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First published online December 20, 2005
doi: 10.1242/10.1242/dev.02214
Review |
1 Department of Genetics, Cell Biology and Development, University of Minnesota,
Minneapolis, MN 55455, USA.
2 The Howard Hughes Medical Institute.
3 Department of Chemical Engineering and Materials Science, University of
Minnesota, Minneapolis, MN 55455, USA.
4 School of Mathematics and Digital Technology Center, University of Minnesota,
Minneapolis, MN 55455, USA.
5 Department of Zoology, 250 North Mills Street, University of Wisconsin,
Madison, WI 53706, USA.
* Authors for correspondence (e-mail: moconnor{at}mail.med.umn.edu and ssblair{at}wisc.edu)
SUMMARY
In the early Drosophila embryo, BMP-type ligands act as morphogens to suppress neural induction and to specify the formation of dorsal ectoderm and amnioserosa. Likewise, during pupal wing development, BMPs help to specify vein versus intervein cell fate. Here, we review recent data suggesting that these two processes use a related set of extracellular factors, positive feedback, and BMP heterodimer formation to achieve peak levels of signaling in spatially restricted patterns. Because these signaling pathway components are all conserved, these observations should shed light on how BMP signaling is modulated in vertebrate development.
Introduction
Key to many developmental processes is the ability of cells to reproducibly
interpret information regarding their spatial position within a developing
field so that patterns and, ultimately, tissues form with the proper
dimensions and connectivity. Nowhere have these processes been more thoroughly
studied than in the early Drosophila embryo and larval imaginal
discs. In each case, morphogens, special classes of signaling molecules that
specify cell fate in a concentration-dependent manner, have emerged as key
components that guide patterning. In recent years, great efforts have been
made to elucidate how cells interpret and respond to morphogen concentration
gradients with specific gene expression outputs. Equally important, however,
is to determine what mechanisms generate extracellular concentration gradients
in the first place. Here, we review recent work on how the gradients formed
by, and the signaling output of, a specific family of morphogens, the bone
morphogenetic proteins (BMPs), are influenced by the formation of ligand
heterodimers and by their binding to extracellular factors. We concentrate
specifically on how these features enhance BMP signaling during early
embryonic patterning and late wing development. We also review recent
experimental data on the existence of positive feedback as an important
additional component for proper BMP signaling in both the embryo and pupal
wings. Furthermore, we discuss how computational modeling has offered insights
into how extracellular gradients form and how the gradients function reliably
in the face of genetic variation. Due to space limitations, we only briefly
allude to related issues from vertebrates (for reviews, see
Balemans and Van Hul, 2002
;
De Robertis and Kuroda, 2004;
Kishigami and Mishina, 2005;
Schier and Talbot, 2005).
The basics of BMP signaling in Drosophila
BMPs belong to the TGFß superfamily of growth and differentiation
factors. Three BMP-type ligands are present in Drosophila:
Decapentaplegic (Dpp), a functional ortholog of vertebrate BMPs 2 and 4, Glass
bottom boat (Gbb), a member of the BMP 5,6,7 subgroup, and Screw (Scw), a
distantly related family member (Newfeld
et al., 1999). In the embryo and wing, ligand dimers signal
through a common set of receptors that include the type II receptor Punt, and
the two type I receptors Saxophone (Sax) and Thickveins (Tkv)
(Fig. 1) (reviewed by
Parker et al., 2004). Upon
ligand binding, Sax and Tkv phosphorylate Mad, the sole Drosophila
BMP Smad. Phosphorylated Mad (pMad) forms a complex with the co-Smad Medea,
which then translocates into the nucleus. Smad proteins either activate or
repress transcription, depending upon the particular complement of co-factors
present.
Dpp as an embryonic morphogen
In the early Drosophila embryo, two major tissues, amnioserosa and
dorsal ectoderm, form from the 40% dorsal-most cells. The amnioserosa derives
from the eight to ten cells that lie adjacent to the dorsal midline, while
dorsal ectoderm derives from more lateral cells. In dpp null mutants,
all dorsal cells acquire a ventral neurogenic fate (reviewed by
Sutherland, 2003). Moreover,
injection experiments have shown that high levels of dpp mRNA convert
all dorsal cells to an amnioserosa fate, whereas moderate levels specify
dorsal ectoderm (Ferguson and Anderson,
1992). Dpp therefore acts as a concentration-dependent morphogen
for the specification of both tissues.
The visualization of pMad levels using a phospho-specific antibody has
shown that pMad accumulates in the nucleus of dorsal cells midway through
cellularization. Initially, anti-pMad staining is low and encompasses the
dorsal-most 18-20 cells, but then it rapidly contracts and strengthens, and by
the onset of gastrulation a sharp, step gradient of pMad has formed
(Fig. 2B), in which pMad levels
are high in the dorsal-most five to nine cells, but rapidly drop off to
undetectable levels in more lateral regions over two to three cell diameters
(Dorfman and Shilo, 2001;
Ray and Wharton, 2001;
Ross et al., 2001). Similarly,
by the end of cellularization, the co-Smad Medea accumulates in the nuclei of
the dorsal-most cells, forming a sharp stripe
(Sutherland et al., 2003).
