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First published online November 21, 2008
doi: 10.1242/10.1242/dev.025635
Institut für Molekularbiologie, Winterthurerstr. 190, CH-8057 Zürich, Switzerland.
* Author for correspondence (e-mail: basler{at}molbio.unizh.ch)
Accepted 15 September 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Brinker, Dpp, Growth, Patterning, Morphogens
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
French et al., 1976;
Metcalf, 1964;
Silber, 1976;
Simpson et al., 1980;
Twitty and Schwind, 1931).
Studies in the chicken limb placed the intrinsic growth programs downstream of
patterning (Wolpert, 1981).
Also, experiments in the Drosophila wing imaginal disc support the
hierarchical relationship between patterning and growth. The wing disc is
first patterned into compartments, which then function as units of size
(Day and Lawrence, 2000;
Martin and Morata, 2006).
Morphogens were shown to be essential for the correct patterning of organs.
Produced at a local source, they spread into the tissue and form a
concentration gradient to specify positional information. Besides their role
in the patterning of the wing, morphogens also regulate wing growth. In
summary, there is evidence that in invertebrates and vertebrates, patterning
controls growth and not vice versa. However, the molecular details of how
patterning and the intrinsic growth programs are linked remain poorly
understood.
The Drosophila wing imaginal disc is a useful model system in
which to study organ patterning and growth. It originates from an embryonic
primordium containing 
40 cells that undergoes a sigmoidal growth period
until it reaches its final size of 30,000-100,000 cells
(Potter and Xu, 2001). Cells
divide with a doubling time of 10-12 hours
(Garcia-Bellido and Merriam,
1971). Although cell divisions occur in clusters of cells and are
therefore spatially and temporally dynamic, growth within the overall disc is
even (Milan et al., 1996). The
wing primordium is patterned by morphogen gradients, with Wingless (Wg) and
Decapentaplegic (Dpp) expressed in stripes along the dorsal-ventral and
anterior-posterior compartment boundaries, respectively. Movement of these
secreted proteins establishes orthogonal concentration gradients along the two
axes (Entchev and Gonzalez-Gaitan,
2002). The graded distribution of the Dpp ligand leads to changes
in the Punt-Tkv receptor complex, which in turn control the ratio of
phosphorylated to unphosphorylated Mad. Phosphorylated Mad (pMad) forms a
complex with Medea, enters the nucleus and, together with its co-factor
Schnurri, represses transcription at the brinker locus
(brk), creating a gradient of brk expression that is
reciprocal to the Dpp gradient. Brk is a transcriptional repressor that acts
negatively in a dosage-dependent manner to establish the nested expression
domains of other genes, such as spalt (sal; spalt
major - FlyBase) and optomotor-blind (omb;
bifid - FlyBase), which position longitudinal veins along the
anteroposterior (AP) axis of the wing
(Campbell and Tomlinson, 1999;
Cook et al., 2004;
Jazwinska et al., 1999;
Marty et al., 2000;
Minami et al., 1999;
Sivasankaran et al., 2000;
Sturtevant et al., 1997). Brk
binds to these Dpp target genes via the sequence GGCGYY
(Cordier et al., 2006) and
uses different molecular mechanisms and co-repressors to regulate their
expression, leading to the differential sensitivity of the target genes to Brk
(Hasson et al., 2001;
Winter and Campbell, 2004).
Another difference between the regulation of omb and sal
expression is that although the expression boundaries of both genes are solely
set by Brk levels, the rate of sal, but not of omb,
transcription requires a direct Dpp input
(Marty et al., 2000). For a
detailed review of the molecular mechanisms of Dpp morphogen readout see
Affolter and Basler (Affolter and Basler,
2007).
Besides its well-described role in patterning, previous studies have
indicated that Dpp also acts as a growth-promoting factor. Hypomorphic
dpp alleles, which lead to a loss of Dpp expression in the wing
imaginal discs, result in small wings
(Spencer et al., 1982;
Zecca et al., 1995), whereas
ectopic expression of Dpp causes abnormally large discs
(Burke and Basler, 1996;
Capdevila and Guerrero, 1994;
Martin-Castellanos and Edgar,
2002). Furthermore, cell clones lacking Dpp signaling fail to
survive, and clones in which the Dpp pathway is uniformly activated overgrow
(Adachi-Yamada et al., 1999;
Burke and Basler, 1996;
Martin-Castellanos and Edgar,
2002; Moreno et al.,
2002). However, key aspects regarding the mechanism of growth
control by Dpp remain to be resolved.
