1 Department of Genetics Cell Biology and Development, and the Developmental
Biology Center, University of Minnesota and the Howard Hughes Medical
Institute, Minneapolis, MN 55455, USA
2 Department of Zoology, University of Wisconsin, 250 North Mills Street,
Madison, WI 53706, USA
* Author for correspondence (e-mail: moconnor{at}mail.med.umn.edu)
Accepted 29 March 2005
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SUMMARY |
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Key words: Tolloid, Bmp, Sog, Crossvein Drosophila, 18w
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Introduction |
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The Bmp1/Tld-like proteases process a number of different extracellular
matrix (ECM) components, including type I to III procollagen
(Kessler et al., 1996),
laminin 5 (Amano et al., 2000
),
biglycan (Scott et al., 2000
),
lysyl oxidase (Uzel et al.,
2001
) and some members of the Tgfß family
(Wolfman et al., 2003
).
However, the best understood developmental patterning of Bmp1/Tld-like
proteins is in the early Drosophila embryo, in which Tolloid (Tld) is
required for specification of dorsal structures
(Shimell et al., 1991
). Tld
acts by cleaving the secreted Bmp antagonist Sog to release the Bmp2/4 homolog
Dpp from an inhibitory complex
(Marqués et al., 1997
).
In Drosophila, graded Dpp activity in dorsal cells is achieved by
diffusion of Sog from its ventrolateral site of synthesis into the dorsal
domain (Ferguson and Anderson,
1992
; François et al.,
1994
; François and
Bier, 1995
; Marqués et
al., 1997
; Srinivasan et al.,
2002
). The diffusion of Sog from ventrolateral cells serves two
purposes. First, it inhibits Dpp activity in dorsolateral regions and second,
it helps transport Dpp (Wang and Ferguson,
2005
; Eldar et al.,
2002
; Shimmi et al.,
2005a
) towards the dorsal side in a complex with Twisted
gastrulation (Tsg), a second extracellular protein that modulates patterning
within dorsal cells (Mason et al.,
1994
; Ross et al.,
2001
). Dorsally expressed Tld proteolytically cleaves Sog and
liberates Dpp from the inhibitory complex
(Marqués et al., 1997
;
Shimmi and O'Connor, 2003
).
Thus, mutations in tld result in a partially ventralized phenotype
that is similar to, but less severe than dpp mutants
(Arora and Nüsslein-Volhard,
1992
; Jürgens et al.,
1984
).
In vertebrates, Bmp1/Tld-like proteases regulate the activity of Chordin, a
Sog homolog, similar to the way Tld regulates Sog activity in
Drosophila embryos. Bmp1 family members can process Chordin
(Piccolo et al., 1997;
Blader et al., 1997
) and
co-overexpression of vertebrate Tld proteases with Chordin can reverse the
dorsalizing effects of Chordin upon early Xenopus development
(Piccolo et al., 1997
; Scott
et al., 1999a). Mutations in the zebrafish mini-fin gene, which
encodes a Tld-related enzyme that is able to cleave Chordin in vitro
(Blader et al., 1997
), exhibit
dorsoventral patterning defects (Connors
et al., 1999
).
The Drosophila genome encodes a second Tld-like protein that maps
immediately upstream of tld. This gene has been referred to as
tolloid-related 1 (tlr; 18w FlyBase) or
tolkin (tok) (Nguyen et
al., 1994; Finelli et al.,
1995
). At the blastoderm stage, tlr expression overlaps
that of tld. However, after embryonic stage 15, tld
expression is no longer seen in any embryonic tissue, while tlr is
heavily expressed in muscles, a subset of cells within the CNS, and in the
corpus allatum of the ring gland (Nguyen
et al., 1994
; Finelli et al.,
1995
). During larval stages tlr and tld show
identical expression patterns within the imaginal discs; however, the
tlr transcript is much more abundant.
Despite several studies, the biological function(s) of Tlr remain ill
defined. Embryos mutant for tlr show no significant defects in dorsal
patterning, although it has recently been demonstrated that the level of Sog
protein present in the dorsal region of tlr tld double mutant embryos
is higher than in tld single mutants
(Srinivasan et al., 2002).
