wg and Hh are packaged into lipoprotein particles
(Panáková et al.,
2005). In larvae, lipophorinenriched particles are probably
synthesized in the fat body and come into contact with signaling cells, where
they are loaded with their lipophilic cargo of modified Wg and modified Hh for
dissemination to receiving cells
(Panáková et al.,
2005). It is now apparent that the correct processing of Wnts as
they move through the secretory pathway, accurate membrane targeting and
packaging at the plasma membrane are intricate and complex processes. Failure
of any number of steps in these processes could lead to a breakdown in the
Wnt/Wg-signaling circuit from signaling to target cells. These observations
show that post-translational maturation of Wg, cellsurface components required
for packaging and dispersal into the extracellular milieu, plays essential
roles in the transmission of the Wg signal. In addition, plasma-membrane
composition and organization, and the molecules that drive these parameters,
are likely to be influential variables in the regulation of Wg cellular
signaling.
The complexity of Wnt signal transmission is not limited to maturation of
the ligand in the sending cells. It now appears obvious why Wg must interact
with both its cognate receptor (Frizzled) and an LDL-like co-receptor (Arrow)
for receipt of the Wg signal. Correct processing of Arrow in the receiving
cells is also essential for Wg signal transduction
(Culi and Mann, 2003). The
processing of both Wg in the sending cells and its receptors in the receiving
cells must occur for appropriate Wg presentation and a productive receptor
interaction, which are required to activate the pathway. It has also been
reported that Wg is differentially degraded in distinct types of receiving
cells in embryos (Dubois et al.,
2001; Piddini et al.,
2005). Clearly, mechanisms governing this process could be
controlled by events both in signaling and in target cells.
Here, we identify a new component in the Wg signaling pathway,
sprinter (srt). Absence of srt7E4
results in the accumulation of the Wg ligand in signaling cells and in an
inability to activate downstream targets of Wg signaling in receiving cells.
Therefore, we hypothesize that srt encodes a factor required for Wg
maturation as Wg moves through the secretory pathway to yield active ligand or
a protein that promotes proper packaging and dissemination of Wg from the
plasma membrane of signal-producing cells. Appropriate maturation of Wg in the
Wg-producing cells could influence its compartmenttargeting in the sending
cells, targeting or binding specificity in receiving cells, or uptake and
endocytic handling in receiving cells. Two recent papers also identify
mutations in this locus, referring to the gene as wntless
(wls) (Bänziger et al.,
2006) or evenness interrupted (evi)
(Bartscherer et al., 2006), and
reach a similar conclusion regarding its role in Wg signaling.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Germline clones
srt germline clone embryos were generated by crossing y w
hs-flp/Y; P{ovoD1-18}3L FRT2A/TM3, Sb males to w;
srt7E4 FRT2A/TM3 virgin females. Resulting
third-instar larvae were then heat-shocked for 1 hour at 37°C to induce
Flp/FRT-mediated recombination, as previously described
(Chou and Perrimon, 1996;
Chou and Perrimon, 1992). The
resulting y w hs-flp/w; P{ovoD1-18}3L FRT2A/srt7E4
FRT2A virgin females were then crossed to w;
srt7E4 FRT2A/TM3 and Df(3L)vin5, ru h gl e
ca/TM3, Sb, Ser males to compare srt7E4
FRT2A/srt7E4 FRT2A germline clone
embryos to srt7E4 FRT2A/Df(3L)vin5
(srt
) embryos. For cuticle preparation, 24- to
36-hour embryos were dechorionated in 50% bleach for 5 minutes, mounted in
Hoyer's media, heated to 55°C for 24 hours and visualized on a Zeiss
Axiophot in darkfield. For antibody staining, embryos were dechorionated and
fixed in 4% formaldehyde, as previously described
(Patel, 1994).