One particularly intriguing issue is how a growth factor that is
distributed in a gradient can lead to uniform growth in the wing disc. Several
models have attempted to answer this conundrum. In one such model (the
threshold model), the proliferation rate is constant for cells in which Dpp
signaling activity exceeds a certain minimal level. Studies with the
growth-promoting gene vestigial (vg) have shown that certain
levels of Dpp and Wg together fuel a feed-forward autoregulation of
vg expression (Zecca and Struhl,
2007a; Zecca and Struhl,
2007b), thus supporting the threshold model. However, the
overproliferation behavior of clones with high experimental Dpp signaling
levels calls for an additional mechanism of growth regulation by Dpp. In a
second model (the cellular fate model), cells along the AP axis of the wing
are programmed for differential sensitivity to Dpp. Such pre-patterning might
occur in a Dpp-dependent or -independent manner. According to a third model
(the inhibitor model), an additional growth-modulatory gradient exists, which
parallels that of Dpp but acts in an antagonistic manner. Although no strong
evidence exists against the cellular fate or inhibitor models, experimental
support for the existence of pre-patterning or inhibitory factors is missing.
Another concept suggests that growth depends on the slope of the Dpp gradient
(Day and Lawrence, 2000;
Gelbart, 1989;
Lawrence and Struhl, 1996). A
recent experimental study has led to a refinement of this model that
incorporates aspects of the cellular fate model
(Rogulja and Irvine, 2005):
the proliferation of medial cells depends on the slope of the Dpp gradient,
whereas proliferation in lateral regions can be triggered by both constant and
graded signaling levels. However, this model fails to explain growth at the
source of Dpp production, where a considerable area is exposed to equal,
saturating levels of the Dpp signal. Moreover, medial clones in which Dpp
signaling was uniformly activated showed no growth disadvantage
(Martin-Castellanos and Edgar,
2002). A completely different explanation for uniform growth
within the wing disc is provided by mechanical feedback models, in which
growth rates differ initially owing to different Dpp levels. These differences
cause mechanical stresses (compression and stretching) that in turn affect
growth rates, resulting in uniform growth
(Aegerter-Wilmsen et al., 2007;
Hufnagel et al., 2007;
Shraiman, 2005).
Besides the problem of how the Dpp gradient directs uniform growth, the
role of Brk in growth regulation remains elusive. The surprising discovery
that the expression boundaries of the Dpp patterning targets sal and
omb are set by Brk raised the question of whether Brk plays an
equally important role in growth. Experiments conducted thus far indicate that
Brk is at least partially involved in growth control. Overgrowth is not only
observed in wing discs with constitutive Dpp signaling
(Capdevila and Guerrero, 1994;
Lecuit et al., 1996;
Nellen et al., 1996), but also
in brk mutant discs (Campbell and
Tomlinson, 1999; Nellen et
al., 1996), and cell clones that lack Dpp signaling, and therefore
fail to survive, can be rescued by eliminating brk function
(Marty et al., 2000). However,
whether Dpp has an additional growth-promoting function in parallel to its
repression of brk remained unresolved.
Here, we experimentally address whether Dpp regulates growth via Brk, and how the graded activity of Dpp can cause uniform growth. Our experiments demonstrate that Dpp regulates growth exclusively by repressing brk, and that the main function of the Dpp-Brk system is to limit proliferation in lateral areas of the wing disc. Second, we confirm that medial and lateral cells in the wing disc react differently to Dpp, and provide evidence that this distinct behavior depends on a mechanism independent of the Dpp-Brk system, effectively ruling out the possibility that the regionally different responses to Dpp are due to an earlier Dpp-dependent fate specification. Finally, we reveal that neither lateral nor medial cells need a Dpp gradient to proliferate. Repression of brk in the medial area and derepression of brk in the lateral area in a non-graded manner are sufficient for uniform growth in the entire disc.
Thus, our results challenge the gradient models, which imply that the juxtaposition of cells with different Dpp signaling levels induces proliferation. Indeed, the data suggest an alternative model in which the Dpp levels along the AP axis set different brk expression levels: high laterally and low medially. The high lateral Brk levels are needed to curb the overproliferation that would otherwise occur in this region.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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yw hsp70-flp hsp70-GFP FRT19A/yw FRT19A.
w tubP-Gal80 hsp70-flp FRT19A/yw hsp70-flp hsp70-GFP FRT19A; tubP-Gal4/UAS-tkvQ235D.
yw brkM68 hsp70-flp FRT19A/yw hsp70-flp hsp70-GFP FRT19A.
yw tubP-Gal80 brkM68 hsp70-flp FRT19A/yw hsp70-flp hsp70-GFP FRT19A; tubP-Gal4/UAS-tkvQ235D.