This led to the speculation that, like Tld, Tlr might recognize Sog as a
substrate. However, the biological relevance of such processing was not
clear.
Null alleles of tlr are largely lethal during larval and pupal
stages but result in a small number of escaper flies in which the posterior
crossvein (PCV) of the adult wing is absent
(Nguyen et al., 1994;
Finelli et al., 1995
). This is
intriguing, as the dpp and sog gene products are reused in
pupal stages of development to help initiate and maintain wing veins
(Posakony et al., 1990
;
Yu et al., 1996
;
de Celis, 1997
;
Conley et al., 2000
;
Ray and Wharton, 2001
;
Ralston and Blair, 2005
).
During pupal stages, dpp is expressed in longitudinal vein primordia
and helps maintain their fate (Posakony et
al., 1990
; de Celis,
1997
), while sog is expressed in complementary intervein
cells and refines vein formation (Yu et
al., 1996
), probably by blocking an auto-activating Dpp feedback
loop. Two smaller veins, called crossveins, develop late in this process, from
tissue that is initially specified to be intervein material. PCV development
is initiated by high levels of Bmp signaling. Therefore, the PCV is especially
sensitive to reductions in such signaling
(Conley et al., 2000
;
Ralston and Blair, 2005
). The
localized, high level of Bmp signaling that initiates PCV development is
probably supplied from ligands produced in the longitudinal veins
(Ralston and Blair, 2005
). The
molecular mechanism responsible for localizing high levels of Bmp signaling
activity to the crossvein primordia is not clear. The tlr mutant
phenotype suggests that Tlr may aid PCV development by cleaving Sog and thus
regulating its activity during crossvein development.
In this report, we provide biochemical evidence that Tlr, like Tld, can
cleave Sog, albeit with different kinetics and site selection specificities.
In addition, we further characterize the tlr loss-of-function
phenotype and find that Tlr activity is required to ensure localized, high
level Bmp signaling during crossvein specification. Moreover, we find that Tlr
activity during crossvein specification is required to cleave Sog, and that
Sog exhibits both agonist and antagonistic activities towards Bmp signaling
during pupal wing patterning. Taken together with recent data indicating that
the source of Bmp ligand for crossvein specification is likely to be
longitudinal vein cells (Ralston and
Blair, 2005), we formulate a model whereby Sog and Tlr
recapitulate the embryonic roles of Sog and Tld in transporting and releasing
Bmp ligands to achieve spatially restricted patterns of signaling. As Tld can
not substitute for Tlr during PCV formation and Tlr can not substitute for Tld
in the embryo, we further speculate that the different catalytic properties of
Tlr and Tld towards a common substrate (Sog) have been matched to their
respective developmental processes: rapid processing for the quickly
developing embryo and slow processing for the more slowly developing PCV.
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Materials and methods |
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Molecular constructs
To make the tlr-HA constructs, a NotI site was introduced
by site-directed mutagenesis (QuickChange, Stratagene) downstream of bp 4729
in the tlr cDNA (GenBank sequence U12634) and was used to insert a
triple HA epitope at the C-terminus of tlr ORF. A processing site
mutant (pmTlr, R516A and R519A) and catalytically dead (cdTlr, S669F) forms
were engineered by site-directed mutagenesis. Astacin domain swapping was
accomplished in two steps. For example for TldastTlr, the sequence
coding for the Tlr astacin domain was PCR amplified (Expand High Fidelity PCR
System, Boehringer Mannheim) using primers that had overhangs homologous to
the flanking sequences in tld cDNA. Then, the PCR product was used as
a pair of matching primers in an amino acid switch step, using QuickChange
site-directed mutagenesis kit (Stratagene) and a tld-based template.
All junctions were verified by sequencing. TldastTlr has the
following composition: Tld amino acids M1-R126, Tlr A520-K719 and then Tld
C327-end. TlrastTld has Tlr amino acids M1-R519, Tld A127-K326 and
then Tlr C720-end. Additional details are available upon request. All tagged
proteins were placed in pRmHaI (Bunch et
al., 1988) for transfections into S2 cells, in pUAST
(Brand and Perrimon, 1993
) to
generate transgenic lines or in Bluescript II SK(+) (Stratagene) for mRNA
synthesis.