Adult and larval clonal analysis
srt-mutant somatic clones were induced by heat-shock in first
instar larvae (Golic, 1991;
Xu and Rubin, 1993). Adult
clones were generated by crossing w; mwh srt7E4
FRT2A/TM3,Sb males to y w hs-flp/y w hs-flp;
M(3)i55 hs-GFP FRT2A/TM3,Sb, and the wings from
non-Sb adult progeny were mounted in Euparal (ASCO Laboratory) and inspected
for phenotypes with a Zeiss Axiophot in brightfield. The presence of
mwh identified the homozygous srt7E4-mutant
tissue. Marked larval clones in wing imaginal discs were obtained by crossing
y w hs-flp/y w hs-flp; M(3)i55 hs-GFP
FRT2A/TM6B to w/Y; srt7E4
FRT2A/TM6B, Tb (Baeg et
al., 2004). Wing discs from Tb+ larvae were
dissected 1 hour following a 1-hour heat-shock at 37°C to induce GFP
expression. Dissected wing discs were then fixed in 4% formaldehyde and
stained, and homozygous srt7E4-mutant tissue was
visualized by the absence of GFP, as previously described
(Baeg et al., 2004).
Antibody labeling
For embryo and wing-disc antibody staining, the following primary antisera
were used: mouse anti-Wg (4D4; Developmental Studies Hybridoma Bank, DSHB)
(Brook and Cohen, 1996) diluted
1:10; guinea pig anti-Sen (from Hugo Bellen, Baylor College of Medicine,
Houston, TX) (Nolo et al.,
2000) diluted 1:1000; mouse anti-Ptc (5E10 from David Strutt,
University of Sheffield, UK) diluted 1:3000; rat anti-Ci (from Robert
Holmgren, Northwestern University, Evanston, IL)
(Motzny and Holmgren, 1995),
1:10; mAb anti-Ac (DSHB) (Skeath and
Carroll, 1991) diluted 1:3; rabbit anti-Hh (from Inge The,
University of Massachusetts Medical School, Worcester, MA)
(Taylor et al., 1993), diluted
1:200; and mouse anti-En (4D9, DSHB)
(Patel et al., 1989) diluted
1:4. Fluorescent secondary antibodies from Jackson ImmunoResearch Laboratories
and AlexaFlours from Molecular Probes were used at a dilution of 1:500. Discs
were mounted in Vectashield mounting media (Molecular Probes) and inspected
using a Zeiss LSM510 confocal microscope. Z-series projections were rendered
as a maximum intensity projection using Zeiss LSM 510 software (version
3.2).
Sprinter-candidate identification
The srt7E4 mutation was uncovered by
Df(3l)vin5 (68A2-3;69A1-3) but excluded from Df(3l)ED4457
(67E2;68A7) and Df(3l)vin4 (68B1-3;68F3-6), placing the mutation on
the left arm of chromosome 3 within the cytological region 68A7 to 68B3. From
these results, the left-most candidate open reading frame (ORF) in this region
was identified as CG7628, a putative phosphate transporter, by the exclusion
of srt from the molecularly defined deficiency ED4457. We
were able to ascertain the right-most candidate ORF as CG6190 using
quantitative PCR to define the left molecular endpoint for Df(3l)vin4
between CG6190 and CG7600, leaving 15 srt candidate ORFs within this
region. As the germline clone screen that identified sprinter
requires that the candidate transcript must be maternally loaded, we further
eliminated candidates within this region by performing RT-PCR with RNA
isolated from unfertilized embryos to determine which of the remaining
srt candidates were maternally loaded. Among the remaining
candidates, we found that six were not maternally loaded, leaving nine
candidates. Lethal excision of P11739 localized to the 5 ' UTR
of Alg10/CG32076 yielded mutations that were found to complement
srt7E4, eliminating this gene as a candidate and reducing
the candidate pool to eight. Identification of srt7E4 as a
nonsense mutation in CG6210, srt mRNA rescue of the mutant phenotype
and srt RNAi induction of the mutant phenotype are described in the
Results. Injection of GFP+ srt7E4 embryos with
dsRNA targeted to three other ORFs among our remaining candidates (CG6207,
CG6190 and CG7616) did not generate a segmentation defect (data not shown).