The embryonic Dpp gradient requires extracellular modulators
Although in the early embryo Dpp activity is highest near the dorsal midline and lower at the lateral boundaries, dpp is transcribed uniformly throughout the entire dorsal domain (Fig. 2A). In other words, the sharp, step distribution forms within a domain of uniform dpp expression. Thus, additional extracellular factors must be involved in shaping the ligand activity gradient.
Mutations in short gastrulation (sog), twisted
gastrulation (tsg) and tolloid (tld) produce
phenotypes similar to, but less severe than, those exhibited by dpp
mutants (Arora and Nusslein-Volhard,
1992). In fact, these mutations all disrupt the step gradient. In
tld mutants, all signaling is reduced, but in sog and
tsg mutants, signaling in dorsolateral regions increases, and
signaling in the dorsal-most cells decreases, producing a broad dorsal region
of pMad that much more closely resembles the pattern of dpp
expression (Ross et al.,
2001). These genes all encode secreted products
(Arora et al., 1994;
Francois et al., 1994;
Mason et al., 1994;
Shimell et al., 1991). Both
Sog and Tsg contain cysteine-rich motifs (CRs) that facilitate the binding of
these proteins to Dpp in a ternary complex that sequesters Dpp from its
receptors (Ross et al., 2001).
Tld is a metalloprotease that can cleave Sog
(Marques et al., 1997;
Shimmi and O'Connor,
2003).
|
), the
flux will facilitate the transport of Dpp out of lateral regions towards the
midline. This should promote Dpp signaling by increasing the Dpp concentration
at the dorsal midline (Holley et al.,
1996; Eldar et al.,
2002; Mizutani et al.,
2005).
In this model, a key component that helps create Sog flux is the processing
of Sog by the metalloprotease Tld (Holley
et al., 1996; Marques et al.,
1997; Shimmi and O'Connor,
2003). This dorsally expressed protease acts locally
(Wang and Ferguson, 2005) to
cleave Sog when bound to Dpp. Released Dpp has two possible fates: it can bind
to its receptor and signal, or it can be recaptured by another Sog/Tsg
complex. When Sog levels are maximal, as in the lateral regions, the
probability of recapture is high, whereas at the midline, where Sog levels are
low, released Dpp is more likely to bind to its receptors and signal
(Eldar et al., 2002;
Mizutani et al., 2005).
Sog/Tsg/Tld-mediated Dpp transport
Although the basic Dpp transport mechanism was proposed over ten years ago,
it is only recently that the distribution of the Dpp protein in the early
embryo has been visualized. In one study, epitope-tagged Dpp was expressed in
its normal dorsal-on ventral-off blastoderm pattern using a transgene
construct driven by the endogenous dpp `hinR' promoter/enhancer
(Shimmi et al., 2005b). The
tagged Dpp accumulates in a profile that is very different from its mRNA
pattern and, ultimately, high levels of Dpp protein amass near the dorsal
midline. To determine whether Dpp was located inside or outside the cell, Wang
and Ferguson (Wang and Ferguson,
2005) developed a novel staining protocol called perivitelline
injection (PVI), in which an antibody is injected into the space between the
cell membrane and the vitelline membrane of live embryos. Under these
conditions, the antibody has no access to the cytoplasm and can only interact
with secreted Dpp. Once again, a stripe of Dpp is seen at the dorsal midline,
confirming that it represents the accumulation of extracellular Dpp,
presumably bound to receptors.
|
;
Wang and Ferguson, 2005).
However, mutations in these genes affect extracellular Dpp accumulation
differently (Wang and Ferguson,
2005). In sog mutants, extracellular Dpp binds to all
cells in the dorsal domain, but the total level of Dpp bound by any one cell
at the midline is lower than normal, whereas in lateral regions it is higher
than normal. Accordingly, pMad staining is broader but weaker at the midline,
resulting in an expanded mid-level target gene expression and the loss of
high-level target gene expression (Ross et
al., 2001). By contrast, tsg mutant embryos accumulate
little extracellular Dpp anywhere in the dorsal domain. In addition,
tsg mutant embryos exhibit lower levels of residual pMad staining
than do sog mutants and, unlike sog mutants, cannot be
rescued by increasing Dpp levels (Wang and
Ferguson, 2005). These observations suggest that Tsg, in addition
to its role as a co-inhibitor and a component of the transport complex, is
likely to have a Sog-independent role in promoting BMP signaling. Recent work
suggests that the zebrafish Tsg homolog also has role in promoting BMP
signaling that is independent of Chordin, the vertebrate Sog homolog
(Xie and Fisher, 2005).
Possible mechanisms include the enhanced binding of Dpp to either its
signaling receptors or to some other cell surface-binding protein, or
inhibition of some, as yet unidentified, extracellular antagonist of BMP
signaling. Hetero- and homodimers produce biphasic signaling
In addition to Dpp, amnioserosa specification also requires Scw, a second
BMP-type ligand (Arora et al.,
1994). Unlike dpp, scw is expressed uniformly, and only
at the blastoderm stage. Mutations in scw result in less severe
phenotypes than do mutations in dpp. Amnioserosa is lost in both
cases, but in scw mutants some dorsal ectoderm is still formed. Dpp
and Scw display an asymmetric relationship in their ability to compensate for
one another in the early embryo, i.e. injection of dpp mRNA can
rescue scw mutants, but scw mRNA cannot rescue dpp
mutants (Nguyen et al., 1998).
