RESEARCH ARTICLE
343
Development 135, 343-351 (2008) doi:10.1242/dev.008920
Drosophila eggshell is patterned by sequential action of
feedforward and feedback loops
Nir Yakoby1,*, Jessica Lembong1,*, Trudi Schüpbach2 and Stanislav Y. Shvartsman1,†
During Drosophila oogenesis, patterning activities of the EGFR and Dpp pathways specify several subpopulations of the follicle cells
that give rise to dorsal eggshell structures. The roof of dorsal eggshell appendages is formed by the follicle cells that express Broad
(Br), a zinc-finger transcription factor regulated by both pathways. EGFR induces Br in the dorsal follicle cells. This inductive signal is
overridden in the dorsal midline cells, which are exposed to high levels of EGFR activation, and in the anterior cells, by Dpp
signaling. We show that the resulting changes in the Br pattern affect the expression of Dpp receptor thickveins (tkv), which is
essential for Dpp signaling. By controlling tkv, Br controls Dpp signaling in late stages of oogenesis and, as a result, regulates its
own repression in a negative-feedback loop. We synthesize these observations into a model, whereby the dynamics of Br expression
are driven by the sequential action of feedforward and feedback loops. The feedforward loop controls the spatial pattern of Br
expression, while the feedback loop modulates this pattern in time. This mechanism demonstrates how complex patterns of gene
expression can emerge from simple inputs, through the interaction of regulatory network motifs.
INTRODUCTION
Pattern formation in development relies on combinatorial
interactions of a small number of signaling pathways, which can act
either in parallel, converging on the enhancers of common target
genes, or regulate each other at the level of signal generation,
reception, transmission and interpretation (Martinez-Arias and
Stewart, 2002). Biochemical and cellular mechanisms of signaling
crosstalk become progressively characterized, but the dynamics of
pathway integration at the tissue level remains poorly understood.
Here we show how the evolutionarily conserved EGFR and BMP
pathways interact in a network that controls epithelial patterning in
Drosophila oogenesis, an established genetic model for studying the
connection between signaling and developmental pattern formation
(Deng and Bownes, 1998; Dobens and Raftery, 2000; Berg, 2005;
Horne-Badovinac and Bilder, 2005).
The three-dimensional shell of the Drosophila egg is derived from
the follicular epithelium in the developing egg chamber (Fig. 1A),
(Spradling, 1993). Dorsal eggshell structures, the respiratory dorsal
appendages and the operculum, are formed by the dorsoanterior
follicle cells, which are patterned by Gurken (Grk), an EGFR ligand
secreted from the oocyte (Schüpbach, 1987), and Dpp, a Bmp2/4like molecule produced by the stretch follicle cells (Twombly et al.,
1996; Peri and Roth, 2000). Each dorsal appendage is formed by two
groups of dorsolateral follicle cells (Dorman et al., 2004; Berg,
2005). The roof of the appendage is formed by cells expressing
Broad (Br), a zinc-finger transcription factor (Deng and Bownes,
1997; Tzolovsky et al., 1999). The floor of the appendage is derived
from the adjacent domain of cells expressing rhomboid (rho), which
encodes an intracellular protease in the Drosophila EGFR pathway
(Ruohola-Baker et al., 1993; Urban et al., 2001). Genetic approaches
1
Department of Chemical Engineering and Lewis-Sigler Institute for Integrative
Genomics, Princeton University, NJ 08544, USA. 2Howard Hughes Medical Institute
and Department of Molecular Biology, Princeton University, NJ 08544, USA.
*These authors contributed equally to this work
†
Author for correspondence (e-mail: [email protected])
Accepted 17 October 2007
have established that the wild-type expression patterns of both br
and rho depend on both EGFR and Dpp signaling (NeumanSilberberg and Schüpbach, 1994; Deng and Bownes, 1997; Peri et
al., 1999; Atkey et al., 2006). However, the origin of the dynamics
of br and rho expression and their connection to eggshell patterning
remained unclear.
Eggshell morphology can be dramatically affected by variations
in the levels of Gurken and Dpp (Neuman-Silberberg and
Schüpbach, 1994; Twombly et al., 1996; Dobens et al., 2000). Given
this degree of sensitivity, it is unclear how these two inductive
signals, which originate from two different tissues, coordinate their
actions in order to establish the elaborate gene expression patterns
and eggshell morphology. One mechanism for coordinating the
actions of the EGFR and Dpp pathways may involve signaling
crosstalk. Indeed, recent studies have shown that EGFR signaling
influences the effects of Dpp by regulating the expression of
inhibitors of Dpp signaling and have demonstrated that this type of
pathway interaction is important for the formation of the Br
expression domain (Chen and Schüpbach, 2006; Shravage et al.,
2007).
Here we demonstrate that the patterning mechanism is
considerably more complex. We found that, in addition to being a
target of Dpp signaling, Br regulates the Dpp pathway in late stages
of oogenesis by controlling the expression of the type I Dpp
receptor thickveins (tkv). The Br-dependent changes in tkv
expression lead to changes in the spatial pattern of Dpp signaling.
