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First published online 13 December 2006
doi: 10.1242/dev.02737
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1 Department of Biological Sciences, Columbia University, New York, NY 10027,
USA.
2 Department of Biochemistry and Molecular Biophysics, Columbia University, HHSC
1104, 701 W. 168th Street, New York, NY 10032, USA.
* Author for correspondence (e-mail: rsm10{at}columbia.edu)
Accepted 13 November 2006
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Drosophila, P-Mad, BMP, dally, Organ size, Decapentaplegic, HSPG
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
These pathways control many aspects of development, including determination of
organ size and morphology. In contrast to the enormous morphological diversity
found in animal body parts, only a handful of morphogen systems are known.
This discrepancy predicts the existence of mechanisms that vary the activities
of these signaling pathways in generating the range of animal structures seen
in nature.
Selector genes, such as the Hox family of transcription factors, create
differences between tissues by altering gene expression
(Mann and Carroll, 2002). We
hypothesized that these transcriptional regulators might control organ size
and morphology by regulating morphogen production, distribution, and/or
transduction. In support of this idea, we and others have previously reported
that the distribution of the morphogen Decapentaplegic (Dpp) is different in
the developing wing and haltere (Crickmore
and Mann, 2006; de Navas et
al., 2006; Makhijani et al.,
2006) (see Fig. S1 in the supplementary material), which are two
serially homologous flight appendages in Drosophila that differ owing
to the activity of a single selector gene, Ultrabithorax
(Ubx) (Lewis, 1978).
Ubx is expressed and required in all haltere cells but not in wing
cells (Beachy et al., 1985;
Lewis, 1978). In the primordia
of both structures (the wing and haltere imaginal discs), Dpp is secreted from
a group of specialized cells called the AP organizer, a stripe of anterior
cells that is adjacent to the anterior (A)-posterior (P) compartment boundary.
AP organizer cells are instructed to produce Dpp in response to the
short-range morphogen Hedgehog (Hh), which is secreted from neighboring
P-compartment cells (Basler and Struhl,
1994; Guillen et al.,
1995; Tabata et al.,
1995).
The Dpp pathway differs in the haltere and wing in several respects. For
example, higher levels of Dpp are made in the wing organizer as compared with
the haltere organizer (Crickmore and Mann,
2006; de Navas et al.,
2006; Makhijani et al.,
2006) (see Fig. S1 in the supplementary material). In addition,
Dpp made in the wing is able to travel further from AP organizer cells than is
Dpp made in the haltere. One reason for the decreased mobility of Dpp in the
haltere is that the levels of the type-I Dpp-receptor Thickveins (Tkv) are
higher in the medial haltere as compared with the medial wing. Higher levels
of Tkv in the haltere limit the spread of Dpp, thus narrowing the Dpp activity
gradient (Crickmore and Mann,
2006), which can be visualized with an antibody directed against
the phosphorylated form of Dpp's downstream transcription factor, Mothers
against Dpp (P-Mad) (Fig. 1A
and see Fig. S1 in the supplementary material)
(Tanimoto et al., 2000). The
Tkv-mediated narrowing of the Dpp activity profile in the haltere contributes
to the smaller size of this appendage as compared with the wing
(Crickmore and Mann, 2006).
In addition to receptors, a second class of molecules, heparin sulfate
proteoglycans (HSPGs), have been reported to affect morphogen diffusion
(Lin, 2004). Unlike receptors,
which are selective for specific ligands, individual HSPGs influence the
mobility of multiple morphogens (Lin,
2004). In this report we show that, in the haltere, Ubx
controls the expression of one such HSPG gene, the glypican dally.
