DEVELOPMENTAL DYNAMICS 236:2721–2730, 2007
RESEARCH ARTICLE
The JAK/STAT Pathway Regulates ProximoDistal Patterning in Drosophila
Aidee Ayala-Camargo,1 Laura A. Ekas,1 Maria Sol Flaherty,1 Gyeong-Hun Baeg,2 and
Erika A. Bach1*
JAK/STAT signaling is thought to control growth and proliferation. However, here we show a novel role for this
pathway in the patterning of Drosophila appendages. Loss of Stat92E function results mainly in ventralizations
and multiplications of the proximo-distal axis in leg and antenna, primarily through the ectopic misexpression
of wingless. We also show that the pathway ligand Unpaired is expressed in two domains in leg and antenna that
abuts those of wingless and decapentaplegic. We report that JAK/STAT signaling represses both wingless and
decapentaplegic, restricting them to their respective domains in leg and antenna. In a reciprocal manner, we
show that wingless and decapentaplegic restrict unpaired to its two domains. Thus, a main function of the
JAK/STAT pathway in leg and antennal development is to promote the formation of a single proximo-distal axis
per disc by constraining the intersection of wingless and decapentaplegic to the center of the disc.
Developmental Dynamics 236:2721–2730, 2007. © 2007 Wiley-Liss, Inc.
Key words: proximo-distal axis; Unpaired; JAK/STAT; leg; antenna; appendage; Drosophila; Wingless; Decapentaplegic
Accepted 14 May 2007
INTRODUCTION
In mammals and Drosophila, the Janus
Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) pathway is a critical regulator of proliferation and has been shown to promote the
formation of tumors (Levy and Darnell,
2002; Arbouzova and Zeidler, 2006). For
instance, an oncogenic allele of jak2
causes hematopoietic cancers and a
dominant active stat3 allele causes tumors in nude mice (Bromberg et al.,
1999; Bowman et al., 2000; Darnell,
2005). Similarly, hyper-activation of the
Drosophila JAK/STAT pathway leads
to dramatic tissue overgrowths that resemble human tumors (Harrison et al.,
1995; Kiger et al., 2001; Tulina and Matunis, 2001; Bach et al., 2003). However, little is known about the role of
this pathway in pattern formation. The
presence of only one JAK and one STAT
in Drosophila, compared to four JAKs
and seven STATs in mammals, make
the fruit fly an ideal system to study the
role of this pathway during development. Three Unpaired ligands (Upd,
Upd2, and Upd3) are thought to bind to
the receptor Domeless (Dome), resulting in its activation and subsequent
trans-phosphorylation and activation of
the associated JAK kinase Hopscotch
(Hop) (reviewed in Arbouzova and Zeidler, 2006). The activated Hop proteins
phosphorylate and activate the transcription factor Stat92E, which then
migrates to the nucleus and acts as a
transcription factor.
Development of the proximo-distal
(P-D) axis is essential to the formation
of mammalian limbs and arthropod
appendages. Significant insights into
P-D axis formation have been made in
Drosophila imaginal discs, groups of
larval epithelia that give rise to adult
structures (Cohen, 1993). The Drosophila leg and antenna are homologous structures based on homeotic
mutations, and both have been used
successfully to study patterning of the
P-D axis (Postlethwait and Schneider-
The Supplementary Material referred to in this article can be viewed at www.interscience.wiley.com/jpages/1058-8388/suppmat
1
Pharmacology Department, New York University School of Medicine, New York, New York
2
Children’s Cancer Research Laboratory, Pediatrics-Hematology/Oncology, New York Medical College, Valhalla, New York
Grant Sponsor: Breast Cancer Alliance, Inc.; Grant Sponsor: Pharmacological Sciences Training Grant (N.I.H.); Grant number: GM06670401.
*Correspondence to: Erika A. Bach, Pharmacology Department, New York University School of Medicine, 550 First Avenue,
MSB 497B, New York, NY 10016-6402. E-mail: [email protected]
DOI 10.1002/dvdy.21230
Published online 11 July 2007 in Wiley InterScience (www.interscience.wiley.com).
© 2007 Wiley-Liss, Inc.
2722 AYALA-CAMARGO ET AL.
man, 1971; Lecuit and Cohen, 1997).
The leg consists of five segments: the
proximal coxa, followed by the trochanter, femur, tibia, and tarsus, and
the distal-most structure, the claw.
The adult antenna consists of six segments: from the proximal a1 to the
distal a6, also called the arista.
Fig. 1. The JAK/STAT pathway is active in young leg and antennal
discs. Unless noted otherwise, discs in all figures are oriented with
anterior to the left and dorsal up. A–D: upd-GAL4, UAS-GFP (upd⬎GFP)
expression in second (A,C) and third instar (B,D) leg and antenna discs.
