Centro de Biología Molecular CSIC-UAM, Universidad Autónoma de Madrid, Madrid 28049, Spain
Author for correspondence (e-mail:
gmorata{at}cbm.uam.es)
Accepted 29 July 2004
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Dpp pathway, Brinker, Growth control, Wing disc, Drosophila
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In normal circumstances, the different parts of an organism grow in a
coherent manner: each organ reaches a size related to the overall size. When,
after experimental (e.g. malnutrition) or genetic [mutations defective in the
Insulin pathway; reviewed by Stocker and Hafen
(Stocker and Hafen, 2000)]
manipulations, the overall body size of Drosophila is altered, all
organs are correspondingly modified, indicating the existence of a general
mechanism that controls growth.
Superimposed with this overall mechanism there have to be other local
processes controlling growth in individual organs and tissues. For example,
the imaginal discs of Drosophila grow by active cell division during
most of the larval period and stop growing at the beginning of pupation
(García-Bellido and Merriam,
1971a). By contrast, the abdominal histoblasts do not divide
during the larval period and start rapid proliferation at the beginning of
pupation (García-Bellido and
Merriam, 1971b
; Madhavan and
Madhavan, 1984
). These two organs use different modes of
growth.
The imaginal discs of Drosophila provide a convenient model with
which to study growth and size control. The wing disc begins cell
proliferation at the first larval instar when it contains 30-50 cells
(Lawrence and Morata, 1977
;
Morata and Garcia-Bellido,
1976
) and reaches the final size at the onset of pupation, with
about 50,000 cells. The proliferation rate appears to be uniform in the
different regions of the disc, and is about 9 hours per division cycle
(Garcia-Bellido and Merriam,
1971a
; Johnston and Sanders,
2003
).
The wing disc contains endogenous factors that promote, as well as others
arrest, growth (Bryant and Simpson,
1984). For example, a young disc will continue growing when
cultured in vivo but will not grow beyond the size corresponding to the mature
disc, even if it is maintained in in vivo culture for several additional days
(Bryant, 1975
;
Kirby et al., 1982
). This is
in contrast to the behaviour of dissociated disc cells or disc fragments under
similar culture conditions, which can grow indefinitely and often
transdetermine (Gehring,
1976
). This indicates the existence of some internal mechanism,
presumably related with the dimensions and the physical integrity of the disc,
that stops growth at the appropriate developmental stage.
The Dpp signalling pathway is a key factor involved in establishing pattern
and growth in the wing disc (Podos and
Ferguson, 1999; Strigini and
Cohen, 1999
). The dpp gene is expressed in a narrow
stripe close to the AP compartment boundary, but the Dpp protein diffuses in
anterior and posterior directions forming a concentration gradient
(Entchev et al., 2000
;
Lecuit et al., 1996
;
Nellen et al., 1996
;
Teleman and Cohen, 2000
;
Zecca et al., 1995
). Through a
well-characterised transduction pathway (reviewed by
Raftery and Sutherland, 1999
;
Tabata, 2001
), the Dpp signal
activates different target genes according to its local concentration. The
local values of Dpp therefore reflect a measure of the distance relative to
the AP border, thus providing a positional cue. Various Dpp targets already
identified, such as spalt (sal), optomotor blind
(omb), vestigial (vg), are positively regulated by
Dpp and appear to be involved in the patterning of specific regions of the
wing (de Celis et al., 1996
;
Grimm and Pflugfelder, 1996
;
Kim et al., 1996
;
Lecuit et al., 1996
;
Podos and Ferguson, 1999
;
Sturtevant et al., 1997
). One
particular target is the transcriptional repressor brinker
(brk), which is negatively regulated by Dpp, but, where active, is
able to block the expression of Dpp target genes
(Campbell and Tomlinson, 1999
;
Jazwinska et al., 1999
;
Minami et al., 1999
);
brk behaves as a general antagonist of the Dpp pathway. Recent
evidence indicates that the Dpp gradient is converted into an inverse gradient
of brk (Muller et al.,
2003
). As the Dpp targets can be activated in absence of Dpp
activity (Marty et al., 2000
),
it can be argued that it is the local levels of brk that determine
the pattern and growth of the disc. In this report, we refer to the Dpp/Brk
gradient as a single biological function, assuming that the intracellular
concentrations of Dpp are converted in the nuclei of the cells into the
corresponding levels of the transcriptional repressor Brk.