The most striking aspect of this regulation is P compartment-specific
repression of dally, which Ubx executes in conjunction with
the posterior selector gene engrailed (en). By carrying out
experiments in both the wing and haltere in which we vary Dally levels in a
compartment-specific manner, we demonstrate that P-specific repression of
dally decreases the P:A compartment size ratio as well as the final
size of the adult appendage. As Dally has been shown to regulate all three
known morphogen systems in the wing (Wg, Hh and Dpp)
(Han et al., 2005;
Lin, 2004), alteration of
dally expression by Ubx is expected to have important
consequences for these signaling pathways. Here, we examine the consequences
of asymmetric dally expression on Dpp signaling in the haltere as
compared with the wing. We show that by downregulating dally
expression in the P compartment, Ubx biases the diffusion of Dpp away
from P cells in the haltere, which is likely to contribute to the decreased
P:A size ratio and overall reduced size of the haltere compared with the wing.
Interestingly, Dpp signaling itself also contributes to dally
repression in the P compartment, demonstrating the presence of a
self-reinforcing loop restricting Dpp diffusion into P cells. These results
provide novel insights into the control of morphogen signaling by HSPGs and
give further support to the hypothesis that selector genes control organ shape
and size by regulation of morphogen signaling, a task which is accomplished by
the spatial and quantitative regulation of morphogen signaling pathway
components.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Dpp::GFP was driven in the AP organizers of the wing and haltere during larval stages by crossing ptc-Gal4; Gal80ts flies to UAS-dpp::GFP flies. The cross was raised at 18°C for two days and then transferred to 30°C until dissection.
UAS-dallyRNAi flies were obtained from Barry Dickson and the RNAi group at IMBA (Vienna, Austria). Embryos from en-Gal4 UAS-dallyRNAi and ci-Gal4 UAS-dallyRNAi crosses were collected at 25°C and shifted to 30°C after 24-48 hours.
Other stocks used: en-Gal4 UAS-GFP; en-Gal4 UAS-lacZ; hh-Gal4 UAS-GFP; ci-Gal4 UAS-GFP (II); FRT42D enE; hsflp; FRT42D Ub-GFP; dpp-lacZ10638; dally-lacZP2; FRT2A dally80; hsflp; FRT2A Ub-GFP; hsflp; Act>y>Gal4; UAS-GFP; tkv-lacZP906; FRT82B Ubx9-22; hsflp; FRT82B Ub-GFP; hsflp; act>y>Gal4 UAS-GFP; dally-lacZP2/TM6B; UAS-dad; UAS-tkvQD (III); UAS-tkv (III); UAS-tkv; dally-lacZP2; tub-Gal4/TM6B; UAS-GFP; act-Gal4; ptc-Gal4 UAS-GFP.
mRNA and protein detection
Standard procedures were followed for antibody staining except for the
extracellular GFP staining for which the protocol of Belenkaya et al.
(Belenkaya et al., 2004) was
used.
Antibodies used: rabbit anti-GFP 1:1000 (Molecular Probes), mouse anti-Wg 1:250 (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), rabbit anti-ß-Gal 1:10,000 (Cappel), guinea pig anti-P-Mad 1:1250 (E. Laufer and T. Jessell, Columbia University, New York, NY), mouse anti-Nub 1:10 (S. Cohen, EMBL, Heidelberg, Germany), mouse anti-Ubx 1:20, mouse anti-En 1:5 (Hybridoma Bank), rabbit anti-Hh 1:1000 (T. Tabata, University of Tokyo, Tokyo, Japan), mouse anti-Dlp 1:50 (Hybridoma Bank).
dally in situ hybridization was performed with a DIG-labeled probe generated using T7 RNA Polymerase to transcribe antisense dally cDNA from PS Bluescript (a gift from H. Nakato).
Size and P:A ratio measurements
All measurements were taken as pixel counts using Adobe Photoshop. For
adult wings and halteres, only the blade or capitellum of females were
measured. All measured animals were raised under non-crowding conditions. Eggs
were collected for 2 hours and grown for 24-48 hours. Approximately 50
first-instar larvae were transferred to fresh tubes and grown until dissection
at wandering or after eclosing. P:A size ratios were calculated by measuring
the fraction of GFP-expressing cells in the Nubbin domain of animals
containing en-Gal4 or ci-Gal4 in combination with either
UAS-GFP alone, or with UAS-GFP plus UAS-dallyRNAi
or UAS-dallyweak. The wing discs shown for the wing P:A
ratio experiments were stained and imaged together and are therefore directly
comparable. All sizes and ratios were measured without knowledge of the
genotype. Error bars are s.e.m.