Furthermore, injection of scw mRNA synergistically enhances Dpp
signaling.
Intriguingly, Shimmi et al. (Shimmi et
al., 2005b) recently showed that Dpp and Scw form heterodimers in
cell culture and in the embryo. Furthermore, they found that, in cell culture,
the heterodimer produces tenfold more signal (pMad phosphorylation) than an
equimolar mixture of Dpp and Scw homodimers. Interestingly, Scw homodimers
exhibit very little signally ability in cell culture, which might explain the
asymmetric rescuing ability of the two ligands in mRNA injection
experiments.
The increased signaling ability of the Dpp/Scw heterodimer may be due to
synergy between the two type I receptors Tkv and Sax. In vitro, the Dpp/Scw
synergistic output required both Tkv and Sax, whereas Dpp homodimers only
required Tkv (Shimmi et al.,
2005b). In the embryo, the injection of activated tkv
mRNA, but not sax, rescues dpp-deficient embryos in a
dose-dependent fashion (Neul and Ferguson,
1998). Furthermore, just as scw mRNA injection augments
dpp signaling, the injection of activated sax mRNA
stimulates activated tkv signaling. This suggests that Scw probably
signals through Sax, whereas Dpp primarily signals through Tkv, and that the
two ligands synergistically activate the two receptors.
In vitro, the Dpp/Scw heterodimer has a higher affinity for Sog and Tsg
than do their homodimers, and, as a result, the heterodimers are more likely
than the homodimers to diffuse toward the dorsal midline as part of a
Dpp/Scw/Sog/Tsg complex. Consistent with this view, it has been shown that in
the absence of Scw, extracellular Dpp homodimers do not localize to the
midline but instead remain broadly distributed, producing a low-level signal
(Shimmi et al., 2005b;
Wang and Ferguson, 2005).
Taken together, these results suggest that the subdivision of dorsal cells
into two tissues results from a biphasic signal that exploits unique aspects
of both homo- and heterodimers. In this scenario, Dpp/Scw heterodimers are
preferentially transported to the dorsal midline in a complex with Sog and
Tsg. There, they are released from the complex by Tld and produce optimal
output through the synergistic activation of a Sax/Tkv heteromeric complex,
resulting in the specification of amnioserosa. By contrast, Dpp and Scw
homodimers are not as efficiently transported as they have a lower affinity
for Sog/Tsg. For this reason, they remain broadly distributed in the dorsal
domain and produce a low-level signal by binding to homomeric complexes of Tkv
and Sax, respectively. The broad, low-level signal pre-patterns the dorsal
ectoderm and suppresses neurogenic activity. However, final specification of
the dorsal ectoderm probably requires a second round of signaling via Dpp, but
not Scw, that occurs later during germ band extension stages and that is
likely to account for the differences in cuticular phenotypes exhibited by the
two mutants (Dorfman and Shilo,
2001).
Although the above model can explain many aspects of dorsal patterning,
evidence against the role of heterodimers in signaling comes from experiments
in which Dpp and Scw are expressed in non-overlapping regions of scw
mutant or dpp scw double-mutant embryos
(Neul and Ferguson, 1998;
Nguyen et al., 1998;
Wang and Ferguson, 2005). As
heterodimer formation is thought to occur in the Golgi during secretion
(Gray and Mason, 1990),
expression of the two ligands in different regions of the embryo should only
allow for homodimer formation. These results demonstrated that at least
moderate levels of signal can be produced by homodimers and led to the
suggestion that some novel higher order receptor complex might contribute to
synergistic signaling. It is interesting to note in this regard that, when BMP
ligands are added to vertebrate cells, the aggregate size of preformed
receptor complexes, which presumably represent tetramers, has been shown to
increase (Hassel et al.,
2003). These observations indicate that additional work will be
required to ascertain the relative contributions of homo- and heterodimers to
the patterning process.
Modeling of BMP embryonic patterning
It has now been directly demonstrated that Dpp ligand accumulates on the surface of cells at the dorsal midline, in agreement with the transport model. However, because of the complexity of the network, it is difficult to predict how the system will behave in the face of genetic perturbations without a quantitative mathematical model that incorporates diffusion and the known kinetic interactions. A desirable characteristic of such models is `robustness', meaning that the output (e.g. the level of signal response) is relatively insensitive to variations in parameters over a physiologically reasonable range. Robust systems have a distinct evolutionary advantage, as they can better cope with naturally occurring fluctuations in the levels of system components.
The first computational model of dorsal patterning demonstrated the
plausibility of a Sog-mediated transport mechanism for BMPs
(Eldar et al., 2002), and
provided a framework within which to explore the issue of pattern robustness.