One of the functions of Dpp is to stimulate growth: cells deficient for the
activity of the Dpp receptor thick veins (tkv) do not proliferate,
even when they are located away from the Dpp source
(Burke and Basler, 1996),
indicating that it stimulates growth at a distance. Conversely, cells with
unrestricted activity of the Tkv receptor proliferate in excess
(Martín-Castellanos and Edgar,
2002
). Other additional evidence for the growth-promoting role of
Dpp comes from experiments in which Dpp activity is forced outside its normal
domain (Burke and Basler, 1996
;
Capdevila and Guerrero, 1994
;
Haerry et al., 1998
;
Zecca et al., 1995
). The usual
outcome is the appearance of outgrowths associated with local
duplications.
As Dpp functions may be mediated by brk, it follows that the
latter has a role in growth control. Indeed, there is evidence that
alterations in brk activity affect growth: brk mutant discs
are bigger than wild type (Campbell and
Tomlinson, 1999), and clones of brk
cells produce local outgrowths (Campbell
and Tomlinson, 1999
; Jazwinska
et al., 1999
; Minami et al.,
1999
). In addition, recent work
(Moreno et al., 2002
) has
shown that in certain circumstances brk is able to trigger programmed
cell death (apoptosis) to eliminate slow dividing cells, a property that may
play a role in regulating growth.
Recent reports (Brennecke et al.,
2003; Harvey et al.,
2003
; Hipfner et al.,
2002
; Jia et al.,
2003
; Kango-Singh et al.,
2002
; Pantalacci et al.,
2003
; Tapon et al.,
2002
; Udan et al.,
2003
; Wu et al.,
2003
) have identified several genes involved in the control of
cell proliferation, notably bantam, hippo (hpo),
salvador (sav) and warts (wts).
bantam encodes a 21 nucleotide microRNA that promotes cell division
and prevents apoptosis (Hipfner et al.,
2002
; Brennecke et al.,
2003
). Genes encoding miRNAs are supposed to be
post-transcriptional regulators, interfering with the function of their target
genes by a mechanism similar to RNA-mediated interference
(Ruvkun, 2001
). Thus,
bantam would be expected to suppress target genes that repress cell
proliferation and promote apoptosis. Indeed, Brennecke et al.
(Brennecke et al., 2003
) have
shown that bantam suppresses the pro-apoptotic gene hid.
In this report, we study the role of the Dpp pathway and brk in the growth of the wing disc. We show that the growth-promoting activity of the Dpp pathway is achieved by repression of brk, which functions as a growth repressor in a concentration-dependent manner. We also show that although brk is able to induce apoptosis, its role in preventing growth in the wing disc is not mediated by massive apoptosis, but by arresting cell proliferation. We present evidence that brk downregulates bantam
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
For gain-of-function experiments, the GAL4 lines used were: nub-Gal4,
C765-Gal4, en-Gal4, ap-Gal4, omb-Gal4
(Calleja et al., 1996) (M.
Calleja and G.M., unpublished) and hh-Gal4 (a gift from T. Tabata).
The UAS lines were: UAS-GFP (Bloomington Stock Center),
UAS-dppD, UAS-dppG;
UAS-dppD (Capdevila and
Guerrero, 1994
), UAS-tkvQD
(Nellen et al., 1996
),
UAS-tkvDN (Haerry et
al., 1998
), UAS-dad
(Tsuneizumi et al., 1997
),
UAS-brk (Jazwinska et al.,
1999
), UAS-p35 (Bloomington Stock center) and
UAS-puc2A (Martín-Blanco
et al., 1998
). Other strains were pucE69
(Martín-Blanco et al.,
1998
), bantam sensor
(Brennecke et al., 2003
) and
the B40 transgene (Muller et al.,
2003
) that reproduced brk expression faithfully. To
induce brk clones in territories where Dpp pathway
is inactivated, larvae of y w brkM68 f36 FRT18A/y w
hs-GFP hsFlp FRT18A; nub-Gal4/UAS-dad were heat shocked at 37°C for
15 minutes at 48-72 hours after egg-laying.
Histochemistry
Fixation and inmunohistochemistry of imaginal discs were carried out as
described (Aldaz et al., 2003).
The following antibodies and dilutions were used: rabbit anti-cleaved caspase
3, 1:50 (Cell Signalling Technology); mouse anti-wg, 1:50 (Hybridoma
Center); rabbit anti-ß-Gal, 1:2000 (Cappel); and rabbit
anti-Phospho-Histone H3, 1:400 (Cell Signalling Technology). Secondary
Antibodies used were purchased from Jackson Inmunoresearch.
The TdT-mediated dUTP nick end-labelling (TUNEL) assay was performed
following the in situ cell death detection kit as described
(Wang et al., 1999). BrdU
staining was carried out as described
(Udan et al., 2003
).
Images were taken in confocal microscopes MicroRadiance (BioRad) or LSM510 META (Zeiss), and subsequently processed using Zeiss LSM Image Browser or MetaMorph and Adobe Photoshop.