To obtain these transgenic animals, the following stocks were used: w
tubP-Gal80 hs-flp FRT19A (stock 5132, Bloomington), tubP-Gal4
(stock 5138, Bloomington), hsp70-GFP
(Vegh and Basler, 2003),
UAS-tkvQ235D (Nellen
et al., 1996) and brkM68
(Jazwinska et al., 1999).
To induce clones, a heat shock of 15 minutes at 37°C was performed 48 hours AEL; 72 hours later the larvae were dissected. Discs were fixed in 4% paraformaldehyde (PFA) and stained with DAPI or propidium iodide. Standard protocols were followed. Twin-spot volumes were calculated as follows: the surface area of a clone was drawn in each z-stack of confocal images using Imaris (Bitplane, Scientific Solutions), and the volume was then calculated by the software. Volume ratios were calculated for each twin-spot individually.
Genotypes for the clonal analysis experiment in wing discs with uniform
Dpp: yw hsp70-flp; UAS-dpp; C765-Gal4/actin5>draf>nuc-lacZ.
Control experiment: yw hsp70-flp; actin5>draf>nuc-lacZ. The
actin5>draf>nuc-lacZ cassette
(Struhl and Basler, 1993),
C765-Gal4 driver (Nellen et al.,
1996) and UAS-dpp construct
(Ruberte et al., 1995) were
described previously. Clones were induced by a heat shock (18 minutes at
34°C) 48 or 72 hours AEL, and larvae were dissected 120 hours AEL. Nuclei
in wing disc cells were stained by DAPI. The volume of clones was calculated
using Imaris.
Driver lines to induce the expression of UAS-brk
(Moreno et al., 2002),
UAS-tkvQ235D (Nellen
et al., 1996), UAS-dpp
(Ruberte et al., 1995) or
UAS-GFP transgenes: C765-Gal4
(Nellen et al., 1996),
esg-Gal4 (Goto and Hayashi,
1999), salE-Gal4
(Mosimann et al., 2006),
omb-Gal4 (Calleja et al.,
1996), en-Gal4 (obtained from the Drosophila
Genetic Resource Center) and act5cP(FRT.y+)-Gal4:PR
(Progesterone receptor) (Rogulja and
Irvine, 2005). tubP-Gal80ts stocks: stock 7108
and 7017 (Bloomington). dpp and brk mutants:
brkM68 (Jazwinska et
al., 1999), brkXA
(Campbell and Tomlinson, 1999),
dpp12 and dpp14
(Spencer et al., 1982).
brkx47-lacZ line
(Campbell and Tomlinson, 1999).
The permissive temperature used for Gal80ts was 18°C and the
restrictive temperature was 29°C.
act5cp(FRT.y+)-Gal4:PR was activated by adding
progesterone to the food to a final concentration of 20 µg/ml.
Immunohistochemistry
Immunostaining was performed using standard protocols. Images were
collected with Leica TCS SP1 and TCS SP5 confocal microscopes. Imaris was used
to analyze the images. z-stacks were projected in the
three-dimensional view.
For BrdU labeling, prior to fixation (4% PFA for 20 minutes), BrdU (0.1 mg/ml) was added to the Ringer's solution and discs were incubated for 30 minutes at room temperature. Before addition of the anti-BrdU antibody, discs were treated for 30 minutes with 2 M HCl. Primary and secondary antibody staining was then performed using standard protocols. For BrdU co-stainings, prior to fixation, BrdU (0.1 mg/ml) was added to the Ringer's solution and the discs were incubated for 30 minutes at room temperature. Anti-pMad and anti-β-galactosidase (β-gal) stainings were performed using standard protocols. After addition of the secondary antibodies, discs were fixed with 4% PFA for 20 minutes, and then treated for 30 minutes with 2 M HCl before the anti-BrdU antibody was added.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). To
explore whether the effects of Dpp on growth are mediated via brk or
whether Dpp regulates growth in a Brk-independent manner, we first performed a
quantitative comparison of the growth behavior of cell clones with an
activated Dpp signaling pathway versus clones lacking brk function.
Since the growth response to altered Dpp signaling levels varies regionally
along the AP axis (Martin-Castellanos and
Edgar, 2002), phenotypes were analyzed separately in the medial
and lateral areas (see Fig.