As a result, the late phase of Dpp signaling in oogenesis has a clear
dorsoventral polarity, in contrast to the early phase of Dpp
signaling, which is uniform along the dorsoventral (DV) axis. We
show that the early and late phases of Dpp signaling control
different spatial domains of the br expression pattern. The early
phase of Dpp signaling represses br in the anterior follicle cells,
whereas the late phase of Dpp signaling limits the duration of br
expression in the cells that form the roof of future dorsal
appendages. We integrate these findings with the results of previous
studies and propose a new model of eggshell patterning, in which
the pattern of Br is established by the sequential action of feedforward and feedback loops.
DEVELOPMENT
KEY WORDS: Cell signaling, Epithelial patterning, Feedforward control, Pattern formation, Drosophila
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MATERIALS AND METHODS
Genetics
The following stocks were used in this study: wild-type OreR, y w; cic flu
(Jimenez et al., 2000), X7;28.20 (which contains four copies of P[w+ grk+]
(Neuman-Silberberg and Schüpbach, 1993), EgfrQY1 (Schüpbach, 1987),
UAS-Dad (a gift from J. Duffy), CY2-Gal4 (Queenan et al., 1997; Goentoro
et al., 2006b), D. pseudoobscura (a gift from V. Orgogozo and D. Stern). D.
virilis, and D. phalerata (a gift from J. Duffy). The FLP/FRT mitotic
recombination system (Xu and Rubin, 1993; Duffy et al., 1998) was used to
generate clones of mutant follicle cells marked by the absence of GFP. The
following genotypes were used to analyze the effects of the EGFR pathway:
a null allele of Ras: e22c-Gal4 UAS-FLP; FRT 82B ras⌬C40b/FRT 82B ubi-GFP
(Hou et al., 1995; James et al., 2002). Analyses of the effects of the BMP
pathway used the following alleles: Med1: e22c-Gal4 UAS-FLP; FRT 82B
Med1/FRT 82B ubi-GFP, and a Mad12 allele: FRT 40A Mad12/FRT 40A
ubiGFP;GR1-Gal4 UAS-FLP, and a tkv12 allele: FRT 40A tkv12/FRT 40A
ubiGFP;GR1-Gal4 UAS-FLP. The Mad12, Med1 and tkv12 mutant lines are
a gift from R. Padgett. Flies were grown on agar cornmeal medium; all
crosses were done at 23°C. In Br overexpression experiments, the UAS-BrZ1 construct (a gift from L. Riddiford) was driven by the Gal80-Gal4, CY2Gal4 expression system (Suster et al., 2004). Mosaic analysis of the effects
of br– cells were done with the brCJ89 FRT19A/FRT19A; e22c-Gal4 UAS-FLP
flies (Chen and Schüpbach, 2006).
The following primers were used to amplify part of the tkv gene from D.
phalerata cDNA; forward primer 5⬘-CCATCATTGTGAGCATGTCC-3⬘
and a reverse primer 5⬘-CGGCATGCATATCATCAAAG-3⬘, in a PCR
(iCycler, BioRad). The product (933 bp) was cloned using a StrataClone
PCR Cloning Kit (Stratagene). The clone was sequenced (GeneWiz) and
BLASTed against the D. melanogaster genome (FlyBase). In situ
hybridization was carried out as previously described (Wang et al., 2006),
but without the RNase digestion step. The digoxigenin-labeled RNA probes
were made from a cDNA clone of br-Z2 (kindly provided by W-M. Deng),
tkv (kindly provided by M. O’Connor), rho (Ruohola-Baker et al., 1993),
and the tkv clone from D. phalerata.
Immunofluorescence and microscopy
Dissection and fixation of ovaries was done as described elsewhere
(Pacquelet and Rorth, 2005). Primary antibodies: mouse anti-Br core
(25E9.D7; 1:100, DSHB), rabbit anti-phosphorylated-Smad1/5/8 (1:3000,
a generous gift from D. Vasiliauskas, S. Morton, T. Jessell and E. Laufer),
rabbit or mouse anti-GFP (1:2000, Molecular Probes and Chemicon
International, respectively), rabbit or mouse Anti-HA monoclonal antibodies
(1:2000, Chemicon) and Hoechst dye to stain the nuclei (1:10,000).
Secondary antibodies: Alexa Fluor and Oregon Green (1:2000, Molecular
Probes). Images were taken with a PerkinElmer RS3 Spinning Disk
Confocal microscope and the Nikon Eclipse E800 compound microscope.
Images were processed with ImageJ (Rasband, 1997-2006) and Photoshop
(Adobe Systems, San Jose, CA). ESEM images were taken as described
elsewhere (Yakoby et al., 2005).
RESULTS
The spatial pattern of Dpp signaling acquires DV
polarity in late stages of egg development
At stages 9-10 of oogenesis, EGFR is activated in a broad DV
gradient (Fig. 1E,F). In older egg chambers the pattern of EGFR
signaling is restricted dorsally and split along the dorsal midline
(Fig. 1G). This transition is relatively well understood and depends
on the change in the identity and the source of the EGFR-activating
ligand. The early pattern is mediated by Gurken, secreted from the
dorsoanterior cortex of the oocyte (Neuman-Silberberg and
Schüpbach, 1993; Pai et al., 2000; Goentoro et al., 2006a). The later
pattern is mediated by Spitz, an EGFR ligand secreted by the two Lshaped stripes of the follicle cells on either side of the dorsal midline
(Ruohola-Baker et al., 1993; Sapir et al., 1998; Wasserman and
Freeman, 1998; Peri et al., 1999).
