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First published online 1 November 2006
doi: 10.1242/dev.02675
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1 Pharmacology Department, New York University School of Medicine, New York, NY
10016-6402, USA.
2 Children's Cancer Research Laboratory, Pediatrics-Hematology/Oncology, New
York Medical College, Valhalla, NY 10595-1690, USA.
* Author for correspondence (e-mail: erika.bach{at}med.nyu.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: STAT, JAK, Unpaired, Drosophila, wingless, Eye imaginal disc, In vivo reporter, Gene expression, Signal transduction
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Two opposing
layers, the peripodial membrane and the disc proper, enclose the disc lumen
and give the disc a sac-like appearance. Discs grow and differentiate during
the three larval instars and evert during pupal development to become
functional in the adult.
The eye imaginal disc is part of the compound eye-antennal disc and
differentiates into the adult retina and head capsule
(Haynie and Bryant, 1986).
Early in development, there is no regional identity in the eye-antennal
primordium (Dominguez and Casares,
2005). The eye field emerges during the second larval instar by
the action of the selector genes eyeless (ey) and twin
of eyeless (Czerny et al.,
1999; Quiring et al.,
1994). These factors are required for the subsequent expression of
the nuclear factor Eyes absent (Eya) in the posterior region of the
eye-antennal disc, which is the presumptive eye field
(Bonini et al., 1993). Eya is
the first of a group of nuclear factors called the `early retinal genes',
which also includes Sine oculis and Dachshund (Dac), that function together in
a network to lock in eye fate (Pichaud et
al., 2001). Decapentaplegic (Dpp), a secreted factor and BMP
family member, is required for Eya expression and is expressed in a similar
pattern to Eya (Kenyon et al.,
2003). Wingless (Wg), a secreted Wnt factor induced by the GATA
transcription factor Pannier (Pnr), acts antagonistically to Dpp and is
expressed in the anterior domain of the eye in a pattern complementary to Dpp
(Cho et al., 2000;
Maurel-Zaffran and Treisman,
2000). By negatively regulating the expression of early retinal
genes, Wg controls the specification of retinal territory
(Baonza and Freeman, 2002). In
early eye development, when most of the cells in the eye disc receive both the
Wg and the Dpp signal, retinal determination cannot take place
(Cavodeassi et al., 1999;
Pichaud and Casares, 2000;
Royet and Finkelstein,
1996).
During second instar, the eye disc becomes divided into dorsal and ventral
compartments, which promotes growth of the entire disc by localized activation
of Notch at the dorsoventral midline. Notch induces expression of Unpaired
(Upd), a secreted mitogenic factor that activates the JAK/STAT pathway and
promotes growth of the eye disc (Bach et
al., 2003; Chao et al.,
2004; Reynolds-Kenneally and
Mlodzik, 2005; Tsai and Sun,
2004). This growth separates wg and dpp domains,
enabling anterior `head' and posterior `eye' regions to form in response to Wg
and Dpp signaling, respectively (Kenyon et
al., 2003). Following partition, wg is localized to the
dorsal and ventral anterior margins of the eye disc and is maintained by Pnr
and the homeodomain transcription factor Homothorax (Hth), respectively
(Maurel-Zaffran and Treisman,
2000; Pichaud and Casares,
2000).
Retinal differentiation begins in third instar at the posterior midline of
the eye disc, where the morphogenetic furrow forms and moves in an anterior
direction across the eye disc (Ready et
al., 1976). The antagonistic relationship between Dpp and Wg
restricts the onset of differentiation to a narrow region at the posterior
margin in the eye disc (Chanut and
Heberlein, 1997; Ma and Moses,
1995; Pignoni and Zipursky,
1997; Treisman and Rubin,
1995). Progression of the furrow requires Hedgehog, which is
expressed in differentiated photoreceptors posterior to the furrow
(Heberlein et al., 1993;
Ma et al., 1993).
We have examined the contribution of the JAK/STAT pathway to regional
specification. In mammals, this pathway is activated by cytokines and growth
factors that bind to specific cell-surface receptors and lead to the
activation of JAK tyrosine kinases that phosphorylate and activate latent
cytosolic STAT transcription factors (reviewed by
Levy and Darnell, Jr, 2002).