Preparation of adult cuticles
The adult flies were dissected in water and cut into pieces. They were then
treated with 10% KOH at 95°C for 3-5 minutes to digest internal tissues,
washed with water, rinsed in ethanol and mounted in Euparal. The preparations
were studied and photographed using a Zeiss photomicroscope.
Bantam sequence analysis
By using the Target Explorer tool
(Sosinsky et al., 2003), we
generated a weight matrix with a set of sequences that have been shown to
interact physically and functionally with Brk protein
(Barrio and de Celis, 2004
;
Rushlow et al., 2001
;
Saller et al., 2002
;
Sivasankaran et al., 2000
). We
searched for these binding sites in a 20 kb fragment of DNA containing the
bantam sequence and found two possible sites (GCAGCGCCAC and
TCAGCGCCAC), 700 bp and 500 bp upstream bantam.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have tested in detail how the growth of the wing disc is affected by
modifications of the Dpp pathway. We have used the Gal4/UAS method
(Brand and Perrimon, 1993) to
alter the active levels of the pathway and have examined their effects on the
size of the disc or of the adult wing. Some constructs allow modification of
the amount or the distribution of the Dpp signal (UAS-dpp), whereas
others permit the interference with Dpp transduction:
UAS-tkvQD, UAS-tkvDN and
UAS-dad. TkvQD is a modified form of the Tkv receptor that
causes a constitutive activity of the pathway
(Nellen et al., 1996
), whereas
TkvDN is a dominant-negative form that causes a reduction of
activity (Haerry et al.,
1998
). daughters against dpp (dad) is a negative
modulator of the pathway; it encodes a Smad protein that interferes with the
phosphorylation of the Mad protein, a Dpp transducer, and with its interaction
with the co-factor Medea (Inoue et al.,
1998
; Tsuneizumi et al.,
1997
). Raising dad levels produces a debilitation or
inactivation of the Dpp pathway (Inoue et
al., 1998
; Muller et al.,
2003
; Tsuneizumi et al.,
1997
).
We have used Gal4 lines that permit the discrimination of the major regions of the wing. The nub-Gal4 and C765 lines drive expression uniformly in the wing region, so we can examine the response of all wing cells to alterations of ligand concentration or of other components of the pathway. One advantage of the use of these lines is that, as alterations are mostly restricted to the wing blade, nearly all the combinations are viable or produce pharate adults, so that the effects can be examined in differentiated wings and in imaginal discs
The general result is that the size of the wing correlates with the activity of the Dpp pathway. Some of the results are shown in Fig. 1. The increase of Dpp signal in nub-Gal4>UAS-dpp (Fig. 1A,B) results in discs in which the wing pouch is bigger than the wild type (Fig. 1C), whereas the inhibition of Dpp activity in nub-Gal4>UAS-dad produces a very small wing pouch (Fig. 1D). The comparison of Fig. 1A,B is of interest because the only difference between the two discs is the amount of Dpp signal; their difference in size illustrates clearly the dependence of growth on the levels of Dpp activity. The effect observed in the discs can also be visualised in adult wings. In the series of genotypes shown in Fig. 1F-I, the gradual decrease in the size of the adult wing correlates with the levels of activity of the Dpp pathway.
|
The previous result suggests that the Dpp pathway affects wing size by
regulating brk activity, and is coherent with the fact that it
represses brk expression
(Campbell and Tomlinson, 1999;
Jazwinska et al., 1999
;
Minami et al., 1999
). Then it
would be expected that there should be a negative correlation between wing
size and brk levels. This is indeed the case, as illustrated in
Fig. 2. This observation
suggests that brk functions as a growth repressor and that the
excessive growth observed in genotypes with high levels of Dpp activity
(Fig. 1A,B,F;
Fig. 2A,B) is due to
suppression of brk in the wing pouch.
|
|
The role of brk on growth can also be demonstrated in
misexpression experiments. We have forced brk activity in various
regions of the disc using the Gal4 lines described above. In the combinations
C765-Gal4>UAS-brk or nub-Gal4>UAS-brk, there is
brk expression in the whole of the wing blade. In all these
combinations, it can be observed that the size of the wing is greatly reduced
(Fig. 4). The degree of the
diminution correlates with the amount of Brk, as illustrated in
Fig. 4 for the combination
C765-Gal4>UAS-brk. At 17°C, the activity of the Gal4 protein
is lower than at 25°C or 29°C
(Brand and Perrimon, 1993), and
this is reflected in the amount of Brk protein synthesised. We observe a clear
difference of size both in differentiated wings
(Fig. 4A,B) and in discs
(Fig. 4C-E) grown at different
temperatures. This result is significant, for it indicates that the Brk
protein represses growth in a concentration-dependent manner.