The expression of upd⬎GFP (green) abuts the Wg domain (red) in both
leg (A,B) and antennal (C,D) discs. E–H: Expression of 10XSTAT92EGFP (abbreviated “10XSTAT-GFP”) (green) and Wg protein (red) in second (E), early third (G), and mid-third (F,H) instar leg and antennal discs,
respectively. The 10XSTAT92E-GFP reporter is expressed throughout
the leg, displaying lower levels on the ventral side where Wg is expressed (E,F). This reporter is also expressed in the distal antenna and
exhibits lower levels in the dorsal, Wg-expression domain (G,H). Scale
bar ⫽ relative size.
Like the vertebrate limb, the Drosophila leg and antenna arise from the
differentiation of three different axes:
antero-posterior (A-P), dorso-ventral
Fig. 2.
JAK/STAT PATHWAY IN DROSOPHILA 2723
(D-V), and P-D. The secreted factor
Hedgehog (Hh) is produced by cells in
the posterior compartment of the leg
and induces the expression of two secreted factors, the Bone Morphogenetic
Protein (BMP) Decapentaplegic (Dpp)
and the Wnt protein Wingless (Wg), in
anterior cells adjacent to the compartment boundary (Basler and Struhl,
1994; Tabata and Kornberg, 1994). The
mutually antagonistic relationship between Dpp and Wg establishes the D-V
axis by creating spatially restricted domains, in which each gene maintains its
own expression pattern (Brook and Cohen, 1996; Jiang and Struhl, 1996;
Johnston and Schubiger, 1996; Penton
and Hoffmann, 1996; Theisen et al.,
1996; Heslip et al., 1997). In addition to
their roles in patterning the D-V axis,
Dpp and Wg determine the P-D axis of
the leg and antenna. The juxtaposition
of dpp- and wg-expressing cells in the
central region of both discs activates
Distal-less (Dll), which encodes a homeodomain protein that is required for the
formation of distal leg and antennal
structures (Cohen et al., 1989; DiazBenjumea et al., 1994; Campbell and
Tomlinson, 1995). Ectopic expression of
Fig. 2. Loss of JAK/STAT signaling results in
patterning defects of the P-D axis in adult leg
and antenna. A–F: Leg phenotypes, anterior is
to the left. Legs and antennae from y hs-flp;
FRT82B CD2 y⫹ flies were labeled “control.” A
control female mesothoracic leg (A). B,C: A
control male prothoracic tibia exhibits rows of
ventral transverse bristles (B). In y stat92E
clones (labeled “stat92E”), there are ectopic
transverse rows of bristles (yellow arrowhead)
immediately adjacent to the normal ones (white
arrowhead) (C). D: y stat92E clones result in a
supernumerary limb that arose from the femur
of a female metathoracic leg and that extends
proximo-distally to a claw (arrowhead). The supernumerary limb is shown at high magnification (D, inset). E,F: There are typically ⬃10 sex
combs in a control male prothoracic tarsus (E).
In y stat92E clones, there are multiple additional
y sex combs (F). G–K: Antennal phenotypes.
Control antenna (G). y stat92E clones lead to an
antenna with two a3–a6 segments (arrowheads)
(H). I, J: Unmarked dome468 (I) or hopC111 (J)
clones result in an antenna that has triplicated
a6 segments (I, arrowheads) or duplicated a3
segments (J, arrowheads). K: y stat92E⫺ clones
in a Minute background result in an antenna
that is missing a3–a6 (red arrowhead) and that
has a leg-like structure (black arrowhead). L:
These defects are fully rescued by expression
of a stat92E transgene (Stat92E FL) (Ekas et al.,
2006) in stat92E clones using the MARCM technique (Lee and Luo, 1999) (D). Trochanter (Tr).
Bar indicates relative size (A,D).
wg on the dorsal side, or dpp on the
ventral side, of the leg disc results in
ectopic expression of Dll and a secondary P-D axis (Campbell et al., 1993;
Struhl and Basler, 1993; Diaz-Benjumea et al., 1994). Lower levels of Wg
and Dpp initiate the transcription of
dachshund (dac), which encodes a nuclear factor required for development of
the femur and tibia (Mardon et al.,
1994; Lecuit and Cohen, 1997). In addition to Dll and Dac, the TALE class
homeodomain protein Homothorax
(Hth) is essential for patterning of the
proximal structures in the leg and antenna (Casares and Mann, 2001; Dong
et al., 2001).
In this study, we address the role of
the JAK/STAT pathway in patterning
by addressing its role in P-D axis formation. Specifically, we find that the JAK/
STAT pathway can repress both wg and
dpp in an autonomous manner, although Stat92E represses wg more robustly than it does dpp. Furthermore,
we show that both wg and dpp restrict
upd to its anterior and posterior domains in leg and antennal discs.