Fig. 1. Dynamics of EGFR and Dpp signaling in the Drosophila
follicular epithelium. (A) Phase contrast image of a stage 10 egg
chamber, showing the follicular epithelium (gray), the oocyte (white), the
nurse cells (blue) and the stretch follicle cells. (B,C) In situ hybridization
images of thickveins (tkv) expression at stage 10B of oogenesis (B, lateral
view; C, dorsal view). (D) Schematic of the tkv pattern in a stage 10B
egg chamber. (E-G) EGFR activation monitored by the nuclear
localization of Capicua (Cic), a transcription factor excluded from the
nucleus in response to EGFR signaling. (E) At stage 10A, Cic is excluded
from nuclei of the dorsal anterior follicle cells (marked by a dotted white
line). (F) At stage 10B, Cic is excluded from the nuclei within a broad
patch of dorsal cells (marked by a dotted white line). (G) Dorsal view of a
stage 12 egg chamber: Cic is excluded from the nuclei in and around
two dorsolateral stripes of the follicle cells. At the same time, nuclear Cic
reappears in the triangular patch of the dorsoanterior cells (marked by
an asterisk). (H-J) Dpp signaling monitored by the phosphorylated Mad
(P-Mad). P-Mad is first detected in the two rows of anterior follicle cells
(H). Later, P-Mad is found in a single row of ventral cells, and in at least
five rows of dorsal cells (marked by a solid white line) (I). After stage 11,
the P-Mad pattern is split along the dorsal midline (J). (K-M) Merged
images of Cic (red) and P-Mad (green). In all images anterior of the egg
chamber is to the left; the yellow dashed line marks the anterior border
of the oocyte; the arrowhead marks the dorsal midline. E,F,H,I,K,L are
lateral views, and G,J,M are dorsal views, of the egg chamber. A,
anterior; D, dorsal; FC, follicular epithelium; NC, nurse cells; P, posterior;
SC, stretch follicle cells; V, ventral.
In contrast to the dynamic pattern of EGFR activation, the pattern
of Dpp signaling is viewed as a static anteroposterior (AP) gradient
that persists through all stages of eggshell patterning (Peri and Roth,
2000; Berg, 2005; Shravage et al., 2007). Before stage 10 of
oogenesis, such a pattern can be readily explained by a model in
which the anteriorly secreted Dpp acts through receptors that are
DEVELOPMENT
In situ hybridization
Signal coordination in tissue patterning
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345
uniformly expressed throughout the follicular epithelium (Peri and
Roth, 2000; Jekely and Rorth, 2003). Later, however, the spatial
pattern of the type I Dpp receptor tkv acquires a clear DV polarity
(Mantrova et al., 1999). In stage 10B egg chambers, tkv is expressed
in a dorsoventral pattern with two lateral patches on both sides of the
dorsal midline (Fig. 1B-D).
As tkv is essential for Dpp signaling in multiple stages of fly
development, we asked whether this change in tkv expression could
lead to the corresponding change in the pattern of the Dpp pathway
activation. Consistent with this prediction, we found that the pattern
of phosphorylated Mad (P-Mad), which is uniform along the DV axis
before stage 10 (Fig. 1H), acquires a clear DV polarity in older egg
chambers. At stage 10B P-Mad was detected in approximately five
dorsal cell rows and in one to two ventral cell rows, respectively (Fig.
1I). At stages 11 and 12, the pattern of Dpp signaling split along the
dorsal midline and shifted posteriorly, which correlates with the
corresponding change in the tkv pattern (Fig. 1C). Thus, after stage
10B of oogenesis, the activation patterns of both the EGFR and Dpp
pathways are split along the dorsal midline (Fig. 1M).
The spatial pattern of tkv regulates the spatial
pattern of Dpp signaling and is sensitive to EGFR
signaling levels
To test whether tkv is essential for Dpp signaling in the follicular
epithelium, we generated GFP-marked clones of tkv– cells and
monitored the resulting change in the P-Mad pattern. We found that
P-Mad signal disappeared in clones of tkv– cells, confirming the
essential role of tkv for Dpp signaling (Fig. 3A-C). Thus, the midline
repression of tkv at stage 10 of oogenesis (Fig. 1C), can account for
the corresponding repression in the wild-type pattern of P-Mad (Fig.
1J).
Based on previous studies of eggshell patterning, the midline gap
in the tkv pattern corresponds to the highest levels of EGFR activation
in the follicular epithelium, whereas the dorsolateral patches
Fig. 2. Dynamics of Dpp signaling in related fly species. (A) The
evolutionary distance between Drosophila melanogaster (D. mel.) and
three other Drosophila species with a different number of dorsal
appendages. (B,C) Early and late patterns of P-Mad (green) and Br (red)
in D. pseudoobscura (D. pse.). The eggshell has two appendages (D), as
in D. melanogaster. (E,F) Early and late P-Mad and Br patterns in D.
virilis (D. vir.). The eggshell has four dorsal appendages (G).
