Download TGF-β/BMP superfamily members, Gbb-60A and Dpp

2723
Development 125, 2723-2734 (1998)
Printed in Great Britain © The Company of Biologists Limited 1998
DEV5189
TGF-β/BMP superfamily members, Gbb-60A and Dpp, cooperate to provide
pattern information and establish cell identity in the Drosophila wing
Ongkar Khalsa*, Jung-won Yoon*, Sonia Torres-Schumann and Kristi A. Wharton†
Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI 02912, USA
*These authors have contributed equally
†Author for correspondence (e-mail: [email protected])
Accepted 24 April; published on WWW 23 June 1998
SUMMARY
Within a developing organism, cells receive many signals
which control their proliferation, fate specification and
differentiation. One group of such proteins is the TGFβ/BMP class of related signaling molecules. Based on
expression studies, multiple members of this class of
ligands must impinge upon the same cells of a developing
tissue; however, the role that multiple TGF-β/BMP ligands
may play in directing the development of such a tissue is
not understood. Here we provide evidence that multiple
BMPs are required for growth and patterning of the
Drosophila wing. The Drosophila BMP gene, gbb-60A,
exhibits a requirement in wing morphogenesis distinct
from that shown previously for dpp, a well-characterized
Drosophila BMP member. gbb-60A mutants exhibit a loss
of pattern elements from the wing, particularly those
derived from cells in the posterior compartment, consistent
with the gbb-60A RNA and protein expression pattern.
Based on genetic analysis and expression studies, we
conclude that Gbb-60A must signal primarily as a
homodimer to provide patterning information in the wing
imaginal disc. We demonstrate that gbb-60A and dpp
genetically interact and that specific aspects of this
interaction are synergistic while others are antagonistic.
We propose that the positional information received by a
cell at a particular location within the wing imaginal disc
depends on the balance of Dpp to Gbb-60A signaling.
Furthermore, the critical ratio of Gbb-60A to Dpp
signaling appears to be mediated by both Tkv and Sax type
I receptors.
INTRODUCTION
monomers. The diversity of biological processes regulated by
TGF-β signaling has often been attributed to the multitude of
molecular interactions possible in this signaling pathway,
including the potential for different monomer combinations,
various associations of multiple type I to type II receptors
(Yamashita et al., 1994), and a variety of interactions between
an increasing number of intracellular proteins, eg. the Smad
proteins (Derynck and Zhang, 1996; Massagué et al., 1997).
An additional level of complexity that must be considered is
that in many developing tissues multiple TGF-β signals must
impinge upon a single cell and possibly signal through the
same pathway. It has been observed that a number of members
exhibit overlapping expression patterns (e.g. Dudley and
Robertson, 1997; Lyons et al., 1995) and the question arises as
to whether a cell can distinguish between different TGF-β
signals and, if so, how is that information interpreted. During
mouse
development
some
degree
of
functional
interchangability has been proposed for different BMP
proteins, the largest group of TGF-β superfamily members
(Dudley and Robertson, 1997). However, the generality of
functional interchangability in other systems has not been
determined. The possibility remains that more than one TGFβ signal contributes to a single developmental process and,
Intercellular signaling is essential for the proper manifestation
of cell growth, fate specification and differentiation during the
development of multicellular organisms. The transforming
growth factor-β (TGF-β) superfamily of signaling proteins are
secreted as dimers and mediate intercellular communication
through their association with heteromeric integral membrane
receptor complexes consisting of type I and type II
serine/threonine kinase receptor proteins (Massagué, 1996).
Ligand binding sets off a cascade of biochemical events, which
culminates in a change to the target cell ranging from
alterations in cell proliferation to the acquisition of specific cell
fates (reviewed in Massagué, 1996). Given the plethora of data
substantiating the importance of these multifunctional
molecules in regulating diverse developmental events in a
number of different species (Roberts and Sporn, 1990;
Kingsley, 1994a; Hogan, 1996), elucidation of the mechanisms
by which TGF-β signaling proceeds and how cells interpret
and distinguish between different signals is crucial to our
understanding of development.
The mature dimer or ligand can exist as a heterodimer as
well as a homodimer, consisting of two different TGF-β-related
Key words: gbb-60A, dpp, decapentaplegic, TGF-β, BMP,
Drosophila, Wing morphogenesis, Imaginal disc, Patterning, Cell
proliferation
2724 O. Khalsa and others
furthermore, that multiple ligands bind to receptors on the same
cell in some way eliciting a different response in that cell. Here
we present evidence indicating that Gbb-60A and Dpp, two
Drosophila BMP members, are both required for proper wing
morphogenesis and that they cooperate to direct normal
development.
Three BMPs have been identified in Drosophila: Dpp, 60A
and Scw (Padgett et al., 1987; Wharton et al., 1991; Doctor et
al., 1992; Arora et al., 1994). Dpp and 60A each define a
separate subfamily exhibiting greatest sequence similarity to
vertebrate BMP2 and BMP4, and BMP5, BMP6, BMP7 and
BMP8, respectively (Wharton et al., 1991; Kingsley, 1994a;
Hogan, 1996). Dpp has been well-characterized and is known
to act as a morphogen during patterning events that take place
at multiple stages of development including embryogenesis
and imaginal disc development. Cells have been shown to
respond to the Dpp signal in a graded manner during the
specification of dorsal embryonic cell fates (Ferguson and
Anderson, 1992; Wharton et al., 1993) and in the specification
of positional values along the anteroposterior (A/P) axis of the
future adult wing (Lecuit et al., 1996; Nellen et al., 1996). The
precursor cells of imaginal structures are set aside during
embryogenesis as separate groups, or discs, of cells that
actively proliferate during the larval period (Cohen, 1993).
Patterning of the imaginal discs occurs during larval
development and differentiation of imaginal structures takes
place during the pupal stage. In the wing imaginal disc, dpp
RNA is expressed in a localized pattern within a stripe of cells
lying just anterior to the anterior/posterior (A/P) compartment
boundary (Gelbart, 1989; Masucci et al., 1990; Posakony et al.,
1991). Support for the proposal that cells adopt different fates
within the wing disc in response to varied levels of a gradient
of Dpp signal emanating from the A/P boundary has been
provided by analysis of receptor mutants and downstream
target genes (Burke and Basler, 1996; Lecuit et al., 1996;
Nellen et al., 1996; Singer et al., 1997). In Drosophila, the
BMP type I receptors encoded by thick veins (tkv) and
saxophone (sax) have been shown to mediate various aspects
of Dpp signaling (Affolter et al., 1994; Brummel et al., 1994;
Nellen et al., 1994; Penton et al., 1994; Xie et al., 1994).
Interestingly, the mutant phenotypes associated with
mutations in dpp and the receptor genes, sax and tkv are not
equivalent (Brummel et al., 1994; Twombly et al., 1996; Singer
et al., 1997), as would be expected if a receptor specifically
transmitted a signal unique to that ligand. These discrepancies
could be explained by the possibility that Sax and Tkv
receptors are activated by a TGF-β family member other than
Dpp. A loss of receptor function could then reflect the loss in
signaling triggered by more than one ligand, either as a
heterodimer or as two homodimers that each bind to the same
receptor. Therefore, the function of a particular TGF-β ligand
cannot be determined based solely on the examination of
receptor function and/or activation of downstream elements, as
such results could incorrectly attribute functions to that ligand
alone, as well as confound the actual molecular mechanism of
signal transduction. As yet it has not been definitively
demonstrated that Sax and Tkv receptors can mediate other
signals such as those elicited by Scw or 60A. However, mutant
analysis and the coexpression of dpp and scw in the Drosophila
embryo has led to the proposal that Scw signals as a Dpp/Scw
heterodimer with a higher ‘activity’ than the Dpp homodimer
(Arora et al., 1994; Raftery et al., 1995) but data indicating
through which receptor such a signal is mediated has not been
reported.