Activated STATs dimerize and accumulate in the nucleus where they alter
transcription of target genes. Studies have elucidated evolutionarily
conserved roles for JAK/STAT signaling, including hematopoiesis, immunity and
proliferation (Arbouzova and Zeidler,
2006; Levy and Darnell, Jr,
2002).
Drosophila serves as an excellent model system to study this
pathway, as flies are highly amenable to genetic manipulation and possess only
one JAK and one STAT gene, compared with the four JAKs and the seven STATs in
mammals (Arbouzova and Zeidler,
2006; Levy and Darnell, Jr,
2002). Genetic studies in Drosophila have uncovered
several components of this pathway, including Upd and two other Upd-like
ligands called Upd2 and Upd3 (Agaisse et
al., 2003; Harrison et al.,
1995; Hombria et al.,
2005); the transmembrane receptor Domeless (Dome), which is
distantly related to the gp130 family of cytokine receptors
(Brown et al., 2001;
Chen et al., 2002); the JAK
Hopscotch (Hop) (Binari and Perrimon,
1994); and the STAT protein Stat92E
(Hou et al., 1996;
Yan et al., 1996). The
JAK/STAT pathway is crucial for diverse processes in Drosophila,
including proliferation and planar polarity in the developing eye
(Bach et al., 2003;
Luo et al., 1999;
Tsai and Sun, 2004;
Zeidler et al., 1999).
In this paper we report that the JAK/STAT pathway regulates regional specification of the eye disc by promoting the formation of the eye field via repression of the wg gene. Specifically, we demonstrate that JAK/STAT signaling negatively regulates wg expression in the eye disc epithelium and in the peripodial membrane and promotes the formation of eye tissue. This is the first demonstration that the repression of wg by the JAK/STAT pathway is an important mechanism in regional specification of the eye imaginal disc. Furthermore, this is the first report of a functional interaction between the JAK/STAT and Wnt signaling pathways, and suggests the possibility that a similar interaction exists in higher organisms.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and wg2.11Z
(Pereira et al., 2006). All
crosses were maintained at 25°C.
We used PCR to generate a full-length stat92E cDNA with
BamHI (5') and NotI (3') ends using the
following primers (restriction sites underlined):
5'-CGCGGATCCATGAGCTTGTGGAAGCGCATCGCCAGCC-3' and
5'-TTTCCTTTGCGGCCGCAAAGTTCTCAAAGTTGTAATCGTATCG-3'.
After digestion with BamHI and NotI, the insert was ligated
into the pBluescript KS-based vector C5HA3. The 5' end of the C5HA3
polylinker includes an ATG immediately upstream of sequence encoding three HA
epitopes, followed by an in-frame BamHI site and the costal2
cDNA (a kind gift of Kent Nybakken, Harvard Medical School, Boston, MA, USA).
At the 3' end, a NotI site immediately precedes and is in frame
with a stop codon. The C5HA3 plasmid was digested with BamHI and
NotI to remove the costal2 sequence. The
3HA-stat92E insert was excised from C5HA3 by digestion with
BssHII. The 3' recessed termini were filled in with Klenow.
This fragment was ligated into UASp
(Rorth, 1996) that had been
cut with BamHI and filled in with Klenow. UASp-3HA-stat92E
transgenic flies were generated as described
(Bach et al., 2003).
Marked clones of mutant cells
Clones were generated using the FLP/FRT system
(Xu and Rubin, 1993).
ey-flp or ey-GAL4, UAS-flp (EGUF) transgenic
animals express FLP in the eye disc
(Newsome et al., 2000). Clones
were marked using the following chromosomes:
P{neoFRT}82B
P{Ubi-GFP(S65T)nls}3R/TM6B,
Tb1 or P{neoFRT}82B
P{arm-lacZ.V}83B/TM6C, Sb1 Tb1. Minute clones were
marked by P{neoFRT}82B M(3)96C, arm-lacZ or
P{neoFRT}82B Ubi-GFP. P{neoFRT}82B
ry506 was used as the control. hop-expressing or
flip-out clones were generated using UAS-hop and P{AyGAL4}25
P{UAS-GFP.S65T}T2; hs-flp MKRS/TM6B, in which FLP is under the control of
the heat-shock promoter (Ito et al.,
1997). Flip-out clones express both Hop and GFP.