(H-K) Dynamics of P-Mad and Br patterns in D. phalerata (D. pha.).
(L-N) Dynamics of tkv expression in D. pha. The eggshell has three
dorsal appendages (O). In all images the arrowhead marks the dorsal
midline; anterior of the egg chamber is to the left.
correspond to the intermediate levels of EGFR activation (NeumanSilberberg and Schüpbach, 1994; Goentoro et al., 2006a). Based on
this correlation between the estimated gradient of EGFR activation
and the wild-type tkv pattern, we hypothesized that high levels of
EGFR signaling downregulate tkv, while intermediate levels of EGFR
signaling induce tkv expression. The midline repression could be due
to Pointed, which is activated by high levels of EGFR signaling and
represses EGFR targets (Fig. 3D) (Morimoto et al., 1996; Deng and
Bownes, 1997; Wasserman and Freeman, 1998; Ward et al., 2006).
This model predicts that a stronger and broader pattern of EGFR
activation should increase the distance between the dorsolateral
patches of tkv, and that a reduced gradient of EGFR activation can
eliminate the dorsolateral patches in the tkv pattern (Fig. 3D). Both
these predictions are supported by in situ hybridization analysis of
tkv expression in mutants with quantitative variations in EGFR
signaling. The distance between the two dorsolateral patches of tkv
expression expanded as the level of the oocyte-derived Gurken
increased (Fig. 3E). At the same time, the dorsolateral patches of tkv
were abolished in the egg chambers with a hypomorphic allele of
EGFR (Fig. 3F). The P-Mad pattern closely followed these
transitions (Fig. 3G,H), once again demonstrating that Dpp signaling
is controlled by the spatial pattern of tkv.
Correlation between the dynamics of Dpp
signaling and Br expression
In the next set of experiments, we used Br, a transcription factor
expressed in the cells that form the roof of future dorsal
appendages, to study the patterning effects of Dpp signaling. Br is
DEVELOPMENT
The AP-to-DV transition in the pattern of Dpp
signaling is conserved across species
In contrast to recently published observations (Shravage et al., 2007),
our results establish that the pattern of Dpp activation undergoes a
clear transition from the purely AP to the DV pattern in late stages of
oogenesis. Remarkably, we found that this transition is conserved in
other fruit fly species (Fig. 2A). The early spatial patterns of P-Mad
were uniform along the DV axis in egg chambers of D.
pseudoobscura, D. phalerata and D. virilis (Fig. 2B,E,H). Later,
however, the P-Mad pattern in each of these species acquired a clear
DV polarity, just as it does in D. melanogaster (Fig. 2C,F,I-K). For
instance, in D. phalerata, the anterior ring of P-Mad in mid-oogenesis
underwent a transition to a ventral band of P-Mad in older egg
chambers (Fig. 2I-K). Importantly, this transition closely followed a
similar transition in the expression of tkv in this species (Fig. 2L-N).
Based on these observations, we made a number of predictions
regarding the origin and significance of the Dpp signaling dynamics
in mid- to late stages of oogenesis. First, based on the correlation
between the spatial patterns of P-Mad and tkv, we predicted that the
DV pattern of tkv controls the spatial pattern of Dpp signaling.
Second, as the DV polarity in oogenesis is due to EGFR signaling,
we predicted that the pattern of tkv depends on EGFR signaling.
Finally, based on the evolutionarily conserved nature of AP-to-DV
transition in the pattern of Dpp signaling, we predicted that this
transition is functionally significant for eggshell patterning. We have
used a combination of gain- and loss-of-function approaches to
directly test each of the predictions in D. melanogaster.
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Fig. 3. The spatial pattern of tkv regulates the spatial
pattern of Dpp signaling and is sensitive to EGFR
signaling levels. (A-C) Dpp signaling is abolished in tkv–
cells. tkv– cells are marked by the absence of GFP (green) and
Dpp signaling is monitored by P-Mad staining (red). (D) Top:
regulatory mechanism proposed for the formation of the
spatial pattern of tkv expression: tkv is positively regulated by
EGFR and negatively regulated by a midline repressor,
potentially Pointed (Pnt), also controlled by EGFR. Bottom:
schematic of the tkv patterns in the wild type, the EGFR
hypomorph (QY1, anterior striped gray), and the mutant
with extra copies of gurken (4PX, solid gray). The dashed line
shows the boundary of the wild-type pattern of tkv; cells
inside the red box express tkv in the wild-type background.
In these cells, tkv expression is lost in both the 4PX and QY1
mutants. (E,F) tkv expression in the 4PX (E) and QY1 (F)
backgrounds. The midline gap is marked by a double
arrowhead and the midline is marked by an arrowhead.
P-Mad (green) in 4PX (G) and QY1 (H) egg chambers,
respectively. The wild-type patterns of Dpp signaling and tkv
expression are shown in Fig. 1. Yellow dashed lines mark the
anterior boundary of the oocyte.
expressing cells (Fig. 1I and Fig. 4B). From stage 11 and onward,
the patterns of P-Mad and Br overlapped quite significantly (Fig.
4C).