A combination of large-scale computation and analytical manipulation
demonstrated that robustness of the patterning response requires several
conditions regarding parameter choices. The conditions for robustness are
that: (1) the processing of Sog by Tld depends on BMPs; (2) free BMPs do not
diffuse; (3) BMPs bind irreversibly to receptors; (4) Sog displaces BMPs from
receptors; and (5) Dpp homodimers are transported by the Sog/Tsg complex,
whereas Scw is transported by Sog. Condition 1 has been shown in vitro
(Holley et al., 1996;
Marques et al., 1997;
Shimmi and O'Connor, 2003),
demonstrating the utility of analyzing a network for robustness requirements.
However, no evidence exists for conditions 3 and 4, and condition 5 is not met
as Dpp and Scw are not independently targeted for transport by Sog and
Sog/Tsg, but are instead likely to be preferentially transported as a
heterodimer (Shimmi et al.,
2005b). Condition 2, that diffusion of Dpp is limited when Dpp is
not bound to a soluble inhibitor, is more controversial. Although it is true
that accumulation of Dpp near the dorsal midline requires Sog/Tsg-mediated
transport, it is not clear what limits the spread of Dpp once it is localized.
Early experimental data supported Dpp immobilization
(Eldar et al., 2002). However,
recent studies of embryonic patterning suggest that, even in the absence of
carrier proteins, Dpp can act over 15-20 cell diameters
(Mizutani et al., 2005), and
that Scw acts at even greater distances
(Wang and Ferguson, 2005). The
disparity between Dpp and Scw is not caused by differences in their intrinsic
diffusion rates because the molecules are of similar size and shape; instead,
it probably reflects differences in production, degradation, and/or binding to
other components of the system.
If Dpp is widely diffusible, how does its distribution evolve into a very
sharp and narrow gradient in the model? The answer is that, even with high
diffusion coefficients, the diffusion length of a ligand can be very short if
other kinetic processes, such as binding to immobile receptors and subsequent
degradation, act upon it. Another model of embryonic patterning allows the
primary ligand to diffuse but incorporates receptor-mediated BMP degradation
as a means to limit its spread (Mizutani
et al., 2005). This model can simulate many of the observed in
vivo distributions of pMad in different genetic backgrounds and on the
appropriate time scales (see Fig.
2E for an example). In essence, this achieves what was
accomplished in the Eldar model by restricting the spread of BMPs, but in a
more realistic manner. Moreover, the model suggests that any soluble
BMP-binding protein can potentially expand the range of BMP action simply by
protecting the ligand from receptor-mediated degradation.
Another interesting experimental result that can be explained by modeling
is that the robustness of the system to changes in gene dosage depends on the
gene. For instance, the embryo is not robust to changes in the levels of
sog gene dosage (Mizutani et al.,
2005), but is to changes in the level of tsg. Similarly,
the embryo is quite robust with respect to changes in scw gene
dosage, but not to dpp. If the conditions 3 to 5 set forth by Eldar
et al. (Eldar et al., 2002)
are not met, then what leads to the robustness of the system to changes in the
concentrations of these proteins? To some extent the answer lies in the
formation of heterodimers. Mathematical analysis of heterodimer formation
demonstrates that it can provide an effective buffer against changes in gene
dosage, at least for one partner of the heterodimer
(Shimmi et al., 2005b). For
instance, robustness with respect to Scw can be explained provided that Scw is
produced in slight excess of Dpp, and that the more effective signaling form
is the heterodimer. Analysis of the local dynamics of Sog/Tsg complex
formation also suggests that this contributes to robustness. Furthermore, a
series of dimerization reactions, in which the output of one step becomes the
input to the next step (e.g. Dpp/Scw binds to Sog/Tsg), has an additive effect
that further reduces the effect of perturbations
(Shimmi et al., 2005b). This
shows that sequential dimerization steps increase robustness in a spatially
homogenous system, but this idea needs to be further analyzed to see whether
the conclusions hold true when diffusion is incorporated into the model.
Positive feedback sharpens Dpp localization
While the mathematical models of extracellular BMP transport can account
for the final distribution of pMad, other regulatory mechanisms probably
contribute to the temporal formation and step-like distribution of this
pattern. The mathematical models with time-independent BMP production predict
that peak signaling originates near the dorsal midline, and, as time
progresses, that pMad signaling widens and increases in intensity
(Fig. 2E). In reality, the pMad
levels increase with time at the dorsal midline, but the width of the region
of high pMad actually contracts towards the midline over the course of about
30 minutes (Fig. 2B), which
suggests that a key component is missing. The enhanced intensity probably
reflects the continued accumulation of Dpp near the midline, but what accounts
for the rapid loss of pMad signal from nearby lateral cells? One possibility
is that it results from an increased Sog concentration in the extracellular
space; this requires, however, that prior signaling is rapidly lost through
the degradation and/or recycling of ligand-activated receptor complexes,
together with Mad degradation (Podos et
al., 2001), and/or Mad de-phosphorylation coupled with nuclear
export. However, recent results suggest an intriguing additional mechanism.