Intensity plots
Images were imported into ImageJ and pixel intensities measured by boxing a
subset of the pouch regions of the discs.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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), Dpp
signaling was also asymmetric relative to its source (the AP organizer) in the
haltere. Specifically, the P-Mad signal was stronger anterior to the AP
organizer (roughly demarcated by the domain of peak P-Mad staining) than it
was posterior to the organizer (Fig.
1A,B,D and see Fig. S1 in the supplementary material). To test if
this asymmetry is due to asymmetric ligand distribution or differences in
signal transduction, we used an extracellular staining protocol to examine the
distribution of a Dpp::GFP fusion protein following its expression in AP
organizer cells (Belenkaya et al.,
2004; Entchev et al.,
2000; Teleman and Cohen,
2000) (Fig. 1C,E).
In wing cells, Dpp::GFP was detected in a broad gradient on both sides of the
AP organizer (Fig. 1C and see
Fig. S2 in the supplementary material). In the haltere, the distribution of
Dpp::GFP is limited in both directions owing to high tkv expression
levels (Crickmore and Mann,
2006), but this restriction is stronger in the P direction
(Fig. 1C,E). Dpp::GFP spread
was abruptly halted a few cell diameters posterior to the haltere AP
compartment boundary, contrasting with a tapering signal seen in the anterior
direction. By contrast, the Gal4 driver used to express Dpp::GFP
(ptc-Gal4) drove nearly symmetrical expression of a UAS-GFP transgene
(see Fig. S1F,G in the supplementary material), demonstrating that the
distribution of Dpp::GFP in the haltere is not due to asymmetric activity of
the ptc-Gal4 driver. In both the wing and haltere, the pattern of
extracellular Dpp::GFP was very similar to the P-Mad pattern, suggesting that
Ubx does not affect Dpp signal transduction downstream of ligand
binding, at least as detected with the anti-P-Mad antibody. Furthermore, in
both the wing and haltere, a similar coincidence of extracellular Dpp::GFP and
P-Mad patterns was observed when Dpp::GFP was expressed in clones
(Crickmore and Mann, 2006). The
correlation between the P-Mad and extracellular Dpp::GFP patterns in both the
wing and haltere allows us to infer extracelluar Dpp ligand distribution by
visualizing P-Mad in the proceeding experiments.
In the wing, Dpp::GFP distribution and P-Mad staining were also asymmetric,
owing to slightly higher levels of Tkv in the P compartment, which impedes
diffusion (see Figs S1, S2 in the supplementary material)
(Tanimoto et al., 2000). By
contrast, because Tkv levels are similar on both sides of the AP boundary of
the haltere (see Fig. S1D in the supplementary material)
(Crickmore and Mann, 2006;
de Navas et al., 2006;
Makhijani et al., 2006), Tkv
levels are unlikely to account for the Dpp signaling asymmetry in this
appendage. We tested this idea directly by providing uniform levels of
UAS-tkv to both the haltere and wing. Under these conditions, P-Mad
staining became symmetric in the wing, but remained asymmetric in the haltere
(Fig. 2). These results suggest
that the more-restricted P-Mad staining in the P compartment of the wild-type
haltere is due to a tkv-independent and haltere-specific anterior
bias in the diffusion of Dpp.