|
|
We first checked the occurrence of apoptosis in cases in which elevated levels of brk cause a large reduction in wing size. In normal wing discs, the levels of apoptosis markers such as TUNEL and the cleaved (active) form of caspase 3 is variable, but low and scattered. In the wing pouch of mature nub-Gal4>UAS-brk wing discs, we find a slight increase of caspase 3 (Fig. 6A,B) and TUNEL (Fig. 6C,D), but most of brk-expressing cells fail to show these markers.
|
The results of the previous experiments were intriguing, because there is
evidence that alterations in brk levels cause JNK-mediated apoptosis
(Adachi-Yamada and O'Connor,
2002; Moreno et al.,
2002
). The former authors have proposed that this form of
apoptosis occurs when there is a disruption in the normal Dpp signalling
gradient (and hence of the Brk gradient). This apoptosis aims to eliminate
cells with disparate Dpp activity levels in order to restore the normal smooth
gradient. The implication is that brk induces JNK activity only where
there is a discontinuity of expression. We tested this by inducing
brk activity with the hh-Gal4, en-Gal4 and ap-Gal4
drivers, and have monitored JNK activity with the puc-lacZ insertion
(Martín-Blanco et al.,
1998
). These experiments generate a sharp discontinuity of
brk at the AP (hh-Gal4>UAS-brk, en-Gal4>UAS-brk) or
the DV (ap-Gal4>UAS-brk) borders. Some of the results are
illustrated in Fig. 7: in
hh-Gal4>UAS-brk puc-lacZ there is a line of puc activity
close to the AP border. Similarly, in ap-Gal4>UAS-brk puc-lacZ
there is puc activity close to the DV boundary, as previously
described by Adachi-Yamada and O'Connor
(Adachi-Yamada and O'Connor,
2002
). The activation of puc in these cases appears to be
non-autonomous, as it affects cells that do not possess brk activity
(Fig. 7C,D).
|
We tested the possibility that the JNK-mediated apoptosis described above
may contribute to the reduction in wing size. Overexpression of puc
has been shown to downregulate the activity of the JNK pathway
(Martín-Blanco et al.,
1998), and also to reduce apoptosis of cells containing high
brk levels (Moreno et al.,
2002
). We therefore constructed flies of genotypes
nub-Gal4>UAS-brk UAS-puc and omb-Gal4>UAS-brk UAS-puc,
and compared them with nub-Gal4>UAS-brk and
omb-Gal4>UAS-brk. We failed to observe any difference in wing
size.
Brk inhibits cell division and downregulates bantam
All the preceding results suggest that the growth inhibition induced by
brk is not mediated by massive apoptosis, but more likely by reducing
the rate of cell proliferation. We have checked the division rate of cells
containing high levels of brk using two different markers of cell
division: the incorporation of BrdU and the staining with an antibody that
recognises the phosphorylated form of Histone 3
(Su et al., 1998). In
wild-type discs, the levels of BrdU and PH3 staining are uniform over the
disc. In nub-Gal4>UAS-brk discs (n=10), both
proliferation markers are less expressed in the wing pouch in comparison with
other regions of the disc (Fig.
8). Similar results are obtained with hh-Gal>UAS-brk
discs (n=27) in which brk is expressed at high levels in the
posterior compartment (Fig. 8).
These results strongly suggest that the principal function of brk is
to reduce the rate of cell proliferation.
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Two different functions of brk
We find that alterations of brk expression may have two different
consequences.
Activation of the JNK pathway
This occurs when an alteration of brk expression generates a sharp
border of brk activity. We have observed this phenomenon both in
experiments inducing ectopic brk activity and in others in which
brk function is eliminated in clones of cells (see
Fig. 7). The local induction of
JNK results in apoptosis that can be visualised by the activation of caspase 3
(Fig. 7G,H).
This local apoptosis induced by Brk is probably the mechanism of cell
elimination during cell competition
(Morata and Ripoll, 1975;
Moreno et al., 2002
) and
suggests that brk is involved in the elimination of slow dividing
cells or of cells that are not able to read or interpret efficiently the Dpp
pathway. This function may be aimed to keep the general fitness of the cell
population (Moreno et al.,
2002
). However, it does not appear to be involved in growth
control, because apoptosis inhibition (by means of puc or
p35 overexpression) does not eliminate the effect on size caused by
Brk.
Alterations of cell proliferation rate
Previous work has already shown that loss of brk activity results
in increased growth: in mutant brk discs there is an enlargement of
the lateral region (Campbell and
Tomlinson, 1999), and cells mutant for brk produce
outgrowths (Campbell and Tomlinson,
1999
; Jazwinska et al.,
1999
; Minami et al.,
1999
) (this work). We show that the cause for the additional
growth associated with the loss or reduction of brk activity is due
to an increase in the cell proliferation rate: brk
clones incorporate BrdU more actively than surrounding cells
(Fig. 3C). Conversely, the
repression of growth caused by elevated levels of Brk is associated with
reduced mitotic activity and BrdU incorporation
(Fig. 8).