1A'-D' for the location of the medial and lateral
twin-spots). For the same reason, the twin-spot technique was used to monitor
the clonal growth behavior (Lee and Luo,
2001). Clones that either exhibit an activated Dpp signaling
pathway (by expressing a constitutively active form of the Dpp receptor Tkv,
TkvQ235D) (Lecuit et al.,
1996; Nellen et al.,
1996), or that lacked brk function (by being homozygous
mutant for the brk-null allele brkM68)
(Jazwinska et al., 1999), were
induced at early second instar [48 hours after egg laying (AEL)] and compared
with sibling control clones at the end of larval development (120 hours AEL).
Owing to the uneven and folded nature of the wing disc epithelium at the
edges, we assessed the volumes of the clones rather than their surface.
Examples of such twin-spot clones are shown for each genotype in
Fig. 1A-D. In a control experiment, we confirmed that wild-type twin-spot clones that differed only in their GFP expression levels grew to equal sizes, irrespective of their position along the AP axis (Fig. 1A,E), indicating that neither the genetic set-up nor the volumetric analysis introduces any experimental distortions.
We then compared twin pairs, of which one clone was wild-type and the other
expressed TkvQ235D. Confirming previous studies,
TkvQ235D-expressing clones grew faster than their wild-type twins
in the lateral area (Martin-Castellanos
and Edgar, 2002). The volume of lateral
tkvQ235D clones was 3.9±1.9-fold larger [values are
shown ± 95% confidence interval (CI)] than the volume of their
corresponding wild-type siblings, whereas medial tkvQ235D
clones exhibited the same volume as their siblings
(Fig. 1B,F). We then analyzed
the growth behavior of brkM68 clones and observed that
they closely reflected the phenotype of tkvQ235D clones.
When located laterally, their volume was 3.8±1.7-fold larger than the
volume of their corresponding wild-type twins; when located medially, they did
not show any extra growth (Fig.
1C,G). The observation that the relative growth differences
between tkvQ235D and wild-type control clones parallel
those of brk mutant versus wild-type clones indicates that the
activation of the Dpp pathway has the same effect on growth as the removal of
Brk function. To more directly corroborate the equivalency of the two
conditions, we set up mitotic recombination events in which the gain of
TkvQ235D and the loss of brk occurred concomitantly in
complementary daughter cells. This led to clone pairs of opposite genotypes,
which could be directly compared in a quantitative manner. As shown in
Fig. 1D,H, no size differences
were observed between tkvQ235D and
brkM68 twins in either lateral or medial regions. Taken
together, these results indicate that, with respect to clonal growth, the
activation of the Dpp pathway is equivalent to the loss of brk. This
is consistent with the notion that Dpp regulates growth in the wing disc
solely by repressing brk and that is does not use a parallel,
brk-independent output.
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;
Rogulja and Irvine, 2005). We
confirmed these observations by expressing Dpp with the C765-Gal4
driver, which is uniformly active in the wing disc from the second instar
onwards (data not shown). In C765>dpp discs, the Dpp signaling
activity was uniformly activated at high levels
(Fig. 2, compare
B',B'' with A',A''; Fig. S1, compare D with C in the
supplementary material). Such discs become larger than the wild-type control
discs, measuring 436±35 µm along the AP axis at third instar, as
compared with 321±15 µm (Fig.
2H). Moreover, the uniform proliferation pattern observed in
wild-type wing discs was lost in C765>dpp third instar discs: BrdU
incorporation was enhanced in the lateral area compared with the medial area
(Fig. 2A,B) (for area
definition see Fig. 3A,B). The
ring of Wg staining indicated that the pouch in C765>dpp discs was
expanded almost to the lateral edges of the wing disc (see Fig. S1B in the
supplementary material). Thus, the overproliferating lateral area partly
overlapped with the wing pouch, leading to its enlargement. Activation of the
Dpp pathway by overexpressing TkvQ235D instead of Dpp yielded
equivalent results (see Fig. S3A,C in the supplementary material).
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)
(see Fig. S1E-J in the supplementary material), but allows development until
pupation. brkXA mutant discs showed the same phenotype as
C765>dpp discs (Fig.
2C,H); they measured 425±20 µm along the AP axis, and
BrdU uptake was enhanced in the lateral region compared with the medial
region. Thus, although Dpp signaling was limited to a narrow gradient along
the AP axis (as assessed by the levels of pMad; see
Fig. 2C',C''), the
growth of brkXA discs was comparable to that of discs with
ubiquitous Dpp signaling.