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). Although Dlp
expression differs between the wing and haltere, its levels were symmetric
along the AP axes of both tissues (see Fig. S1H in the supplementary
material), making it unlikely that dlp is responsible for asymmetric
Dpp signaling in the haltere. In the wing disc, dally expression, as
monitored by a dally-lacZ enhancer trap
(Fig. 1A)
(Fujise et al., 2001) and in
situ hybridization (see Fig. S3 in the supplementary material)
(Fujise et al., 2001), was high
in the AP and dorsoventral (DV) organizers and was largely symmetric with
respect to the source of Dpp production at the AP organizer
(Fig. 1A and see Fig. S3 in the
supplementary material) (Fujise et al.,
2001). By contrast, in the haltere, dally expression was
asymmetric with respect to the AP organizer: dally levels were much
lower on the P side of the AP organizer than they were on the A side
(Fig. 1A,B,D and see Fig. S3 in
the supplementary material). Most strikingly, dally-lacZ expression
was almost undetectable in P cells that lie immediately adjacent to the AP
boundary in the haltere (Fig.
1B,D). We refer to these cells as the domain of dally
hyper-repression. In the A compartment of the haltere, dally
expression was intermediate medially and high laterally. These findings
suggest that differences in dally expression between the wing and
haltere may contribute to the asymmetric Dpp signaling observed along the AP
axis of the haltere.
dally levels and Dpp signaling
dally is known to influence the diffusion of Wg, Hh and Dpp, all
of which function as morphogens in the wing
(Han et al., 2005;
Lin, 2004) (see Fig. S4 in the
supplementary material). Thus, the altered pattern of dally
expression in the haltere might influence some or all of these pathways.
Because of its role in appendage growth, we examined in detail how the pattern
of dally expression in the haltere affects Dpp signaling.
Dpp signaling is promoted by Dally activity. For example, P-Mad levels are
increased in Dally-overexpressing clones
(Fujise et al., 2003) (see Fig.
S4B in the supplementary material) and decreased in dally-mutant
clones (Belenkaya et al., 2004)
(see Fig. S4A in the supplementary material). We therefore hypothesized that
the dally expression pattern in the haltere (in particular the domain
of dally hyper-repression) might be responsible for the reduced Dpp
diffusion and signaling seen in the P compartment of the haltere. Strong
support for this idea comes from the observation that increasing Dally levels
in the P compartment by driving UAS-dally with en-Gal4
caused peak P-Mad staining to extend far into the P compartment of the haltere
(Fig. 3B,C and see Fig. S5 in
the supplementary material) (Makhijani et
al., 2006). Similar results were obtained by expressing
UAS-dally with hh-Gal4 (data not shown). Interestingly, the
severity of this result depended on the UAS-dally construct used,
which is likely to reflect the different levels of Dally they produce (see
Materials and methods). Another interesting observation derived from this
experiment is that, in addition to increasing posterior Dpp signaling, driving
either of the UAS-dally constructs with en-Gal4 or
hh-Gal4 created a non-autonomous reduction in P-Mad labeling in the A
cells immediately adjacent to the AP boundary
(Fig. 3B,C and see Fig. S5 in
the supplementary material; data not shown). Thus, increased Dally levels in
the P compartment cause an increase in posterior Dpp signaling, and seem to do
so at the expense of A compartment signaling. This observation suggests that
the Dpp produced in the A compartment more readily travels into or is retained
within the cells that express higher levels of dally. The
non-autonomous reduction in P-Mad levels in the A cells of these discs argues
against a role for Dally in promoting the stability of Dpp, as such effects
would be predicted to be strictly cell-autonomous. We conclude that
artificially high posterior Dally levels bias Dpp mobility in the posterior
direction. By extension, these data suggest that the domain of dally
hyper-repression in the P compartment of the wild-type haltere functions to
hinder the posterior diffusion of Dpp, thereby biasing its diffusion in the
anterior direction. We further tested this idea by monitoring P-Mad levels in
wing discs in which Dally levels were reduced in the P compartment.
Consistently, driving UAS-dallyRNAi with en-Gal4 decreased
the fraction of P-Mad staining detected in P cells
(Fig. 4B). Thus, in this
experiment, as in the wild-type haltere, Dpp appears to diffuse from its
source in a direction that is determined by the levels of Dally in the
surrounding tissue. We have observed similar non-autonomous alterations in
P-Mad staining in cells surrounding UAS-dally-expressing cells when
driven with ap-Gal4 or in flip-out clones (data not shown),
demonstrating that the effects of altering Dally levels are not
regionspecific.
