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First published online 3 August 2006
doi: 10.1242/dev.02513
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Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, RI 02912, USA.
* Author for correspondence (e-mail: kristi_wharton{at}brown.edu)
Accepted 28 June 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Bmp signaling, Dpp, Gbb, Tkv, Sax, Bmp type I receptor, Wing imaginal disc, Morphogen
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Tgfß
superfamily members elicit their effects by binding to heteromeric receptor
complexes composed of two types of transmembrane serine/threonine kinase
receptor proteins: type I and type II (Luo
and Lodish, 1996; Weis-Garcia
and Massagué, 1996). Biochemical evidence, as well as
structural data, indicate that the basic signaling unit consists of a single
ligand dimer in contact with two type I and two type II transmembrane
serine-threonine kinase receptors (Kirsch
et al., 2000a; Kirsch et al.,
2000c). Upon ligand binding, the constitutively active type II
receptor phosphorylates the type I receptor
(Wrana et al., 1994;
Luo and Lodish, 1996) that
then phosphorylates a cytoplasmic effector molecule belonging to a family of
transcriptional regulators, the receptor-mediated Smads (R-Smads)
(Eppert et al., 1996;
Hoodless et al., 1996;
Attisano and Wrana, 2002).
Phosphorylated R-Smads form heteromeric complexes with a related co-Smad, and
the R-Smad/co-Smad complex translocates into the nucleus, where it, with other
transcriptional regulators, influences target gene transcription
(Massagué and Wotton,
2000; Moustakas et al.,
2001; ten Dijke and Hill,
2004).
Patterning of the Drosophila wing imaginal disc requires a Bmp
activity gradient. The graded activation of Bmp signaling along the AP axis
can be visualized using an antibody that recognizes the phosphorylated form of
Mad (pMad), the Drosophila R-Smad
(Tanimoto et al., 2000). Cells
within the wing pouch respond to graded levels of pMad by activating three Bmp
target genes, spalt (sal), optomotor blind
(omb) and vestigial (vg), at different distances
from the AP boundary (Lecuit et al.,
1996; Nellen et al.,
1996; Minami et al.,
1999; Kirkpatrick et al.,
2001), and by repressing brinker (brk)
expression, a direct Bmp target (Muller et
al., 2003). Proper patterning of the adult wing has been known for
some time to require the Bmp ligand encoded by decapentaplegic
(dpp) and, more recently, a second Bmp encoded by glass bottom
boat (gbb) (Spencer et al.,
1982; Ray and Wharton,
2001a). dpp is expressed in a stripe of anterior
compartment cells abutting the AP compartment boundary
(Blackman et al., 1991) and
gbb is expressed more broadly throughout much of the wing pouch
(Khalsa et al., 1998). Despite
its broad expression, loss-of-function studies indicate that Gbb produced by
cells along the AP boundary is absolutely required for patterning
(Ray and Wharton, 2001a). In
the absence of Gbb, Dpp exhibits only short-range signaling and the breadth of
the Bmp activity gradient requires gbb function, indicating that Gbb
provides the long range nature of the Bmp activity gradient
(Bangi and Wharton, 2006). A
reduction of Dpp, however, profoundly influences the peaks of the gradient
and, thus, cell fate specification in the central domain.
The two Bmp type I receptors, Thick veins (Tkv) and Saxophone (Sax), have
different requirements in wing patterning. Although sax is reported
to be ubiquitously expressed in the wing pouch
(Brummel et al., 1994),
tkv, which is regulated by both Hh and Bmp signaling, shows a more
complex expression pattern with higher expression in the posterior compartment
and a downregulation near the AP boundary
(Funakoshi et al., 2001;
del Alamo Rodriguez et al.,
2004). Tkv is essential for all Bmp signaling in the wing disc
based on the fact that eliminating tkv function leads to the absence
of pMad (Tanimoto et al.,
2000) (L. Soares and K.W., unpublished). However, although Sax is
not absolutely required for wing patterning
(Ray and Wharton, 2001a), the
reduction in sal expression seen in small sax-null clones
(Singer et al., 1997)
indicates that Sax must contribute in some way to the mediation of Bmp signals
in the wing imaginal disc.