If, as suggested by the above results, Dpp solely affects growth by
regulating brk, the levels and spatial distribution of Dpp signaling
should be irrelevant in brk mutant discs or in discs expressing
brk constitutively. To test the first of these predictions, we
analyzed the growth phenotype of third instar discs lacking both brk
and dpp. To circumvent embryonic lethality of dpp mutants,
we used the dpp disc alleles dpp12 and
dpp14 (dpp12/14), which lack Dpp
expression only in the wing imaginal discs and not in the embryo
(St Johnston et al., 1990).
Strikingly, although no Dpp signaling activity was detected in
brkXA dpp12/14 discs
(Fig. 2D',D''), they
overgrew (435±23 µm) and exhibited enhanced BrdU incorporation in
lateral as compared with medial areas (Fig.
2D,H). In the reciprocal experiment, we tested whether discs
constitutively expressing Brk mimic the reduced size of discs mutant for
dpp, regardless of their Dpp signaling levels. Indeed, discs
expressing Brk uniformly and at high levels under the escargot
(esg)-Gal4 driver, which is active uniformly during wing
disc development, measured only 128±16 µm along the AP axis,
resembling dpp12/14 discs (124±9 µm)
(Fig. 2E,F,I). Even massive and
ubiquitous Dpp signaling (by means of UAS-tkvQ235D under
esg-Gal4 control) could not rescue the phenotype of such discs
(Fig. 2G,I). Consistent with
these results, expressing Brk and TkvQ235D under C765-Gal4
led to the same phenotype as when expressing only Brk. However, owing to the
later activation of the C765-Gal4 driver, discs were larger than
dpp12/14 discs (see Fig. S4 in the supplementary
material).
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Uniform activation of the Dpp pathway enhances lateral and reduces medial growth
The patterns of BrdU incorporation described above confirmed previous
observations (Rogulja and Irvine,
2005) and suggest that in wing discs with ubiquitous Dpp
signaling, cell proliferation is enhanced in the lateral regions (compared
with the medial area). The extended shape of the wing disc along the AP axis
also suggests that the growth rate of lateral cells is higher than in
wild-type discs. However, as BrdU is only a marker for cells in S phase, these
results alone are not conclusive evidence for a decrease in the growth rate of
medial cells, as compared with the situation in wild-type discs. We therefore
examined the growth rates in discs with uniform Dpp signaling in more detail.
First, we used the anti-phosphohistone H3 (pH-H3) antibody to identify cells
in M phase, which represent proliferating cells. The lower percentage of cells
found in M phase, as compared with cells in S phase, renders the pH-H3 assay
more appropriate for quantification. Furthermore, the reduced variability in
the pH-H3 (relative to the BrdU) assays enabled a more reliable comparison of
the results from discs of different larvae. C765>dpp discs showed
an increase in pH-H3-positive cells in the lateral area, and a decrease in the
medial area, confirming the BrdU results
(Fig. 3B,C) (for area
definition see Fig. 3A).
Quantification of the pH-H3-positive cells demonstrated that both the increase
in proliferation levels in the lateral areas and the decrease in the medial
areas, as compared with the wild type, were statistically significant
(Fig. 3D). The earliest time
point at which we observed differences between medial and lateral cells was at
the mid-third instar stage, 96 hours AEL (see Fig. S2 in the supplementary
material). As an additional confirmation of these observations, we used an
independent quantitative assay to compare the proliferation behavior of medial
and lateral cells. Randomly positioned, neutral lacZ-expressing
clones were induced both in C765>dpp discs and in wild-type discs
at 48 hours and 72 hours AEL and allowed to grow until 120 hours AEL. The
volumes of these clones were recorded separately for medial and lateral
regions (Fig. 3A,E-G). In
agreement with the other assays, clone size, and hence growth, was enhanced in
the lateral area and reduced in the medial area of C765>dpp discs,
as compared with wild-type discs, in a manner that was statistically
significant.
The two distinct areas of growth in the wing disc are defined by a Dpp-independent mechanism
The experiments above show that the wing disc consists of at least two
distinct cell populations, the medial and lateral, which exhibit different
proliferation rates under conditions of uniform Dpp signaling. In principle,
two different explanations could account for the existence and divergent
behavior of medial and lateral cells. The Dpp gradient itself could provide
early positional information along the AP axis, causing the subdivision of
cells into a mirror-symmetric arrangement of lateral, medial and again lateral
cells. Later in development, these distinct cell populations react differently
to experimental, uniformly high Dpp signaling levels. Alternatively, a
Dpp-independent system might determine medial and lateral regions. To
distinguish between these two scenarios, we sought to analyze discs that were
never exposed to graded Dpp signaling during their entire developmental
history. This criterion is fulfilled by discs of
dpp12/14;brkXA genotype. As mentioned
above and shown in Fig. 2D, the
proliferation of lateral cells in such discs is enhanced, indicating that the
two populations can form in the absence of a detectable Dpp gradient
(Fig. 2D',D'').