In situ hybridization, antibody staining and microscopy
Sense and antisense upd riboprobes were generated as previously
described (Bach et al.,
2003).
Antibody stainings were performed as previously described
(Bach et al., 2003). We used
the following primary antibodies: rat anti-Elav (1:50), mouse anti-Wg (1:20),
mouse anti-ß-galactosidase (1:50), mouse anti-Discs large (Dlg) (1:50),
mouse anti-Dac (1:100) (all from Developmental Studies Hybridoma Bank), rabbit
anti-Hth (1:300) (a kind gift of H. Sun)
(Pai et al., 1998), rabbit
anti-ß-galactosidase (1:100) (Cappel) and rat anti-HA (1:100) (Roche). We
used fluorescent secondary antibodies (Jackson Laboratories) at 1:250. We
collected fluorescent images (at 25 x magnification) using a Zeiss LSM
510 confocal microscope, scanning electron micrographs (at 100x) using a
Leo SEM (Zeiss) (Harvard School of Public Health), and bright field pictures
of adults (at 2.5x or 5x) and of discs (at 20x) using a
Zeiss Axioplan microscope with a Nikon Digital Sight DL-UL camera.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). An upd-GAL4, UAS-GFP
(upd>GFP) reporter revealed that upd was expressed in
cells residing next to the optic stalk in first and second instar eye discs
(Fig. 1A,B). However,
upd mRNA was not detected after early third instar, suggesting a
function in the early eye disc (Fig.
1C,D). Upd is likely to be the only Upd-like molecule involved in
eye development. A upd2-null mutant is viable and fertile and has no
eye defects, whereas upd3 is not expressed in the eye disc (data not
shown).
Upd is a secreted molecule that acts cell non-autonomously
(Bach et al., 2003;
Tsai and Sun, 2004). To assess
the range of Upd activity, we generated a reporter called
10XSTAT92E-GFP that specifically reflects JAK/STAT pathway activity,
as evidenced by the loss of reporter expression in stat92E clones
(Bach et al., 2006). Although
Upd is synthesized only by cells at the posterior midline, it has long-range
effects. The 10XSTAT92E-GFP reporter was activated uniformly
throughout the posterior domain of the eye to the lateral margins in first and
second instar eye discs (Fig.
1E-H). Using this reporter, we demonstrated that the JAK/STAT
pathway is not active in the eye disc after early third instar
(Bach et al., 2006). These data
indicate that JAK/STAT signaling is active only during the early stages of
larval eye disc development. We next assessed the spatial and temporal
relationship between JAK/STAT signaling and other pathways, such as Dpp and
Wg, that are also active in early eye development. We used a dpp-LacZ
reporter and a wg-lacZ enhancer trap (wgP) that
faithfully recapitulate expression of the endogenous genes
(Blackman et al., 1991;
Kassis et al., 1992). In early
larval eye development, the 10XSTAT92E-GFP reporter partially
overlapped with dpp but abutted the wg expression domain,
suggesting functional interactions between these pathways
(Fig. 1E-H).
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). These
data indicate that the early, restricted expression of Upd to the posterior
midline is essential for proper formation of the eye field.
Stat92E is required for eye development
The consequences of loss of Stat92E during eye development have not been
reported. To address this issue, we generated mutant stat92E clones
in the eye using the stat92E85C9 allele, which results
from an R to P substitution at position 442 in the DNA-binding domain
(Silver and Montell, 2001).