Until stage 10B of oogenesis, the dynamics of br transcript and Br
protein levels appear to be highly correlated in space and time (Fig.
4D-F). Low levels of br transcript were detected in all follicle cells
before stage 9; br was then repressed in the dorsal midline and
anterior cells, and began to emerge in the roof cells. At later stages,
however, br is downregulated in the roof cells (Fig. 4G) (Deng and
Fig. 4. Spatial and temporal correlation between the
wild-type patterns of Dpp signaling and Br expression.
(A-C) P-Mad (green) and Br expression (red) at different
stages of oogenesis; see text for details. (D-G) The spatial
pattern of br transcript at different stages of oogenesis.
Stage 9, low level of expression throughout the follicular
epithelium (D); stage 10A, repression in the anterior and
dorsal midline follicle cells (E); stage 10B, expression in the
roof cells (F); stage 11 and onward, no expression
throughout the follicular epithelium (G). (H-J) A mosaic egg
chamber with a large Medea– (Med–) clone that spans the
lateral and anterior regions of the follicular epithelium; Med–
cells are marked by the absence of GFP (H, green). The clone
generates ectopic Br expression in the anterior cells, but does
not disrupt the lateral expression of Br (I). (J) Enlarged view of
Br expression in the boxed area in H. (K-M) A clone of Mad–
cells, which spans both the roof cell domain and the dorsal
midline, does not affect Br levels in the dorsal midline cells
and does not disrupt Br expression in the roof cells. Br
expression is shown in red; Mad– cells are marked by the
absence of GFP (green). A similar effect can be seen with a
lateral of clone Med– cells (N-P). Midline levels of Br are also
not affected by removal of Med in the dorsal midline (not
shown). In all images the dorsal midline is marked by an
arrowhead, anterior is to the left, and the clones are outlined
by a dotted line.
DEVELOPMENT
initially expressed at low levels throughout the oocyte-associated
follicular epithelium; it is then repressed in the anterior and dorsal
midline cells (Fig. 4A) (Deng and Bownes, 1997). The P-Mad
domain borders, but does not overlap the Br domain at this stage
(Fig. 4A). At later stages of oogenesis, Br is expressed at high
levels in two dorsolateral patches of the follicle cells, called the
‘roof’ cells (Fig. 4B,C). High levels of Br in the roof cells became
clear at stage 10B of oogenesis. At this stage, the P-Mad domain
expanded posteriorly and overlapped the most anterior row of Br-
Fig. 5. Dpp signaling limits the duration of br expression in the
roof cells. (A) Schematic of the dynamics of br levels in the roof cells in
the wild type (dark gray). We predicted that the pulse-like dynamics of br
expression in the roof cells should become sustained in the absence of
Dpp signaling (light gray). (B) Uniform expression of Dad, the inhibitory
Smad, changes the spatial pattern of Br protein: Br is ectopically
expressed in the anterior cells. (C) At the same time, the duration of br
expression in the roof cells is increased, compared with the wild-type
pattern of br expression (Fig. 4G). This agrees with the schematic
prediction in Fig. 5A. (D,E) Egg chambers with uniformly inhibited Dpp
signaling. (D) Changes in the spatial pattern of Br in the anterior cells
generate a predictable change in the expression of rho, which was
previously shown to be repressed by Br. (E) Schematic of rho (blue) and Br
(red) patterns. (F) Patterning defects in egg chambers with uniformly
inhibited Dpp signaling lead to defects in eggshell morphology: EM
image of Dad overexpression eggshell shows flat dorsal appendages and
reduced operculum size. (G) Wild-type rho expression; (H) schematic of
wild-type rho (blue) and Br (red) patterns; (I) wild-type eggshell
morphology. In B,C,D,G anterior is to the left; the dorsal midline is
marked by an arrowhead; yellow dashed lines mark the anterior
boundary of the oocyte. DA, dorsal appendages; op, operculum.
Bownes, 1997). Thus, while the levels of Br protein in the roof cells
are sustained, the expression of br transcript in these cells is transient,
and can be viewed as a pulse in time. We will return to this difference
in our analysis of the connection between Dpp signaling and br.
Dpp signaling represses Br in the anterior follicle
cells
Previous studies of the Dpp effects on Br expression in oogenesis
led to two different models. Results of experiments with the
manipulation of the Dpp levels or intracellular inhibitors of Dpp
signaling suggested that Dpp acts as a repressive signal (Deng and
Bownes, 1997; Dequier et al., 2001; Chen and Schüpbach, 2006).
However, studies of Br pattern in egg chambers with clones of tkv–
and Medea– (Med–, a co-Smad essential for Dpp signal transduction)
cells concluded that Dpp signaling provides a positive input for Br
expression (Peri and Roth, 2000; Shravage et al., 2007). Given these
conflicting results and the dynamic character of Br expression, we
revisited the effect of Dpp on Br.