Since it was theoretically possible that the hypomorphic dpp disc
alleles might still provide some early graded distribution of Dpp, we also
examined discs from esg>dpp larvae. esg is expressed
uniformly at high levels during embryogenesis in the wing disc precursors
(Fuse et al., 1994), as well
as during all larval stages of wing development in the regions of interest
(Fig. 4A). Hence, the
esg>dpp transgene combination leads to saturating Dpp signaling
activity in all cells from the very beginning of disc development. Strikingly,
third instar wing discs still showed a differential proliferation pattern in
medial versus lateral regions (Fig.
2D, Fig. 4B).
Moreover, no significant size difference could be detected between discs with
early uniform Dpp signaling
(dpp12/14;brkXA double-mutant discs
and esg>dpp discs) and discs with uniform Dpp signaling from
second instar onwards (C765>dpp)
(Fig. 4C). Expressing
TkvQ235D instead of Dpp resulted in the same phenotype (see Fig.
S3B,C in the supplementary material). Together, these experiments argue for
the existence of a second AP patterning system that acts independently of Dpp
to specify the distinct growth behaviors of medial and lateral cells.
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).
However, during wing disc development, proliferation occurs in a normal manner
at the source of Dpp expression, where cells are exposed to saturating and
equal Dpp signaling levels. This prompted us to reassess the gradient model by
analyzing the proliferation pattern in a situation in which the Dpp pathway is
uniformly activated in the medial area of the wing primordium.
TkvQ235D was expressed under the control of the sal-Gal4
driver, which is active in the medial area from early second instar
(Fig. 5A'') until third
instar (Fig. 5B'').
Intensity measurements for pMad and brk-lacZ confirmed uniform and
full activation of the Dpp pathway within the sal domain
(Fig. 5D): brk-lacZ
expression was fully repressed within this domain, and the brk-lacZ
gradient observed in wild-type discs was lost
(Fig. 5, compare C with D). The
uniform and high brk expression observed lateral to the sal
domain can be attributed to the ability of Tkv to sequester Dpp and thus
hinder its movement (Belenkaya et al.,
2004; Crickmore and Mann,
2006; Lecuit and Cohen,
1998). As shown in Fig.
5B, despite uniform activation of the Dpp pathway - and hence the
absence of a Dpp signaling gradient - in the sal domain, close to
normal growth was observed. The sal expression domain expanded over
time (Fig. 5, compare A with B,
second instar discs compared with third instar discs), and
sal>tkvQ235D third instar discs were approximately the
size of control discs (305±11 µm along the AP axis compared with
321±15 µm). Importantly, BrdU staining was uniform in
sal>tkvQ235D discs, indicating even growth throughout
the entire wing disc (Fig.
5B'). Expression of tkvQ235D under the
omb-Gal4 driver, which is also downstream of Dpp signaling but
expressed in a broader domain, led to similar results (see Fig. S5 in the
supplementary material). Together, these results demonstrate that uniform Dpp
signaling levels do not prevent cell proliferation in the medial area and thus
strongly argue against the gradient slope model.
Ectopic Dpp pathway activity in lateral cells causes reduced proliferation in the residual disc
The experiments above indicate that the slope of the Dpp gradient does not
play a major role in determining the growth rates of the wing disc. However,
the need for differences in Dpp signaling levels between adjacent cells was
proposed to explain the reduced medial proliferation rates in discs with
uniform Dpp signaling (Rogulja and Irvine,
2005). An alternative explanation for this phenomenon is that
ectopic Dpp signaling in the lateral area causes reduced proliferation in the
medial area. To test this idea, we analyzed proliferation in discs that
exhibit the endogenous Dpp gradient in the medial area, but ectopic Dpp
signaling in the lateral area. One way to obtain such a situation is the
generation of TkvQ235D-expressing clones.