Through these reciprocal inhibitory interactions, the expression domains of
upd, wg, and dpp become mutually exclusive. Thus, we show that JAK/STAT
signaling regulates the development of
one central P-D axis per leg or antennal
disc by constraining the expression domains of dpp and wg.
RESULTS
Upd Expression and JAK/
STAT Pathway Activity Are
Detected Throughout Leg
and Antennal Imaginal Disc
Development
We recently reported that upd is expressed in two distinct domains in leg
and antennal discs, one in the anterior
and the other in the posterior compartment (Bach et al., 2007). In both
discs, upd-expressing cells abut the
Wg and Dpp domains (Fig. 1A–D; see
also Fig. 6G). An upd2 null mutant is
viable and fertile and has no antennal
or leg defects, while upd3 is not expressed in either disc (data not
shown). These data suggest that these
factors do not act in these discs or
have redundant roles with Upd. To
assess the range of Upd activity, we
used a 10XSTAT92E-GFP reporter
that specifically reflects JAK/STAT
pathway activity in vivo (Bach et al.,
2007). This reporter is activated
throughout larval development in leg
and antennal discs (Bach et al., 2007).
Interestingly, its expression is lowest
in the Wg-expressing domains (Fig.
1E–H). These data indicate that in
both discs the JAK/STAT pathway is
activated by Upd, which acts over a
long range. Additionally, the mutual
spatial and temporal exclusion of cells
with JAK/STAT activity from those
that express Wg suggests that a negative regulatory interaction exists between these pathways.
Loss of JAK/STAT Pathway
Activity Leads to Defective
Patterning of the P-D Axis
To assess the role of stat92E in the developing leg and antenna, stat92E
clones were randomly induced and
marked by the absence of the yellow (y)
gene product in the adult. In the leg, y
stat92E clones are associated with supernumerary appendages and incomplete duplications (Fig. 2A,D, arrowhead and inset, and data not shown).
Furthermore, stat92E clones can promote the formation of ventro-lateral
structures like ectopic transverse bristles of the prothoracic legs next to the
normally positioned ventral ones (Fig.
2B,C yellow arrowhead). In addition,
other ventro-lateral structures, such as
the sex combs in the male prothoracic
leg, are increased in number when they
reside within y stat92E clones (Fig.
2E,F). The formation of supernumerary
limbs and of both ectopic transverse
bristles and sex combs has been observed when ectopic wg expression is
present on the dorsal side of the leg disc
(Campbell et al., 1993; Struhl and
Basler, 1993).
In the antenna, y stat92E clones exhibit supernumerary structures, including the duplication or triplication of segments a2-a6 (Fig. 2G,H arrowheads
and data not shown). Other phenotypes,
such as reduced or absent medial and
distal segments (a3–a6), as well as antenna-to-leg transformations, are also
observed (Fig. 2K, arrowheads). These
defects are fully rescued by co-expression of a full-length stat92E transgene
(Ekas et al., 2006), indicating that they
were specifically due to the loss of JAK/
STAT signaling (Fig. 2L). Lastly, we de-
2724 AYALA-CAMARGO ET AL.
termined that mutant clones of dome or
hop, genes that act upstream of
stat92E, result in antennal phenotypes
similar to those observed in stat92E
clones, including the formation of supernumerary segments (Fig. 2I,J, arrowheads). Taken together, these data
indicate that the JAK/STAT pathway
regulates P-D patterning in both the leg
and antenna.
Stat92E Is Required for the
Proper Expression of Major
P-D Patterning Genes
To determine if the stat92E adult phenotypes arise during larval development, we examined the effect of loss of
stat92E activity in leg and antennal
imaginal discs. First, we examined the
expression of major P-D patterning
genes. In the leg, Dll and Dac are expressed in a central and medial domain, respectively, while Hth is expressed in the proximal domain (Fig.
3A; See Supplemental Fig. 1A, which
can be viewed at www.interscience.
wiley.com/jpages/1058-8388/suppmat).
Third instar leg discs carrying stat92E
clones often contain at least one secondary P-D axis (Fig. 3B, arrowhead).
These ectopic P-D axes are only found
on the dorsal side of the leg disc but can
form in either the anterior or posterior
compartment (Fig. 3B,D, arrowheads).
Bric-a-brac (Bab), a BTB/POZ-domain
protein required for the development of
distal joints in the leg and antenna
(Godt et al., 1993; Couderc et al., 2002),
is also observed in these ectopic P-D
axes (Fig. 3C,D, arrowhead). Consistent
with a lack of defects in the coxa and
trochanter in adult legs containing
stat92E clones, we found that Hth expression is not altered in stat92E clones
in the leg disc (Supplemental Fig.
1A,B).