Protein production and purification
Drosophila S2 cells were used for producing recombinant proteins
as described previously (Shimmi and
O'Connor, 2003). The cleared supernatant (conditioned medium) was
used directly for in vitro cleavage assays and western blotting. Cell pellets
were disrupted by boiling in SDS-loading buffer, followed by centrifugation
and the clarified supernatant analyzed by western blotting as the soluble cell
pellet fraction.
For in vitro cleavage assays, mixtures of proteins were incubated for the indicated times at 25°C in the presence of 1 x reaction buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2.5 mM CaCl2, 1 µM ZnCl2 and CompleteTM, EDTA-free protease inhibitor cocktail, Roche).
Immunoprecipitation and immunoblotting
Protein samples were separated by SDS-PAGE on 4-12% NuPAGE gels
(Invitrogen) and transferred onto PVDF membranes (Millipore). Primary
antibodies were used at the following dilutions: anti-HA 12CA5 (Roche) 1:2000;
anti-Myc A14 (Santa Cruz) 1:1000; rat anti-Gbb (raised against the C-terminus
of Gbb, kindly provided by Mike Hoffmann and Grace Boekhoff-Falk) 1:500.
Immune complexes were visualized using HRP-conjugated secondary antibodies
(Jackson Immuno Research Laboratories) and ECL Super Signal (Pierce).
Alternatively, for simultaneous multiple detection, anti-rat Alexa Fluor 680
and anti-mouse IRDye 800 secondary antibodies were used at 1:5000 dilution,
followed by scanning with Odyssey Infrared Imaging System (Li-cor
Biosciences).
For Sog-Tld/Tlr co-immunoprecipitation, equivalent amounts of wild-type and variant Tld-HA and Tlr-HA proteins in conditioned media were mixed with Sog-Myc for 3 hours at room temperature in a final volume of 250 µl. The mixtures were diluted with an equal volume of 0.4% BSA in mock conditioned media, followed by immunoprecipitation at 4°C overnight with Affinity Matrix Mono HA.11 (Covance). The samples were washed five times in ice-cold PBS and analyzed as above. For Dpp-Gbb heterodimer analysis, equivalent amounts of individually expressed and co-expressed ligands were preincubated for 2 hours at room temperature in a final volume of 1 ml. The mixtures were divided for immunoprecipitation with Affinity Matrix Mono HA.11 (Covance) and with anti-Flag M2-Agarose (Sigma) at 4°C overnight. The samples were washed with ice-cold PBS and eluted with non-reducing SDS-loading buffer to prevent the dissociation of antibodies from matrix. Prior to electrophoresis and analysis as described above, ß-mercaptoethanol was added to the samples.
RNA localization and immunohistochemistry
In situ hybridization to pupal wings was performed with digoxigenin-labeled
RNA probes and visualized with alkaline phosphatase precipitates as previously
described (Conley et al.,
2000). Specimens were stained using anti pMad (gift from P. ten
Dijke) at 1:1000 dilution followed by anti-rabbit HRP (Jackson Immuno Research
Laboratories) at 1:2000 and visualized using the fluorescein TSA kit (NEB) or
as described by Ralston and Blair (Ralston
and Blair, 2005
).
Secondary axis assay
Xenopus embryos were obtained by in vitro fertilization of eggs
with testes homogenates. Embryos were dejellied in 2% cysteine and staged. For
secondary axis induction assays, synthetic capped mRNAs were injected into one
of the two adjacent ventral blastomeres of four-cell stage embryos in the
equatorial region. The injection volume was up to 5 nl at the concentrations
indicated. mRNAs for injections were transcribed using either T7 or SP6
Message Machine kits (Ambion).
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Results |
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To examine the abilities of these proteins to process Sog, we used an in
vitro assay system that was previously developed to characterize Sog cleavage
by Tld (Marqués et al.,
1997; Shimmi and O'Connor,
2003
). In these studies, it was determined that, in the presence
of Dpp, Tld processes Sog at one minor (II) and three major sites (I, III and
IV, Fig. 1C). Processing occurs
preferentially at sites I and III, although the selection of cleavage site can
be influenced by the presence of Tsg, another small secreted protein that
makes a tripartite complex with Dpp and Sog
(Ross et al., 2001
;
Shimmi and O'Connor, 2003
). We
find that Sog is also an in vitro substrate for Tlr, and that Tlr processes
Sog in a ligand-dependent reaction (Fig.