Fig. 6A shows a lateral clone
in which the hormone-inducible Gal4, Gal4:PR, drives TkvQ235D
expression. Upon induction, Dpp signaling activity within the clone is
uniformly high, but the Dpp activity gradient outside of the clone is
unaffected (Fig. 6A'). In
contrast to the autonomous increase in proliferation within the clone,
proliferation was reduced in the rest of the disc
(Fig. 6A). Next, we directly
compared the non-autonomous influence of lateral Dpp signaling on medial cells
in the presence and absence of a Dpp signaling gradient. We used an
engrailed (en)-Gal4 transgene to drive
TkvQ235D expression in the P compartment, and the
Gal80ts system for temporal control. In such
en>tkvQ235D discs we assayed proliferation 24 and 48
hours after induction, and observed an overproliferation of posterior lateral
cells (Fig. 6B-D) (for area
definition see Fig. 3A,B).
Importantly, in these discs only cells in the anterior half of the medial area
are exposed to a Dpp gradient, whereas cells in the posterior medial half
exhibit saturating and uniform Dpp pathway activity. Despite this difference,
proliferation was significantly reduced in the medial parts of both the
anterior and posterior halves of the discs as assessed by BrdU
(Fig. 6B,C). For the 48-hour
induction time point, proliferation was also assayed by pH-H3 staining and
quantified (Fig. 6D,E) (CIs of
pH3-positive cells in wild-type discs do not overlap with the CIs of
pH3-positive cells in the medial areas and the posterior lateral area of
en>tkvQD discs; P<0.0001). Besides the
reduction of proliferation in the medial areas, proliferation was also reduced
in the anterior lateral region (P=0.0004), indicating that the
growth-curbing signal acts on all cells within the disc except those with an
additional growth advantage. Taken together, our results provide an
alternative explanation for the reduced growth rates in the medial area of
discs with uniform Dpp pathway activity. It seems that this effect is not
caused by the loss of the gradient, but rather by a non-autonomous effect of
overproliferating lateral cells on medial cells. This model is now also in
agreement with our finding that only in the medial area are uniform and high
Dpp signaling levels sufficient for normal growth.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Lawrence,
2001; Vincent and Briscoe,
2001) in various embryonic organ systems. The tight link between
organ patterning and organ growth raised the notion that morphogens also
determine cell proliferation rates and final tissue size. This latter aspect
of the morphogen concept, however, is not well understood. Indeed, it is not
clear whether the nuclear response to morphogen signals that directs the
transcription of patterning genes also regulates growth. And what property of
a morphogen signaling system explains how uniform growth rates can ensue in
response to a graded input? Here we address these questions in the
experimental system of the Dpp gradient, a key determinant in pattern
formation and growth of the Drosophila wing.
Brk plays an equally important, and essential, role in patterning and growth
Studies from the past decade have shown that the Dpp gradient in the wing
disc does not define the expression boundaries of subordinate patterning genes
directly via its nuclear mediators, but does so indirectly by setting up an
inverse gradient of the transcriptional repressor Brk
(Affolter and Basler, 2007).
Here we have investigated the potential role of this indirect mechanism in
growth regulation and found that it is equally important, and essential, for
the ability of Dpp to promote growth. Clones of cells with a constitutively
active Dpp signaling pathway exhibited qualitatively and quantitatively the
same growth behavior as brk- clones, overgrowing when
located in the lateral area. Moreover, the phenotype of discs in which Brk
levels can no longer be regulated by Dpp (because brk is either
lacking genetically, or controlled by a heterologous promoter) are insensitive
to experimentally varying Dpp signaling levels. Thus, our experiments
demonstrate that the growth output of the Dpp pathway is entirely funneled
through the regulation of the brk gene.
|
The Dpp-Brk system evens out regional differences in wing disc growth
Discs lacking both dpp and brk functions grow to a larger
size than wild-type discs. Importantly, in this state, in contrast to the
normally uniform profile, cell proliferation also occurs unevenly across the
disc, with higher rates in the lateral areas and lower rates in the medial
area. Based on this difference, we conclude that the Dpp-Brk system is not a
growth promoter but is rather a growth-modulatory system, ironing out inherent
regional differences in proliferation rates.
The origin of the regional proliferative differences in discs devoid of the Dpp-Brk system is unknown. Since such discs lack Dpp, as the only agent known to impose mirror-symmetric differences along the AP axis, no pre-patterning mechanism that depends on it can be postulated. The smooth transitions to higher proliferation rates between medial and lateral areas would be consistent with a diffusible factor that acts in a concentration-dependent manner. This hypothetical factor could originate, for example, at the border between the disc proper and the adjacent peripodial membrane and promote growth laterally. Alternatively, the factor could be a growth inhibitor with high activity in the center of the disc and low activity peripherally. Expression of the factor could be controlled by Hedgehog in a Dpp-independent manner. But this is pure speculation because to date there is no evidence for the existence of such a factor(s) in the developing wing discs.