Similarly, additional P-D axes are
observed in antennal discs containing
stat92E clones. In the third instar antenna, Hth is expressed in a1, a2, and
faintly in a3, while Dll is expressed in
a2–a6 (Supplemental Fig. 1C) (Dong
et al., 2000). We show one disc that
contains three P-D axes, as assessed
by Dll expression (Fig. 3E, asterisks).
One of these ectopic axes occurs
within the stat92E clone, while the
bifurcation that generated the other
two axes follows the stat92E clone
boundary (Fig. 3E,E’). We presume
that comparable antennal discs would
give rise to the duplicated or triplicated antennal structures similar to
those shown in Figure 2H–J. In addition, we also observed absent or reduced Dll expression in large stat92E
clones, and a concomitant expansion
in Hth expression throughout the
proximal and distal antenna (Supplemental Fig. 1C,D). These results are
consistent with the adult phenotypes
where a3–a6 segments are most frequently lost (Fig. 2K). To assess
whether Dll and Hth can be directly
regulated by JAK/STAT signaling, we
used the flip-out technique to make
random hop-expressing clones, which
result in ligand-independent, autonomous activation of Stat92E (Struhl
and Basler, 1993; Ekas et al., 2006). In
hop-expressing clones, the expression
patterns of Dll and Hth are not altered, arguing that this pathway does
not directly affect the expression of
these factors (Fig. 3F,F’; Supplemental Fig. 1E,E’).
JAK/STAT Signaling
Autonomously Represses wg
Expression
The defects observed in stat92E clones
in leg and antennal discs and their
corresponding adult structures suggested that wg was ectopically expressed. In wild type leg discs, Wg
protein is expressed in a wedge in ventral anterior cells close to the A-P
boundary, while dpp, as assessed by
expression of the dpp-LacZ reporter
(Blackman et al., 1991), is synthesized
by dorsal anterior cells adjacent to
this boundary and at lower levels by
ventral anterior cells (Fig. 4A). In wild
type antennal discs, Wg and dpp domains are reversed relative to the leg
(Fig. 4C). As expected, ectopic Wg is
expressed within stat92E clones on
the dorsal side of the leg disc. Moreover, we observe a secondary P-D axis
adjacent to the ectopic Wg domain, as
assessed by ectopic non-autonomous
dpp expression and local outgrowths
(Fig. 4B, arrowhead). Similarly, ectopic Wg is also seen in stat92E antennal clones, as is an additional P-D
axis, and non-autonomous ectopic expression of dpp (Fig. 4D, arrowhead).
Furthermore, the ectopic Wg protein
observed in stat92E clones reflects autonomous ectopic transcription of the
wg gene, as monitored by the enhancer trap wgP (Kassis et al., 1992)
(Fig. 4E,E’ arrowhead). We note that
the lateral posterior domain in the antennal disc appears to be the most
sensitive to loss of JAK/STAT signaling, as ectopic wg is always observed
in stat92E clones in this region (Fig.
4E).
We next attempted to identify the
region of the wg gene regulated by
Stat92E. The 3⬘ cis genomic region of
the wg gene regulates wg expression
in many imaginal discs (Pereira et al.,
2006). These authors found that a
⬃2-kb enhancer called wg2.3Z was
the smallest enhancer from this 3⬘ cis
genomic region that could partially recapitulate wg expression in leg and
other ventral discs (Supplemental Fig.
2A). We were unable to detect wg2.3Z
expression in leg discs by immuno-fluorescence using antibodies specific for
␤-galactosidase (data not shown).
However, we could detect its expression in wild type discs by X-Gal staining (Supplemental Fig. 2B,D). This
enabled us to ask whether wg2.3Z is
ectopically expressed in discs containing stat92E clones. Indeed, this reporter is ectopically expressed in these
discs and in regions where we observe
re-patterning and overgrowth (Supplemental Fig. 2C,E, yellow arrowheads). However, we were unable to
determine if Stat92E regulates this
wg enhancer autonomously.
The data presented to this point
suggest that Stat92E represses wg.
Therefore, we reasoned that ectopic
activation of the JAK/STAT pathway
should lead to wg repression. To address this issue, we assessed whether
hop-expressing clones could autonomously repress expression of wg, as
monitored by the wgP enhancer trap.
As expected, in both leg and antennal
discs, ectopic activation of JAK/STAT
signaling represses wg in an autonomous manner (Fig. 5A,A’ and data not
shown). These results indicate that
JAK/STAT signaling represses wg.
JAK/STAT Signaling Also
Represses dpp Expression
We also find that dpp can be autonomously expressed in stat92E clones
(4/24 discs), although at a lower penetrance than ectopic expression of wg.