Wild-type embryos with both maternal and zygotic srt gene expression
injected with 543 bp srt dsRNA hatched with no apparent defects when
compared to a dsRNA targeted to wg, a control zygotic target (data
not shown). This suggests that the srt maternal contribution could
not be knocked down sufficiently to yield in embryonic lethality.
Quantitative PCR
Homozygous-mutant GFP-negative Df(3L)vin4, ru h gl e ca embryos were
enriched by sorting 16- to 20-hour embryos from Df(3L)vin4, ru h gl e ca/TM3,
Sb, Twi-GFP under a fluorescence stereo microscope. Genomic DNA was prepared
from both GFP- (vin4/vin4) and GFP+ (vin4/TM3, Sb,
Twi-GFP) embryos, and was used to perform Q-PCR in a BioRad iCycler using the
QuantiTect SYBER Green PCR kit (Qiagen). DNA sequences present in the ORFs
that are deleted from the vin4 chromosome are less represented in
GFP- than in GFP+ genomic-DNA samples, resulting in a
higher threshold of detection. Typically, an ORF was determined to be within
vin4 if the ct between GFP- and GFP+
genomic-DNA samples was greater than three cycles. Primer sequences used in
this experiment are available upon request. By these criteria, the left end
point for the Df(3L)vin4 was defined by determining that CG6190 was outside
of, and CG7600 was within, the deleted region of vin4.
RT-PCR
Total RNA was isolated from 16- to 20-hour unfertilized and control
fertilized w1118 embryos using Ultraspec (Biotecx).
Presence of the message from each ORF within 68A8:68C1 was detected with the
Super Script One-Step RT-PCR kit (Invitrogen). Srt maternally loaded candidate
ORFs were defined as those present in both unfertilized and fertilized
embryos, as compared to zygotic controls that are only present in RNA derived
from fertilized embryos. Evaluation of each ORF was determined from three
independent RNA samples.
RNA rescue and RNA interference in srt7E4 germline clone embryos
Germline clone females generated in mass as described above were crossed to
w/Y; srt7E4 FRT2A/TM3, GAL4-twi2.3,
UAS-2xEGFPAH2.3, Sb Ser. Embryos from this cross 0- to 1-hour
old were aligned on double-stick tape, desiccated, covered with halocarbon oil
and injected at the midline with either srt mRNA or dsRNAs at a
concentration of 1 mg/ml. Following an approximately 48-hour incubation period
at 18°C, the embryos and any hatched larvae were scored for GFP
fluorescence and separated into GFP+ and GFP- groups.
The samples were briefly washed with heptane to remove the halocarbon oil,
mounted in Hoyer's media and visualized as above. srt polyadenylated
message was prepared in vitro using mMessage mMachine T7 Ultra kit (Ambion)
from linearized pOT2-GH01813 (full-length srt cDNA, isoform A). dsRNA
against CG6210/srt, CG6207, CG6190, CG7616 and wg were
prepared using the Megascript kit (Ambion) from PCR templates derived from
w1118 genomic DNA. Primer sequences are available upon
request.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Chou and Perrimon, 1992;
Chou et al., 1993). Using this
method, only those female germ cells in which both homologs of a gene are
mutant are able to complete oogenesis and produce fully mature eggs. Hence,
fertilization of these eggs by heterozygous mutant fathers allows us to
monitor embryonic development in the absence of maternal and zygotic
contribution of the gene in question. Examination of the cuticles from
srt7E4 germline clone embryos, (hereafter referred to as
srt7E4 embryos), results in the loss of naked cuticle to
yield a `lawn of denticles' phenotype (Fig.