1B). Analysis of fragment sizes
(Fig. 1B,C) and microsequencing
(M.S. and M.B.O., unpublished) reveals that Tlr processes Sog at the same
three major sites as does Tld. However, Tlr preferentially cleaves Sog at
sites I and IV in the absence of Tsg, and Tsg promotes cleavage at site III
(Fig. 1B). We also find that
the pmTld and cdTlr variant proteins were unable to process Sog
(Fig. 1D, lanes 4 and 5),
indicating that, like Tld, Tlr activity requires removal of the pro-domain as
well as an intact catalytic domain.
To further compare the catalytic activities of Tld and Tlr for Sog
processing, we performed time course processing assays. Protease levels were
quantified by western analysis of the similarly C-terminal triple HA-tagged
enzymes (Fig. 2A, insert). As
shown in Fig. 2A, Tlr processed
Sog less efficiently at all time points compared with Tld, despite the fact
that there is fivefold more Tlr in the reaction. Reactions were carried out in
the presence of 3 x1010 M Dpp (R&D Systems), a
concentration shown previously to stimulate approximately half maximal levels
of processing (Shimmi and O'Connor,
2003). Using the bacterial recombinant Dpp conferred the advantage
of allowing accurate quantification of the ligand introduced in reactions;
however, with this recombinant ligand, Sog processing is limited to sites I
and III. When quantifying Sog processing by Tlr and Tld, the time taken for
the disappearance of the full-length Sog (120 kDa band) and the appearance of
the 50 kDa processing product appeared approximately doubled in the case of
Tlr (Fig. 2A, lower panels).
Coupled with the five-fold excess of Tlr relative to Tld in these reactions,
the results suggest that under these in vitro conditions, Tlr processes Sog
approximately one order of magnitude slower than Tld. Addition of even higher
amounts of Tlr (10-fold and 25-fold more than Tld) to the Sog processing
reaction slightly increased the disappearance of the full-length Sog (120 kDa
band) and the appearance of the 110 kDa product
(Fig. 2B), but had no effect
upon the production of the 50 kDa product (not shown). To address the
possibility that the Dpp requirements for the two enzymes might be different,
we carried out the Sog processing in the presence of three times more Dpp (1
x109 M). This increased Dpp concentration did not
significantly alter the rate of Sog processing by Tlr
(Fig. 2C, compare with 2B),
indicating that the Dpp concentration was not limiting under these conditions.
Therefore, the slow kinetics of Sog processing by Tlr appears to stem from a
slow proteolytic activity and not from inefficient use of the required
co-substrate. Even in co-transfection experiments, where Sog, Dpp, and Tld or
Tlr were expressed in the same cells, Tlr was a less effective enzyme than Tld
(not shown).
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Bmp heterodimers are the preferred catalyst for Tld and Tlr processing of Sog
The catalytic role of Bmps in stimulating Sog processing is thought to be
caused by a Bmp-induced conformational change within Sog that better presents
the substrate to the Tld protease domain. In the embryo, both Dpp and another
Bmp family ligand, Screw (Scw), are required for proper dorsal patterning. In
imaginal and pupal tissues, Dpp and the Bmp family ligand Glass bottom boat
(Gbb) are required for proper patterning
(Khalsa et al., 1998;
Wharton et al., 1999
;
Ray and Wharton, 2001
). It has
recently been determined that the primary patterning ligand in the early
embryo is likely to be a heterodimer of Dpp and Scw formed by intermolecular
disulfide bridge during ligand processing and secretion
(Shimmi et al., 2005a
). In
these studies, Dpp/Scw heterodimers were found to stimulate Sog processing
better than either homodimer. In imaginal and pupal tissues Dpp/Gbb
heterodimers have been suggested to play important patterning roles
(Ray and Wharton, 2001
). We
therefore tested the relative abilities of Bmp homodimers and heterodimers to
stimulate processing of Sog by Tlr. We generated homodimers by transfection of
individual expression plasmids in Drosophila S2 cell cultures.