We next specifically examined leg
JAK/STAT PATHWAY IN DROSOPHILA 2725
Fig. 3. Expression of proximal and distal markers in stat92E clones in
leg and antennal discs. stat92E clones were generated in a Minute (B, D)
or non-Minute (E) background and lack GFP (green). All discs are third
instar. In a wild type leg disc, Dac (blue) is expressed in the femur, tibia,
and first tarsal segment, while Dll (red) is expressed in all tarsal segments
and distal tibia (A). In a leg disc that contains large stat92E clones, a
secondary P-D axis is observed in the posterior compartment (B, arrowhead). In a wild type leg disc, Bab (red) is expressed in 4 concentric rings
within the Dll domain (C). A stat92E clone in the anterior compartment of
the leg disc is associated with a secondary P-D axis, in which Bab is
ectopically expressed (D, arrowhead). A stat92E clone that runs through
the D-V axis in an antennal disc is associated with a triplicated P-D axis,
as assessed by Dll (red) staining (E, asterisks). The expression of Hth
follows the edge of the clone ventro-laterally and marks the proximal
edge of the ectopic axes (E’). hop-expressing clones (abbreviated hop
FO and marked by GFP) do not alter the expression of Dll (red) (F,F’).
D, dorsal; V, ventral; A, anterior; P, posterior.
Fig. 4. wg and dpp are ectopically expressed in stat92E clones. All discs
are third instar. stat92E clones were generated in a Minute (B,F) or nonMinute (D,E) background and lack GFP (green). Wild type discs (A,C). A–E:
Wg is ectopically expressed in stat92E clones in leg and antennal discs. In
a wild type leg disc, dpp (blue) is expressed in the dorsal domain, while Wg
protein (red) is expressed ventrally (A). In a leg disc containing a dorsal
anterior stat92E clone, Wg is ectopically expressed (B, arrowhead), which
leads to a duplicated axis, as assessed by the local outgrowth and ectopic,
non-autonomous dpp expression adjacent to the ectopic Wg (B). In a wild
type antennal disc, dpp is expressed ventrally, while Wg is expressed
dorsally (C). In an antennal disc containing stat92E clones, Wg expression
expands into the ventral domain and is associated with a secondary P-D
axis, in which ectopic non-autonomous expression of dpp is observed (D).
In an antennal disc containing a stat92E clone in the lateral posterior
domain, the wg gene (wgP in red), is ectopically expressed within the clone
(E,E’ arrowhead). F: dpp is ectopically expressed within stat92E clones. A
stat92E clone in the dorsal lateral domain of the posterior compartment of
a leg disc leads to ectopic expression of dpp (blue) in an autonomous
manner (F,F’ arrowhead). wgP (wg-lacZ) (anti-␤-Gal) is red in E and dpplacZ, (anti-␤-Gal) is blue in A–D and F. Magenta lines mark one side of the
clone boundary in F,F’.
2726 AYALA-CAMARGO ET AL.
various locations in the leg disc. We
find that dpp can be repressed in dorsal hop flip-out clones located at the
lateral edge of the dpp expression domain but not in those located close to
the A-P boundary (Fig. 5B,B’ arrowhead, and data not shown). Previous
work has shown that loss of wg expression or signaling in the leg disc
leads to ectopic expression of dpp in
the ventral domain (Brook and Cohen,
1996; Jiang and Struhl, 1996). However, we find that dpp is never expressed within ventral hop-expressing
clones located in or adjacent to the wg
expression domain (21 discs examined) (Fig. 5C,C’ arrowheads, and
data not shown). Lastly, we find that
the effects of Stat92E on wg and dpp
are not due to alteration in Hh expression in the absence of Stat92E (Supplemental Fig. 1F,G). Taken together
these results suggest that JAK/STAT
signaling represses dpp. However, the
fact that supernumerary P-D axes and
local repatterning are always associated with stat92E clones on the dorsal
side of the leg disc suggests that the
repressive effects of Stat92E are
greater on the wg gene than on the
dpp gene.
wg and dpp Restrict upd
Expression
Fig. 5. wg and dpp are repressed by JAK/STAT signaling. All discs are third instar. A,A’: A large
hop-expressing clone (abbreviated hop FO and marked by GFP and an arrowhead in A’) in the
ventral leg disc strongly represses the wgP enhancer trap (red) in an autonomous manner. B,B’:
hop-expressing clones in the dorsal domain of the leg disc can repress dpp (magenta) in an
autonomous manner (arrowhead) on the lateral edge of the dpp expression domain. C,C’: dpp
(magenta) is not expressed in hop-expressing clones (arrowheads) in the ventral domain of the leg
disc that reside both within and adjacent to the wg expression domain. wgP (wg-lacZ) (anti-␤-Gal)
is red in A; dpp-lacZ, (anti-␤-Gal) is magenta in B,C. White boxes in A–C indicate regions that are
magnified at 2⫻ in A’–C’.
discs in which dpp was ectopically expressed in an autonomous manner
(n ⫽ 13 discs) and we made the following observations: (1) in all cases, dpp
is ectopically expressed only in the
dorsal domain and never in the ventral domain (13/13 discs); and (2)
small patches of ectopic dpp are observed at the dorsal lateral margin of
the disc, close to the boundary with
the proximal region that gives rise to
the trocanter and coxa (10/13) (Fig.