Localized injection of activated tkv, but not of wild-type
tkv, mRNA leads to the accumulation of extracellular Dpp, implying
that the activation of BMP signaling enhances future ligand-receptor
interactions (Wang and Ferguson,
2005). One explanation for this observation is that the initial
BMP signal activates a target gene whose product either reduces the
interaction of ligand with an inhibitory component, or aids in further ligand
capture by receptors. Consistent with this is the finding that blocking signal
transduction with medea mutants also blocks the sharpening of
extracellular Dpp. At present, the identity of the induced factor remains
unknown, but a signaling-induced, cell-surface BMP-binding protein (CSBBP)
could produce the observed contraction of pMad signaling and lead to a
step-like distribution of surface-localized ligand. To see how this works,
however, we must first introduce a different system, wing vein
development.
BMP signaling during vein development
Recent studies indicate that Sog and other extracellular regulators of BMP activity promote BMP signaling in a quite different developmental context, the specification of a subset of Drosophila wing veins. Although the constraints of this system have so far prevented the type of direct assessment of ligand movement performed in the early embryo, mutants affecting venation have been used to identify an additional extracellular component that provides a nice example of the type of positive feedback predicted to exist in the embryo.
Wing veins arise as stripes of cells in the wing imaginal disc just before
and after pupa formation. Each vein is positioned by a slightly different
mechanism (Bier, 2000;
de Celis, 2003), but, for our
purposes, we will divide the veins into two categories, the longitudinal veins
(LVs) and the crossveins (CVs), on the basis of their orientation and timing
of development (Fig. 3). The
precursors of the LVs, those that run along the proximodistal axis of the
wing, first appear in larval wing discs, whereas the anterior and posterior
CVs (ACV and PCV), those that bridge the LVs, do not appear until the early
stages of pupal development (Conley et
al., 2000).
BMP signaling plays at least two different roles in vein development. The
first is to position the LVs along the anteroposterior axis of the wing disc.
During larval development, Dpp is expressed in a stripe down the midline of
the wing disc, forming a long-range gradient of BMP signaling. A subset of the
LVs are positioned in response to specific levels of BMP signaling, and
reductions in BMP signaling can either shift the positions of these veins or
lead to gaps. There is no evidence that Sog, Tsg-like or Tld-like proteins
modulate BMP activity at this stage
(Shimmi et al., 2005a;
Yu et al., 1996).
During pupal stages, the expression of dpp changes: it is lost
from the midline stripe, and now appears in all of the LVs
(de Celis, 1997;
Yu et al., 1996). This Dpp
acts locally to maintain the previously specified LV fate; its loss leads to
the `shortvein' dpp phenotype
(de Celis, 1997;
Posakony et al., 1990;
Ray and Wharton, 2001).
However, Dpp also acts as a long-range signal for the initial specification of
the CVs. BMP signaling is activated in the prospective CV regions prior to the
appearance of other known vein-promoting signals; manipulations that inhibit
BMP signaling block the formation of the CVs, often with minimal effects on LV
development, leading to a `crossveinless' phenotype
(Conley et al., 2000;
Ralston and Blair, 2005). CV
development has thus provided a sensitive assay for studying BMP
signaling.
Signaling in the ACV is prefigured by dpp expression in a stripe
that intersects the ACV (Ralston and
Blair, 2005). However, localized BMP signaling in the incipient
PCV is not initially accompanied by a higher expression of ligand within the
PCV itself (Fig. 3C). Rather,
this signaling requires the expression of dpp in the adjacent LVs,
and thus the movement of Dpp from the LVs into the PCV region
(Ralston and Blair, 2005). In
this respect, the discrepancy between the regions of ligand expression and
signaling is even more extreme in the PCV than in the early embryo.
Sog, Tolloid-related and Crossveinless
As in the embryo, Sog is required for this long-range Dpp signaling in the
PCV; removing endogenous Sog causes a loss of signaling in the PCV and a
crossveinless phenotype (Serpe et
al., 2005; Shimmi et al.,
2005a). This was surprising, as the initial studies of Sog in the
wing suggested just the opposite; strong overexpression of Sog led to loss of
the PCV (Yu et al., 1996), and
co-expression of Sog and Tsg led to a loss of signaling during even the early
stages of Dpp signaling in the larval imaginal disc
(Ross et al., 2001). However,
although high levels of Sog can inhibit signaling in the wing, low levels of
overexpression actually stimulate signaling distant from the site of
misexpression (Shimmi et al.,
2005a), much as occurs after localized misexpression of
sog in the embryo (Ashe and
Levine, 1999). A truncated form of Sog that contains only the
first two CRs can also stimulate BMP signaling in the developing wing
(Yu et al., 2004).
Mosaic analysis indicates that Sog acts over a long range in the pupal
wing, consistent with it having a role in transporting Dpp
(Shimmi et al., 2005a). The
parallel with the embryo also extends to Sog's partners. As in the embryo,
signaling in the PCV requires a Tolloid family protease, in this case
Tolloid-related (Tlr, also known as Tolkin)
(Finelli et al., 1995;
Nguyen et al., 1994;
Serpe et al., 2005), and the
presence of the Tsg family member Crossveinless (Cv or Tsg2)
(Shimmi et al., 2005a;
Vilmos et al., 2005).