Previous experiments suggested that Gbb signaling is primarily mediated by
Sax, whereas Dpp is primarily mediated by Tkv, based on the fact that the wing
phenotype associated with overexpression of Gbb or Dpp is better suppressed by
co-expression of the dominant-negative (DN) receptor, DN-Sax or DN-Tkv,
respectively (Haerry et al.,
1998). These respective phenotypic suppressions are similar to
demonstrated preferred binding affinities, i.e. Dpp and its vertebrate
ortholog Bmp2 have high binding affinity for Tkv, and Gbb, or its vertebrate
ortholog Bmp7, cannot compete with Bmp2 for Tkv binding
(Penton et al., 1994). A
similar receptor-ligand binding preference has been observed among the
vertebrate orthologs (Yamashita et al.,
1995; Nishitoh et al.,
1996; Chalaux et al.,
1998; Ebisawa et al.,
1999; Piek et al.,
1999).
Curiously, other data do not agree with the proposal that Sax is the
primary mediator of Gbb during wing patterning, despite the likelihood that
Gbb and Sax interact with high affinity. Primarily, unlike what would be
expected of a receptor and its ligand, loss of sax function does not
result in a wing phenotype similar to the loss of gbb function
(Singer et al., 1997;
Khalsa et al., 1998;
Ray and Wharton, 2001a).
Furthermore, reducing sax gene dose does not enhance the gbb
partial loss of function wing phenotype, whereas a similar reduction in
tkv gene dose clearly compromises Gbb signaling
(Khalsa et al., 1998). These
inconsistencies prompted us to examine the role of Sax as a potential mediator
of Gbb signaling more closely.
If as the functional data suggest, Sax is not absolutely necessary for
mediating Gbb signals, what role does Sax play in wing patterning? We found in
the complete absence of sax function that alterations in pMad
distribution and target gene expression were instead consistent with an
increase in Gbb activity, not a loss of Gbb signaling. In addition to this
apparent antagonistic function, our experiments also revealed that Sax
positively mediates Gbb signaling. To account for both positive and negative
functions of Sax, we propose a mechanism for signal transduction that depends
on whether Sax complexes with itself or Tkv. The endogenous Bmp activity
gradient in the wing disc is dependent not only on the mediation of Dpp and
Gbb by the Tkv, but also on the dual function of Sax as a modulator of ligand
availability. In both Drosophila and vertebrates, a different
mechanism for modulating ligand activity has been shown to involve secreted
molecules in other Bmp-dependent processes
(Massagué and Chen,
2000; Miyazono,
2000). The modulation of Tgfß/Bmp ligand availability by a
signaling receptor has not been observed before, and we discuss the
implications of this dual role for Sax in shaping the wing disc Bmp activity
gradient.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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),
UASdppw, A9-Gal4
(Haerry et al., 1998),
MS1096 (Capdevila,
1994), UASsax and UAStkv1 (gifts from M.
O'Connor) are homozygous viable. The brkX47-lacZ enhancer
trap recapitulates brk wing disc expression
(Campbell and Tomlinson, 1999).
All other alleles are described in FlyBase
(www.flybase.net).
gbb and dpp overexpression
Ligand and receptor overexpression was achieved using the Gal4-UAS system
(Brand and Perrimon, 1993).
Progeny overexpressing gbb in a background compromised for signaling
component genes were generated by yw/Y;*/Bl;UASgbb
X wA9-Gal4 (*=+, UASsax, nub b pr sax4,
FRTG13 sax4, Df(2R)H23, tkv8cn bw sp,
tkv7cn bw sp, tkv5, b pr Mad12, or
FRT43 shn3.3) and y w/Y;
*UASgbb/TM3,Sb X w A9-Gal4
(*=punt135 or Med1). Wings
from the following genotypes were mounted (DPX, EM sciences) and scored: w
A9-Gal4/y w;*/+;UASgbb/+ versus w A9-Gal4/y
w;Bl(+)/+;UASgbb/+ and w A9-Gal4/y w;UAS-gbb/+ versus w
A9-Gal4/y w;*UAS-gbb/+. Wings from progeny co-expressing
gbb and tkv were compared: w A9-Gal4/w;UAStkv1/UASgbb, w
A9-Gal4/w;UASgbb/TM3,Sb and w A9-Gal4/w;UASgbb/+ were scored.
dpp overexpression crosses were carried out similarly; however, the
final cross was raised for 2 days at 25°C, then 18°C until eclosion.