Given the nature of the Brk protein, it would be expected that its role in
growth be mediated by transcriptional repression of genes involved in cell
division and proliferation. Our results indicate that it acts as a repressor
of bantam (Fig. 9),
although this control may not be direct. Given that Bantam protein is itself a
post-transcriptional regulator of cell division genes
(Brennecke et al., 2003), this
observation suggests that Brk occupies a high position in the genetic
hierarchy controlling cell proliferation. Its activity links Dpp signalling
and cell proliferation.
Control of growth by the Brk gradient
Our results, and those of others (Burke
and Basler, 1996;
Martín-Castellanos and Edgar,
2002
), have established that the Dpp pathway is involved in the
control of growth of the wing (and of other appendages; data not shown). The
activity of the Dpp pathway has a positive effect on growth, and, furthermore,
we find that the growth response of the disc correlates with its levels of
activity. This graded response is of interest, as it suggests that growth
control mechanisms recognise different concentrations of inducing or
repressing factors. This result has implications in the understanding of these
mechanisms; classically, it has been argued that proliferation in the imaginal
discs is a response to confrontation of cells with different positional values
(French et al., 1976
;
Haynie and Bryant, 1976
). Our
results in the wing disc do not support this view, as they suggest that growth
is a lineal response to Dpp/Brk activity.
Our results also indicate that the role of Dpp on growth is mediated by brk. The simplest view is that as the Dpp gradient is converted into an inverse Brk gradient, the concentration-dependent stimulus of Dpp on growth should be converted into a concentration-dependent repression by Brk. Our demonstration (Fig. 4) that the effect of Brk on wing size depends on the amount of protein supports this view.
There are several arguments that implicate brk as a principal
factor controlling growth. First, loss of brk activity leads to
increased proliferation (Fig.
3A-C). This is consistent with previous observations
(Campbell and Tomlinson, 1999)
showing that brk wing discs are bigger than wild-type discs.
Furthermore, this excessive proliferation can occur in absence of Dpp
activity. Fig. 3D shows two
overgrowing brk mutant clones originated from the wing pouch of
nub-Gal4>UAS-dad discs in which Dpp function is obliterated or
much reduced. Second, increased or ectopic brk levels block or reduce
growth, even though brk does not alter dpp expression
(Fig. 4A-E). And, third, the
stimulation caused by the Dpp pathway on growth requires repression of
brk. This is demonstrated by our finding that the presence of Brk
protein suppresses the excessive growth caused by Dpp hyperactivity
(Fig. 5).
Together, these observations indicate that growth does not require direct
input from Dpp, but simply its repression of brk. However, the
repression of brk by Dpp is an important developmental phenomenon
because in the absence of such control brk would become
constitutively active, thus repressing all or the majority of Dpp targets.
Recent work (Muller et al.,
2003) has identified two control elements in the brk
regulatory region: a Dpp-regulated silencer that contains binding sites for
the Mad/Medea complex; and a constitutive enhancer. This enhancer is probably
responsible of the generalised brk expression in the absence of Dpp
activity.
What is the role of brk in normal development? Our results
demonstrate that Brk has the properties of a growth repressor and can perform
this function all over the wing. However, in wild-type wing discs,
brk is expressed only in the lateral region and therefore its
repressing role is limited to this region. This is agreement with the
observation that brk clones overgrow only on the
sides of the disc (Campbell and Tomlinson,
1999; Jazwinska et al.,
1999
; Minami et al.,
1999
) (this work).
The restriction of the role of brk to the lateral region is
intriguing, because if it were the only repressor it would be expected that
the central region, where there is no brk activity, would grow more
than the lateral one. The overall growth of the different wing regions is
uniform; not only does clone size fail to change in the different wing regions
(Garcia-Bellido and Merriam,
1971a) but BrdU incorporation and PH3 staining are also uniform
(Milan et al., 1996; Johnston and Sanders,
2003
). This suggests that there another factor located in the
centre of the disc should exist that represses growth in the absence of
brk. This hypothetical gene would fulfil in the centre of the wing
the role that brk performs in the lateral region.
In principle, a candidate could be daughters against dpp
(dad), a Dpp target that is expressed at high levels in the centre of
the disc. We have observed that dad overexpression reduces growth.