Heterodimers were generated by co-transfection of two Bmp-ligand expression
plasmids. This produces a significant proportion of heterodimers together with
individual homodimers (Fig. 3A)
(Shimmi et al., 2005a
).
|
Tlr suppresses Sog gain-of-function phenotypes in vivo
As Tlr can process Sog in vitro, we tested whether it could also function
as a modulator of Sog activity in vivo. In Xenopus embryos, injecting
sog mRNA into the ventral marginal zone blocks Bmp signaling and
produces a secondary dorsoventral axis
(Holley et al., 1995;
Piccolo et al., 1997
). We used
this assay to determine if Tlr can reverse the effects of Sog. As shown in
Fig. 4A, ventral injection of 2
ng of sog mRNA induces secondary axes in
61% (n=83) of
the resulting tadpoles. However, when sog mRNA was co-injected
together with an equimolar amount of tld or tlr mRNA,
secondary axis induction was reduced to 11% (n=120) and 16%
(n=149), respectively. These results suggest that like Tld, Tlr is
able to process Sog in Xenopus embryos.
|
Tlr is required for Bmp signaling during the pupal crossvein formation
Previous analysis of tlr mutants found that these animals die
mostly in the larval and pupal stages of development. However, rare escapers
exhibit loss of the crossveins (Nguyen et
al., 1994) (Fig.
5B). Crossvein formation is initiated by high levels of Bmp
signaling beginning 19 hours after pupariation (AP)
(Conley et al., 2000
;
Ralston and Blair, 2005
). We
therefore examined the expression of tlr transcript during pupal wing
development. For comparative purposes, we also examined tld
expression in pupal wings. As illustrated in
Fig. 5A we found that
tlr is expressed in the intervein regions, while tld was not
detectable above background. The absence of detectable tld expression
suggests that Tlr is the major contributor of Tld-like activity to the
venation process during pupal development.
To determine directly whether tlr is required for Bmp signaling in
the developing crossveins, we assayed for accumulation of the phosphorylated
form of Mad (pMad) in the crossvein region of
tlrex[2-41]/tlrE1 mutants during pupal
wing development. In wild-type wings dissected at 19 hours AP, pMad signal is
evident in a broad region around the PCV
(Fig. 5B); by 24 hours AP this
region starts to narrow and by 30 hours AP a narrow stripe forms corresponding
to the PCV (Conley et al.,
2000). In
tlrex[2-41]/tlrE1 mutant wings, pMad
is severely reduced at 19 hours AP (Fig.
5B). As an internal reference, we note that pMad accumulation in
the longitudinal veins and in the axons and nuclei of the sensory neurons
along the anterior margin is not affected in
tlrex[2-41]/tlrE1 mutants. By 24 hours
AP, pMad nuclear staining was completely lost in the PCV region of almost all
tlrex[2-41]/tlrE1 mutant wings (20/22
wings).
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One way of explaining this result is to posit that Sog has both an
inhibitory and positive effect on Bmp signaling during PCV development. There
is precedent for this in the early embryo: Sog is required to specify
amnioserosa, the dorsal-most tissue that requires high levels of Bmp signal.
It has been proposed that this positive function arises because Sog aids in
the transport of ligand so that it is concentrated at the dorsal midline
(Holley et al., 1995;
Marqués et al., 1997
;
Eldar et al., 2002
;
Shimmi et al., 2005a
). Recent
results indicate that signaling in the PCV depends on Dpp produced in adjacent
longitudinal veins (Ralston and Blair,
2005
), and thus a similar transport role for Sog might be required
in the pupal wing.