An entirely different explanation for our experimental observations could
be a growth-regulatory mechanism that depends on mechanical forces. It has
been proposed that during growth, mechanical compression of cells increases in
the center, while cells in the peripheral regions become stretched
(Aegerter-Wilmsen et al., 2007;
Hufnagel et al., 2007).
Assuming a growth-stimulatory role for stretching and a growth-inhibitory role
for compression, growth would be facilitated in the peripheral regions during
normal development, and Brk would counter this advantage and thus ensure
uniform growth (Aegerter-Wilmsen et al.,
2007; Martin et al.,
2004). In the absence of the Dpp-Brk system, the amount and
distribution of mechanical stresses are likely to differ significantly, which
in turn could feed back on growth and lead to the observed differences between
the lateral and medial regions of the disc.
Cell proliferation under conditions of uniform Dpp signaling
Here we have confirmed and extended previous findings that in wing discs
with uniform Dpp signaling, lateral cells proliferate faster, and medial cells
slower, than cells of wild-type discs
(Martin et al., 2004;
Rogulja and Irvine, 2005). The
inhibition of cell proliferation in the medial region is an important pillar
for the model which proposes that it is the slope of the Dpp morphogen
gradient that serves as the driving force behind medial wing cell
proliferation during normal development
(Rogulja and Irvine, 2005).
Contradicting the proposed requirement for disparate Dpp signaling activities
among adjacent cells, we found that when uniform pathway activity is
established in, and limited to, the medial area, no deficit in cell
proliferation rates occurs. Indeed, the medial domain of discs with such even
Dpp signaling levels expands, and proliferation is uniform
(Fig. 5). This finding is
consistent with results from our twin-spot analysis, which showed that the
growth rates of medial tkvQ235D and
brkM68 clones are identical to those of wild-type clones
(Fig. 1). Thus, the transient
effect of additional proliferation at clonal boundaries observed by Rogulja
and Irvine (Rogulja and Irvine,
2005) seems to be more important for situations such as wound
healing, in which cells of different Dpp signaling levels become juxtaposed,
than for the normal growth of a wild-type wing disc. We found that a reduction
in proliferation rates in the medial area only occurs when Dpp activity is
driven in the lateral area, independent of the presence or absence of a Dpp
signaling gradient. Ectopic Dpp pathway activation in lateral cells is not
only necessary, but also sufficient, to impede proliferation of medial cells.
Thus, overproliferating lateral cells appear to exert a
proliferation-retardant effect on other cells. Whether this effect underlies a
mechanism also used to control proliferation rates during wild-type
development, or whether it is `only' a back-up mechanism used if something
goes wrong during development (e.g. wound healing and regeneration), is not
known. Moreover, as noted earlier, the mechanistic nature of the communication
between lateral and medial cell populations remains speculative. It is
possible that high Dpp signaling in lateral cells not only provides them with
a growth advantage, but also causes the expression of a factor that spreads
within the entire disc to reduce proliferation of cells without an additional
growth advantage. Other possible explanations include the competition among
wing cells for a limiting proliferation factor (whereby ectopic
Dpp-transducing cells prevail), or the negative impact that overproliferating
cells might exert on remaining cells via metabolic side-products or increased
mechanical compression. These models would also be consistent with the
observation that proliferation is reduced in all cells of the wing disc except
those with an additional growth advantage.
Growth regulation of the wing disc by the Dpp-Brk system
Based on our observations and the above conclusions we summarize
Dpp-mediated growth control in the wing disc as follows
(Fig. 7). The disc consists of
at least two different cell populations, medial and lateral, which have
distinct abilities to proliferate. The Dpp signal is required to even out
these growth differences and establish a uniform pattern of cell proliferation
within the wing primordium. Medial cells must sense high levels of Dpp to shut
down brk expression, which consequently promotes medial
proliferation. Lateral cells have a growth advantage and must receive little
or no Dpp input to allow brk expression. The action of Brk curbs
lateral proliferation. We do not know how intermediate Brk levels affect the
proliferative behavior of cells situated between lateral and medial cells.
However, we can conclude from our results that differential pathway activity
between neighboring cells is not necessary to direct proliferation, as
constitutively high Dpp levels in the medial area and nil or low levels in the
lateral areas are sufficient for uniform and normal cell proliferation rates
throughout the disc.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/24/4003/DC1
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ACKNOWLEDGMENTS |
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TOP
SUMMARY
INTRODUCTION
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RESULTS
DISCUSSION
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