However, this appears to be achieved by allowing high brk levels
(Fig. 2G,H) subsequent to
slackening of Dpp activity (Tsuneizumi et
al., 1997), indicating that the effect of dad is mediated
by brk. Thus, dad appears to be a Dpp modulator with no
direct role in growth. Our finding that brk clones
containing high levels of dad activity can overgrow
(Fig. 3D) also supports this
view.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Adachi-Yamada, T. and O'Connor, M. B. (2002). Morphogenetic apoptosis: a mechanism for correcting discontinuities in morphogen gradients. Dev. Biol. 251, 74-90.[CrossRef][Medline]
Adachi-Yamada, T., Fujimura-Kamada, K., Nishida, Y. and Matsumoto, K. (1999). Distorsion of proximodistal information causes JNK-dependent apoptosis in Drosophila wing. Nature 400,166 -169.[CrossRef][Medline]
Aldaz, S., Morata, G. and Azpiazu, N. (2003).
The Pax-homeobox gene eyegone is involved in the subdivision of the thorax of
Drosophila. Development
130,4473
-4482.
Barrio, R. and de Celis, J. F. (2004).
Regulation of spalt expression in the Drosophila wing blade in response to the
Decapentaplegic signaling pathway. Proc. Natl. Acad. Sci.
USA 101,6021
-6026.
Brand, A. H. and Perrimon, N. (1993). Targeted
gene expression as a means of altering cell fates and generating dominant
phenotypes. Development
118,401
-415.
Brennecke, J., Hipfner, D. R., Stark, A., Russell, R. B. and Cohen, S. M. (2003). bantam encodes a developmentally regulated microRNA that controls cell proliferation and regulates the proapoptotic gene hid in Drosophila. Cell 113, 25-36.[Medline]
Bryant, P. (1975). Pattern formation in the imaginal wing disc of Drosophila melanogaster: fate map, regeneration and duplication. J. Exp. Zool. 193, 49-78.[Medline]
Bryant, P. J. and Simpson, P. (1984). Intrinsic and extrinsic control of growth in developing organs. Q. Rev. Biol. 59,387 -415.[Medline]
Burke, R. and Basler, K. (1996). Dpp receptors
are autonomously required for cell proliferation in the entire developing
Drosophila wing. Development
122,2261
-2269.
Calleja, M., Moreno, E., Pelaz, S. and Morata, G.
(1996). Visualization of gene expression in living adult
Drosophila. Science 274,252
-255.
Campbell, G. and Tomlinson, A. (1999). Transducing the Dpp morphogen gradient in the wing of Drosophila: regulation of Dpp targets by brinker. Cell 96,553 -562.[Medline]
Capdevila, J. and Guerrero, I. (1994). Targeted expression of the signaling molecule decapentaplegic induces pattern duplications and growth alterations in Drosophila wings. EMBO J. 13,4459 -4468.[Abstract]
de Celis, J. F., Barrio, R. and Kafatos, F. C. (1996). A gene complex acting downstream of dpp in Drosophila wing morphogenesis. Nature 381,421 -424.[CrossRef][Medline]
Entchev, E. V., Schwabedissen, A. and Gonzalez-Gaitan, M. (2000). Gradient formation of the TGF-beta homolog Dpp. Cell 103,981 -991.[Medline]
French, V., Bryant, P. J. and Bryant, S. V. (1976). Pattern regulation in epimorphic fields. Science 193,969 -981.[Medline]
García-Bellido, A. and Merriam, J. (1971a). Parameters of the wing imaginal disc development of Drosophila melanogaster. Dev. Biol. 24, 61-87.[Medline]
García-Bellido, A. and Merriam, J. R. (1971b). Clonal parameters of tergite development in Drosophila. Dev. Biol. 26,264 -276.[Medline]
Gehring, W. (1976). Developmental genetics of Drosophila. Ann. Rev. Genet. 10,209 -252.[CrossRef][Medline]
Grimm, S. and Pflugfelder, G. O. (1996). Control of the gene optomotor-blind in Drosophila wing development by dacapentaplegic and wingless. Science 271,1601 -1604.[Abstract]
Haerry, T. E., Khalsa, O., O'Connor, M. B. and Wharton, K.
A. (1998). Synergistic signaling by two BMP ligands through
the SAX and TKV receptors controls wing growth and patterning in Drosophila.
Development 125,3977
-3987.
Harvey, K. F., Pfleger, C. M. and Hariharan, I. K. (2003). The Drosophila Mst ortholog, hippo, restricts growth and cell proliferation and promotes apoptosis. Cell 114,457 -467.[Medline]
Hay, B. A., Wolff, T. and Rubin, G. M. (1994).
Expression of baculovirus P35 prevents cell death in Drosophila.
Development 120,2121
-2129.
Haynie, J. L. and Bryant, P. J. (1976). Intercalary regeneration in imaginal wing disk of Drosophila melanogaster. Nature 259,659 -662.[Medline]
Hipfner, D. R., Weigmann, K. and Cohen, S. M.