1C), as compared with the normal patterning of wild-type embryos
(Fig. 1A). As Wg and Hh
signaling exist in a feedback loop during embryonic epidermal patterning, this
result suggests that either the Wg or the Hh pathway is disrupted
(Martinez-Arias et al., 1988;
Perrimon, 1994). The
srt7E4 phenotype can be rescued to larval and adult stages
of development if the father contributes a wild-type copy of the gene,
although some die during embryonic development with mild segment-polarity
defects (Fig. 1B).
srt7E4 is a strong loss-of-function mutation, because its
embryonic cuticle phenotype is just as severe whether the father contributes a
srt or srt7E4 chromosome
(Fig. 1D). Zygotic
srt7E4/srt transheterozygotes are pupal
lethal, suggesting maternally contributed Srt function perdures through the
larval stages of development (see Fig. S2 in the supplementary material).
Loss of sprinter in embryos blocks signaling by preventing Wingless secretion
Because of the signaling feedback loop that exists between the Wg and Hh
pathways during embryonic development, it is difficult to determine which
pathway is specifically disrupted (DiNardo
et al., 1988; Martinez-Arias
et al., 1988; Heemskerk et
al., 1991; Bejsovec and
Martinez Arias, 1991). We examined the expression of Wg in both
paternally and non-paternally rescued srt7E4 embryos, and
found that Wg accumulated in the cells that express it (compare wild type
Fig. 2A,D with
Fig. 2B,C,E,F). This result
indicates that mutation in srt obstructs normal Wg secretion and thus
prevents normal Wg signaling to yield the embryonic `lawn of denticles'
phenotype (Fig. 1C,D).
Low levels of Wg secretion rescues downstream signaling in embryos
Because we had observed that paternally rescued embryos reached adult
stages of development, we compared the Wg-secretion phenotypes at stages 9 and
13 of embryonic development in nonpaternally and paternally rescued
srt7E4 embryos with wild-type embryos
(Fig. 2A,D). Non-paternal and
paternal rescue was distinguished by the presence of the TM3,
Twist-GFP balancer (data not shown). Surprisingly, we detected little
difference in the accumulation of Wg in stage 9 non-paternally
(Fig. 2B) and paternally
(Fig. 2C) rescued
srt7E4 embryos, although some Wg was detected in endocytic
vesicles in the receiving cells in the paternally rescued
srt7E4 embryos (Fig.
2C, arrowheads). Even by stage 13, when Wg had completely faded
from epidermal cells and could only be detected in the neuroblasts of
non-paternally rescued srt7E4 embryos
(Fig. 2E)
(Ingham and Hidalgo, 1993), Wg
accumulation was still significant in paternally rescued stage-13 embryos
(Fig. 2F). Through the later
stages of embryonic development, paternally rescued srt7E4
embryos gradually approached, but never completely achieved, wild-type levels
of Wg secretion (data not shown). Very little Wg is released from paternally
rescued srt7E4 embryos, but this is sufficient to maintain
expression of Engrailed (En), an epidermal target for Wg signaling in
receiving cells (Fig. 3B)
(Ingham and Martinez Arias,
1992; Perrimon,
1994), to ultimately yield larva capable of reaching adult stages
of development. This suggests only a minimal level of secreted Wg is necessary
to maintain downstream Wg signaling events required for embryonic patterning.
En expression fades from the epidermis of stage 9 non-paternally rescued
srt7E4 embryos (Fig.
3A) and is completely lost shortly there after (data not
shown).
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;
Sánchez-Herrero et al.,
1996; Slusarski et al.,
1995). Wg is secreted from the dorsoventral boundary of the wing
imaginal disc to mediate patterning of the wing margin
(Couso et al., 1994). The
effect of srt7E4 homozygous mutant tissue, marked by
multiple wing hairs (mwh), was examined in adult wings
(Fig. 4)
(Golic, 1991;
Xu and Rubin, 1993). We
consistently observed that clones of srt7E4-mutant tissue
at the wing margin resulted in the loss of both sensory bristles
(Fig. 4B,D) and margin wing
vein (Fig. 4C,E), a phenotype
diagnostic of Wg-pathway disruption and consistent with our embryonic data.