The sign of the effect of Dpp signaling on Br can be deduced from
changes of the Br pattern in response to the local perturbations of the
Dpp pathway. For example, if Dpp acts as a repressive signal, then
negative perturbations of Dpp signaling are expected to lead to
ectopic Br expression in the anterior cells. To test this prediction, we
generated the GFP-marked clones of cells lacking Medea, and
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347
Fig. 6. EGFR represses Br in the midline and is required for Br
expression in the roof cells of Drosophila. (A-D) Experiments with
genetic mosaics with GFP-marked clones of ras– cells reveal regionspecific effects of EGFR signaling on Br expression. The dorsal midline is
marked by an arrowhead in all images. (A) Merged image of Br
expression (red) and GFP (green). (B) Midline ras– clones induce cellautonomous ectopic expression of Br. (C,D) Lateral clones of ras– cells
lead to cell-autonomous loss of Br expression in the roof cells. (E) A
model proposed for the regulation of Br expression by EGFR: the spatial
pattern of Br is established by the feedforward loop controlled by EGFR
signaling. Br is induced in a wide dorsal domain. This effect is
overridden in the dorsal midline cells, where Pointed, induced by high
levels of EGFR signaling, represses Br.
examined their effect on the expression pattern of Br. We found that
anterior Med– clones generated cell-autonomous ectopic Br
expression (Fig. 4H-J). We found similar results in experiments with
anterior clones tkv– cells (see Fig. S1 in the supplementary material)
and in the egg chambers expressing tkv-RNAi construct (see Fig. S2
in the supplementary material) (Crickmore and Mann, 2006; Dietzl
et al., 2007). Based on this, we concluded that Dpp signaling
represses Br in the anterior follicle cells. This is consistent with the
anticorrelated patterns of Br and P-Mad in these cells (Fig. 4A,B).
Dpp signaling limits the duration of br expression
in the roof cells
In contrast to their clear effect in the anterior of the egg chamber,
Med– and Mad– clones did not affect Br expression in the roof cells
(Fig. 4K-P). Furthermore, we observed no defects in Br expression
in the roof cells in egg chambers where the Dpp pathway was
uniformly inhibited by overexpressing Dad, an inhibitory Smad
(Fig. 5B). The only change in the Br pattern in this background
amounted to its expansion to the anterior boundary of the follicular
epithelium (Fig. 5B). These observations would be consistent with
the model in which the effect of Dpp is repressive. Indeed, removal
of repressor in the region that already expresses Br (roof cells) can
only increase the level of expression, and could not be detected on
the background of already strong Br expression level.
The appearance of P-Mad in the roof cells correlates with the
disappearance of br transcript in this region (Fig. 4C,G). Based on
the repressive effect of the Dpp pathway in the anterior cells, we
hypothesized that the late (DV) phase of Dpp signaling is responsible
for the downregulation of br transcript in the roof cells. This predicts
that in the absence of Dpp signaling, the transient pulse-like pattern
of br expression in the roof cells should be transformed into a
sustained temporal pattern (Fig. 5A). In agreement with this
prediction, we found that in egg chambers with uniformly inhibited
Dpp signaling, br was still expressed at high levels in the roof cells
at stage 11 of oogenesis, when it is already abolished in the wild type
DEVELOPMENT
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Development 135 (2)
Fig. 7. Br controls Dpp signaling in the follicle cells by regulating the expression of tkv. (A-D) Uniform expression of Br-Z1 isoform in the
follicular epithelium leads to ectopic expression of tkv and Dpp signaling. (A-C) Br (A) and P-Mad (B) patterns in a stage 12 egg chamber
overexpressing Br-Z1. P-Mad signal is detected in the midline and anterior follicle cells, where it is absent in the wild-type egg chamber at this stage.
The image in C is a merge of Br (red) and P-Mad (green) patterns. (D) Overexpression of the Br-Z1 isoform generates ectopic expression of tkv: tkv is
expressed in the dorsal midline (denoted by the arrowhead), where it is repressed in the wild type (see also Fig. S3 in the supplementary material).
(E-K) Clones of br– cells (marked by an arrow) generate defects in the spatial patterns of Dpp signaling and tkv expression. The arrowhead marks
the dorsal midline. (E-J) P-Mad pattern (red) in mosaic epithelial layers with the GFP-marked clones of br- cells. (K) Defective pattern of tkv (denoted
by the arrow) in the egg chamber with a clone of br– cells. Dorsal view; the arrowhead marks the midline. (L) Model summarizing the results of
experiments in A-K: Br regulates tkv, which is essential for Dpp signaling. (M) ESEM image of the eggshell morphology induced by the
overexpression of the Br-Z1 isoform.
Midline repression of Br is controlled by EGFR and
independent of Dpp signaling
We found that negative perturbations of the Dpp pathway did not
affect Br repression in the midline cells. Specifically, clones of tkv–,
Med–, Mad– cells and uniform expression of Dad did not generate
ectopic Br in the midline (Fig. 4K-P, Fig. 5B,C). This shows that the
midline repression of Br is independent of Dpp signaling. The midline
repression of Br was attributed to Pointed, which is activated by high
levels of EGFR signaling (Deng and Bownes, 1997). At the same
time, the induction of Br in the roof cells is also dependent on EGFR
signaling (Deng and Bownes, 1997; Atkey et al., 2006). This suggests
that high levels of EGFR signaling repress, whereas intermediate
levels of EGFR activate, Br expression. This model agrees with the
results of our own clonal analysis experiments. Using genetic mosaics
with clones of cell lacking ras, which is essential for EGFR signaling,
we found that EGFR affects Br in a region-specific manner: the
midline clones of ras– cells led to ectopic expression of Br, while ras–
clones in the dorsolateral cells greatly reduced Br expression (Fig. 6AD). Taken together with our analysis of the effects of Dpp signaling
on Br (Fig. 4H-P), these results suggest that the rising phase of br
expression in the roof cells is due to EGFR signaling, whereas its
shutdown is due to Dpp signaling.