(2002). The bantam gene regulates Drosophila growth.
Genetics 161,1527
-1537.
Inoue, H., Imamura, T., Ishidou, Y., Takase, M., Udagawa, Y.,
Oka, Y., Tsuneizumi, K., Tabata, T., Miyazono, K. and Kawabata, M.
(1998). Interplay of signal mediators of decapentaplegic (Dpp):
molecular characterization of mothers against dpp, Medea, and daughters
against dpp. Mol. Biol. Cell
9,2145
-2156.
Jazwinska, A., Kirov, N., Wieschaus, E., Roth, S. and Rushlow, C. (1999). The Drosophila gene brinker reveals a novel mechanism of Dpp target gene regulation. Cell 96,563 -573.[Medline]
Jia, J., Zhang, W., Wang, B., Trinko, R. and Jiang, J.
(2003). The Drosophila Ste20 family kinase dMST functions as a
tumor suppressor by restricting cell proliferation and promoting apoptosis.
Genes Dev. 17,2514
-2519.
Johnston, L. A. and Sanders, A. L. (2003). Wingless promotes cell survival but constrains growth during Drosophila wing development. Nat. Cell Biol. 5, 827-833.[CrossRef][Medline]
Kango-Singh, M., Nolo, R., Tao, C., Verstreken, P., Hiesinger, P. R., Bellen, H. J. and Halder, G. (2002). Shar-pei mediates cell proliferation arrest during imaginal disc growth in Drosophila. Development 129,5719 -5730.[CrossRef][Medline]
Kim, J., Sebring, A., Esch, J. J., Kraus, M. E., Vorwerk, K., Magee, J. and Carroll, S. B. (1996). Integration of positional signals and regulation of wing formation and identity by Drosophila vestigial gene. Nature 382,133 -138.[CrossRef][Medline]
Kirby, B., Bryant, P. and Schneiderman, H. (1982). Regeneration following duplication in imaginal wing disc fragments of Drosophila melanogaster. Dev. Biol. 90,259 -271.[Medline]
Lawrence, P. and Morata, G. (1977). The early development of mesothoracic compartments in Drosophila. An analysis of cell lineage and fate mapping and an assesment of methods. Dev. Biol. 5640 -51.[Medline]
Lecuit, T., Brook, W. J., Ng, M., Calleja, M., Sun, H. and Cohen, S. M. (1996). Two distinct mechanisms for long-range patterning by Decapentaplegic in the Drosophila wing. Nature 381,387 -393.[CrossRef][Medline]
Madhavan, M. M. and Madhavan, K. (1984). Do larval epidermal cells possess the blueprint for adult pattern in Drosophila? J. Embryol. Exp. Morph. 82, 1-8.
Martín-Blanco, E., Gampel, A., Ring, J., Virdee, K.,
Kirov, N., Tolkovsky, A. M. and Martinez, A. A. (1998).
puckered encodes a phosphatase that mediates a feedback loop regulating JNK
activity during dorsal closure in Drosophila. Genes
Dev. 12,557
-570.
Martín-Castellanos, C. and Edgar, B. A. (2002). A characterization of the effects of Dpp signaling on cell growth and proliferation in the Drosophila wing. Development 129,1003 -1013.[Medline]
Marty, T., Muller, B., Basler, K. and Affolter, M. (2000). Schnurri mediates Dpp-dependent repression of brinker transcription. Nat. Cell Biol. 2, 745-749.[CrossRef][Medline]
Milan, M., Campuzano, S. and García-Bellido, A.
(1997). Developmental parameters of cell death in the wing disc
of Drosophila. Proc. Natl. Acad. Sci. USA
94,5691
-5696.
Minami, M., Kinoshita, N., Kamoshida, Y., Tanimoto, H. and Tabata, T. (1999). brinker is a target of Dpp in Drosophila that negatively regulates Dpp-dependent genes. Nature 398,242 -246.[CrossRef][Medline]
Morata, G. and García-Bellido, A. (1976). Developmental analysis of some mutants of the bithorax sistem of the Drosophila. Rouxs Arch. Dev. Biol. 179,125 -143.