4F,F’ arrowhead).
Our results indicate that the loss of
Stat92E function can result in ectopic
expression of dpp within the stat92E
clone boundary. To assess if JAK/
STAT signaling leads to autonomous
repression of dpp, we examined dpp
expression in hop-expressing clones at
It is not known how upd becomes restricted to two domains as in leg and
antennal discs (Fig. 6A, arrowheads).
We observed that the cells producing
upd, dpp, and wg do not overlap in
either leg or antennal discs (Fig. 6G
and data not shown). These mutually
exclusive expression domains, together with our results that Stat92E
represses wg and dpp, raise the possibility that there are multiple negative
regulatory interactions between these
factors. To investigate this issue, we
examined upd mRNA in leg discs that
had temperature-sensitive mutations
in key P-D axis patterning genes.
Notch (N) signaling is necessary and
sufficient to induce transcription of
the upd gene in the eye disc (Chao et
al., 2004; Reynolds-Kenneally and
Mlodzik, 2005). Consistent with these
published reports, we observed loss of
upd at the posterior margin of Nts eye
discs at the restrictive temperature
(data not shown). In contrast, upd remains in its two domains in leg and
JAK/STAT PATHWAY IN DROSOPHILA 2727
antennal discs when N signaling is
reduced, thus ruling out the hypothesis that N regulates upd in these tissues (Fig. 6C, arrowheads, and data
not shown). However, we observed
that upd is negatively regulated by
Hh signaling, as upd mRNA is found
throughout hhts2 leg discs at the restrictive temperature (Fig. 6B). To investigate whether the repressive effects of Hh were due to Wg and/or
Dpp, we examined upd expression in
hetero-allelic combinations that behave as wgts or dppts alleles (van den
Heuvel et al., 1993; Kenyon et al.,
2003). When wg signaling is reduced,
we find that upd expression collapses
into a single domain that is aberrantly
localized to the ventral leg disc (Fig.
6D, arrowhead). Similarly, a single
upd domain that localized to the dorsal leg disc is observed when dpp signaling is reduced (Fig. 6E, arrowhead). Consistently, we also find that
Upd activity is significantly altered in
the absence of dpp signaling. In a
dppts leg disc, Wg is ectopically expressed in the dorsal domain, which is
consistent with previous reports
(Brook and Cohen, 1996; Jiang and
Struhl, 1996; Penton and Hoffmann,
1996). Moreover, the 10XSTAT92EGFP reporter is ectopically expressed
at the dorsal-most region of these
discs, consistent with the dorsal expression of upd in these discs, but is
lost from the central region of the disc
(compare Fig. 1E to 6F). Taken together, our results indicate that both
wg and dpp repress upd and hence
restrict its expression to two domains
in the leg disc (Fig. 6H).
Fig. 6. Wg and Dpp restrict the upd expression domains. In A–E, upd mRNA is detected by in situ
hybridization. B–E show leg discs from animals shifted to the restrictive temperature. A: In a WT leg
disc, upd mRNA has two expression domains, one in the anterior and the other in the posterior
compartment (arrowheads). B: In a hhts2 leg disc, upd is ectopically expressed throughout the disc.
C: In a Nts leg disc, upd expression is not affected and two upd expression domains are still
observed (arrowheads). D: In a wgIL114/CX3 mutant disc, only one upd expression domain, aberrantly
localized to the ventral part of the disc, is observed (arrowhead). E: In a dpphr4/hr56 mutant disc, only
a single domain of upd expression, localized to the dorsal part of the disc, is observed (arrowhead).
F: In a dpphr4/hr56 mutant leg disc, Wg (marked by Wg protein staining in red) and the 10XSTAT-GFP
reporter are ectopically expressed in the dorsal domain. The 10XSTAT-GFP reporter is no longer
expressed in the central part of the disc (compared with Fig. 1E). G: Wg, dpp, and upd are
expressed in mutually exclusive domains in the antennal disc. Wg protein (red); dpp-lacZ (anti-␤
galactosidase blue) and upd⬎GFP green. H: Model of the role of the JAK/STAT pathway in P-D axis
formation in a third instar leg disc. Thickness of the line is intended to reflect the strength of the
inhibitory interaction. See text for details. Proximal (Pr) and Distal (Di).
DISCUSSION
Numerous studies have documented
the function of the JAK/STAT pathway
in proliferation and growth control.