Each experiment was repeated multiple times with 200-400 wings/genotype scored
each time.
tkv and sax overexpression
sax was overexpressed at different levels by raising y w/Y;
UASsax X w A9-Gal4, w MS1096-Gal4 or w;1348-Gal4 at
25°C or 29°C, and in a gbb mutant background: wings of the
w *-Gal4/y w; UASsax bw gbb4/bw
gbb5I females were scored, where * indicates A9 or
MS1096. Wings of all progeny were scored. Different levels of tkv
overexpression were achieved by crossing w/Y; UAStkv1 X w A9/w;
UAStkv1/+ at 25°C or 29°C. For each cross, wings from both sexes
and with one versus two copies of UAStkv1 were scored.
Clonal analysis
sax-null clones were generated in the following genotypes: (1) for
adult wing, whsFLP122/yw;
FRTG13sax4shaIN/FRTG13M; (2)
in gbb mutant background, whsFLP122/yw;
FRTG13 gbb4/FRTG13 sax4
shaIN gbb4; and (3) with brk-lacZ in wing
discs, w brkX47-lacZ/w hsFLP122;
FRTG13shaINsax4/FRTG13Mubi-GFPx2.
Clones were induced by heat-shock (37°C) at various times after egg lay to
generate different size clones. A recessive marker, shavenoid
(shaIN), which removes or reduces the trichomes on the
wing blade (Lawrence et al.,
1986), was used to identify wing clones.
Immunohistochemistry
sax4/Df(2R)H23, Df(2R)H23/+ and
gbb1/+ larvae with brk-lacZ were
selected making use of CyOGFP. Everted larvae were fixed in 4%
formaldehyde for 10 minutes, blocked in 1% NGS and 0.1% Triton-X in PBS for an
hour and incubated overnight at 4°C with mouse anti-ß-galactosidase
antibody (Cappel, 1:3000) or rabbit anti-PS1 (anti-pMad, 1:2000). Secondary
antibodies (Alexa 488, 594 and 633, Molecular Probes) were used at 1:3000.
Discs were mounted in 80% glycerol, 0.5% N-propyl galate in PBS. Within an
experiment, fluorescence images were captured at identical settings with a
LeicaTCS-SP2-AOBS confocal microscope, 3D reconstructions were made using
DeskVOX [Jürgen Schulze in the Computer Science Department at Brown
University
(http://www.calit2.net/~jschulze/)].
Fluorescence intensity profiles were obtained using Metamorph across the
regions of the wing disc shown in each figure.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Based on the results and interpretations of previous experiments using DN
receptors (Haerry et al.,
1998), increasing levels of wild-type Sax were expected to
facilitate Gbb signaling and enhance this phenotype. Similarly, reducing Sax
levels should suppress the gbb overexpression wing phenotype by
lowering the number of receptors available for the increased level of Gbb
ligand to signal. However, in striking contrast to these predictions, we found
that overexpression of wild-type Sax strongly suppressed gbb
overexpression (Fig. 1A,B).
Thus, increasing Sax levels appears to inhibit Bmp signaling attributed to
excess Gbb. To ensure that Gbb overexpression did not saturate the signaling
system by limiting factors essential for Bmp signaling and thus, indirectly
prevent Sax from further increasing signaling, we tested the effect of
overexpressing Tkv. Increasing Tkv levels enhanced the gbb
overexpression phenotype (Fig.
1C), clearly demonstrating that the ability of Sax to suppress Gbb
signaling in this assay cannot be explained simply by the limitation of a
general Bmp signaling component, but rather, reflects an ability of excess Sax
to block Gbb signaling.
Making use of a cell culture assay for Bmp signaling
(Muller et al., 2003), we
found that transfection of Drosophila S2 cells with DNA encoding Dpp or Gbb
stimulated signaling in a dose-dependent manner, making use of endogenously
expressed components (see Fig. S1 in the supplementary material).
Co-transfection of constructs encoding Dpp and Tkv or Gbb and Tkv led to an
increase in signaling, while co-transfection with Sax led to reduced signaling
consistent with our in vivo studies indicating that Sax is able to block Bmp
signaling.
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). By
contrast, reducing the dose of sax enhances the gbb
overexpression phenotype (Fig.