The Stat92E85C9 protein is nonfunctional, as evidenced by the lack
of activation of the 10XSTAT92E-GFP reporter in
stat92E85C9 clones (data not shown)
(Bach et al., 2006). We induced
stat92E85C9 mutant clones in the eye disc using
ey-flp, in which the yeast FLP recombinase is expressed under the
control of the eye-specific enhancer of ey
(Newsome et al., 2000). To
generate large patches of stat92E mutant tissue we also used a
Minute mutation, which causes slow growth and recessive lethality in
cells possessing the wild-type chromosome
(Morata and Ripoll, 1975).
When used with ey-flp and a mutation on an FRT chromosome, the
Minute technique produces an eye composed almost entirely of
homozygous mutant tissue. Adults carrying a control chromosome in a
Minute background (hereafter referred to as + M+)
did not exhibit pupal lethality or eye or head cuticle defects
(Fig. 2A,I). By contrast, less
than 10% of animals carrying stat92E85C9 clones in a
Minute background (hereafter referred to as stat92E
M+) eclosed from their pupal cases
(Fig. 4G). The few adults that
eclosed exhibited a small or ablated eye and abnormal head cuticle
(Fig. 2B,C). Instead of the
smooth head cuticle seen in control animals, stat92E85C9
M+ adults displayed a ridged cuticle that had a disorganized
bristle pattern (Fig. 2A-C).
Non-eclosed stat92E85C9 M+ pupae were
frequently headless or exhibited severe eye defects and expansion of the head
cuticle (Fig. 2J,K). Similar
phenotypes and significant hatching defects were observed with another
stat92E hypomorphic allele, stat92E397, whereas
less severe phenotypes were observed with stat92E06346
(Fig. 2F,G). We also observed
defects in stat92E clones in a non-Minute background.
Frequently, stat92E clones within the eye field developed into
ectopic head cuticle or formed ambiguous outgrowths
(Fig. 2H). The stat92E
mutant eye and head cuticle phenotypes were due specifically to the loss of
Stat92E function. Expression of a wild-type stat92E transgene
(UAS-3HA stat92E) fully rescued all stat92E mutant
phenotypes (Fig. 2D). However,
misexpression of this transgene (ey>3HA-stat92E) in a
wild-type background did not have a phenotype
(Fig. 2E). Taken together,
these data suggest that Stat92E, activated by a Upd/Dome/Hop signal, promotes
the formation of the eye field. In the absence of Stat92E, cells which are
normally part of the eye field form other tissues.
Loss of JAK/STAT signaling results in abnormal eye disc morphology and ectopic wg expression
To determine if the stat92E phenotypes observed in the adult arise
during larval eye development, we examined the effect of loss of
stat92E activity in the eye imaginal disc. Indeed,
stat92E85C9 M+ eye discs display morphological
defects consistent with adult stat92E mutant phenotypes. The dorsal
domain in stat92E85C9 M+ eye discs was
morphologically abnormal and was marked by protrusions, an elongated
appearance and an ectopic eye field (Fig.
3B,C,E and data not shown). In 5% of stat92E85C9
M+ discs, the morphogenetic furrow failed to initiate (see
Fig. S1 in the supplementary material). In about 85% of these discs, the
furrow moved only through the ventral portion of the eye disc
(Fig. 3B,C). Ommatidia in
stat92E85C9 M+ adult eyes are almost entirely
of ventral origin. In the eye disc, dorsal cells express the homeodomain
protein Mirror (Mirr) (McNeill et al.,
1997). The lack of dorsal ommatidia in stat92E mutants is
not due to altered expression of mirr, but due rather to an inability
of the furrow to move through the dorsal, mirr-positive cells in the
stat92E85C9 M+ eye disc (see Fig. S1 in the
supplementary material).