In this paper, we provide evidence that 60A (henceforth
referred to as gbb-60A) is required for the growth of imaginal
tissues and for patterning of the adult wing. We examine the
expression of gbb-60A in the imaginal discs and show that high
levels of Gbb-60A protein expression is in a pattern
complementary to dpp expression. We demonstrate that Gbb60A and Dpp signal together to accomplish the wild-type
pattern of the adult Drosophila wing. The Tkv and Sax
receptors are implicated as mediators of this synergistic
signaling. Furthermore, several pieces of evidence suggest that
Tkv may directly mediate the Gbb-60A signal. These data
provide the first functional evidence that concerted signaling
from two different TGF-β/BMP ligands is required in the
control of a specific developmental process.
MATERIALS AND METHODS
Drosophila melanogaster strains and crosses
Mutant alleles used in this study are described in Flybase (1996). gbb60A1 is a null allele and gbb-60A4 is a hypomorphic allele (K. A. W.,
K. deCastro, E. Borod and J. Cook, unpublished data). Recombinant
lines were generated that removed a secondary mutation present on
the original gbb-60A4 chromosome. All lines behave similarly; line
10 (gbb-60A4 R10) was used in experiments presented here. All
recombinants between alleles of dpp, sax, tkv and gbb-60A were tested
for the presence of each allele independently. All crosses were done
on a cornmeal-sugar media at 25˚C. Percent viability was determined
by dividing the number of individuals in the Cy+ class by half of those
in the Cy balancer class and multiplying by 100. Wings and legs were
mounted in DPX mountant (EM Sciences).
Generation of gbb-60A1 mutant clones
Clones of genetically marked cells were made using the FLP/FRT
system (Golic and Lindquist, 1989; Xu and Harrison, 1994). gbb-60A
null clones were generated in the progeny of a cross of: yw1118/Y;
P{>whs>}G13 sha1 gbb-60A1/SM6a males to yw1118 P{hsFLP};
P{>whs>}G13 P{y+} sha+ gbb-60A+ females. Adults were
transferred every 12 hours. FLP recombinase was induced by heat
shock (37˚C) for 50 minutes at 36 hours, 48 hours and 60 hours after
egg lay (AEL). Clones were identified based on the shavenoid (sha)
recessive marker, sha1, which removes or reduces the tricomes on the
wing blade (Lawrence et al., 1986). A total of 158 wings were
examined.
Construction of UAS-gbb-60A transgenic lines
A 1.6 kb EcoRI fragment from cDNA16 which contains the entire
gbb-60A coding region (Wharton et al., 1991) was cloned into the
ApoI site of pUAST (Brand and Perrimon, 1993). Four transgenic
lines were established by injection of UAS-gbb-60A and, when driven
by GAL4 24B and hsGAL4 (Brand and Perrimon, 1993), each line
was tested for its ability to rescue gbb-60A mutant phenotypes.
RNA in situ hybridization and immunohistochemistry
Whole-mount in situ hybridization to RNA in imaginal discs using
digoxigenin-labeled RNA probes was done as described in Tautz and
Pfeifle (1989) with the following modifications. Tissues were treated
with proteinase K (50 µg/ml) for 3.5 minutes at room temperature and
samples were hybridized O/N 55˚C. Sense and antisense RNA probes
were made using cDNA16 (Wharton et al., 1991) as a template.
Immunohistochemistry with polyclonal rabbit anti-60A (Doctor et al.,
1992) and monoclonal mouse anti-β-galactosidase (Sigma) was done
Gbb-60A to Dpp ratio critical for patterning 2725
by the following procedure: imaginal discs were fixed in 2%
formaldehyde in PBS for 30 minutes and blocked in 0.2% saponin,
1% normal goat serum in PBS. Imaginal discs were incubated
overnight at 4˚C with anti-60A (7.7:1000). In double labelling
experiments, dpp expression was detected by mouse anti-βgalactosidase (1:1000) in H1-2 dpp-lacZ/+ (Blackman et al., 1991)
larvae. Secondary antibodies were obtained from Jackson
Immunological Laboratories (Cy3 and FITC) and used at a dilution
of 1:500. Discs were mounted in 80% glycerol, 100 mM Tris (pH 8.0),
0.5% n-propyl galate. Signals were detected by epifluoresence using
a Nikon FXA or by laser scanning microscopy on a Zeiss LSM410.
Wild-type (Canton S) and gbb-60A mutant third instar larvae were
dissected and stained with DAPI in order to visualize imaginal versus
polyploid larval tissue. Everted larvae were fixed in a 3:1 ethanol:acetic
acid. Samples were stained in 0.05% DAPI (in 0.1M Tris buffer,
pH7.5) 10 minutes RT and mounted in 50% glycerol (in PBS).
RESULTS
Mutations in gbb-60A result in reduced imaginal
discs
In order to gain a full understanding of the function of the 60A
gene during development and to investigate the potential for
combined signaling from two different TGF-β/BMP family
members, we had previously isolated mutations in the 60A
gene (see Materials and Methods). Individuals homozygous for
null alleles of the 60A gene, such as gbb-60A1, exhibit
embryonic defects in gut morphogenesis and result in early
larval lethality (K. A. W., K. deCastro, E. Borod and J. Cook,
unpublished data). Allelic combinations between a
hypomorphic 60A allele, gbb-60A4, and a null allele or a
deficiency deleting the 60A locus result in later larval lethality
with only rare adult escapers (<1%). Third instar larvae of such
a genotype (gbb-60A4/gbb-60A1) appear transparent and
smaller than wild-type larvae (Fig. 1A,B). These larvae
develop more slowly than wild type and never attain wild-type
size. The transparency appears to be due to a defect in both the
quantity and quality of the fat body, as well as a dramatic
reduction in imaginal tissues (Fig. 1C,D). In addition to a
reduction in imaginal disc tissue, other tissues that normally
proliferate during larval development, such as areas of the brain
(Fig. 1D), are also reduced. The 60A locus has been named
glass bottom boat (gbb) in light of the remarkable transparency
of the mutant larvae and we will refer to it as gbb-60A in this
paper.
Mutations in gbb-60A affect imaginal disc
derivatives
The rare adult escapers of the genotype gbb-60A4/gbb-60A1
exhibit small misshapen wings, which lack the posterior cross
vein (PCV), much of longitudinal vein L5, distal portions of
L4 and the posterior half of the anterior cross vein (ACV) (Fig.
2C). The mutant wings are small, narrow and pointed with a
loss of intervein material, especially between veins L2/L3,
L4/L5 and posterior to L5. In addition to the change in wing
shape and the loss of veins, a thinning of vein L2 and some
ectopic vein material in the intervein region flanking L2 is
common. No abnormalities are found along the margin in the
position or type of wing margin bristles, however, ectopic
margin bristles were often seen along a vein or in intervein
tissue in the distal regions of the wing (not shown). Defects in
Fig. 1. gbb-60A is required for proliferation of imaginal tissues.
(A) Wild-type third instar larva. (B) gbb-60A1/ gbb-60A4 mutant
larva. DAPI staining reveals a dramatic reduction in the size of
imaginal discs in dissected gbb-60A mutant (D) as compared to wildtype (C) larvae. A reduction in the size of the gbb-60A1/ gbb-60A4
larval brain is also evident (*, ventral ganglia). Arrows indicate wing
and eye/antennal imaginal discs.
these mutant flies are not limited to the wings: a reduction in
the size of the eye, with the presence of supernumerary
vibrissae, an increase in the number of thoracic bristles, the
presence of misshapen leg segments and female sterility have
all been observed (S.T.-S. and K. A. W., unpublished data).
Subsequent to our initial observations of the lethality and
phenotype of the gbb-60A hypomorphic allele, gbb-60A4, we
have removed by recombination a secondary mutation present
on the gbb-60A4 chromosome (see Materials and Methods).
This secondary mutation exacerbated only the lethality
associated with the gbb-60A4 mutation and had no effect on
adult phenotypes. Once the secondary mutation was removed,
it was possible to produce viable adults that were homozygous
for the gbb-60A4 mutation at a significantly higher frequency
than that observed for the gbb-60A4/gbb-60A null genotype
(see Fig. 5I). We have verified by sequence analysis that the
viable adults are indeed homozygous for the gbb-60A4
mutation (data not shown). The mutant wing phenotype of the
gbb-60A4 homozygous adult is similar to, but less severe than,
that observed for the rarer gbb-60A4/Df or gbb-60A4/gbb-60A1
adults (Fig. 2B,C). In gbb-60A4 homozygotes, the overall shape
of the wing is broader and less pointed, more like wild type.