Like Tld, Tlr can cleave Sog in vitro and its loss leads to loss of
signaling in the PCV, probably through the accumulation of excess full-length
Sog (Serpe et al., 2005).
Although the excess Sog can presumably transport Dpp, it apparently sequesters
Dpp from its receptor. Indeed, lowering endogenous Sog levels can rescue the
tlr mutant phenotype. The embryonic protease Tld cannot substitute
for Tlr in the wing. This may be explained by the slower kinetics of Tlr
activity observed in vitro; such kinetics may be required for the movement of
the intact Sog-Cv-ligand complex over the longer time scale of the developing
wing.
Loss of the Tsg-like protein Cv also leads to loss of BMP signaling in the
PCV and a crossveinless phenotype. Cv acts with Sog to bind ligand in
vitro, and thus Cv might be required to form the transport complex; this is
consistent with rescue experiments indicating that Cv acts over a long range
in the wing (Shimmi et al.,
2005a; Vilmos et al.,
2005). In addition, Cv can substitute for Tsg in the early embryo
and Tsg can substitute for Cv in the wing, although they differ in the
strength of their genetic interactions with ligands
(Shimmi et al., 2005a;
Vilmos et al., 2005).
Heterodimers and crossveins
A final parallel with the embryo is that signaling in the PCV may be driven
partly or wholly by ligand heterodimers. Although scw is not
transcribed at pupal stages (Arora et al.,
1994), another BMP-like ligand, Gbb, is expressed at this time
(Khalsa et al., 1998;
Wharton et al., 1999). Loss of
either dpp or gbb blocks signaling in the PCV
(Ralston and Blair, 2005). Gbb
is expressed ubiquitously in the pupal wing
(Conley et al., 2000);
however, mosaic analysis indicates that the PCV is only disrupted when Gbb is
removed from the adjacent LVs (Ray and
Wharton, 2001), the same cells that express dpp.
|
). Why
then would a Dpp/Gbb heterodimer be more effective at signaling within the
PCV? As in the embryo, there may be an effect on transport. Because the
Dpp/Gbb heterodimer binds to the Sog-Cv complex with a higher affinity than
homodimers, it may be preferentially transported over long distances
(Shimmi et al., 2005a).
Heterodimers may also decrease the levels of uncleaved Sog, as cleavage of Sog
by Tld or Tlr requires the presence of ligand, and Dpp/Gbb heterodimers more
effectively stimulate processing than does either homodimer
(Serpe et al., 2005). Thus,
the simplest model is that Sog and Cv form a complex with a Dpp/Gbb
heterodimer and help to carry it into the PCV region where Sog is cleaved by
Tlr, freeing the Dpp/Gbb heterodimers for signaling
(Fig. 3E).
|
One BMP-binding protein that is crucial for signaling in the CVs, but whose
function in the early embryo has not been examined, is Crossveinless 2 (Cv-2).
Loss of Cv-2 causes loss of BMP signaling in the developing CVs
(Conley et al., 2000). Cv-2
contains five N-terminal CR domains (Fig.
4A), similar to the BMP-binding CRs of Sog, followed by a partial
von Willebrand Factor D (VWFD) domain. Vertebrates have a Cv-2 homolog with a
larger VWFD domain that includes a Trypsin inhibitor-like cysteine-rich (TIL)
domain (Binnerts et al., 2004;
Coffinier et al., 2002;
Coles et al., 2004;
Kamimura et al., 2004;
Moser et al., 2003).
Vertebrates also have large `Kielin-like' proteins, which contain varying
numbers of CRs and lengths of VWFD domains, and CRIM1, which has CR domains
and a transmembrane-spanning segment (Fig.
4A) (Kolle et al.,
2000; Lin et al.,
2005; Matsui et al.,
2000).
All Cv-2 and Kielin-like proteins so far tested are secreted and bind BMPs,
presumably through their CR domains
(Binnerts et al., 2004;
Coffinier et al., 2002;
Coles et al., 2004;
Lin et al., 2005;
Matsui et al., 2000;
Moser et al., 2003),
indicating that Cv-2 might promote signaling by aiding ligand transport,
perhaps as part of the Sog-Cv complex. However, mosaic analyses indicate that
cv-2 expression, unlike that of sog and cv, is
required locally within the crossvein itself. Thus, Cv-2 is not a long-range
transporter but is likely to act as a co-factor to concentrate ligand near the
receiving cells or to free it from the Sog-Cv complex. This is consistent with
the behavior of chick Cv-2 and mouse Kielin/Chordin-like protein (KCP) in
vitro, where conditioned medium containing either protein enhances BMP
signaling and, in the case of KCP, the binding of BMP7 to its receptor
(Kamimura et al., 2004;
Lin et al., 2005). Likewise,
studies in chick, Xenopus and mice suggest that Cv-2 and KCP have
agonist roles (Coles et al.,
2004; Lin et al.,
2005), but these proteins have also been reported to antagonize
BMP in various overexpression and in vitro assays
(Binnerts et al., 2004;
Coles et al., 2004;
Matsui et al., 2000;
Moser et al., 2003). There may
be several reasons for this. As with Sog, high levels of Cv-2 overexpression
may sequester ligand. It may also be that localized processing and/or
co-factors are required for Cv-2 to promote BMP activity.