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;
Ma et al., 1993). However, in
stat92E M+ eye discs, dpp was lost from the
dorsal region of the disc, whereas its ventral expression appeared normal
(Fig. 3A-C). wg is
expressed in dorsal and ventral margin cells, located between the peripodial
membrane and the disc proper, and in cells adjacent to them in wild-type eye
discs (Fig. 3D)
(Cho et al., 2000). By
contrast, wg was expanded throughout the disc epithelium in
stat92E M+ eye discs, as well as in eye discs hemizygous
for the amorphic mutation hop M13
(Fig. 3E,G and data not shown).
wg was also ectopically expressed in mosaic stat92E clones
(Fig. 3F). The increased
expression domain of the wg gene in discs lacking JAK/STAT signaling
led to a dramatic increase in Wg protein
(Fig. 3C). In stat92E
M+ eye discs expressing a stat92E transgene, ectopic
Wg protein was not observed (data not shown). The presence of a ventral furrow
in stat92E M+ eye discs is consistent with high dorsal
levels of Wg, which is observed with ectopic clonal expression of wg
in the eye disc (data not shown) (Treisman
and Rubin, 1995).
In wild-type adults, wg is expressed around the periphery of the
eye and in the lateral head cuticle (Fig.
3H) (Heslip et al.,
1997). However, ectopic wg was apparent in
stat92E clones within the adult eye field, often at the base of
ectopic outgrowths (Fig. 3I).
Similar outgrowths are observed in shaggy clones, which behave as if
they have transduced the Wg signal (Heslip
et al., 1997). The hypothesis that JAK/STAT signaling functions
upstream of Wg is further supported by the observation that the
stat92E ablated eye phenotype was identical to that observed in
ey>wg adults, in which wg was expressed throughout the
developing eye (compare Fig. 2C with
2L). Although these results indicate that JAK/STAT signaling is an
important regulator of wg expression, wg was not ectopically
expressed in all cells lacking stat92E
(Fig. 3E-G,I). This may reflect
the time of induction of stat92E clones, which are constantly
generated in an ey-flp background. Alternatively, another factor may
be acting redundantly with Stat92E to regulate wg.
Ectopic JAK/STAT signaling autonomously represses wg expression
stat92E mutants exhibit phenotypes characteristic of expanded
wg expression. However, the antagonistic relationship between
dpp and wg raises the possibility that Stat92E regulates
dpp, and wg expansion occurs as a secondary effect. We
therefore looked at the ability of ectopic JAK/STAT signaling to either induce
dpp or repress wg. Ectopic expression of hop using
the flip-out technique resulted in ligand-independent, autonomous activation
of Stat92E. Ectopic hop clones did not induce dpp in the eye
disc (Fig. 4A). However, they
did autonomously repress wg in both the dorsal and ventral eye
(Fig. 4B). Therefore, JAK/STAT
signaling regulates wg, and dpp is altered as a consequence.
This is further supported by the ability of upd expressed throughout
the eye disc (ey>upd) to repress wg. In
ey>upd discs, dorsal wg was completely eliminated and
ventral wg was substantially repressed
(Fig. 4C). Furthermore,
ey>upd discs exhibited precocious furrow initiation from the
lateral margins, a phenotype also observed in wg mutants
(Fig. 4C)
(Treisman and Rubin, 1995).
This result is consistent with the reduction in the inter-eye distance
displayed by ey>upd adults
(Fig. 1J). To determine if
JAK/STAT regulation of wg is specific to the eye disc, we examined
hop flip-out clones in other imaginal discs. Indeed, hop
expressing clones also repressed wg in the notal region of the wing
disc, indicating that the JAK/STAT pathway can repress wg in other
tissues (Fig. 4D).
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Stat92E negatively regulates a 263 bp region of the wg gene
Although the majority of functions attributable to STATs involve
transcriptional activation, at least one STAT protein, Dd-STATa, acts as a
functional repressor (Mohanty et al.,
1999). We therefore assessed the ability of Stat92E to directly
repress wg. A reporter called wg2.11Z, in which
ß-galactosidase is driven by a 263 base pair enhancer from the 3'
cis wg genomic region, was sufficient to recapitulate wg
expression in the dorsal margin of the disc proper and in the dorsal
peripodial membrane (Fig. 5A,B)
(Pereira et al., 2006). This
reporter was ectopically expressed in mosaic stat92E clones as well
as in stat92E M+ clones in a manner similar to that
observed for wgP (Fig.
5C,D). Moreover, wg2.11Z was repressed autonomously in
hop-expressing clones (Fig.