To determine if Sog is also required to achieve peak levels of Bmp signaling in the PCV, we generated sog heteroallelic combinations with reduced Sog protein level and monitored PCV formation. We find that the survival rate of the sogP129D/sogYL26 animals was reduced to 31% (20 progeny eclosed compared with 64 of the control genotype sogP129D/FM7); of these, 15% (n=40 wings) exhibited partial loss of the PCV (Fig. 7C). The rest of the escapers (85%) exhibited more subtle alterations in which the PCV appeared as a fragmented line instead of a continuous refined structure. For the stronger allelic combination sogP129D/sogP1 genotype, the survival rate was further reduced to 12% (15 adults compared with 124 sogP129D/FM7 animals) and the phenotype was stronger: 46% of the surviving progeny (n=30 wings) exhibited various degrees of loss of the PCV (Fig. 7D,D'). These results indicate that, as in the embryo, Sog is required to establish peak levels of Bmp signaling during PCV specification.
The disruption of the PCV appeared to correlate with the extent of
remaining Sog protein, i.e. stronger sog allelic combinations or
misexpression of stronger/activated tld transgenes exhibited stronger
loss of PCV structures (this study)
(Shimmi and O'Connor, 2003).
As a further test of this issue, we used different combinations of
tld and tlr misexpression, each of which on their own did
not affect PCV formation but which in combination exhibited loss of the PCV.
Thus, as shown in Fig. 7E-G,
expression of two copies of a particular UAS-tlr line using the
A9-Gal4 driver did not affect PCV formation. Likewise expression of
one copy of a particular UAS-tld transgene produced only mild effects
(a detached PCV in 16% of the wings n=102). However, when the
tld and tlr lines were co-overexpressed, the frequency of
PCV perturbation was greatly increased to 82% (n=192) and the
severity of the phenotype was also increased
(Fig. 7G). Taken together,
these results suggest that an inappropriate increase in total Sog processing
activity disrupts the PCV specification most probably by interfering with the
positive role of Sog in Bmp signaling within the pupal wing.
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Discussion |
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|
Previously, we speculated that this difference in activity might reflect
differences in activation of the proteases at the level of pro-peptide
removal. Like all the members of the Bmp1 family, Tld and Tlr are secreted as
pro-enzymes; the processing of the pro-peptide is necessary for the activation
of proteolytic activity, as the N-terminal end of the astacin motif is buried
inside the catalytic domain forming an internal salt bridge
(Bode et al., 1992). Mutations
at the processing site rendered the enzymes inactive, whereas removal of the
pro-peptides produced activated forms of Tld and Tlr. Tlr has a much longer
pro-peptide that could either aid or inhibit activation in a tissue-specific
manner. However, the inability of Tlr to rescue Tld mutants does not appear to
result from an inefficient activation step. We have previously shown that Tld
activation, both in the embryo and in S2 cells, is very inefficient
(Marqués et al., 1997
)
with most of the protein found in the pro-enzyme state. By contrast, we show
here that pro-peptide removal from Tlr is very efficient in S2 cells, and the
same is true when Tlr is ectopically expressed in the embryo (M.S. and M.B.O.,
unpublished).
Instead, it seems likely that the difference in kinetics of Sog processing by Tlr is the reason behind the inability of Tlr to rescue tld mutants. We show here that Tlr is much less efficient at cleaving Sog in vitro than Tld. Given the rapid developmental time of early embryogenesis, where patterning by Bmps during cellularization occurs within approximately a 30 minute time window, the slower kinetics of Sog processing by Tlr may not support proper patterning. Indeed, our recent computational work has shown that a three to fourfold reduction in kinetic properties of Tld will completely disrupt the patterning process (D. Umulis, H. Othmar and M.B.C., unpublished).
Although the slow processing kinetics of Tlr towards Sog may prevent it
from functioning effectively in early embryonic patterning, this property may
be exactly what is required to achieve proper formation of the PCV. Unlike
patterning in the early embryo, formation of the PCV, as assessed by profile
of pMad accumulation, occurs over at least a 7 hour time frame
(Conley et al., 2000). The
slower processing rate of Tlr towards Sog may be required to achieve the
appropriate balance of Sog destruction and diffusion (see below) that is
necessary for proper patterning to occur. Consistent with this view, we find
that overexpression of various UAS-tld lines under the control of the
A9-Gal4 driver in a tlr mutant background did not rescue PCV
formation. In fact, in many cases overexpression of an activated Tld, or
co-expression of wild type tld and tlr together produced
loss of the crossvein tissue in a wild-type background. We envision that,
under these conditions, the increased level of enzymatic activity results in
over digestion of Sog, a situation that would phenocopy sog
hypomorphs. Consistent with this view, we find that hypomorphic sog
allelic combinations also result in the loss of the PCV. In addition, it has
recently been shown that large sog null clones can also result in
loss of the PCV (Shimmi et al.,
2005b
).