However, we report here for the first
time the crucial role of the JAK/STAT
pathway in P-D axis formation during
development. Specifically, we demonstrate that one of the main functions of
the JAK/STAT pathway is to restrict wg
gene expression to a small domain in
Drosophila appendages. Moreover, we
show that Stat92E represses dpp in leg
discs, although less robustly than
Stat92E represses wg. Furthermore, we
show for the first time that upd is subject to negative regulation by both Wg
and Dpp. Our results are consistent
with a model in which Upd is restricted
by the repressive actions of Wg and Dpp
to two reciprocal groups of cells in the
anterior and posterior compartments of
leg and antennal discs. Upd also acts at
a distance to restrict wg and dpp expression to their appropriate domains
in leg and antennal discs and ultimately promotes the formation of a single, central P-D axis per disc (Fig. 6H).
It is not yet known whether the mutual
antagonism of upd-wg and upd-dpp is
direct or indirect. Additional experiments will be needed to address these
issues. Nevertheless, this study shows
for the first time that these three major
patterning factors (Upd, Wg, and Dpp)
cross-regulate one another and that this
regulation is crucial for proper P-D patterning of leg and antennal discs.
Although we show that Stat92E autonomously represses both wg and
dpp, it must be stressed that Stat92E
appears to more strongly repress wg
than it does dpp. This conclusion is
supported by the following observations. First, ectopic wg is observed
more frequently than ectopic dpp in
stat92E clones. Second, stat92E clones
only give rise to P-D axis duplication
and overgrowth on the dorsal side of
2728 AYALA-CAMARGO ET AL.
the leg disc. We presume that Stat92E
repression of dpp may be cell-type specific, as we have previously shown
that Stat92E represses wg but not dpp
in the eye disc (Ekas et al., 2006). In
contrast, our work demonstrates that
the repression of wg by activated
Stat92E is a broad mechanism utilized during pattern formation in
imaginal discs and one that has recently been highlighted (Tolwinski,
2007). We have demonstrated the repression of wg by Stat92E in the eye
and notal region of the wing disc
(Ekas et al., 2006), as well as antennal
and leg discs (this study). In the eye
disc, we have identified a small (263
bp) enhancer that is negatively regulated by Stat92E in an autonomous
manner (wg 2.11Z) (Ekas et al., 2006).
The inability to look at the wg2.3Z
enhancer within stat92E clones precludes us from making conclusions
about autonomous regulation of wg by
Stat92E in leg and antennal discs
through this enhancer (this study).
Nevertheless, the wg2.11Z enhancer
is contained within wg2.3Z, which
leaves open the possibility that
Stat92E regulates these two enhancers by the same molecular mechanism
(Ekas et al., 2006; Pereira et al.,
2006). Canonical Stat92E binding
sites are not present in either wg enhancer, which suggests that Stat92E
does not directly repress wg but
rather acts through another factor. In
the simplest scenario, activated
Stat92E induces the expression of a
repressor that acts directly on wg in
eye, wing (notal region), antennal, and
leg discs. However, we cannot rule out
the possibility that Stat92E represses
wg by distinct mechanisms in different discs. Moreover, there may be
cryptic Stat92E binding sites in these
wg enhancers, through which Stat92E
may directly repress wg. Future work
will be needed to address these issues.
Do Mammalian STATs
Repress BMP and Wnt Genes
in Limb Formation?
Our study raises the possibility that
the JAK/STAT pathway also plays an
important role during mammalian development by negatively regulating
the expression of vertebrate Wnts and
BMPs. Furthermore, our study also
highlights the question of whether
Wnts and BMPs regulate expression
of cytokines/growth factors that activate JAK/STAT signaling during development. While it is not currently
known whether the JAK/STAT pathway functions during mammalian
limb development, BMP and Wnt
genes do play fundamental roles in
this process (Capdevila and Izpisua
Belmonte, 2001). The elucidation of
the roles of the JAK/STAT pathway
during development have been hampered by the early embryonic lethality
of stat3-deficient mice, and likely genetic redundancy between Stat3 and
Stat5, which are most similar to
Stat92E (Takeda et al., 1997; Levy
and Darnell, 2002). The effects of a
stat3/stat5 double knock-out mouse
have not been reported. The generation of mice with conditional single
stat3 or with double stat3 and stat5
deficiency only in the limb bud will be
required to address this issue. Nevertheless, given the homology between
molecules involved in limb development in Drosophila and mammals and
the critical role of Stat92E in Drosophila appendage development presented
here, our observations are likely to
provide new insights into the role of
STAT proteins in limb development in
higher organisms.