1D), indicating that a reduction in endogenous Sax receptor leads
to a further increase in Gbb signaling. Taken together, these findings suggest
that excess Gbb signaling is mediated by endogenous Tkv but antagonized by
endogenous Sax. In order to test whether Sax also antagonizes Dpp function, we took a similar approach and, as expected, reduction of tkv gene dose resulted in a strong suppression of the dpp overexpression phenotype (Fig. 1F,H). A mild suppression was also seen when the sax gene dose was lowered (Fig. 1G), indicating that lowering endogenous Sax compromises excess Dpp signaling.
The strong suppression of dpp overexpression by reduced Tkv levels supports the current proposal that Tkv is the primary mediator of Dpp signaling. The ability of tkv/+ to efficiently suppress gbb overexpression indicates that Gbb signaling is also dependent on endogenous Tkv. As indicated by studies on the relative binding affinities of Dpp and Gbb for Tkv and Sax (see Fig. S2 in the supplementary material), both Dpp and Gbb are sensitive to Tkv levels and the higher sensitivity of Dpp is consistent with its higher affinity. Endogenous Sax, however, can antagonize the ability of Gbb to signal. No other Bmp signaling component was found to behave antagonistically towards Gbb signaling (Fig. 1I).
Antagonizing function of Sax limits the range of the Bmp activity gradient
From the experiments presented thus far, several important conclusions can
be drawn: (1) increasing Sax levels in the developing wing inhibits Gbb
signaling caused by excess ligand activity; (2) the ability of Sax to block
Gbb signaling is also evident when endogenous levels of Sax are reduced; (3)
Sax is the only Bmp signaling component tested thus far that antagonizes Gbb
signaling; and (4) although Tkv shows a preference for Dpp, both Gbb and Dpp
signals are mediated by Tkv. These conclusions predict that in the complete
absence of Sax, Bmp signaling should still occur. Furthermore, when Sax levels
are lowered, a change in Bmp signaling activity (pMad) should be evident given
the potential for ligand availability to be affected. We examined the
distribution of pMad and the expression of the Bmp target gene brk in
wing discs with varying doses of sax to get a better understanding of
how Sax levels may effect Bmp signaling globally
(Fig. 2).
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As one would predict for a morphogen gradient, the Bmp activity (pMad) gradient is very sensitive to the ligand levels that generate it. When gbb dose is halved (gbb1/+), we consistently saw in a sampling of discs a slight narrowing of pMad distribution and a corresponding failure to repress brk expression in the more central domain of the wing pouch (Fig. 2D). Taken together, the fact that the profile of the pMad gradient changes with a corresponding expansion or retraction of brk repression as the dose of sax or gbb changes, indicates that the ability of endogenous Sax to antagonize Gbb signaling affects the shape of the Bmp activity gradient. The fact that obvious patterning defects are not apparent in the adult wings of sax or gbb heterozygotes indicates that the developing wing is able to self-regulate with time at some level across the disc at either the total amount of ligand produced (L. Soares and K.A.W., unpublished) or at the level of signaling thresholds required for fate specification.
Sax as a signaling receptor in the wing
Although global changes in Sax levels reveal its novel antagonistic
function, they also show a lowering in pMad levels near the AP boundary where
it normally peaks in wild-type discs (Fig.
2B). These observations indicate that Sax plays some role in
mediating Bmp signals, consistent with the reduction in sal
expression seen in small sax clones and the ability of such clones to
generate pattern duplications of L2 and L5
(Singer et al., 1997). If the
anterior border of the sax clone falls between L2 and L3, an ectopic
L2 may form at the anterior boundary of the clone
(Fig. 3A) consistent with the
idea that reducing Bmp signaling in the clone would lead to a shift in the
gradient, and thus, a change in target gene expression, such that cells at the
border take on a fate typical of those closer to the AP boundary
(Fig. 3F). Yet, we previously
showed that sax clones encompassing an entire compartment had little
effect on the overall wing pattern, suggesting that global removal of Sax does
not compromise Bmp signaling (Ray and
Wharton, 2001b). Given this apparent contradiction, we looked more
closely at the ability of Sax to promote signaling and investigated the
possibility that Sax could both promote, as well as antagonize signaling in
vivo.