In the Xenopus assay, we found that Tlr is only slightly less
efficient than Tld at reverting secondary axis induction caused by Sog.
Although we do not know how well each enzyme is activated in this animal, it
should be noted that the developmental time period over which the patterning
process functions in Xenopus is long compared with early
Drosophila development. The longer time frame may enable the less
efficient protease to produce a similar biological response. Our protease
domain swap experiments suggest that the reduced processing rate did not
involve evolution of intrinsic differences in the catalytic abilities of the
protease domain itself, but rather changes in the way that the Sog substrate
initially interacts with the enzyme. In summary, we propose that during
evolution there was selection for particular biophysical properties of these
two enzymes to properly match the developmental time frame over which the
patterning mechanisms operate. We cannot exclude however, that other
differences besides kinetic activity might also play a role in providing
functional specificity. For example, it is possible that variation in cleavage
site selection might also contribute to the different biological activities of
Tlr and Tld. It is worth noting in this regard that different fragments of Sog
have been shown to have both positive and negative effects when overexpressed
in the wing (Yu et al., 2000;
Yu et al., 2004
). However the
in vivo roles of endogenous Sog fragments have not been defined.
Recapitulation of the Bmp-mediated dorsal patterning mechanism during specification of the PCV
Our results suggest that a proper balance of Sog and protease activity is
necessary to pattern the PCV. Interestingly, the same situation holds true in
the early embryo. In this case, Sog plays both positive and negative roles in
patterning the dorsal domain (Holley et
al., 1995; Ashe and Levine,
1999
; Marqués et al.,
1997
). It is required in dorsolateral regions to block Bmp
signaling, but it also acts as an agonist to achieve peak levels of Bmp
signals at the dorsal midline. Two types of models have been proposed to
account for these dual activities. In one model, the different cleavage
fragments of Sog are thought to provide either agonist or antagonist function,
but the details of the mechanism are unclear
(Ashe and Levine, 1999
;
Yu et al., 2000
). In the other
model, both functions are proposed to come about as a result of Sog providing
a transport mechanism that spatially redistributes Bmp ligands from the
lateral region to the dorsal most cells
(Holley et al., 1995
;
Ross et al., 2001
;
Shimmi and O'Connor, 2003
;
Shimmi et al., 2005a
). This
transport mechanism also requires the activity of Tsg, a small cysteine-rich
secreted protein which has been shown to form a tripartite complex with Sog
and Dpp (Ross et al., 2001
;
Shimmi and O'Connor, 2003
).
The prevailing view is that as Sog diffuses into the dorsal domain it forms a
high affinity complex with Tsg and Dpp. This complex is unable to bind to
receptors and is responsible for the antagonistic activity of Sog. At the same
time, the complex protects Dpp from degradation and receptor binding allowing
it to diffuse and accumulate dorsally where it is released by Tld processing.
The ability of Sog to redistribute the Bmp ligands accounts for the agonist
function of Sog. Recent computational analyses have provided additional
support for this model (Eldar et al.,
2002
) (D. Umulis, H. Othmar and M.B.C., unpublished).
We propose that the same type of mechanism may be responsible for
patterning the PCV. Recent analysis has provided evidence that the
longitudinal veins act as the source of Dpp for PCV specification. Dpp is
thought to diffuse from these veins into a PCV competent zone
(Ralston and Blair, 2005). The
exact mechanism by which the competent zone is specified is not clear, but low
levels of Sog expression are required
(Ralston and Blair, 2005
;
Shimmi et al., 2005b
).
tlr is expressed within the PCV competent zone during the initial
stages of crossvein development (A.R. and S.S.B., unpublished), suggesting the
Tlr:Sog ratio will be higher in this region. Furthermore, because processing
of the Sog/Dpp complex by Tld-like enzymes is dependent on the Dpp
concentration (Shimmi and O'Connor,
2003
) (this work), the complex will be most efficiently processed
in the center of the competent zone.