Morata, G. and Ripoll, P. (1975). Minutes: mutants of drosophila autonomously affecting cell division rate. Dev. Biol. 42,211 -221.[Medline]
Moreno, E., Basler, K. and Morata, G. (2002). Cells compete for decapentaplegic survival factor to prevent apoptosis in Drosophila wing development. Nature 416,755 -759.[CrossRef][Medline]
Muller, B., Hartmann, B., Pyrowolakis, G., Affolter, M. and Basler, K. (2003). Conversion of an extracellular Dpp/BMP morphogen gradient into an inverse transcriptional gradient. Cell 113,221 -233.[Medline]
Nellen, D., Burke, R., Struhl, G. and Basler, K. (1996). Direct and long-range action of a DPP morphogen gradient. Cell 85,357 -368.[Medline]
Pantalacci, S., Tapon, N. and Leopold, P. (2003). The Salvador partner Hippo promotes apoptosis and cell-cycle exit in Drosophila. Nat. Cell Biol. 5, 921-927.[CrossRef][Medline]
Podos, S. D. and Ferguson, E. L. (1999). Morphogen gradients: new insights from DPP. Trends Genet. 15,396 -402.[CrossRef][Medline]
Raftery, L. A. and Sutherland, D. J. (1999). TGF-beta family signal transduction in Drosophila development: from Mad to Smads. Dev. Biol. 210,251 -268.[CrossRef][Medline]
Rushlow, C., Colosimo, P. F., Lin, M. C., Xu, M. and Kirov,
N. (2001). Transcriptional regulation of the Drosophila gene
zen by competing Smad and Brinker inputs. Genes Dev.
15,340
-351.
Ruvkun, G. (2001). Molecular biology. Glimpses
of a tiny RNA world. Science
294,797
-799.
Saller, E., Kelley, A. and Bienz, M. (2002).
The transcriptional repressor Brinker antagonizes Wingless signaling.
Genes Dev. 16,1828
-1838.
Sivasankaran, R., Vigano, M. A., Muller, B., Affolter, M. and
Basler, K. (2000). Direct transcriptional control of the Dpp
target omb by the DNA binding protein Brinker. EMBO J.
19,6162
-6172.
Sosinsky, A., Bonin, C. P., Mann, R. S. and Honig, B.
(2003). Target Explorer: an automated tool for the identification
of new target genes for a specified set of transcription factors.
Nucleic Acids Res. 31,3589
-3592.
Stocker, H. and Hafen, E. (2000). Genetic control of cell size. Curr. Opin. Genet. Dev. 10,529 -535.[CrossRef][Medline]
Strigini, M. and Cohen, S. M. (1999). Formation of morphogen gradients in the Drosophila wing. Semin. Cell Dev. Biol. 10,335 -344.[CrossRef][Medline]
Sturtevant, M. A., Biehs, B., Marin, E. and Bier, E.
(1997). The spalt gene links the A/P compartment boundary to a
linear structure in the Drosophila wing. Development
124, 21-32.
Su, T. T., Sprenger, F., DiGregorio, P. J., Campbell, S. D. and
O'Farrell, P. H. (1998). Exit from mitosis in Drosophila
syncytial embryos requires proteolysis and cyclin degradation, and is
associated with localized dephosphorylation. Genes
Dev. 12,1495
-1503.
Tabata, T. (2001). Genetics of morphogen gradients. Nat. Rev. Genet. 2, 620-630.[CrossRef][Medline]
Tapon, N., Harvey, K. F., Bell, D. W., Wahrer, D. C., Schiripo, T. A., Haber, D. A. and Hariharan, I. K. (2002). salvador Promotes both cell cycle exit and apoptosis in Drosophila and is mutated in human cancer cell lines. Cell 110,467 -478.[Medline]
Teleman, A. and Cohen, S. (2000). Dpp gradient formation in the Drosophila wing imaginal disc. Cell 103,971 -980.[Medline]
Tsuneizumi, K., Nakayama, T., Kamoshida, Y., Kornberg, T., Christian, J. and Tabata, T. (1997). Daughters against dpp modulates dpp organizing activity in Drosophila wing development. Nature 389,627 -631.[CrossRef][Medline]
Udan, R. S., Kango-Singh, M., Nolo, R., Tao, C. and Halder, G. (2003). Hippo promotes proliferation arrest and apoptosis in the Salvador/Warts pathway. Nat. Cell Biol. 5, 914-920.[CrossRef][Medline]
Wang, S. L., Hawkins, C. J., Yoo, S. J., Muller, H. A. and Hay, B. A. (1999). The Drosophila caspase inhibitor DIAP1 is essential for cell survival and is negatively regulated by HID. Cell 98,453 -463.[Medline]
Wolff, T. and Ready, D. F. (1991). Cell death in normal and rough eye mutants of Drosophila. Development 113,825 -839.[Abstract]
Wu, S., Huang, J., Dong, J. and Pan, D. (2003). hippo encodes a Ste-20 family protein kinase that restricts cell proliferation and promotes apoptosis in conjunction with salvador and warts. Cell 114,445 -456.[Medline]
Zecca, M., Basler, K. and Struhl, G. (1995). Sequential organizing activities of engrailed Hedhehog and decapentaplegic in the Drosophila wing. Development 121,2565 -2578.