Large clones in the posterior wing, and those encompassing veins 3 and 4,
which would be predicted to yield Hh-specific phenotypes, appeared normal,
suggesting that the loss of srt7E4 does not disrupt Hh
signaling in this tissue (data not shown). Furthermore, except for the
wing-margin defects, we observed no other effect of
srt7E4-mutant tissue on wing development. These results
suggest that srt blocks Wg signaling during wing development.
The srt7E4 mutation blocks Wg signaling by preventing Wg secretion in wing discs
We further explored the effect of the srt7E4 mutation
on Wg signaling by examining the expression of molecular markers in third
instar larval wing discs. In this tissue, Wg is secreted from the dorsoventral
boundary (Fig. 5A), and
diffuses dorsally and ventrally to activate Senseless (Sen;
Fig. 5B, blue)
(Nolo et al., 2000) and
Achaete (Ac; Fig. 5J, red)
(Couso et al., 1994;
Phillips and Whittle, 1993) in
adjacent target cells. We generated srt7E4-mutant tissue
in third instar wing discs, which were distinguished by the absence of GFP
(Fig. 5F,I,M,N, green)
(Golic, 1991;
Xu and Rubin, 1993).
Fig. 5D,G shows the
side-by-side expression pattern of Wg in wild-type (within outline) and
srt7E4-mutant (excluded from outline) tissue at the
dorsoventral boundary. In srt7E4-mutant tissues, the
intensity of Wg staining is significantly higher because the protein is
accumulating in the cells that express it, as was observed in embryos. There
is also a loss of Sen expression in the adjacent receiving cells
(Fig. 5E,H), as compared with
wild-type tissue (Fig. 5B).
However, some srt7E4-homozygous Sen-expressing mutant
cells are detected within the clone, suggesting that srt is not
required in target cells to receive the Wg signal
(Fig. 5E, excluded from
outlined heterozygous cells and Fig.
5H, arrows). These cells do not express Wg
(Fig. 5G, arrows), confirming
that they are exclusively target cells. In the anterior compartment, we do
detect some low-level Sen expression in target cells that abut
srt7E4-mutant Wg-signaling cells, indicating that
srt7E4 may not completely block Wg signaling
(Fig. 5E). Similar results were
obtained for Ac, a Wg anterior compartment target
(Couso et al., 1994;
Phillips and Whittle, 1993).
Ac expression is lost when srt7E4-mutant tissue crosses
the Wg-expressing cells at the dorsoventral boundary
(Fig. 5K, red). These results
demonstrate that the action of Srt is non-cell autonomous, which would be
expected for a gene product acting on a secreted ligand. Hence, the
extracellular movement of Wg produced from a few srt/+-heterozygous
cells present at the dorsoventral boundary
(Fig. 5E) is sufficient to
activate the expression of Wg target genes, as observed in
srt7E4 embryos. These results suggest that
srt7E4 blocks the secretion of Wg from its producing
cells, thereby preventing the activation of Wg downstream targets in adjacent
receiving cells.
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) (Fig. 5M-O).
Notably, the large clones of srt7E4 encompassing almost an
entire compartment did not distort the overall wing-disc structure, suggesting
that srt is not essential for cell survival or cell proliferation in
the wing pouch (Fig. 5F,O). If
srt were necessary in the wing disc for Hh or other signaling
pathways, such as Decapentaplegic (Dpp), such large clones would be expected
to have a significant effect on cell growth and, as a result, on wing-disc
morphology.
srt7E4 has no effect on Hh secretion or on the activation of the downstream targets of Hh in wing discs
In order to confirm that srt is specific for Wg secretion and
signaling, we examined Hh expression and the activation of its downstream
targets in the wing disc (Fig.