Consequences of posterior dally repression for appendage development
If dally repression in the posterior haltere alters morphogen
signaling, it would be predicted to influence the development of the
appendage. Below we show that asymmetric dally expression levels do
indeed impact organ development. For example, Dpp target gene expression in
the haltere changes in accordance with the altered Dpp signaling levels seen
when UAS-dally is supplied to posterior haltere cells (see Fig. S6 in
the supplementary material).
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0.48) is much less than that of a wild-type wing (1), possibly
owing to decreased morphogen signaling in the haltere P compartment.
Consistent with this idea, driving UAS-dallyweak with
en-Gal4 increased the P:A size ratio of the appendage-forming region
of the haltere disc from 0.48 to 0.59 (Fig.
3D). In addition to the increased P:A size ratio, we found that
the overall size of the adult haltere was reproducibly 5% larger in
en-Gal4 UAS-dallyweak animals compared with control
animals (Fig. 3E). These
limited effects on relative compartment size and entire organ size, when
compared with the dramatic effects on Dpp signaling in en-Gal4
UAS-dally flies, suggest that Ubx works through means in
addition to dally repression to limit the P:A size ratio and overall
size of the haltere. For example, Ubx also represses wg
expression along the DV boundary of the haltere P compartment
(Weatherbee et al., 1998) (see
Fig. S1E in the supplementary material). Thus, wg expression might
become limiting for growth when dally expression is increased.
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To test whether the P:A ratio and/or overall size altering capabilities of dally repression are specific to P cells, we knocked-down Dally levels in the A compartment of the wing by driving UAS-dallyRNAi with ci-Gal4. P-Mad levels were increased in the P compartments of these discs as compared with control discs, which were co-stained and imaged identically and were therefore directly comparable (Fig. 4A,F). We interpret this observation to suggest that Dpp mobility is biased away from A cells as a consequence of low Dally levels in this compartment. P-Mad was also increased in the AP organizers (dpp-expressing cells) of these discs (Fig. 4, compare F with A). We suggest that, because Dally levels are lower throughout the A compartments of these discs, Dpp signaling is more tightly restricted to the cells that produce Dpp. The P:A ratio of ci-Gal4 UAS-dallyRNAi discs increased from 0.93 to 1.39 and vein L4 was shifted anteriorly in adult wings, findings that complement the reduction of the P:A ratio of en-Gal4 UAS-dallyRNAi discs (Fig. 4G,H). However, in contrast to the en>dallyRNAi experiments (Fig. 4D,E), the final size of ci>dallyRNAi wings was not significantly different from that of control wings (Fig. 4H,I). This result suggests that knocking down Dally levels from either compartment of the wing is able to shift the P:A size ratio, but only a P-specific reduction in Dally levels reduces overall organ size. These data support the conclusion that the Ubx-mediated repression of dally in the P compartment of the wild-type haltere contributes to the reduced P:A size ratio and overall reduced size of this appendage.
Regulation of dally in the haltere
We next turned our attention to how Ubx generates the haltere
pattern of dally expression. dally is activated by Hh and Wg
(Fujise et al., 2001) and
repressed by Dpp (Fig. 5B)
(Fujise et al., 2003). In the
wing, Dpp signaling, as monitored by P-Mad levels, peaked in the cells on
either side of the AP organizer, whereas the cells that produce Dpp had lower
levels of Dpp signaling (see Fig. S1A,B in the supplementary material). By
contrast, Hh signal transduction was highest in the AP organizer, where Dpp
signaling is low (see Fig. S1A,B in the supplementary material). Consequently,
in the wing, dally expression is high in the AP organizer (due to
positive Hh input) and low on either side of the organizer (due to negative
Dpp input) (Fig. 5A). Dpp
signaling also repressed dally in the haltere
(Fig. 5B,F). However, because
of high Tkv levels in the medial haltere, Dpp diffusion is limited, resulting
in peak Hh and Dpp signaling coinciding in the same cells (Crickmore and Mann,
2006). Therefore, opposing positive (Hh) and negative (Dpp) inputs into
dally occur in AP organizer cells of the haltere, which is likely to
contribute to the intermediate dally levels observed in this region
of the disc (Fig. 5A').