The ACV is often complete (54% of gbb-60A4 individuals
versus 0% of gbb-60A4/gbb-60A1) and longitudinal veins L4
and L5 are longer.
Given that gbb-60A4 retains some gbb-60A function, we
examined the affect of complete loss of gbb-60A function on
wing patterning and morphogenesis through the production of
2726 O. Khalsa and others
Fig. 2. gbb-60A signaling is required for
normal wing morphogenesis. (A) Wild-type
wing. Longitudinal veins L2, L3, L4, L5,
anterior and posterior crossveins (ACV and
PCV, respectively) and the anterior/posterior
(A/P) boundary are denoted. (B,C) Adult
viable gbb-60A mutants exhibit a loss of both
vein (indicated by arrows) and intervein
material. (B) gbb-60A4 R10/ gbb-60A4.
(C) gbb-60A4 R10/ gbb-60A1 results in
greater loss of L5, L4 and the posterior
portion of ACV. Some variability is seen in
the degree of loss for L5: 43% have a longer
L5 as shown in Fig. 6A. (D-I) Adult wings
containing sha gbb-60A1clones. Clone
boundaries on the dorsal wing surface (dotted
line) and the ventral wing surface (dashed line) are demarcated. Large clones (D) or multiple clones (E) in the anterior and posterior
compartments with significant dorsal/ventral overlap produce phenotypes similar to B and C, except distal gaps in L3 are often observed (see
rightmost vertical arrow). (F) Ectopic vein material (arrow) is observed when a small dorsoventral clone is located anterior to L3. (G) An
example of the non-autonomous effect resulting from gbb-60A clones. All wings are shown with anterior to the top and distal to the right.
null clones. The FLP-FRT-mediated recombination system
(Golic and Lindquist, 1989; Xu and Harrison, 1994) was used
to generate clones of cells null for gbb-60A function in the
imaginal discs (Fig. 2D-G). Consistent with a potential role for
gbb-60A in proliferation, gbb-60A null clones are never
recovered in the adult wing if induced during the early period
of cell proliferation (before 36 hours AEL); however, clones in
all parts of the wing were recovered from later inductions.
Clones located in both the anterior and posterior
compartments, as well as clones limited to the posterior
compartment, exhibited defects in the same pattern elements
affected in hypomorphic wings: loss of the PCV, and portions
of L5 and L4 (Fig. 2D,E). Clones often also resulted in the loss
of distal L3 at the margin. The majority (>80%) of the clones
that exhibited a mutant phenotype covered a portion of the
wing where vein material would normally be found. Clones
strictly limited to the anterior compartment or along the A/P
boundary exhibited no wing defects with the exception of very
small clones that were located near the ACV (7% of the total
clones restricted to the anterior compartment). These clones
resulted in ectopic vein material anterior to L3 (Fig. 2F). In
several wings vein loss was observed in wild-type tissue at a
distance from the gbb-60A− patch (Fig. 2G). In each case that
this non-autonomous effect was found, multiple clones were
present in the wing, one in the posterior compartment and
another along the A/P boundary, sites that otherwise did not
produce abnormalities. This type of vein loss was never
observed in the generation of control clones.
Expression of gbb-60A in the imaginal discs is
complementary to that of dpp
The expression of gbb-60A in imaginal discs was examined by
both whole-mount RNA in situ hybridizations (Fig. 3A,C,E)
and antibody staining using a Gbb-60A antibody (Fig.
3B,D,F). gbb-60A RNA is expressed in the wing disc (Fig. 3A)
mainly in the posterior compartment in the pteropleural and
medial regions extending into the progenitors of the scutellum.
High levels are found within the wing pouch in cells belonging
to both the posterior and anterior compartments with higher
levels in the posterior. A small amount of expression is found
within the hinge region. In the eye/antennal disc, gbb-60A
RNA is highest anterior to the morphogenetic furrow and in
the medial regions with lower levels of expression posterior to
the morphogenetic furrow (Fig. 3C). gbb-60A is expressed
throughout the posterior compartment of the leg imaginal discs
and within the ventral anterior compartment (Fig. 3E).
Gbb-60A protein is generally detected (Fig. 3B,D,F) at
locations coincident with gbb-60A RNA with one major
exception: the presence of a stripe of markedly lower Gbb-60A
protein running through the middle of the wing pouch (Fig.
3B). This ‘stripe’ of reduced expression is coincident with the
prominent stripe of dpp RNA expression in the cells anterior
to the anterior/posterior (A/P) boundary of the wing imaginal
disc (Fig. 4A-C). We also observe a weak but consistent
reduction in Gbb-60A expression along the dorsal/ventral
boundary from which the cells of the wing margin are derived
(Fig. 4A). High levels of Gbb-60A expression exists in a
pattern complementary to the localized expression of dpp in
the other imaginal discs as well. For example, Gbb-60A
expression is absent or at very low levels in the morphogenetic
furrow of the eye disc, the site of dpp expression (Figs 3, 4DF). In addition, the regions of low or absent Gbb-60A
expression in the antennal and leg imaginal discs are the sites
of dpp expression (Fig. 4D-F; data not shown). It seems
Gbb-60A to Dpp ratio critical for patterning 2727
therefore appropriate that the abbreviation of glass bottom boat
(gbb) is a mirror image of the letters dpp, thus reflecting the
observed complementary pattern of gbb-60A and dpp
expression. We do not intend to suggest that gbb-60A and dpp
expressing cells are mutually exclusive, especially since the
reagents available to assay for the presence of these BMP
ligands do not allow such resolution. However, based on the
reagents available our data indicate that the predominant RNA
and protein expression of gbb-60A and dpp appear to be in
different regions of the imaginal discs with some cells in the
overlapping areas in which the co-expression of gbb-60A and
dpp may occur.
While in most discs the expression of gbb-60A RNA and
protein does not differ significantly, we cannot exclude the
possibility that in the wing disc the apparent absence of Gbb60A along the A/P boundary reflects the inability of a Gbb60A polyclonal antibody to recognize the Gbb-60A protein in
these cells perhaps due to some particular structural
conformation of Gbb-60A, such as a heterodimer, in these
cells. We believe this is very unlikely since a second polyclonal
antibody directed at a different portion of the protein, the
amino-terminal pro domain as opposed to the ligand domain
(Doctor et al., 1992), also reveals the same reduced staining
along the A/P boundary (data not shown).
Fig. 3. gbb-60A is expressed in a discrete pattern in imaginal discs.
RNA in situ hybridizations performed on imaginal discs from third
instar larvae using a gbb-60A antisense probe are shown (A,C,E). In
general, Gbb-60A protein expression (B,D,F) follows the RNA pattern
except that it appears more diffuse. A stripe (bracket) of reduced or
absent Gbb-60A protein is apparent anterior to the anterior/posterior
boundary of the wing disc (B). Wing and leg imaginal discs (A,B,E,F)
are shown dorsal up and posterior to the right. Eye/antennal discs are
shown lateral to the left and posterior down (C,D). The location of the
morphogenetic furrow is indicated by an arrowhead (C,D).
Fig. 4. High levels of Gbb-60A are complementary to
the expression of dpp. Confocal images of a wing
disc (A-C) and epifluoresence images of an
eye/antennal disc (D-F) demonstrate that low levels
of Gbb-60A protein (red) are coincident with the
localized expression of dpp RNA (green) along the
A/P boundary (bracket) and in the morphogenetic
furrow (arrowhead). Arrow marks the dorsal/ventral
boundary in (A). (A-C) Wing discs: dorsal up and
posterior right. (D-F) Eye/antennal discs: lateral left
and posterior down.