6). The anterior compartment and anterior-posterior compartment
boundary is defined by the expression of Cubitus interruptus (Ci;
Fig. 6B,E)
(Han et al., 2005). In
contrast to Wg, Hh does not accumulate in posterior
srt7E4-mutant ligand-expressing cells. Furthermore, we
observe no disruption in Hh targeting to the anterior receiving cells,
visualized as punctate staining in the Ci-expressing cells, regardless of
whether the Hh was derived from or received by wild-type or
srt7E4-mutant cells
(Fig. 6A,D). This result
indicates that srt is not required for the delivery or the receipt of
the ligand in efficient Hh signal transduction. This conclusion is supported
by the fact that the anterior expression of Patched, a target of Hh signaling
in the wing disc, is similar to wild-type expression
(Fig. 6G)
(Capdevila et al., 1994;
Strigini and Cohen, 1997)
whether posterior Hh-expressing cells or anterior Hh-receiving cells are wild
type or srt7E4 mutant
(Fig. 6H). Similar results were
obtained for the anterior compartment expression of Engrailed, another target
of Hh signaling in the wing disc (data not shown)
(Blair, 1992;
Blair and Ralston, 1997). Based
on these results, we conclude that srt does not play a role in any
aspect of the Hh signal transduction pathway in the wing disc. Therefore, we
propose that the embryonic `segment polarity' defects observed in the srt
mutant result from Wg-signaling defects.
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Sprinter is predicted to encode an evolutionarily conserved multi-transmembrane-spanning protein of unknown function
The srt/CG6210 genomic locus is composed of three exons with two
possible splice variants to encode novel proteins of 594 (isoform A) and 562
(isoform B) amino acids that include or exclude exon 2. Both splice variants
are expressed in Drosophila, as the EK288129 and CK00022 ESTs exclude
the second intron, whereas the srt GH01813 cDNA used in our rescue
experiment includes it. Indeed, we have found that both splice variants are
expressed in S2R+ cells (data not shown). Our analysis of the amino
acid sequence suggests that Srt is composed of four to eight transmembrane
domains. The signal sequence constitutes the first transmembrane domain
because it does not have a good consensus-signal peptidase-cleavage site
(Bendtsen et al., 2004). The
next four hydrophobic sequence elements all represent potential transmembrane
domains, but are either too short to traverse the membrane or are weakly
hydrophobic, reducing the likelihood that they are within the membrane
(Fig. 7E, gray bars). The next
three hydrophobic regions of the Srt protein are probably transmembrane
domains (Fig. 7E, black bars).
Based on these observations, we hypothesize that Srt has four transmembrane
domains with a large N-terminal globular extracellular/luminal domain that has
two potential N-linked glycosylation sites
(Fig. 7D), although several
other topologies are clearly possible
(Puntervoll et al., 2003).
This predicted structure places the Trp492
srt7E4 nonsense mutation within the last transmembrane
domain to yield either a truncated protein or one that is earmarked for
degradation through nonsense-mediated decay of the message or through the
breakdown of the misfolded protein. We also noticed that, although Flybase
(www.flybase.org)
has srt/CG6210 annotated as a multi-drugresistance-related protein
(MRP), our analysis of the Srt amino acid sequence indicates that the only
commonality between these proteins is that they are
multi-transmembrane-spanning proteins. Hence, the current annotation of
srt/CG6210 in Flybase as MRP is incorrect.
Sequence comparison of Srt to all protein databases reveals that its
closest known relative is found in Drosophila pseudoobscura sharing
87% identity and 91% similarity along its length. In Drosophila
melanogaster, the closest relative of Srt is encoded by CG13409, located
at cytological region 94A, and has only 22% identity and 42% similarity. Srt
shows much stronger homology to protein sequences from its evolutionarily
distant relatives, suggesting that Srt is unique in Drosophila. Fig.