5E). These data indicate that Stat92E can regulate dorsal
wg expression through the wg2.11Z enhancer. wg2.11Z
does not contain any Stat92E binding sites (TTC(N)3GAA), strongly
suggesting that Stat92E does not repress dorsal wg directly, but
rather regulates another factor which represses wg
(Hou et al., 1996;
Yan et al., 1996).
Stat92E does not act through known wg regulators to repress wg in the disc proper
Expression of the wg2.11Z enhancer is maintained by pnr
in dorsal peripodial cells (Pereira et
al., 2006). We therefore assessed whether pnr expression
is altered in the absence of JAK/STAT signaling using a pnr>gfp
reporter, which mimics expression of the endogenous pnr gene
(Singh and Choi, 2003). In
wild-type eye discs, pnr was expressed in dorsal anterior peripodial
cells and in dorsal margin cells in the disc proper
(Fig. 6E). In
hopM13 eye discs, pnr expanded throughout the
dorsal posterior peripodial membrane, whereas it remained unchanged in the
disc proper (Fig. 6F). These
data suggest that Stat92E negatively regulates expression of pnr in
peripodial cells. To test this hypothesis, we assessed pnr>gfp
expression when JAK/STAT signaling was ectopically activated. We used the
GMR-upd transgenic line, in which ectopic Upd activates the
10XSTAT92E-GFP reporter in anterior cells adjacent to the furrow
(Bach et al., 2006;
Bach et al., 2003). In
mid-third-instar eye discs, pnr was expressed in peripodial cells
that reside `above' dorsal cells both anterior and posterior to the furrow
(Fig. 6G). By contrast, in
GMR-upd discs, the pnr expression domain was significantly
repressed and now resided only `above' dorsal cells anterior to the furrow
(Fig. 6H). These data suggest
that when stat92E function is lacking in peripodial cells,
pnr is ectopically expressed and may induce wg expression in
these cells via the wg2.11Z enhancer.
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). Dac
and Wg mutually repress each other's expression
(Baonza and Freeman, 2002;
Treisman and Rubin, 1995). Dac
expression was reduced in stat92E M+ discs; however, its
loss did not coincide with the boundary of stat92E clones but rather
with regions of the eye disc that contained high levels of ectopic wg
(Fig. 6A,B). This suggests that
the loss of Dac expression is an indirect consequence of ectopic wg,
as has been reported previously (Baonza and
Freeman, 2002). The observation that Dac was not induced by clones
expressing ectopic hop, strongly supports the conclusion that Dac
expression is lost as a result of the ectopic Wg expressed in stat92E
M+ discs (data not shown). Wg positively regulates expression
of the hth gene. In addition, ectopic Hth can increase expression of
existing wg in both the dorsal and ventral margins of the eye disc
(Pichaud and Casares, 2000).
Hth was expressed at the anterior edge of the third instar eye disc
(Fig. 6C). By contrast, in
stat92E M+ discs, Hth expanded into the posterior dorsal
and ventral domains (Fig. 6D).
Clones expressing ectopic hop cannot induce Hth expression,
suggesting that the effects on hth are an indirect consequence of the
ectopic Wg observed when JAK/STAT signaling is impaired (data not shown).
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|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our results conflict with those of a previous study, which reported that
JAK/STAT signaling does not repress wg in the eye disc. This
conclusion was reached on the basis of wild-type Wg protein expression in eye
discs that contained ectopic upd-expressing clones
(Zeidler et al., 1999).
However, we found that in the absence of stat92E, wg was ectopically
expressed in both dorsal and ventral halves of the eye disc. It is likely that
our examination of the wg gene using the wgP
enhancer trap is a more sensitive measure of wg expression than
monitoring Wg protein. Zeidler and colleagues also reported that the JAK/STAT
pathway negatively regulates mirr expression. This conclusion was
drawn after finding a preponderance of dorsal, mirr-positive
ommatidia in adult eyes containing unmarked upd loss-of-function
clones (Zeidler et al., 1999).
However, using marked clones, we showed that Mirr is expressed normally in eye
tissue that is largely homozygous mutant for stat92E. Moreover, we
demonstrated that stat92E M+ adult eyes are largely
composed of Mirr-negative ommatidia, which indicates their ventral origin.