Dpp and Gbb-60A signaling are required together
for normal wing morphogenesis
The structures in the wing that are affected most dramatically
by mutations in gbb-60A, the PCV and L5 are those that are
least sensitive to the reduction or absence of dpp (Posakony
et al., 1991; deCelis, 1997; Sturtevant et al., 1997). The ACV
and longitudinal veins L2 and L4, lying on either side of the
A/P boundary, have been shown to be most sensitive to the
loss of Dpp signaling (Segal and Gelbart, 1985; Posakony et
al., 1991; deCelis, 1997) consistent with the proposal that
Dpp organizes wing pattern via a morphogen gradient
emanating from the A/P boundary. (It has been proposed that
longitudinal vein L3 is fated by a different mechanism and as
a result is not sensitive to the level of Dpp signaling
(Sturtevant, 1997).)
2728 O. Khalsa and others
I
80
,
% viability
60
40
20
0
+
gbb4
gbb4
dppd12 gbb4
+
dppd5 gbb4
gbb4
gbb4
(763)
gbb4
+
gbb4
dppd12 gbb1
+
(718)
dppd5 gbb1
gbb4
gbb1
gbb1
(>1000)
Df
(536)
(537)
(646)
(106)
Fig. 5. gbb-60A and dpp interact synergistically. (A-D) Genetic
combinations involving the dppd alleles, dppd5 and dppd12, with gbb-60A4
homozygotes or the heteroallelic gbb-60A4/ gbb-60A1 result in enhanced
wing phenotypes. (A) dppd5 gbb-60A4/ + gbb-60A4R10. (B) dppd12 gbb60A4 / + gbb-60A4 R10. The complete loss of the ACV is indicated by an
arrow. (C) dppd5gbb-60A1/ + gbb-60A4 R10. (D) dppd12 gbb-60A1/ + gbb60A4 R10. (E) Wild-type male prothoracic leg. (F) Male prothoracic leg
from dppd12 gbb-60A1/ + gbb-60A4 R10. The five tarsal segments
(bracket) in the wild-type male prothoracic leg are indicated (E). Loss of
the ACV (arrow) in the dpp heteroallelic combination dpps4/ dppd6 (G) is
suppressed by gbb-60A null allele, dpps4 + / dppd6 gbb-60A1 (H). All
wings were taken from females and photographed at the same
magnification. (I) The percentage viability of dppd gbb-60A/ + gbb-60A
genotypes is indicated. gbb-60A is abbreviated gbb. The total number of
individuals scored in the viaibility crosses is shown in parenthesis.
Since mutations in the gbb-60A locus indicate that gbb-60A
is essential for establishing cell identity in the wing, especially
within the posterior compartment, we investigated the
possibility that Gbb-60A and Dpp signal together to provide
positional information for the entire wing. No dominant
genetic interactions were observed between alleles of gbb-60A
and dpp, therefore, recombinant chromosomes were
constructed using alleles of dpp which result in an overall
lowering of dpp expression in the imaginal discs (dppd5, dppd6
and dppd12) (Masucci et al., 1990), and gbb-60A alleles, gbb60A4 and gbb-60A1. Individuals heterozygous for dppd alleles
(dppd/+) are phenotypically wild type. As homozygotes or in
various heteroallelic combinations, these dpp alleles can be
lethal or can generate adults with appendage defects ranging
from minor loss of vein material to truncations of the entire
appendage (Spencer et al., 1982).
An individual heterozygous for a dppd allele and
homozygous or transheterozygous for gbb-60A alleles (dppd
gbb-60A4/ + gbb-60A4 or dppd gbb-60A4/ + gbb-60A1) exhibits
wing phenotypes qualitatively different than that observed in
the gbb-60A mutant alone (Fig. 5). gbb-60A mutants
heterozygous for dppd5 exhibit a significant loss of L4 (>60%
lack more than half of L4) and a more frequent loss of the ACV.
gbb-60A mutants heterozygous for a more severe dpp allele,
dppd12, not only show a greater loss of L4, ACV and L2 vein
material (>90% lack more than half of L4 and 100% lack the
entire ACV including a thinning of L2) but also a loss of
intervein tissue, especially between L2/L3 and L4/L5 as
exhibited by a reduction in the overall size of the wing (Fig.
5B,D). In a small number of individuals (3%), a gap at the
distal end of L3 is apparent. Interestingly, in the dpp gbb-60A/
+ gbb-60A genotypes, we do not observe a greater loss of L5
but in fact a reduction in the loss of this posteriormost vein.
100% of dppd12 gbb-60A4/ + gbb-60A1 exhibit only a gap in
Gbb-60A to Dpp ratio critical for patterning 2729
L5 with >K of the vein being complete, while 100% of gbb60A4/gbb-60A1 lack >K of L5 with >60% lacking >G of L5.
These phenotypes are not only observed with dpp regulatory
mutants (dppd alleles) but also with alleles that affect activity
of the dpp protein (Wharton et al., 1993). For example, dpphr56
gbb4/ + gbb4 exhibits wing phenotypes similar to dppd12 gbb60A4/ + gbb-60A4.
In addition to defects in wing patterning, abnormalities were
noted in the proximal/distal organization of the legs (Fig. 5F),
a process in which dpp is known to play a central role (Jiang
and Struhl, 1996; Lecuit and Cohen, 1997). The common
defects exhibited by the dppd12 gbb-60A1/ + gbb-60A4
genotype are truncations and/or apparent fusions of the distal
most tarsal segments of the male prothoracic leg (Fig. 5E,F).
A decrease in the viability of dpp gbb-60A/ + gbb-60A
individuals as compared to that observed for gbb-60A
mutations alone was also observed (Fig. 5I). The adult viability
of gbb-60A4 homozygotes decreases from 74.5% to 10.1% of
the expected size class with the inclusion of a single mutant
allele of dpp (dppd12 gbb-60A4/ + gbb-60A4).
These data indicate that heteroallelic combinations of gbb60A and dpp result in phenotypes more pronounced and distinct
from phenotypes observed as a result of mutations in either gene
alone. These new phenotypes are not simply additive. Individual
pattern elements are affected differently as a result of these
heteroallelic combinations, for example, a greater loss of L4 is
seen while the loss of L5 is reduced. In an attempt to test further
the apparent synergistic, as well as antagonistic, nature of the
gbb-60A/dpp interaction, we examined several other allelic
combinations. In one case, we investigated the effect of
lowering gbb-60A function in a viable dpp mutant. Wings
derived from dpps4/dppd6 individuals lack only the ACV (Fig.
5G) presumably due to the fact that, in this mutant, dpp
expression is absent specifically from the primordial cells for
that wing vein (deCelis, 1997). In this genotype lowering the
dose of gbb-60A by one copy (dpps4 +/dppd6 gbb-60A1) results
in a restoration of the ACV (Fig. 5H) not a further loss of other
vein material as seen above with other dpp/gbb-60A
combinations (Fig. 5A-D). However, similar to the other
dpp/gbb-60A interactions, the size of these wings is reduced and
the viability of these individuals is significantly reduced (15%
of expected as compared to control crosses). These results
suggest that the requirement for both dpp and gbb-60A in earlier
stages of larval development, presumably at times of high cell
proliferation, is not met in the dpps4 +/dppd6 gbb-60A1
genotype. Yet the suppression of the ACV defect indicates that
gbb-60A does not solely act to modulate levels of Dpp signaling
but rather as the ratio of gbb-60A to dpp changes across the
wing imaginal disc the specification of a pattern element is
affected. Different relative levels of Gbb-60A to Dpp signaling
would result in different positional information. At some points
within the developing wing, the readout may be synergistic
while at other sites antagonistic.