As in the embryo, pMad accumulation in the CVs refines from a broad to a
narrow domain (Fig. 3A,B), and
this is paralleled by a similar increase and refinement in cv-2
expression in the CVs (Conley et al.,
2000) (Fig. 3D).
cv-2 expression is responsive to BMP signaling, and thus likely
provides positive feedback that aids in the refinement process (A. Ralston,
PhD thesis, University of Wisconsin, 2004;
Fig. 3D). However, other
factors must be present that initially increase the movement or accumulation
of ligand from the LVs into the PCV region, or that raise the sensitivity of
those cells to signaling. Although in the embryo the ventrolateral expression
pattern of sog is sufficient to provide directionality to gradient
formation, this is not the case in the PCV. In the pupal wing, sog
mRNA expression is reduced in the developing PCV
(Fig. 3F), but clones lacking
sog do not induce ectopic signaling
(Ralston and Blair, 2005;
Shimmi et al., 2005a;
Yu et al., 1996). cv
expression is slightly higher at vein boundaries and tlr is higher in
the intervein (Fig. 3G,H), but
uniform overexpression of either does not significantly alter PCV signaling
(Serpe et al., 2005;
Shimmi et al., 2005a;
Vilmos et al., 2005). Uniform
overexpression of Cv-2 does not expand signaling outside the PCV, either alone
or in combination with Sog and/or Cv (Fig.
3J) (Conley et al.,
2000; Ralston and Blair,
2005; Vilmos et al.,
2005) (A. Ralston, PhD thesis, University of Wisconsin, 2004). Nor
is the cue likely to be provided by changes in receptor expression. Sax is not
required for formation of the PCV (Ray and
Wharton, 2001; Singer et al.,
1997). Tkv expression is reduced in the PCV
(Fig. 3I), which could in
theory increase ligand diffusion, but this reduction is apparently the result,
not the cause, of heightened signaling
(Ralston and Blair, 2005).
One obvious place to look for additional factors that modulate BMP
signaling is the other crossveinless mutations, several of which are
uncharacterized. However, not all of these have provided an obvious link to
BMP signaling. crossveinless c (cv-c) encodes a RhoGAP
protein (Denholm et al.,
2005), and signaling in the PCV is reduced in cv-c
mutants (A. Ralston, PhD thesis, University of Wisconsin, 2004). This is
intriguing, as reductions in Cdc42 activity can induce ectopic CVs
(Baron et al., 2000;
Genova et al., 2000), but the
connection between small GTPase activity and PCV development is, as yet,
poorly understood, and could be quite indirect.
Positive feedback and bi-stability
The positive feedback potentially provided by molecules like Cv-2 not only increases signaling globally, but in theory can create the increasingly sharp step-gradients observed in both the embryo and the PCV by producing spatial bi-stability. Here, spatial bi-stability means that the response to the extracellular BMP distribution divides a region into a stable high signaling zone and a stable low signaling zone separated by a sharp boundary (i.e. the spatial distribution of signaling is step-like). Bi-stability frequently arises in models of complex networks, particularly in those that include positive-feedback loops, in which the balance between competing processes can lead to multiple steady states for a given set of conditions. A typical response diagram that illustrates bi-stability is shown in Fig. 4B. Without positive feedback, the level of BMP-bound receptor is fixed by the binding equilibrium (on-off rate, dotted line Fig. 4B), but intracellular positive feedback can shift the equilibrium curve and lead to bi-stability (S-shaped curve, Fig. 4B). For low levels of BMP, the level of BMP bound to its receptor follows the binding curve equilibrium (dotted line, Fig. 4B) until a point (red dot, Fig. 4B) where the lower steady state ceases to exist and only the upper stable branch is accessible. Thus, regions with BMP levels higher than the limit of stability for the lower branch will adopt a high signaling fate, while cells below that point will adopt a low signaling fate (spatial bi-stability). Thus, cells can re-interpret the extracellular gradient at the level of BMP-bound receptor (red line, Fig. 4B) and produce a step-like response in space to a more gradual change in BMP levels.
Previous analysis of the Patched/Hedgehog patterning system suggests that
positive feedback on receptor expression can lead to a spatial bi-stability
(Eldar et al., 2003). However,
it is unlikely that Tkv or Sax is the positively regulated target of BMP
signaling in Drosophila. Misexpression of Tkv does not significantly
affect the pMad output in the embryo
(Mizutani et al., 2005;
Wang and Ferguson, 2005), and,
in the wing disc and pupal wing, BMP receptors are actually downregulated by
BMP signaling (de Celis, 1997;
Lecuit and Cohen, 1998;
Ralston and Blair, 2005;
Tanimoto et al., 2000).
However, a positively regulated co-receptor or a CSBBP, like Cv-2, can play
the same role (Fig. 5). Vertebrate Cv-2, Keilin-like and CRIM1 proteins could also act in a similar
manner.