Thus, our data indicate that mirr is not regulated by JAK/STAT
pathway activity.
Stat92E repression of the wg gene
Previous work has shown that the 3' cis region of the
wg gene regulates its expression in imaginal discs. Several
wg mutations that specifically affect imaginal disc development, as
well as discspecific enhancers, map to this region
(Baker, 1988;
Couso et al., 1993;
Neumann and Cohen, 1996;
Pereira et al., 2006;
van den Heuvel et al., 1993).
In this study, we showed that Stat92E negatively regulates dorsal wg
through a small enhancer (wg2.11Z) in the 3' cis
genomic region of the wg gene. This enhancer is ectopically expressed
in stat92E and hop mutants and is autonomously repressed by
ectopic activation of Stat92E. The DNA binding preferences of Stat92E and
other STAT proteins have been well characterized
(Hou et al., 1996;
Seidel et al., 1995;
Yan et al., 1996). Because
there are no Stat92E binding sites in the wg2.11Z enhancer, we favor
the interpretation that Stat92E does not directly repress dorsal wg
but rather acts through another factor. This repressor may be encoded by a
direct Stat92E target gene, because wg is autonomously repressed by
the JAK/STAT pathway. However, we cannot rule out the possibility that Stat92E
regulates wg through other transcription factors, such as Dorsal or
vHNF-4, which have putative sites in wg2.11Z
(Pereira et al., 2006). It is
also possible that there are cryptic Stat92E binding sites in this wg
enhancer, through which Stat92E may directly repress wg. Additional
experiments will be needed to test these possibilities.
We also demonstrated that Stat92E represses ventral wg in the eye
disc epithelium. This is presumably independent of the wg2.11Z
enhancer, which recapitulates wg expression in the dorsal but not the
ventral eye disc (Pereira et al.,
2006). Moreover, we showed that Stat92E negatively regulates
pnr in peripodial cells. In the absence of JAK/STAT signaling,
pnr is dramatically expanded into the posterior peripodial membrane.
However, we stress that because pnr is an intracellular protein, the
ectopic pnr in the peripodial membrane cannot account for the ectopic
wg observed in the disc proper of stat92E mutants.
Currently, we do not know whether Stat92E regulates wg in the ventral
eye disc epithelium and the peripodial membrane in the same manner as in the
dorsal eye. All three wg expression domains may be regulated by the
same as yet unidentified factor. Alternatively, Stat92E may regulate
wg expression domains through different mechanisms. For example,
dorsal wg may be regulated indirectly, whereas ventral and peripodial
wg may be regulated directly by Stat92E. The wg gene
3' cis genomic region contains one putative Stat92E binding
site, which resides downstream of the wg2.11Z enhancer. Therefore, it
is possible that Stat92E regulates ventral and peripodial wg through
this site. Future work will be needed to address these issues.
Do mammalian STATs repress Wnt genes?
Our study raises the possibility that the JAK/STAT pathway negatively
regulates expression of vertebrate Wnts. Interestingly, both pathways are
active in the developing vertebrate eye. One recent study indicates that
activation of canonical Wnt signaling in the developing chick eye inhibits
retinal progenitor gene expression and the differentiation of retinal neurons
(Cho and Cepko, 2006). This
led to the proposal that canonical Wnt signaling is required for peripheral
eye development, which is similar to the role of wg in peripheral eye
tissue in Drosophila. In contrast to Wnt signaling, the roles of the
JAK/STAT pathway in the development of the vertebrate eye are poorly
understood. However, numerous cytokines that activate this pathway, such as
ciliary neurotrophic factor, are expressed in the developing retina and induce
proliferation of lens cells and the expression of glial intermediate filament
protein (Potts et al., 1998;
Wang et al., 2002). It is
therefore possible that, in mammals, activation of the JAK/STAT pathway in the
developing retina inhibits Wnt expression, thus promoting retinal progenitor
gene expression and the differentiation of retinal neurons.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/23/4721/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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