Correct balance of Gbb-60A and Dpp signaling is
essential to normal patterning
If cells across the wing disc indeed respond to different levels
of Gbb-60A versus Dpp signaling, we reasoned that by
changing the level of one ligand we should be able to generate
a patterning mutant phenotype. We should then be able to
potentially rescue that phenotype by changing the level of the
Table 1. UAS-Gbb-60A rescues gbb-60A mutants
UAS-gbb-60A
GAL4 24B
hsGAL4
Genotype
% Rescue
gbb-60A1/gbb-60A4
gbb-60A1/gbb-60A4
70.9
42.4
other ligand in an attempt to restore the balance of signaling
elicited by the two ligands to that found in wild type. To test
this hypothesis, we first made a UAS-Gbb-60A construct
which contained the entire gbb-60A coding region and
generated multiple transgenic lines. We have demonstrated that
these UAS-Gbb-60A lines produce functional Gbb-60A since
gbb-60A mutations can be rescued to adulthood when the
expression of UAS-Gbb-60A is driven by hsGAL4 or by
GAL4-24B (Table 1). GAL4-24B directs expression mainly in
the embryonic mesoderm (Brand and Perrimon, 1993), the site
of high levels of gbb-60A expression in the embryo (Doctor,
1992; K. A. W., unpublished data). In addition to the rescue of
lethality, the mutant wing phenotype associated with gbb-60A
viable mutants (Fig. 2) was rescued when hsGAL4 was used
to direct expression. Using the same experimental conditions,
we were unable to rescue gbb-60A mutants (lethality or wing
phenotype) with previously reported UAS-60A lines
(Staehling-Hampton et al., 1994).
Since Gbb-60A is normally expressed at a very reduced level
along the A/P boundary, we investigated the effect of
overexpressing Gbb-60A in this region. Wings from UAS-Gbb60A/ptc-GAL4 exhibit ectopic vein material anterior to the A/P
boundary (Fig. 6B). If dpp is also mis-expressed (UAS-gbb60A/UAS-dpp/ptc-GAL4) this mutant phenotype is rescued
(Fig. 6C). The effect of raising Gbb-60A signaling along the
A/P boundary where Dpp is normally very high results in
mispatterning. If the original balance of higher Dpp to Gbb-60A
along the A/P boundary is re-established by adding more Dpp
the wild-type positional information is restored. When
ectopically expressed, the UAS-dpp (gift from Mike O’Connor)
used in these studies fails to produce any mutant phenotypes.
Other examples of the effects of manipulating the relative levels
of Gbb-60A and Dpp is shown when ectopic expression is more
widespread using 71B-GAL4 (Fig. 6E,F) or A9-GAL4 (Fig.
6H,I). In each case some but not all of the defects in pattern
elements caused by ectopic expression of gbb-60A are rescued
by also expressing dpp. Given that our loss-of-function
interaction data fail to demonstrate a simple antagonistic
relationship between gbb-60A and dpp (indeed quite the
opposite), we must conclude that these ectopic expressions
reflect a change in the balance of dpp to gbb-60A activity or
signaling. Which specific elements of the ectopic Gbb-60A
mutant phenotype are rescued clearly depends on the pattern of
ectopic expression during the entire period of wing patterning
as well as the endogenous levels of Dpp and Gbb-60A.
gbb-60A alleles genetically interact with mutations
in BMP type I receptor genes, tkv and sax
The Dpp signal has been shown to be mediated by two different
BMP type I receptors, Tkv and Sax, during wing
morphogenesis as well as during other stages of development
(Xie et al., 1994; Brummel et al., 1994; Nellen et al., 1994;
Penton et al., 1994; Twombly et al., 1996; Singer et al., 1997).
To address whether either or both of these receptors are
important in mediating the signals resulting from the actions of
2730 O. Khalsa and others
Fig. 6. The relative levels of gbb-60A and dpp are critical to wing development. Expression pattern of UAS-lacZ directed by ptc-GAL4 (A),
GAL4-71B (D) and GAL4-A9 (G) in the wing imaginal disc as assayed by X-gal staining. Wings exhibit dominant phenotypes when UAS-gbb60A is ectopically expressed using ptc-GAL4 (B), GAL4-71B (E) and GAL4-A9 (H). A decrease in intervein tissue between L3/L4
accompanied by ectopic vein material is seen when Gbb-60A is misexpressed along the A/P boundary (B, arrow). Wing blisters and ectopic
vein material are apparent when UAS-gbb-60A is ectopically expressed in the wing pouch (E). Excessive vein formation is seen in both the
anterior and posterior compartments of the wing (H). Aspects of the dominant phenotypes shown in (B,E,H) are rescued or suppressed by
simultaneous expression of UAS-dpp (C,F,I).
Gbb-60A and Dpp, we investigated the possibility of a genetic
interaction between alleles of gbb-60A and alleles of tkv or sax.
Recombinants were constructed between gbb-60A4 or gbb-60A1
and several alleles of tkv and sax. The addition of Df(2L)tkv2,
a chromosomal deficiency that removes the tkv locus (Penton et
al., 1994), into a gbb mutant background results in a severe
mutant wing phenotype with a dramatic loss of both the PCV
and ACV and most of L4 and L5 (Fig. 7A, B). In addition, distal
gaps are present in L2 and L3. A less extreme phenotype is seen
with tkv6, a hypomorphic allele that retains significant receptor
function (data not shown). Interestingly, the more severe
phenotype of tkv2 gbb-60A1/ + gbb-60A4 has qualities that are
distinct from those observed in dpp gbb/ + gbb individuals,
notably the effect on L5. Furthermore, unlike the genetic
interactions observed with dpp alleles, the viability of tkv gbb60A1/ + gbb-60A4 is not reduced but in fact is considerably
increased when compared to gbb-60A1/gbb-60A4 (Fig. 7E).
These data indicate that the interaction that we observe between
tkv and gbb-60A cannot be explained solely as a secondary
consequence of lowering Dpp signaling readout by mutating a
receptor that mediates Dpp signaling. These data suggest that
Tkv is able to mediate Gbb-60A signaling and that it may do
so in different ways at different times during development.
We also analysed the effect of reducing the gbb-60A copy
number in an individual compromised for functional Tkv
receptor. tkv427 behaves as a hypomorphic allele. As a
homozygote, tkv427 is phenotypically wild type and exhibits no
defects in the wings; however, in trans to a null, Df(2L)tkv2, a
slight thickening of wing veins is observed (Fig. 7C). This
ectopic vein phenotype is thought to reflect a feedback loop in
which dpp fails to be downregulated properly by tkv in the vein
primordia resulting in the response of boundary intervein cells
to the vein-promoting Dpp signal, and thus the formation of
vein outside of the vein proper (deCelis, 1997). We observe
that reducing gbb-60A in this tkv mutant background (tkv427
+/Df(2L)tkv2 gbb-60A1) produces a further thickening of wing
veins (Fig. 7D). This result suggests that gbb-60A may play a
role in vein differentiation itself and/or in the tkv/dpp feedback
loop important in defining the boundaries of the vein.
Genetic combinations used to investigate the potential
interaction between gbb-60A and sax alleles indicate a
reduction in viability for gbb-60A mutant genotypes containing
a single copy of a sax null allele (sax gbb-60A1/ + gbb-60A4)
(Fig. 7E). This reduction in viability suggests that lowering
both gbb-60A and sax compromises development. The wing
phenotype of the few viable adults recovered was similar to a
very severe gbb-60A mutant wing phenotype shown in Fig. 2C,
with a substantial loss of L5, complete loss of the PCV and
ACV and loss of half of L4. Clearly the levels of Gbb-60A
signaling and Sax function are dependent on one another.
DISCUSSION
gbb-60A is required for growth and patterning of
imaginal tissues
Our gbb-60A mutant analysis indicates that the gbb-60A gene
is required for proper wing morphogenesis, a process requiring
Gbb-60A to Dpp ratio critical for patterning 2731
E
E
50
% viability
40
30
20
10
+ gbb4
tkv2 gbb1
(407)
+ gbb4
(283)
tkv6 gbb1
+ gbb4
(618)
sax5 gbb1
(718)
+ gbb4
(497)
sax4 gbb1
gbb1
both growth and patterning of wing imaginal tissues. The
requirement for gbb-60A in growth of imaginal discs is
exemplified by the dramatic reduction of imaginal tissues
observed in gbb-60A mutant larvae (Fig. 1D), which
contributes to a transparent phenotype for which we have
named the 60A gene glass bottom boat (gbb). Our mutant
analysis further demonstrates that lowering gbb-60A function
results in the reduction of the size of the adult wing (Fig. 2).