To test this idea, we expressed uniform tkv levels in the wing to
mimic the haltere tkv pattern. This manipulation caused the peak Hh
and Dpp signaling domains to align, as shown by the single stripe of P-Mad
staining in the AP organizer (Fig.
2A, Fig. 5C)
(Crickmore and Mann, 2006). The
resulting dally-lacZ pattern in the A compartment is similar to that
found in the A compartment of the wild type haltere
(Fig. 5C). Thus, we conclude
that the Ubx-dependent upregulation of tkv in the haltere is
sufficient to account for the A-compartment differences in dally
expression between the wing and haltere. Interestingly, a similar
tkv-mediated effect also impacts upon the transcription of
dpp, which, like dally, is activated by Hh signaling and
repressed by Dpp signaling (Fig.
5G) (Crickmore and Mann,
2006).
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) (see Fig. S1E in the supplementary material), removing a
positive input into dally expression. The strongest repression of
dally in the haltere (dally hyper-repression) occurred in
the P cells immediately adjacent to the A compartment. These were the only
cells to show detectable P-Mad staining in the posterior haltere
(Fig. 1B), suggesting a role
for Dpp signaling in dally hyper-repression. In support of this idea,
dally was derepressed in this region when Dpp signaling was clonally
inhibited (Fig. 5F).
Interestingly, the repression of dally by Dpp in the posterior
haltere demonstrates that the AP asymmetry in Dpp diffusion is
self-reinforcing because any Dpp that is transduced in the P compartment
further contributes to dally repression. In summary, as depicted in
Fig. 5G, dally is
repressed in the P compartment of the haltere by the convergence of
en and Ubx and the absence of DV wg. The domain of
dally hyper-repression results from an additional repressive input
from Dpp signaling. As this stripe of P cells in which dally is
hyper-repressed abuts the Dpp producing cells of the A compartment, we suggest
that it is responsible for biasing the movement of Dpp in the anterior
direction and for the ensuing consequences to haltere development. |
DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). Here we show that the HSPG dally is
repressed in the P compartment of the haltere and that this regulation
decreases the P:A ratio and overall size of the haltere. We show that
posterior dally repression causes Dpp diffusion to be biased away
from P cells, generating an AP asymmetry in Dpp signaling. The findings
reported here therefore provide another instance wherein Ubx controls
the extracellular signaling environment of the developing haltere and thereby
distinguishes it from the wing.
The movement of most or all signaling molecules through tissues is
regulated by HSPGs, including glypicans such as dally. In contrast to
receptors, HSPGs control the distribution of multiple signaling molecules.
Regulation of HSPG expression and activity by selector genes is therefore a
potentially very powerful mechanism for shaping signaling pathway activation
profiles and molding organ shapes and sizes. However, the promiscuity of HSPGs
also makes it difficult to assign the morphological consequences of their
expression patterns to the alteration of individual signaling pathways.
Indeed, we think it likely that the altered dally expression pattern
in the haltere has implications for Hh, Wg and Dpp signaling (see Fig. S4 in
the supplementary material) (Han et al.,
2005; Lin, 2004),
all of which control growth and patterning. Here, we have focused on the
relationship between dally expression and Dpp signaling.