Conclusions and perspectives
The data reviewed here show that we now have a reasonably complete
understanding of how the molecules and their interactions lead to the spatial
distribution of BMPs in the early Drosophila embryo, and, to a lesser
extent, in the PCV of the pupal wing. However, there are still unanswered
questions that will continue to drive research in this area over the next few
years. Not the least of these is the identification of new players, such as
those responsible for positive feedback in the embryo and the spatial
regulation of signaling in the PCV. The Drosophila genome encodes
several uncharacterized proteins that contain CRs like those known to bind
BMPs. We also need to factor in new findings about interactions between the
known players and other extracellular elements. For instance, Tsg, Sog, and
its vertebrate ortholog Chordin, have been shown to interact with cell-surface
components such as integrins, proteoglycans and heparin, and these
interactions may influence gradient formation
(Araujo et al., 2003;
Jasuja et al., 2004;
Larrain et al., 2003).
|
; Little and Mullins,
2004; Oelgeschlager et al.,
2000; Oelgeschlager et al.,
2003; Ross et al.,
2001; Scott et al.,
2001; Xie and Fisher,
2005; Zakin and De Robertis,
2004). The precise biochemical mechanism responsible for the
agonist activity has not been explained, but may be related to the promotion
of BMP accumulation that has been noted in the Drosophila embryo
(Wang and Ferguson, 2005).
There are also some intriguing differences between Drosophila and
vertebrate proteins that might give them different properties. The Tsg protein
of Drosophila can bind heparin, whereas the vertebrate proteins do
not (Jasuja et al., 2004;
Mason et al., 1997). Similarly, in Drosophila, Sog is processed in at
least three positions by Tld in a ligand-dependent fashion, whereas, in
vertebrates, Chordin is processed at only two major sites, and this processing
does not depend on Chordin forming a complex with BMPs
(Marques et al., 1997;
Piccolo et al., 1997;
Scott et al., 1999;
Shimmi and O'Connor, 2003). Do
these differences account for the inability of Chordin to have agonist
activity when expressed in flies (Decotto
and Ferguson, 2001)? Lastly, individual fragments of Sog have been
found to have either antagonistic or agonistic function when overexpressed in
the wing (Yu et al., 2004;
Yu et al., 2000). Do these
fragments play a role in the endogenous modulation of BMP activity? Presumably
rescue experiments employing mutant versions of these sites, together with the
production of Sog/Chd chimeric proteins, will provide definitive answers to
each of these issues in the near future.
The biochemical mechanism of receptor synergism is also an important issue.
Are Smads more efficiently recruited to the Tkv/Sax-containing complex than
either homomeric complex? Is there a novel cross phosphorylation of the two
receptors in a heteromeric complex that contributes to the synergism? Are
there intracellular regulators of receptor activity that differentially bind
to the different receptor complexes? Alternatively, the heteromeric receptor
complex may be routed through a different signaling endosome that persists and
signals longer than homomeric receptor complexes do. It is also important to
determine whether the synergism is even a necessary component of the early
developmental process, as overexpression of one isoform of Tkv has been shown
to partially rescue sax mutations
(Brummel et al., 1994).
Finally, is receptor synergism a feature of vertebrate systems? Two type I BMP
receptors exist in vertebrates, and BMP heterodimers have been implicated in
regulating several developmental events
(Butler and Dodd, 2003;
Schmid et al., 2000). In
addition, heterodimers can produce stronger signals in vertebrate cell culture
systems than homodimers can (Aono et al.,
1995).
For developmental processes, bi-stable behavior has several implications.
What determines whether the cell will have a high or low signal-reception
fate? With positive feedback it is entirely possible that cells adopt distinct
fates based on the history of their exposure to a changing extracellular
morphogen gradient instead of on an absolute concentration at a given point in
time (Dillon and Othmer,
1999). A last issue, raised by cell culture signaling assays, is
whether stochastic influences have to be considered when modeling the
embryonic patterning mechanism. Previous studies indicate that BMP
responsiveness in cell culture is saturated at the 10-nanomolar level
(Shimmi and O'Connor, 2003).
If this holds true in the embryo, then it extrapolates to only several
thousand BMP molecules in the perivitelline space. Such a low number would
make patterning susceptible to stochastic fluctuations
(England and Cardy, 2005).
Once again, a solution might be positive feedback, which should dampen
stochastic influences on signaling output
(Dillon and Othmer, 1999).
Because computational analysis shows that step gradients in morphogen
interpretation can form in the absence of feedback, might buffering against
stochastic fluctuations be the primary reason that positive feedback is
employed in this system? Measuring the actual levels of ligands in the
perivitelline space is therefore crucial to obtaining a more complete
understanding of the patterning mechanism.
ACKNOWLEDGMENTS
We thank Guillermo Marques and MaryJane Shimell for critical comments on the manuscript. S.S.B. is supported by grants from the NSF and the NIH, H.O. by a grant from the NIH, and D.U. by a NIH Biotechnology Training Grant. M.B.O. is an Investigator with the Howard Hughes Medical Institute.
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*x). As the level of BMP increases, the
level of BMP-bound receptor follows the on/off equilibrium solution until it
reaches a limit point (LP) where the lower equilibrium solution ceases to
exist. For levels of BMP above this point, the level of BMP-bound receptor
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