The reduction in wing tissue is suggestive of a failure of
imaginal cell proliferation. These results are similar to what
has been found for mutations in dpp, as well as in a number of
other genes that encode BMP signaling components in
Drosophila (Spencer et al., 1982; Raftery et al., 1995;
Brummel et al., 1994; Burke and Basler, 1996; Singer et al.,
1997). These data taken together suggest that BMP signaling
in Drosophila plays a role in the positive control of cell
proliferation, opposite to the negative effect on cell growth
attributed to TGF-β itself (Massagué et al., 1990; Roberts and
Sporn, 1990). Studies in progress will delineate in which cells
and at what times during development gbb-60A is required for
proliferation, as well as how the signal is mediated.
With the identification of a gbb-60A hypomorphic allele, we
have been able to partially bypass the requirements for gbb60A in early stages of development which when not met lead
to lethality. Thus, we have been able to elucidate a requirement
for gbb-60A in wing morphogenesis (Fig. 2). The generation
of clones null for gbb-60A function reaffirmed this requirement
for gbb-60A in the specification of pattern elements within the
wing. Our analysis of loss-of-function mutations demonstrates
that the posterior portion of the wing is most sensitive to the
level of gbb-60A function with the PCV and vein L5 most
sensitive to a reduction of gbb-60A, followed by L4, the ACV,
L3 and least sensitive, L2 (Fig. 2). In addition, the nonautonomous effect of gbb-60A− clones suggests that disrupting
gbb-60A levels in one part of the disc must alter how cells in
other regions of the disc interpret their position (Fig. 2G).
0
gbb4
Fig. 7. Alleles of gbb-60A genetically interact with tkv and sax receptor mutant alleles. (A) gbb60A1/ gbb-60A4. (B) Df(2L)tkv2gbb-60A1/gbb-60A4 results in a more severe wing phenotype but
less severe lethality (E). Note gaps in L2 and L3 (arrows). (C) Slight thickening of veins (*) in
tkv427/Df(2L)tkv2. (D) tkv427+/Df(2L)tkv2gbb-60A1results in an enhancement of vein thickenings
(*). (E) Percentage viability of gbb-60A1/gbb-60A4 in conjunction with receptor mutant alleles.
The number of individuals scored is shown in parenthesis.
Complementary expression pattern of gbb-60A and
dpp suggest these ligands exist primarily as
homodimers
The expression of gbb-60A RNA and protein are consistent
with the requirement that we have identified for gbb-60A in the
wing. The pattern of Gbb-60A protein expression in the wing
imaginal disc as revealed by a Gbb-60A polyclonal antibody
(Fig. 3B) is intriguing in light of the highly localized
expression of dpp RNA in the cells just anterior to the A/P
boundary of the wing imaginal disc (Gelbart, 1989; Masucci
et al., 1990; Posakony et al., 1991). The highest levels of
expression observed for these two BMPs not only complement
one another in the wing imaginal disc but also in the leg and
eye/antennal discs (Figs 3, 4). One implication of the mainly
non-overlapping expression domains of gbb-60A and dpp is
that these two BMPs may exist predominately as homodimers.
Heterodimer formation would only take place in cells where
Gbb-60A and Dpp were co-expressed and given the localized
expression of dpp RNA this could only occur along the A/P
boundary. Further evidence of a role for Gbb-60A as a
homodimer is provided by the defects that we observe in gbb60A null clones in posterior cells, which do not express dpp
and do not exhibit a mutant phenotype when null for dpp. The
loss of pattern elements within these clones indicates that Gbb60A must act as a homodimer in these cells.
Ratio of Gbb-60A to Dpp signaling is critical to
normal wing morphogenesis
An increasing number of TGF-β/BMP family members have
been implicated as key players in directing a number of
developmental processes including cell proliferation,
patterning and differentiation. Given that multiple TGFβ/BMP members have been found to be expressed within the
same developing tissue (see Hogan, 1996; Kingsley, 1994b), it
is of interest to understand the specific function of each of these
members as well as the mechanism by which they signal. Thus
2732 O. Khalsa and others
far, we know very little about the role of more than one ligand
in a tissue. In some cases the expression domains overlap,
raising the possibility of heterodimer formation and therefore
the potential for eliciting a different response than that
acheived by the homodimer. Varied responses within a tissue
may not only result from homo/heterodimer differences but
also from multiple homodimers signaling in an additive or
perhaps antagonistic manner. Furthermore, should the
signaling from multiple homodimers be synergistic, other
responses could be achieved. Empirical evidence to support
this possibility has not previously been presented. Rather, in
the mouse, it has been suggested that the loss of BMP7 can be
compensated for by BMP2 which is expressed in the same
tissue (Dudley and Robertson, 1997). In this paper, we provide
data indicating that, in addition to the fact that Gbb-60A has a
role in wing morphogenesis distinct from that of Dpp, Gbb60A and Dpp signal together to pattern the adult wing. Both
genes are also required earlier in larval development and again
the process appears to depend on the presence of both ligands.
In order to investigate the potential interplay between Gbb60A and Dpp signaling, we examined the possiblity that alleles
of both genes may genetically interact. The genetic interaction
that we observed between dpp and gbb-60A resulted in a
change in viability as well as a change in the presence or
absence of pattern elements in the adult. Given the nature of
the molecules encoded by these genes, the genetic interaction
that we observe could be interpreted in several ways.
One interpretation is that gbb-60A somehow modulates dpp
activity such that in the presence of Gbb-60A the output of Dpp
signaling is boosted. Given the dramatic morphogenetic
organizing properties exhibited by dpp and thus far not
observed for gbb-60A, we could speculate that the role of gbb60A may be to merely fine tune the effectiveness of Dpp
signaling. A simplistic model to explain such a modulation of
Dpp signaling could either be that a different response is
attained by the Dpp/Gbb-60A heterodimer versus the Dpp
homodimer. Alternatively, the two ligands could signal
independently as homodimers and Gbb-60A signaling
somehow facilitates Dpp signaling, in which case we would
expect the functions of dpp and gbb-60A to be additive.
Another interpretation is that for the most part Dpp and Gbb60A signal independently and where the signals overlap cells
are able to differentiate between the relative levels of these
signals, read the relative levels of these two ligands and
respond accordingly. The important issue being that the
response elicited is dependent on the balance of Dpp to Gbb60A signaling at that location in the developing tissue. This
model would allow for both homodimer and heterodimer
signaling as well as the complex response that we observe in
the interactions between dpp and gbb-60A loss-of-function
alleles, which exhibits aspects of both synergy and antagonism.
Dpp and Gbb-60A signaling could be additive in some cells
but more importantly a shift in the balance of these ligands
would provide new or different positional information.
Our data provide strongest support for the second model
based on the following reasons. Genetic interactions between
alleles of gbb-60A and dpp were identified by mutant wing
phenotypes, which include the loss of pattern elements not
normally absent in phenotypes produced by mutations in either
gene alone or by the copy number of that mutation alone (Fig.
5). In addition to the more severe wing phenotype, we observed
an increase in the degree of lethality of dpp gbb-60A/ + gbb60A genotypes (Fig. 5I) beyond what would be expected if their
signaling were additive. The effect of dpp− on the percent
viability observed for gbb-60A mutants is not limited to alleles
of dpp that alter dpp transcriptional levels but is also apparent
in genotypes where the reduction of Dpp protein activity is
affected (S. S.-T., unpublished data). Therefore, lowering the
level of Dpp signaling in a gbb-60A mutant background results
in an exacerbation of the degree of lethality normally observed
for either genotype alone. The qualitative changes in the wing
phenotype of dpp/gbb-60A interacting genotypes is indicative
of the fact that the role of gbb-60A is not merely to enhance
the Dpp signal in an additive manner but that the relationship
between Dpp and Gbb-60A signaling must be much more
complex. If Dpp and Gbb-60A signaling were simply additive,
we would predict that in a gbb-60A mutant the effective activity
of Dpp would just be lowered and thus the first pattern element
to be affected would be those that are normally most sensitive
to the loss of dpp, such as the ACV, L2 and L4. This is not the
case, as the pattern elements most sensitive to the lowering of
gbb-60A are the PCV, L5 and then L4. In addition, as the levels
of both dpp and gbb-60A are lowered, we see a restoration of
L5 indicating that the ratio of Gbb-60A to Dpp is the important
criteria not the absolute levels of both ligands. Furthermore, if
Gbb-60A enhanced the Dpp signal, we would expect that
increasing both dpp and gbb-60A would lead to phenotypes
associated with elevated Dpp signaling. Instead, phenotypes
induced by ectopic expression of gbb-60A are partially
suppressed by ectopic expression of dpp (Fig. 6) further
supporting our proposal that the balance of Gbb-60A and Dpp
signaling activity is the mitigating factor in establishing
positional information.