EXPERIMENTAL
PROCEDURES
D. melanogaster Stocks
These stocks are described in FlyBase:
y; stat92E85C9; stat92E397; dome468;
hopC111; dpp-lacZ; wg-lacZ (wgP);
UAS-hop; UAS-gfp; P{AyGAL4}25
P{UAS-GFP.S65T}T2; hs-flp MKRS/
TM6B; hs-flp; ey-FLP; FRT82B ubiGFP(S65T)nls/TM6B, Tb1; FRT82B
M(3)96C, Ubi-GFP; Nts; hhts2/TM6B,
Tb; a wg temperature sensitive hetero-allelic combination wgIL114/CX3,
referred to as wgts in the text; a dpp
temperature-sensitive hetero-allelic
combination dpphr4/hr56, referred to as
dppts in the text. We also used y, hsFLP; FRT82B hsCD2, y⫹/TM2 and y,
hs-FLP; FRT82B hsCD2, y⫹ Minute/
TM2 (Casares and Mann, 2001), upd2
null allele (Hombria et al., 2005);
upd3-Gal4, UAS-GFP (Agaisse et al.,
2003); 10XSTAT92E-GFP (Bach et al.,
2007), UAS-3HA-stat92E, referred to
as “Stat92E FL” in text and figures
(Ekas et al., 2006), and wg2.3Z
(Pereira et al., 2006).
stat92E Clones
stat92E85C9 and stat92E397 were used
interchangeably as they produce identical phenotypes (Silver and Montell,
2001; Ekas et al., 2006). We induced
stat92E clones randomly using hs-flp
or in the eye-antennal disc using eyflp. Collections of larvae of the genotype y, hs-FLP; FRT82B stat92E/
FRT82B hsCD2, y⫹ were heat shocked
for 2 hr at 37°C during first or second
instar. The adult legs and antennae
were dissected and mounted in a 2:l
mixture of Canada balsam and methyl
salicylate. To provide stat92E cells
with a growth advantage, we employed the Minute technique, which
allows mutant cells to grow faster
than surrounding heterozygous cells
and to encompass the majority of the
compartment in which they arise (Morata and Ripoll, 1975). To rescue
stat92E antennal phenotypes, we used
the MARCM technique (Lee and Luo,
1999) to drive expression of the
UAS-3HA-stat92E transgene only in
stat92E clones.
hop and dome Clones
We used dome468, a strong hypomorphic allele, and hopC111, an amorphic
allele, and generated clones in the
eye-antennal disc using the ey-Gal4
UAS-flp, GMR-hid technique (Stowers and Schwarz, 1999). This system
relies upon the presence of a recessive
cell lethal mutation on the chromosome arm of interest to generate large
clones in the antenna.
Temperature-Sensitive
Experiments
Flies of the appropriate genotype were
allowed to lay eggs at the permissive
temperature 18°C for 2 days. Their
offspring were then shifted to the restrictive temperature 29°C for 2 days,
and then they were returned to 18°C.
For the Nts experiments, we isolated
male larvae for the dissection of discs.
For hhts2 experiments, Tb⫺ larvae
were dissected. The wg and dpp alleles were maintained over a compound chromosome SM6⬃TM6B and
Tb⫺ larvae were isolated for dissec-
JAK/STAT PATHWAY IN DROSOPHILA 2729
tion. Discs were fixed and sense and
anti-sense upd ribo-probes were generated according to the protocol in
Bach et al. (2003).
Antibody Staining and
Microscopy
Antibody stainings were performed as
described (Bach et al., 2003). We used
mouse anti-Wg (1:15), mouse anti-␤galactosidase (1:20), mouse anti-Dac
(1:200) (all from the Developmental
Studies Hybridoma Bank); rabbit anti-Hth (1:1,000) (Pai et al., 1998);
mouse anti-Dll (1:500) (Duncan et al.,
1998); rabbit anti-␤-galactosidase (1:
2,000) (Cappel); rabbit anti-Hh
(1:1,000) (Rogers et al., 2005); rat antiBab II (1:1,000) (Couderc et al., 2002).
We used fluorescent secondary antibodies at 1:250 (Jackson Laboratories). We collected fluorescent images
(at 25⫻) using a Zeiss LSM 510 confocal microscope, and bright field pictures of adult legs or antennae (at 10,
20, or 40⫻) using a Zeiss Axioplan
microscope with a Spot camera.
ACKNOWLEDGMENTS
We thank R. Mann, J. Treisman, F.
Casares, J. Hombria, H. Agaisse, H.
Sun, I. Duncan, F. Laski, E. Rogers,
and the Bloomington Stock center and
the Developmental Studies Hybridoma Bank (University of Iowa, Department of Biological Sciences, Iowa
City, IA 52242) for fly stocks and antibodies. We are indebted to J. Treisman, R. Dasgupta, and members of
the Bach lab for reading the manuscript and for insightful comments.
L.A.E. was supported in part from an
NIH institutional training grant.
E.A.B was supported in part by a
Young Investigator Award from the
Breast Cancer Alliance, Inc. and
Whitehead Fellowship for Junior Faculty.
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