8E shows the alignment of the Drosophila Srt isoform B relative to
nematode, frog and human. Overall, the Drosophila Srt isoform B
shares 43% identity and 62% similarity with human Srt (hSrt). Whereas some
regions in the N-terminus and the majority of C-terminal regions of Srt
diverge from its vertebrate relatives, there is a high level of conservation
that extends throughout the central region of Srt. The most N-terminal amino
acids, including the signal sequence/first putative transmembrane domain, are
fairly well conserved across species, even in the absence of a good consensus
peptidase cleavage site, supporting the hypothesis that this constitutes a
transmembrane domain (Bendtsen et al.,
2004).
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Srt function
Based on our results, we believe that the primary function of Srt in the Wg
pathway is to support the maturation of activate Wg ligand. In this capacity,
it is possible that Srt acts in post-translationally processing Wg, in the
targeting of Wg to the plasma membrane or in the release of active Wg from the
membrane. As porcupine mutants, as well as point mutations in the Wg
protein itself, yield similar Wg-retention phenotypes
(Manoukian et al., 1995;
Dierick and Bejsovec, 1998;
van den Heuvel et al., 1993),
and because porc is required for the post-translational processing of
Wg (Kadowaki et al., 1996;
Tanaka et al., 2002;
Zhai et al., 2004), a role for
Srt in the post-translational processing of Wg is one possible function of
Srt. In this role, we would predict that Srt might act as an enzyme that
either participates in known post-translational changes to the Wg protein,
such as glycosylation or palmitoylation, or identifies a new
post-translational alteration in Wg that is required for its maturation. In
addition to catalyzing the palmitoylation of Wg, the action of Porc is
required to target Wg to lipid rafts in the plasma membrane
(Zhai et al., 2004). This
observation suggests that membrane targeting might occur by an active process
mediated by specific protein(s). Another possible function of Srt could be as
a Wg-specific chaperone protein that promotes proper folding and shuttles Wg
through the secretory pathway to the plasma membrane once posttranslational
processing is complete. Indeed, there is precedent for the need of
protein-specific chaperones in the Wg pathway. In order for functional Arrow -
the Wg low-density lipoprotein co-receptor - to reach the plasma membrane, it
requires the activity of a specific chaperone protein, Boca
(Culi and Mann, 2003). Recent
studies suggest that at least some Wg protein is loaded into lipoprotein
particles during larval development
(Panáková et al.,
2005), which may be required for the movement of lipid-modified Wg
in the extracellular space to establish its morphogenetic gradient in the
wing. These lipoprotein particles are exogenously synthesized in the fat body
and must be loaded with their lipid-modified cargo in the cells that express
the ligand (Panáková et al.,
2005). Hence, there must be protein(s) present at the plasma
membrane that catalyzes this process. Sprinter may be localized within
membrane rafts, at the ready to load palmitoylated Wg into arriving
lipoprotein particles for dissemination to the Wg target cells. Another
potential role for Sprinter could be to act indirectly on Wg by supporting the
posttranslational maturation or subcellular targeting of the proteins that
directly regulate these processes, although a physical interaction between Wg
and Srt (also known as Wls or Evi) has been reported
(Bänziger et al., 2006).
As a Wg-specific secretory chaperone, numerous other functions of Srt could be
imagined, and we are actively trying to determine the specific role of Srt
among these possibilities.