According to this model, there is limited processing of Sog and therefore
limited release of Dpp from its inhibitor in tlr mutants. Conversely,
Sog also supplies a positive function for PCV formation, probably by providing
a transport mechanism for Dpp, accounting for the partial loss of the PCV in
hypomorphic sog mutants and complete loss of the PCV in large
sog-null clones (Shimmi et al.,
2005b). The partial reversion of the tlr mutant phenotype
by introduction of hypomorphic sog alleles is also consistent with
the view that it is the balance between these two factors that is crucial for
proper patterning. Interestingly, this is the way in which Sog was originally
identified as an inhibitor of Dpp signaling in the embryo: weak sog
alleles were isolated as partial suppressors of tld mutations
(Ferguson and Anderson, 1992
).
One difference is that, in our case, lowering Sog levels is able to revert a
null mutation instead of a hypomorphic condition, as was the case in the
embryo. There are at least two possibilities that can explain this suppression
effect. First, although these animals may be null for tlr, there
could be some low level tld expression in the pupal wing. If so, then
these wings would not be devoid of all Sog-processing activity and therefore
lowering Sog levels might enable the limited amount of Tld to provide the
proper production-destruction balance. Alternatively, neither Sog nor Tlr may
be absolutely required for PCV formation. Instead their functions may be
simply to ensure that the patterning occurs reproducibly. Thus, in the absence
of both Sog and Tlr, partial PCV formation may occur as a result of some Bmp
ligand accumulating in the correct position. However, under these
circumstances, the patterning mechanism would be unreliable and would produce
different results on case-by-case basis. To prevent this from occurring, we
posit that evolution has selected for supplementary regulatory controls
involving Sog and Tlr to ensure that the PCV always forms completely and
reliably at the correct position.
Two additional observations make the comparison between formation of the
PCV and establishing the high point in embryonic Bmp signaling even more
compelling. First, in the embryo, Tsg is required to enable Sog to bind to Dpp
and Scw to achieve peak levels of Bmp signaling. Although Tsg and Scw are not
transcribed in the pupal wing, we have recently determined that a Tsg-related
gene, encoded by the crossveinless (cv) locus, is expressed
in the pupal wing (Shimmi et al.,
2005b; Vilmos et al.,
2005
). As cv mutants exhibit a crossveinless phenotype,
it seems likely that Cv functionally substitutes for Tsg during PCV formation.
Second, Gbb, another Bmp-like ligand, may functionally replace Scw as
gbb hypomorphic mutations lack the PCV and associated Bmp signaling
(Wharton et al., 1999
;
Ralston and Blair, 2005
).
A major distinction between embryonic amnioserosa development and PCV
formation is that PCV specification also requires the activity of Cv2, a
protein that contains cysteine-rich repeats similar to those found in Sog,
while amnioserosa specification does not, despite the expression of Cv2 in
those cells (Conley et al.,
2000) (M.S. and M.B.O., unpublished). Vertebrate homologs of Cv2
can bind Bmps, and act variously as agonists or antagonists of Bmp signaling
in different assays (Kamimura et al.,
2004
; Coffinier et al.,
2002
; Binnerts et al.,
2004
). It is not clear by what mechanism Cv2 promotes Bmp
signaling during PCV formation. It is also not clear why Cv2 is not required
in the early embryo, even though it is expressed in dorsal blastoderm cells
(M.S., A.R., S.S.B. and M.B.O., unpublished).
Finally, Tlr plays additional roles during development, besides processing
Sog for specification of the PCV. In contrast to cv and cv2
null mutations, which result in homozygous viable and fertile flies
(Shimmi et al., 2005b;
Vilmos et al., 2005
) (M.S. and
M.B.O., unpublished), most tlr mutant animals die during larval
stages (Nguyen et al., 1994
;
Finelli et al., 1995
) when
there is no known requirement for Sog. In addition, although reducing Sog
levels did suppress the PCV defect observed in the tlr mutant escaper
flies, it did not increase the frequency of eclosing animals. Therefore Tlr
may be required for processing of some other essential component(s) during
Drosophila development.
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ACKNOWLEDGMENTS |
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