Br expression in the roof cells is controlled by a
negative-feedback loop
The results of the previous sections suggest that EGFR signaling can
induce both br and its repressor (Dpp signaling) in the roof cells
(Fig. 3D, Fig. 6E). Two models can be formulated based on these
results. In one model, the expression of br would be controlled by
an incoherent feedforward loop, a network in which the input
induces both the target and its repressor in the roof cells (Alon,
DEVELOPMENT
(Fig. 5C and Fig. 4G, respectively). Furthermore, in this background
the br pattern expanded at the expense of the cells that would
normally express rho and contribute to the floor of dorsal appendages
(Fig. 5D,E) (Ward et al., 2006). This change in the two-dimensional
arrangement of cell fates led to eggshells with greatly reduced
operculum and deformed dorsal appendages (Fig. 5F-I). This shows
that that uniform inhibition of Dpp signaling in mid-stages of
oogenesis does not cause a developmental delay, and that the
observed changes of br expression reflect the altered pattern of Dpp
signaling. Uniform expression of tkv-RNAi (Crickmore and Mann,
2006; Dietzl et al., 2007) led to similar effects: we observed longer
persistence of br in the roof cells and ectopic br in the anterior of the
follicular epithelium, and defects in dorsal eggshell structures (see
Fig. S2 in the supplementary material). A significant fraction of tkvRNAi eggshells had ectopic dorsal appendages (see Fig. S2 in the
supplementary material), consistent with the model in which br
marks the roof of dorsal appendages (Berg, 2005). Taken together,
these data show that Dpp represses Br in the anterior and limits the
duration of br expression in the roof cells, and that this mode of
regulation is important for proper eggshell patterning.
Signal coordination in tissue patterning
DISCUSSION
Our results provide new insights into the dynamics and function of
the Dpp pathway in oogenesis. First, we demonstrate that, contrary
to the current model of Drosophila eggshell patterning (Berg,
2005; Shravage et al., 2007), the pattern of Dpp signaling in
oogenesis is not static, and undergoes a clear transition from purely
AP to DV pattern in late stages of eggshell patterning. This
transition reflects the change in the expression of the type I Dpp
receptor and is conserved in fly species separated by more than 40
million years of evolution. Second, we show that the early and late
patterns of Dpp signaling control the dynamic pattern of br, a
transcription factor expressed in the roof of future dorsal
appendages. While the AP phase of Dpp signaling represses br in
the anterior region of the egg chamber, the DV phase of Dpp
signaling limits the duration of br expression in the roof cells.
Third, we establish that, in addition to being regulated by Dpp, Br
actively controls Dpp signaling, thus regulating its own repression
via a negative-feedback loop.
The Br expression pattern is established by
sequential action of feedforward and feedback
loops
Our results, together with the previously published data, lead to a
new model for the dynamics of Br expression in the roof cells (Fig.
8A). Within the framework of this model, the rising phase of Br
expression is due a to an incoherent feedforward loop, a network in
which the input activates both the target and its repressor (Alon,
2007). In this case, the feedforward loop, formed by EGFR, Pointed,
and Br, determines the spatial pattern of Br (Fig. 6E). This pattern is
then modulated in time by a negative-feedback that depends on the
A
349
Grk
EGFR
Pnt
B
br
P-Mad
Br
Tkv
Dpp
time
S9
S10A
S10B
S11
br
Br
pnt
tkv
P-Mad
1
Fig. 8. Model for the dynamic regulation of Br by EGFR and Dpp
pathways. (A) A revised model of Br regulation by EGFR and Dpp
signaling. The rising phase of Br expression in the roof cells is due to a
feedforward loop activated by EGFR signaling (blue). The shutdown of
br expression in the roof cells is due to a negative-feedback loop (red),
which is formed by Br and Dpp signaling. See text for details.
(B) Schematic diagram of the spatial and temporal patterns of the main
components in the model. See text for details.
Br-mediated increase of tkv expression and Dpp signaling. The
feedforward part of the model (outlined in blue) is supported by the
previously published gain- and loss-of-function experiments with
Pointed and EGFR signaling (Morimoto et al., 1996; Deng and
Bownes, 1997; Yamada et al., 2003), and by our analysis of Br
expression in ras– mosaics (Fig. 6). The negative-feedback loop
(outlined in red) is supported by the correlation of patterns of Br,
Tkv, and P-Mad (Fig. 3), by the previously published experiments
with manipulation of the levels of Dpp (Deng and Bownes, 1997;
Dequier et al., 2001), by our analysis of Br protein and br transcript
in the Dpp pathway loss-of-function experiments (Fig. 4H-P, Fig. 5),
and by the effects of br– clones and Br overexpression on tkv and
Dpp signaling (Fig. 7).