BMP type I receptors, Tkv and Sax, mediate
signaling
We investigated the possibility that BMP type I receptors, Tkv
and Sax, mediate the signaling by both Gbb-60A and Dpp.
Heteroallelic combinations sax gbb-60A1/ + gbb-60A4 or tkv
gbb-60A1/ + gbb-60A4 exhibit more extreme mutant
phenotypes than those observed in gbb-60A genotypes alone
(Fig. 7). Since Dpp and Gbb-60A signaling is linked in some
cases, the tkv/gbb-60A or sax/gbb-60A interactions could
reflect an indirect effect of compromising the efficiency of Dpp
signaling by lowering the pool of receptors that are known to
mediate the Dpp signal. Several aspects of the tkv/gbb-60A
genetic interactions suggest that this is not wholly the
explanation for the enhanced phenotypes. First, in Df(2L)tkv2
gbb-60A1/ +gbb-60A4 individuals, we observe a severe loss of
veins including L3, a pattern element that is never affected in
dpp mutants and has been postulated to be determined
independently of Dpp signaling (Sturtevant et al., 1997).
Second, if the interaction that we observe is a secondary
consequence of reducing Dpp signaling, we would expect the
viability of the Df(2L)tkv2 gbb-60A1/+ gbb-60A4 genotype to
be much reduced as is seen in dpp gbb-60A1/+ gbb-60A4
genotypes (Fig. 5I), yet it is significantly increased. Third, a
similar enhancement of the tkv427/Df(2L)tkv2 vein thickening
phenotype as induced by gbb-60A1/ + has been observed by
increasing, not decreasing, dpp+ through the addition of a dpp
transgene (Penton et al., 1994). Thus, different levels of Gbb60A and Dpp affect signaling through Tkv in dramatically
Gbb-60A to Dpp ratio critical for patterning 2733
different ways at different stages of development. These pieces
of data do not negate the possibility that Gbb-60A boosts or
enhances the Dpp signal at some level and that this is mediated
through Tkv in some fashion, perhaps as a heterodimer with
higher affinity or by some other as yet unknown mechanism.
This possibility is consistent with data suggesting that Tkv
mediates a higher level of Dpp signaling (Singer et al., 1997).
However, our results indicate that, for some developmental
events, it is likely that Tkv mediates the Gbb-60A signal alone
and may be responsible for mediating the antagonism between
these two ligands observed in some aspects of their genetic
interaction. The mechanism by which this differential
mediation of signal remains to be elucidated.
Despite the unique roles that dpp and gbb-60A each play, the
observed genetic interactions indicate that Dpp and Gbb-60A
signaling can be dependent upon one another and that they must
signal together in a complex manner involving both synergy
and antagonism to achieve normal wing morphogenesis as well
as viability. gbb-60A, like dpp, has multiple requirements
during development, in addition to its role in growth, patterning
and vein promotion in the developing wing. Given the
expression pattern of gbb-60A and dpp in other imaginal discs
and the phenotypic consequences of their genetic interaction in
the adult leg (Fig. 5F), it is likely that gbb-60A and dpp function
together in other tissues as well, to direct various developmental
programs. The molecular implications of the functional data
presented here can be quite straightforward. The complex
signaling of gbb-60A and dpp must be mainly accomplished by
Gbb-60A and Dpp homodimers, however, some degree of Gbb60A/Dpp heterodimer signaling can not be eliminated by our
data. Regardless, cells in the developing wing are clearly able
to interpret different levels of Gbb-60A versus Dpp. Numerous
protein/protein interactions at different points in the
transduction of TGF-β/BMP signals contribute to the diversity
of effects elicited by these signaling molecules. The mechanism
responsible for the interpretation of different ligands is
unknown and remains to be examined. However, at one level
both Tkv and Sax receptors appear to mediate Gbb-60A and
Dpp signals and in one scenario a difference in binding
affinities for the two different ligands could account for
different signaling readouts. Experiments using activated and
dominant negative receptors support the notion that Tkv and
Sax mediate Dpp and Gbb-60A signaling at differential levels
(T. E. Haerry, O. K., M. B. O’Connor and K. A. W.,
unpublished data). TGF-β receptors are thought to form
tetrameric signaling complexes consisting of two type I and two
type II receptors, however, as yet it is not known whether two
different type I receptors can both contribute to that complex.
If this were the case, it is conceivable that Gbb-60A and Dpp
could signal through complexes containing both Tkv and Sax.
Therefore, various combinations of these ligands and type I
receptors could form to elicit different types of output. When
considering ligand/receptor interactions it is important to
remember that the presence of proteases and other binding
proteins may dictate the availability of the ligand to bind to a
receptor complex, as well as with what affinity it binds (LopezCasillas et al., 1994; Nakato et al., 1995). Having demonstrated
that two different BMPs are both required for a single
developmental process, we can now begin to dissect the
signaling network by which these two ligands accomplish their
task.
We thank Laura Dickinson for technical assistance with RNA in
situ hybridizations. We thank Bruce Reed, Matt Singer and Michael
O’Connor for sending fly stocks and John Doctor for Gbb-60A
antibodies. O. K. and J. Y. have been supported in part by NIH
GM07601 training grant and this work was done in partial fulfillment
of the requirements for a PhD. This research was supported in part by
NSF IBN-9205808, IBN-9604769 and an ACS Research Grant DB32. K. A. W. was supported by an Established Investigatorship from
the American Heart Association and with funds contributed in part by
the AHA, Maine Affiliate.
REFERENCES
Affolter, M., Nellen, D., Nussbaumer, U. and Basler, K. (1994). Multiple
requirements for the receptor serine/threonine kinase thick veins reveal
novel functions of TGF-ß homologs during Drosophila embryogenesis.
Development 120, 3105-3117.
Arora, K., Levine, M. and O’Connor, M. (1994). The screw gene encodes
a ubiquitously expressed member of the TGF-ß family required for
specification of dorsal cell fates in the Drosophila embryo. Genes Dev. 8,
2588-2601.
Blackman, R. K., Sanicola, M., Raftery, L., Gillevet, T. and Gelbart, W.
M. (1991). An extensive 3′ cis-regulatory region directs the imaginal disk
expression of decapentaplegic, a member of the TGF-β family in
Drosophila. Development 111, 657-666.
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.
Brummel, T. J., Twombly, V., Marqués, G., Wrana, J. L., Newfeld, S. J.,
Attisano, L., Massagué, J., O’Connor, M. B. and Gelbart, W. M. (1994).
Characterization and relationship of dpp receptors encoded by the
saxophone and thick veins genes in Drosophila. Cell 78, 251-261.
Burke, R. and Basler, K. (1996). Dpp receptors are autonomously required
for cell proliferation in the entire developing Drosophila wing. Development
122, 2261-2269.
Cohen, S. M. (1993). Imaginal disc development. In The Development of
Drosophila melanogaster, pp. 747-841. Cold Spring Harbor Laboratory Press
deCelis, J. F. (1997). Expression and function of decapentaplegic and thick
veins during the differentiation of the veins in the Drosophila wing.
Development 124, 1007-1018.
Derynck, R. and Zhang, Y. (1996). The mad way to do it. Curr. Biol. 6,12261229.
Doctor, J. S., Jackson, D., Rashka, K. E., Visalli, M. and Hoffmann, F. M.
(1992). Sequence, biochemical characterization, and developmental
expression of a new member of the TGF-ß superfamily in Drosophila
melanogaster. Dev. Biol. 151, 491-505.