It has been shown that Dpp signaling is increased in
dally+ clones (Fujise
et al., 2003) and decreased in dally- clones
(Belenkaya et al., 2004) (see
Fig. S4 in the supplementary material). These and other findings have
suggested that Dally participates in the control of Dpp mobility. Our results
add to these earlier observations by suggesting that variations in the levels
of Dally between the cells of a tissue influence the direction and extent of
Dpp diffusion. Specifically, we propose that in addition to simply being
promoted by Dally, Dpp mobility is biased towards cells with higher Dally
levels. This idea derives mainly from the observation that Dally can influence
Dpp movement in a cell-non-autonomous manner. For example, we found that when
Dally levels are increased in the haltere P compartment, there is a shift in
Dpp signaling from the A to the P compartments, as visualized by the levels of
P-Mad. Similarly, knocking down Dally levels in the P compartment of the wing
influences the extent and levels of P-Mad in the A compartment. If
discontinuities in Dally levels can non-autonomously influence Dpp signaling
across compartment borders, it follows that differences in Dally levels
between cells within a compartment can also shape the Dpp signaling landscape.
This might be important for wild-type wing development, where graded Dpp
signaling represses dally, resulting in an inverse dally
gradient that increases towards the lateral edge of the disc
(Fig. 1A). We suggest that this
inverse dally gradient helps to attract Dpp to more lateral regions
of the disc. Accordingly, in a dally-mutant wing disc, the Dpp
gradient is less broad than in a wild-type wing disc (data not shown)
(Fujise et al., 2003). It is
possible that other HSPGs control the mobility of signaling molecules in a
similar manner.
We have shown that altering dally levels in either the A or P compartment changes relative compartment size, but that only P compartment dally levels are relevant for total organ size. We suggest two possible explanations that link the P-specific dally repression seen in the haltere to a reduction in final organ size. Both of these scenarios (which are not mutually exclusive) focus on the role of P cells in producing Hh, which diffuses into A cells to instruct Dpp production and, consequently, controls final organ size. Importantly for both models, we find that there is in fact less Hh detected in the P compartment of the wild-type haltere as compared with the wing (see Fig. S1 in the supplementary material). In the first model, the repression of dally reduces overall Hh production simply by reducing the size of the P compartment, which is a consequence of reduced Dpp signaling. In this scenario, fewer Hh-producing P cells result in less total Hh production from the P compartment, and therefore less Dpp produced in the A compartment. The logic of this potential mode of size regulation is interesting: a selector gene (Ubx) restricts growth factors (Wg and Dpp) from the pool of cells (the P compartment) that produces another growth factor (Hh). In the second scenario, dally repression may directly reduce the amount of Hh in the P compartment that can be transported into the A compartment. In support of this idea, we found Hh staining to be reduced in clones of cells where Dally levels are reduced through UAS-dallyRNAi (see Fig. S4 in the supplementary material).
Together, dally and dlp influence the mobility of all
known morphogens in Drosophila
(Lin, 2004). In addition to
the compartmental regulation of dally, we also note that Dlp levels
are generally lower throughout the haltere as compared with the wing. The
haltere also lacks the domain of dlp repression seen at the DV
boundary of the wing (see Fig. S1 in the supplementary material). Finally, we
also noticed that the expression of Notum-lacZ, an enhancer trap into
a gene that encodes an HSPG-modifying enzyme
(Han et al., 2005;
Kirkpatrick et al., 2004;
Kreuger et al., 2004), is
different between the wing and haltere (M.A.C and R.S.M., unpublished). The
combined alteration of dally, dlp and Notum levels in the
haltere is likely to have consequences for any signaling molecule that uses
HSPGs for transport. When these observations are combined with those of our
earlier work showing that the levels of both Dpp and its receptor are
regulated differently in the haltere and wing
(Crickmore and Mann, 2006;
de Navas et al., 2006;
Makhijani et al., 2006), and
the observation that wg is repressed in the posterior haltere
(Weatherbee et al., 1998) (see
Fig. S1 in the supplementary material), a picture emerges in which selector
genes alter the expression of multiple components of multiple signaling
pathways to change morphogen signaling landscapes between tissues and thereby
modify organ shapes and sizes. We hypothesize that the summation of all
signaling pathway changes may be sufficient to understand the size and shape
differences between fundamentally similar epithelia such as the wing and
haltere imaginal discs.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/2/327/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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