We found that the effect of globally removing sax function depends on the dose or level of gbb function. Complete loss of sax can enhance a gbb partial loss-of-function wing phenotype (see Fig. S3 in the supplementary material), indicating that Sax must mediate some Gbb signaling. This requirement was only revealed when gbb function is compromised. Small sax clones near the AP boundary show a reduced level of pMad (Fig. 4B,C). This reduction in pMad provides a direct readout for the requirement of Sax for signaling and indicates that endogenous Tkv alone cannot compensate for the loss of Sax to maintain wild-type levels of signaling. The level of Bmp signaling must be below what is necessary to repress brk expression in some sax mutant cells (Fig. 4C). Those that are near the AP boundary are less likely to show ectopic brk, indicating that loss of sax function must not impact Bmp signaling as significantly in these cells as in the periphery of the wing disc. An interesting feature of these small sax clones is the increase in pMad levels in cells immediately outside the clone, clearly evident from pMad intensity profiles taken across the sax clone (Fig. 4B'',C''). This increase in Mad-mediated signaling is consistent with the idea that ligand produced by the cells of the sax null clone is no longer trapped or bound and is able to move to and signal in surrounding cells. Thus, it is possible to visualize both the antagonistic and positive signaling functions of Sax in the wing disc.
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If the localized increase in Bmp signaling is indeed due to an increase in available Gbb, then ectopic veins associated with sax clones should not form when the total level of functional Gbb available for signaling is reduced. Null sax clones induced in a genetic background compromised for Gbb function that are posterior to L5 or anterior to L2 never (n>50) lead to vein duplications (Fig. 3D). Thus, the formation of such ectopic veins is sensitive to Gbb function and any increase in mutant Gbb protein due to the loss of Sax does not produce levels of pMad high enough to specify an ectopic L2. Together, these data reveal that endogenous Sax must normally limit the amount of endogenous Gbb available for signaling and that local changes in Sax levels can impact the Bmp activity gradient and, thus, wing patterning.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Dual function of Sax likely depends on its receptor partner
Given that Tkv is required for all Bmp signaling in the wing disc, the
simplest explanation for the fact that Sax signaling appears to depend on the
presence of Tkv is that Sax can only promote signaling in a receptor complex
also containing Tkv. Three different forms of Bmp receptor complexes can
potentially form in wing disc cells, those composed of two type II receptor
molecules and either two Tkv, two Sax or one molecule of each: Tkv-Tkv,
Sax-Sax and Tkv-Sax (Fig. 5A).
Overexpressing Tkv or Sax in wing disc cells enabled us to shift the balance
between the relative levels of these two molecules, artificially enriching for
the formation of receptor complexes homomeric for type I molecules Tkv-Tkv or
Sax-Sax. Disrupting the balance of endogenous Tkv to Sax levels by
overexpressing Sax immediately reveals the antagonistic function of Sax,
consistent with the idea that excess Sax could be sequestering ligand in
Sax-Sax receptor complexes which signal either very poorly or not at all.
However, overexpression of Tkv, enriching for Tkv-Tkv complexes with high
affinity for Dpp and lower affinity for Gbb, leads to increased signaling
given sufficient ligand. The third receptor complex, Tkv-Sax, probably
accounts for the contribution of Sax to the promotion of Bmp signaling and
probably signals in vivo more efficiently than Tkv-Tkv, based on the fact that
pMad levels are lower inside clones devoid of Sax than the pMad levels seen in
cells at an equivalent position along the AP axis elsewhere on the disc (see
Fig. 4,
Fig. 5B). Loss of Tkv, by
definition, eliminates signaling by both Tkv-Tkv and Tkv-Sax, leaving only
Sax-Sax containing receptor complexes, which are clearly unable to elicit a
pMad-mediated signal on their own. Thus, our model predicts that removing Sax
function results in two opposing consequences: (1) a reduction in total Bmp
signaling caused by loss of Tkv-Sax complexes; and (2) an increased
availability of Bmp ligand and potential signaling caused by loss of Sax-Sax
complexes (Fig. 5C). Several
biochemical studies support the putative existence of functional Sax-Tkv
receptor complexes. Heteromeric complexes involving different vertebrate type
I receptors have been shown to contribute to a single signaling receptor
complex (Gilboa et al., 2000;
Kirsch et al., 2000b) and in
Drosophila S2 cells both Sax and Tkv appear to be necessary to
produce a synergistic signal (Shimmi et
al., 2005).