Dudley, A. T. and Robertson, E. J. (1997). Overlapping expression domains
of bone morphogenetic protein family members potentially account for
limited tissue defects in BMP7 deficient embryos. Devel. Dynam. 208, 349362.
Ferguson, E. L. and Anderson, K. v. (1992). decapentaplegic acts as a
morphogen to organize dorsal-ventral pattern in the Drosophila embryo.
Cell 71, 451-461.
The Flybase Consortium (1996). Flybase: the Drosophila database. Nucl.
Acids Res. 24, 53-56.
Gelbart, W. M. (1989). The decapentaplegic gene: a TGF-β homologue
controlling pattern formation in Drosophila. Development 1989
Supplement, 65-74.
Golic, K. G. and Lindquist, S. (1989). The FLP recombinase of yeast
catalyzes site-specific recombination in the Drosophila genome. Cell 59,
499-509.
Hogan, B. L. M. (1996). Bone morphogenic proteins: multifunctional
regulators of vertebrate development. Genes Dev. 10, 1580-1594.
Jiang, J. and Struhl, G. (1996). Complementary and mutually exclusive
activities of Decapentaplegic and Wingless organize axial pattern during
Drosophila limb development. Cell 86, 401-409.
Kingsley, D. M. (1994a). The TGF-β superfamily: new members, new
receptors, and new genetic tests of function in different organisms. Genes
Dev. 8, 133-146.
Kingsley, D. M. (1994b). What do BMPs do in mammals? Clues from the
mouse short-ear mutation. Trends in Genet. 10, 16-21.
2734 O. Khalsa and others
Lawrence, P. A., Johnston, P. and Morata, G. (1986). Methods of marking
cells. In Drosophila, a Practical Approach (D. B. Roberts). pp. 229-242.
Oxford: IRL Press
Lecuit, T., Brook, W., Ng, M., Calleja, M., Sun, H. and Cohen, S. (1996).
Two distinct mechanisms for long-range patterning by decapentaplegic in
the Drosophila wing. Nature 381, 387-393.
Lecuit, T. and Cohen, S. M. (1997). Proximal-distal axis formation in
theDrosophila leg. Nature 388, 139-145.
Lopez-Casillas, F., Patne, H. M., Andres, J. L. and Massague, J. (1994).
Betaglycan can act as a dual modulator of TGF-β access to signaling
receptors: mapping of ligand binding and GAG attachment sites. J. Cell Biol.
124, 557-568.
Lyons, K. M., Hogan, B. L. M. and Robertson, E. J. (1995). Colocalization
of BMP7 and BMP2 RNAs suggests that these factors cooperatively
mediate tissue interactions during murine development. Mech. Dev. 50, 7183.
Massagué, J. (1996). TGF-β signalling: receptors, transducers and mad
proteins. Cell 85, 947-950.
Massagué, J., Cheifetz, S., Boyd, F. T. and Andres, J. L. (1990). Mediators
of TGF-β action: TGF-β receptors and TGF-β binding proteoglycams. Ann.
NY Acad. Sci. USA 593, 59-72.
Massagué, J., Hata, A. and Liu, F. (1997). TGF-β signalling through the
Smad pathway. Trends Cell Biol. 7, 187-192.
Masucci, J. D., Miltenberger, R. J. and Hoffmann, F. M. (1990). Patternspecific expression of the Drosophila decapentaplegic gene in the imaginal
disks is regulated by 3’ cis-regulatory elements. Genes Dev. 4, 2011-2023.
Nakato, H., Futch, T. A. and Selleck, S. B. (1995). The division abnormally
delayed, dally, gene: A putative integral membrane proteoglycan required
for cell division patterning during postembryonic development of the
nervous system in Drosophila. Development 121, 3687-3702.
Nellen, D., Affolter, M. and Basler, K. (1994). Receptor serine/threonine
kinase implicated in the control of Drosophila body pattern by
decapentaplegic. Cell 78, 225-237.
Nellen, D., Burke, R., Struhl, G. and Basler, K. (1996). Direct and longrange action of a DPP morphogen gradient. Cell 85, 357-368.
Padgett, R., St. Johnston, R. and Gelbart, W. (1987). A transcript from a
Drosophila pattern gene predicts a protein homologous to the transforming
growth factor-β family. Nature 325, 81-84.
Penton, A., Chen, Y., Staehling-Hampton, K., Wrana, J. L., Attisano, L.,
Szidonya, J., Cassill, J. A., Massagué, J. and Hoffmann, F. M. (1994).
Identification of two bone morphogenetic protein type I receptors in
Drosophila and evidence that Brk25D is a decapentaplegic receptor. Cell
78, 239-250.
Posakony, L. G., Raftery, L. A. and Gelbart, W. M. (1991). Wing formation
in Drosophila melanogaster requires decapentaplegic gene function along
the anterior-posterior compartment boundary. Mech. Dev. 33, 69-82.
Raftery, L., Twombly, V., Wharton, K. and Gelbart, W. (1995). Genetic
screens to identify elements of the decapentaplegic signaling pathway in
Drosophila. Genetics 139, 241-254.
Roberts, A. B. and Sporn, M. B. (1990). The transforming growth factor-βs.
In Peptide Growth Factors and Their Receptors I. (ed. Michael B. Sporn
and Anita B. Roberts). pp. 419-472. Springer-Verlag.
Segal, D. and Gelbart, W. M. (1985). Shortvein, a new component of the
decapentaplegic gene complex in Drosophila melanogaster. Genetics 109,
119-143.
Singer, M. A., Penton, A., Twombly, V., Hoffmann, F. M. and Gelbart, W.
M. (1997). Signaling through both type I DPP receptors is required for
anterior-posterior patterning of the entire Drosophila wing. Development
124, 79-89.
Spencer, F. A., Hoffmann, F. M. and Gelbart, W. M. (1982).
decapentaplegic: A gene complex affecting morphogenesis in Drosophila
melanogaster. Cell 28, 451-461.
Staehling-Hampton, K., Jackson, P. D., Clark, M. J., Brand, A. H. and
Hoffmann, F. M. (1994). Specificity of bone morphogenetic protein-related
factors: Cell fate and gene expression changes in Drosophila embryos
induced by decapentaplegic but not 60A. Cell Growth, Differen. 5, 585-593.
Sturtevant, M. A. B., Marin, E. and Bier, E. (1997). The spalt gene links
the A/P compartment boundary to a linear adult structure in the Drosophila
wing. Development 124, 21-32.
Tautz, D. and Pfeifle, C. (1989). A non-radioactive in situ hybridization
method for the localization of specific RNAs in Drosophila embryos reveals
a translational control of the segmentation gene hunchback. Chromosoma
98, 81-85.
Twombly, V., Blackman, R. K., Jin, H., Graff, J. M., Padgett, R. and
Gelbart, R. W. (1996). The TGF-β signaling pathway is essential for
Drosophila oogenesis. Development 122, 1555-1565.
Wharton, K. A., Ray, R. P. and Gelbart, W. M. (1993). An activity gradient
of decapentaplegic is necessary for the specification of dorsal pattern
elements in the Drosophila embryo. Development 117, 807-822.
Wharton, K. A., Thomsen, G. H. and Gelbart, W. M. (1991). Drosophila
60A gene, another transforming growth factor-β family member, is closely
related to human bone morphogenetic proteins. Proc. Natn. Acad. Sci. USA
88, 9214-9218.
Xie, T., Finelli, A. L. and Padgett, R. W. (1994). The Drosophila saxophone
gene: a serine-threonine kinase receptor of the TGF-β superfamily. Science
263, 1756-1759.
Xu, T. and Harrison, S. D. (1994). Mosaic analysis using FLP recombinase.
Vol. 44. Drosophila melanogaster: Practical Uses in Cell and Molecular
Biology. (ed. S. Lawrence, B. Goldstein and Eric A. Fyrberg). San Diego:
Academic Press.
Yamashita, H., ten Dijke, P., Franzen, P., Miyazono, K. and Heldin, C. H.
(1994). Formation of hetero-oligomeric complexes of type I and type II
receptors for transforming growth factor-β. J. Biol. Chem. 269, 2017220178.