Localization of Srt within the secretory pathway could be predictive of its
function. Srt localization in lipid rafts at the plasma membrane would suggest
involvement in generating Wg-loaded lipoprotein particles. However, ER or
Golgi localization could indicate a role in Wg maturation as it moves through
the secretory pathway. Although determination of the subcellular localization
of Srt awaits specific antibodies, we have observed that, in
srt7E4-mutant tissues, there is shift in the cellular
distribution of Wg toward the basolateral surface of wing-disc cells - the
surface of Wg extracellular gradient formation in target cells
(Baeg et al., 2004;
Greco et al., 2001;
Strigini and Cohen, 2000)
without disruption of Golgi localization. This would suggest that the
srt block to Wg maturation occurs within the ER or a post-Golgi
compartment of the secretory pathway (E.M.S., unpublished).
srt specificity to the Wg pathway
We have found that the activity of the protein encoded by srt
disrupts the Wg pathway, but has no effect on Hh signaling. During wing-disc
development, Hh is expressed in the posterior compartment and moves anteriorly
to activate its anterior-compartment targets that promote cell growth and
patterning of the wing (Méthot and
Basler, 1999;
Sánchez-Herrero et al.,
1996; Slusarski et al.,
1995). In our analysis of the developing wing, there is no
indication that srt plays a role in Hh signaling. Large clones
encompassing the adult wing blade show no alteration in the separation of wing
veins 3 and 4 observed in Hh-signaling mutants. Furthermore, homozygous mutant
wing discs from a probable srt-null allele show normal wing-disc size
and morphology. In the wing disc, there was no detectable accumulation of Hh
in the srt7E4 mutant cells that express it, as was
observed in Wg-producing cells. Additionally, we found that Hh is visualized
in its anterior-compartment receiving cells within punctate vesicles typical
of Hh in wild-type tissues (Tabata and
Kornberg, 1994). Regardless of whether the protein was derived
from or received by srt7E4 mutant cells, the distribution
of Hh in the wing disc appeared similar to wild-type cells. Furthermore, we
also looked at the anterior-compartment expression of Ptc, a sensitive readout
for Hh target cells in the wing (Capdevila
et al., 1994; Strigini and
Cohen, 1997). Again, no matter where the
srt7E4 homozygous mutant tissue was located with respect
the anteriorposterior axis, Ptc expression looked wild type. Therefore, at
both the level of ligand expression and activation of downstream targets, we
found no indication that srt functions in Hh signal transduction
during wing-disc development. Although our data do not directly address the
possible role of srt in Hh signaling during embryonic development,
based on these observations in wing discs it appears unlikely that
srt affects Hh signaling during embryonic development. Hence, we
hypothesize that the phenotypes observed in the developing embryo are
specifically due to disruption of the Wg pathway.
Establishing the specificity of srt for Wg is important because
lipid-modified ligands, such as Wg and Hh, have been shown to be present in
the form of lipoprotein particles
(Panáková et al.,
2005). As one possible function of Srt could be the targeting or
loading of Wg into lipoprotein particles for dissemination to receiving cells,
Srt might serve a similar function for the dissemination of lipidmodified Hh
to receiving cells. The fact that our results support the conclusion that
srt is specific to Wg maturation would argue that this is not the
case. These observations suggest that either Srt does not play a role in this
process or that Wg and Hh use different protein assemblies to support this
function. Analogous to the relationship between srt and Wg, Hh
accumulates in dispatched (disp) mutant Hh-expressing cells
(Burke et al., 1999). Perhaps
Srt and Disp serve similar roles in the maturation of active Wg and Hh ligand,
respectively. Lack of an effect on Hh secretion shows that Srt is not a
general secretion factor for this class of ligands. It was also important to
demonstrate specificity for the Wg pathway, as srt7E4 was
isolated in an embryonic screen (Chou and
Perrimon, 1996) and selected based upon its `lawn of denticle'
phenotype, which could either arise as a result of the loss of Wg or Hh
signaling.
Although we have demonstrated that srt is not required for Hh
signaling, it is possible that srt might be required in other signal
transduction pathways. Examination of adult clones revealed no indication that
srt disrupts other signaling pathways. However, it is likely that
srt is required for the maturation of other Drosophila Wnts
- a conclusion supported by the finding that multiple human Wnts depend on Srt
function (Bartscherer et al.,
2006; Bänziger et al.,
2006).
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/24/4901/DC1
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ACKNOWLEDGMENTS |
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