It is important to note that increasing wild-type Tkv levels in the presence versus absence of excess ligand results in very different phenotypic outcomes. In contrast to Sax, increasing Tkv in the presence of excess ligand leads to a larger increase in Bmp signaling. However, at endogenous ligand levels, as Tkv levels are experimentally increased, we see a loss of Bmp signaling that is indicative of the preference of Tkv for binding Dpp over Gbb (Fig. S2). Clearly, both Gbb and Dpp become limiting in the presence of excess Tkv, with low level Tkv overexpression preferentially limiting Dpp-dependent signaling, while higher levels of overexpression limit both. Clearly, although overexpression of ligand and receptor together reveals a significant difference in the signaling ability of Tkv and Sax, overexpression of receptor alone in the absence of increased ligand appears to reflect only receptor ligand-binding preference.
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Ligand specificity of different receptor complexes in wing disc
The fact that both Dpp and Gbb are dependent on Tkv for signaling has
significant implications regarding the Bmp activity gradient, given that
removal of Tkv at any point along the gradient results in the loss of both Gbb
and Dpp signaling, not just Dpp signaling. When both ligands are present at
similar levels, the higher affinity of Dpp for Tkv means the contribution of
Dpp to total Bmp signaling will be more significant than that of Gbb, and
movement of Dpp across the wing disc will be affected more strongly by Tkv
than that of Gbb. Thus, Gbb should and does contribute more significantly to
the low points of the Bmp activity gradient
(Ray and Wharton, 2001b;
Bangi and Wharton, 2006),
especially as competition with Dpp for binding to Tkv will also be lower in
these regions.
Our findings from receptor and ligand overexpresion experiments suggest
that both the antagonistic and signal promoting functions of Sax impact Gbb
signaling most significantly because of their preferential interaction. For
example, although localized loss of Sax from the peripheral cells of the wing
pouch leads to ectopic induction of brk, loss in more central cells
does not, suggesting that the relative contribution of Sax to overall Bmp
signaling is less in the central cells where Tkv must contribute more
significantly given the higher level of Dpp near the AP boundary. The greater
contribution of Sax to total signaling in the more peripheral cells of the
wing pouch is consistent with its higher affinity for Gbb and the long-range
nature of Gbb versus Dpp (Bangi and
Wharton, 2006). Similarly, removal of Sax from just anterior
compartment cells results in brk repression in both the anterior and
posterior compartments (data not shown) suggesting that in the absence of Sax,
anteriorly expressed Gbb can signal to the posterior-most cells of the wing
pouch to effectively repress brk expression beyond its normal domain.
This result indicates that endogenous Sax normally functions to not only
restrict the level of Gbb signaling but also the range of Gbb. The role that
Sax plays in promoting Gbb function, in particular, is detected only when
sax function is completely eliminated and gbb function is
also significantly compromised (see Fig. S3 in the supplementary
material).
Given that Tkv is also required for mediating Gbb signals, of the two proposed receptor complexes that could mediate Gbb signaling (Tkv-Tkv and Tkv-Sax), which is preferentially used by Gbb in wild-type cells? It is clear that Tkv-Sax complexes are not obligatory for Gbb signaling as Gbb signaling is not abolished in sax mutants. The fact that removing Sax does not cause a gbb loss-of-function phenotype indicates that enough Gbb is made available by the loss of Sax antagonism and can signal to compensate for losing that region of total signaling that Sax normally promotes. The fact that pMad levels within a sax clone are lower then endogenous levels indicates that signaling in the clone cells containing only Tkv-Tkv is less efficient than the neighboring cells that have wild-type levels of both Sax and Tkv (Fig. 5D).
A synergy has been observed between co-expressed constitutively active (CA)
Tkv and Sax in the early embryo (Neul and
Ferguson, 1998; Nguyen et al.,
1998) and between Tkv and Sax in S2 cells in response to Dpp-Scw
heterodimers, as only Dpp homodimers are able to signal efficiently in the
absence of Sax (Shimmi et al.,
2005). We have detected a likely, albeit minimal, contribution of
Dpp-Gbb heterodimers to long-range wing patterning
(Bangi and Wharton, 2006)
making it is possible that Tkv-Sax complexes could respond to Dpp-Gbb
heterodimers and such complexes could be particularly efficient at signaling.