Dynamics of two-dimensional patterning in the
model
We distinguish four phases in the dynamics of the Br pattern (Fig.
8B). (1) Low levels of Br before stage 9 of oogenesis are
independent of EGFR signaling and insensitive to repression by
Dpp. (2) Following the formation of the DV gradient of EGFR
activation, Br is repressed in the midline and in the dorsoanterior
cells. The midline repression is due to Pointed, a transcription factor
induced by high levels of EGFR activation in the dorsal midline
(Morimoto et al., 1996; Deng and Bownes, 1997; Yamada et al.,
2003). The dorsoanterior repression is due to the early phase of Dpp
signaling, which reflects the anterior secretion of Dpp and uniform
expression of Tkv (Peri and Roth, 2000; Jekely and Rorth, 2003).
DEVELOPMENT
2007). In this case, the input is EGFR signaling, the target is br, and
its repressor is Dpp signaling in the roof cells. Alternatively, br
expression in the roof cells could be controlled by a negativefeedback loop, whereby Br, activated by EGFR signaling, would
induce tkv and Dpp signaling, which would then downregulate br.
Note that although both of these networks are consistent with the
pulse-like dynamics of br in the roof cells (Fig. 3 and Fig. 5A), only
the negative-feedback network provides a natural way for generating
an ordered induction of br and its repressor (Dpp signaling).
To discriminate between these two models, we tested whether tkv
expression and Dpp signaling in the roof cells are controlled by Br.
For this we overexpressed Br in the follicular epithelium and
examined the effect on the spatial pattern of Dpp signaling and tkv
expression (Fig. 7M). As a result, we found ectopic Dpp signaling
in the anterior follicle cells, including those in the dorsal midline
(Fig. 7A-C), probably due to the ectopic expression of tkv (Fig. 7D
and see Fig. S3 in the supplementary material). This suggests that
Br expression in the dorsal follicle cells is sufficient for the
induction of tkv. In a complementary experiment, we observed that
P-Mad signaling disappeared in GFP-marked clones of br– cells
(Fig. 7E-J). Furthermore, we found systematic disruptions in the
spatial pattern of tkv in egg chambers with clones of br– cells (Fig.
7K). This qualitative change of a very robust two-patched wild-type
pattern of tkv expression strongly suggests that Dpp signaling
depends on the Br-mediated expression of tkv (Fig. 7L). In
combination with the repressive effect of Dpp signaling on br
expression, these results provide strong evidence for the negativefeedback control of br levels in the roof cells (Fig. 8A). At the same
time, these results show that the effects of the EGFR activation on
the pattern of tkv expression and Dpp signaling (Fig. 3) are
mediated by Br.
RESEARCH ARTICLE
RESEARCH ARTICLE
(3) Levels of Br begin to rise in the roof cells. Changes in the Br
pattern have two effects on the spatial pattern of Dpp signaling:
higher levels of Br lead to higher levels of tkv in the roof cells.
Second, the dorsoanterior and midline repression of Br generates a
corresponding repression of tkv. (4) As a result, the anteriorly
produced Dpp can diffuse over the ‘Tkv-free’ area to the roof cells.
A combination of the arrival of the anteriorly produced ligand and a
higher level of receptor expression leads to a higher level of Dpp
signaling in the roof cells and subsequent repression of br. Another
layer of regulation is provided by Brk, a transcriptional repressor of
Dpp signaling, which is induced by Gurken and repressed by Dpp
signaling in the dorsal follicle cells. Brk antagonizes the repressive
effect of Dpp in the roof cells until the level of Dpp signaling in the
roof cells becomes high enough to repress Brk expression (Chen and
Schüpbach, 2006; Shravage et al., 2007).
The network characterized in our study can interact with a number
of previously discovered feedback loops (Queenan et al., 1997;
Wasserman and Freeman, 1998; Ghiglione et al., 1999; Reich et al.,
1999; Peri and Roth, 2000; Ward et al., 2006). For instance, Argos,
which provides negative-feedback control of EGFR signaling in the
dorsal midline, is a potential target of Dpp signaling (Queenan et al.,
1997; Wasserman and Freeman, 1998; Klein et al., 2004). Future
work is required to explore the extent to which this feedback loop,
which had been proposed to affect dorsal midline patterning,
interacts with the mechanism established in this paper.
We thank Mark Lemmon, Yury Goltsev, Mike Levine, Joe Duffy, Cyrill
Muratov, Gerardo Jimenez and all members of the Shvartsman lab for
helpful discussions and comments on the manuscript. We are grateful to
Dan Vasiliauskas, Susan Morton, Tom Jessell and Ed Laufer for providing the
P-Mad antibody. We also thank Oliver Grimm, Gerardo Jimenez, Rick
Padgett, Mike O’Connor, Joe Duffy, Celeste Berg, Wu-Min Deng, Lynn
Riddiford and Laura Nilson for fly stocks and reagents. We thank Maria Pia
Rossi, Sam Laurencin and Kara Spiller for help with ESEM imaging. This work
has been supported by the following NIH grants: P50 GM071508 and R01
GM078079.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/2/343/DC1
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Signal coordination in tissue patterning