Given the dual function of Sax, the relative levels of Sax to Tkv are likely
to be crucial for establishing a synergistic interaction. The ability of
Tkv-Sax containing complexes to mediate ligand homodimers has not yet been
determined in vivo and it is also not yet completely clear if the antagonism
by Sax can affect heterodimers as well as homodimers. Our data indicate that
the ability of Sax to promote signaling must reside with Tkv-Sax-containing
complexes and the strong contribution of Gbb to the low points of the gradient
with a minimal contribution by Dpp leaves open the possibility that Dpp-Gbb
can signal, in addition to Gbb-Gbb, to cells far from the AP boundary.
Sax as a general modulator of Bmp signaling
Overexpression studies in the follicle cells of the Drosophila
ovary produce the same results as those described in
Fig. 1, indicating that the
ability of Sax to block Gbb signaling is not limited to the developing wing
(F. Cernilogar, R. Ray and K.W., unpublished). However, in contrast to studies
in the wing disc, loss of sax from the follicle cells, as well as the
embryonic midgut and neuromuscular synapse produces mutant phenotypes
indicative of a loss of ligand function
(Nellen et al., 1994;
Twombly et al., 1996;
Khalsa et al., 1998;
Rawson et al., 2003). It is
possible that the contribution of Sax to signal promotion in these tissues may
be stronger than its antagonistic function. The phenotypic outcome of
sax loss of function in a particular process probably depends on the
relative numbers of Sax-Sax and Sax-Tkv complexes on the cell surface and the
relative binding affinity of a given Bmp ligand for these two complexes. What
regulates the composition of type I receptors in a signaling complex is not
yet known.
Possible molecular explanations for unique role of Sax receptor
The ability of the Sax to block Bmp signaling may reflect its requirement
to have input from another molecule to activate its kinase domain. When
activated by in vitro mutagenesis, Sax and its vertebrate orthologs Alk1/Alk2
(Acvrl1 and Acvr1 - Mouse Genome Informatics) are able to phosphorylate Bmp
specific R-Smads (Chen and Massagué,
1999), but ligand-induced activation of Sax or Alk1/2 kinase has
not been reported. Interestingly, a ligand-induced Bmp receptor complex
containing Alk2 and ActRII is unable to phosphorylate Smad1
(Macias-Silva et al., 1998).
Furthermore, Alk1 has been shown to require a different type I receptor (Alk5)
to activate its kinase domain (Goumans et
al., 2003). Although Macias-Silva et al.
(Macias-Silva et al., 1998)
suggest that the Alk2/ActRII complex might be unstable in vitro, it is also
possible that activation of Alk2 (and of its Drosophila ortholog Sax)
may depend on its partner type I receptor and/or which ligand is bound, or
some other protein. Although Gbb fails to activate Sax-Sax, perhaps another
Bmp ligand (i.e. Scw) can. Similarly, endoglin, related to the co-receptor
betaglycan (Lopez-Casillas et al.,
1993; Lopez-Casillas et al.,
1994; Letamendia et al.,
1998), could be important in modulating Alk1-dependent signaling
given that mutations in either gene give rise to hereditary hemorrhagic
telangiectasia (van den Driesche et al.,
2003). Sax may require a different type I receptor partner, i.e.
Tkv, to activate its kinase or transduce a signal, and such a requirement may
be a universal feature of the Alk1/Alk2/Sax subgroup of Bmp type I
receptors.
Role of Sax as an antagonist for the establishment of a robust Bmp activity gradient
The robustness of morphogen gradients may depend on negative-feedback
mechanisms to buffer against environmental and genetic fluctuations. Clearly,
Sax plays a crucial role in modulating the range of the Bmp activity gradient
from analysis at both the level of Bmp-dependent target gene expression and
the final pattern of the adult wing. The identification of the antagonistic
nature of a Bmp type I receptor to modulate signaling activity by sequestering
ligand without transducing a signal provides a new mechanism that contributes
to the robustness of the Bmp activity gradient. We propose that the dual
function of Sax is crucial for buffering the wing disc Bmp activity gradient
against local fluctuations in ligand levels (environmental, genetic or
experimentally induced). Whether this mechanism of signal modulation is
evolutionarily conserved remains to be determined, but the fact that the
vertebrate Sax orthologs Alk1 and Alk2 have been shown biochemically to
exhibit antagonistic behaviors in vitro is interesting. Detailed analysis of
these orthologs in developmental contexts will be crucial to determine whether
the robustness of vertebrate Bmp activity gradients also depends on the
modulation of ligand availability by specific receptors.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/17/3295/DC1
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ACKNOWLEDGMENTS |
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