1 Departamento de Histologia e Embriologia, Universidade Federal do Rio de
Janeiro, 21941-970, Rio de Janeiro, Brazil
2 Section of Cell and Developmental Biology, University of California, San
Diego, La Jolla, CA 92093-0349, USA
* Author for correspondence (e-mail: haraujo{at}histo.ufrj.br)
Accepted 9 May 2003
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SUMMARY |
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Key words: Drosophila, Sog, Integins, Bmps
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INTRODUCTION |
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During early pupal development, Decapentaplegic (Dpp), the
Drosophila homolog of vertebrate Bmp2/Bmp4, and the Bmp-binding
protein Short gastrulation (Sog), the homolog of vertebrate chordin, function
antagonistically to ensure the formation of straight continuous veins
(Haerry et al., 1998;
Lecuit et al., 1996
;
Sturtevant and Bier, 1995
;
Yu et al., 1996
;
Zecca et al., 1995
).
Throughout this period, dpp is expressed in vein primordia, while
sog is expressed in a complementary intervein pattern
(Yu et al., 1996
). Sog also
opposes Bmp signaling during dorsoventral patterning of the early embryo,
which involves zygotic (Biehs et al.,
1996
; Francois et al.,
1994
; Marques et al.,
1997
) as well as maternal
(Araujo and Bier, 2000
)
functions of this pathway. sog encodes a secreted molecule with
domains resembling thrombospondin and procollagen
(Francois and Bier, 1995
;
Francois et al., 1994
) and has
been shown to bind Dpp (Ross et al.,
2001
). It has been suggested that regulated cleavage of Sog
generates different forms with distinct activities
(Yu et al., 2000
). Cleavage at
three sites by the metalloprotease Tolloid (Tld) inactivates Sog
(Marques et al., 1997
), while
alternative cleavage at a different site, which occurs in the presence of the
co-factor Twisted Gastrulation (Tsg), results in the production of truncated
forms of Sog referred to as Supersog, which antagonize a broader spectrum of
Bmp activities than intact Sog (Yu et al.,
2000
). Chordin is also subject to proteolysis by Xolloid, the
vertebrate homolog of Tolloid (Piccolo et
al., 1997
). Because all of these molecules interact
extracellularly, it is important to understand how the activities and
localization of these factors are regulated in the extracellular milieu.
Binding of growth factors to specific proteins or to the ECM is one type of
mechanism for regulating the availability or dispersion of growth factors in
different developmental contexts. ECM proteins may sequester growth factors in
an inactive form, as well as modulate cellular responses to them
(Streuli, 1999). Several ECM
proteins such as Collagen, Fibronectin, Thrombospondin, Noggin and Chordin
bind to TGFß or to members of the bone morphogenetic protein (Bmp)
subfamily (Piccolo et al.,
1996
; Taipale and Keski-Oja,
1997
; Zimmerman et al.,
1996
). Such binding may activate or reduce growth factor activity
and/or availability. Several extracellular matrix molecules and their
receptors have been described in Drosophila (for a review, see
Brown, 2000
). Among these
proteins, integrins are expressed during embryogenesis, larval and pupal
stages and perform functions including muscle attachment, morphogenesis of the
midgut, and adhesion between the two surfaces of the wing
(Brabant et al., 1996
;
Brower et al., 1995a
;
Brower et al., 1995b
;
Martin-Bermudo and Brown,
1996
; Roote and Zusman,
1995
; Wilcox et al.,
1989
). During pupal development, integrins are expressed in
intervein cells and perform a central role in regulating apposition (i.e.
alignment and adhesion) of the dorsal and ventral surfaces of the wing
(Brabant et al., 1996
;
Fristrom et al., 1993
;
Wilcox et al., 1989
). Three
integrin subunits are known to be required for adhesion between the two wing
surfaces: ßPS integrin, encoded by the myospheroid
(mys) gene, is expressed on both wing surfaces during pupal
development; and two
-integrins,
PS1, encoded by the
multiple edematous wing (mew) gene, and
PS2, encoded
by the inflated (if) gene, are expressed on the dorsal and
ventral wing surfaces, respectively, during early wing development
(Brabant et al., 1996
;
Brower et al., 1995b
).
Functional integrin molecules are composed of one ß-subunit combined with
one of the
-subunits, and consist of a large extracellular domain and a
small cytoplasmic tail. Mutations in any of these integrin genes cause
blisters in the adult wing, a phenotype characteristic of a lack of apposition
between the wing surfaces (Brower et al.,
1995b
; Brown et al.,
1989
; Wilcox et al.,
1989
; Zusman et al.,
1990
).
In this report, we show that in addition to their well established adhesive
function, integrins play another role during pupal vein development to
modulate Bmp signaling. This modulation of Bmp activity may be mediated, at
least in part, by integrins binding and/or regulating the activity or
diffusion/distribution of the Sog protein. Genetic analysis indicates that the
role of integrins in modulating Bmp signaling involves the well studied
ßPS and PS1 subunits, and another less extensively characterized
PS3 subunit (Grotewiel et al.,
1998
; Stark et al.,
1997
), which we show is also expressed in dorsal cells of the
pupal wing. We find that Sog diffuses into provein domains from adjacent
intervein cells, but does so only on the dorsal surface. Moreover, we find
that this dorsal specific diffusion of Sog into provein regions depends on the
activity of both ßPS and
PS1 integrin subunits. We discuss these
results in light of current models for regulated Sog processing and
recycling.
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MATERIALS AND METHODS |
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Df 3R slo3, which deletes both tld and
tlr. Detailed characteristics of all alleles can be found in FlyBase.
Enhancer piracy sog lines (e.g. sogEP2,
sogEP3, sogEP7,
sogEP8, sogEP9,
sogEP11) are described elsewhere
(Yu et al., 1996).
Production and analysis of mitotic clones
Clones of cells mutant for X-linked genes were induced by mitotic
recombination in animals homozygous for FRT 18A and heterozygous for FRT18A
mys or FRT18A mew chromosomes. mys clones were
generated using the allele mysXB87 and the marker
multiple wing hair (mwh), by use of mwh- flies
containing a copy of the mwh gene on the first chromosome. Clones
were generated in a wild-type background or in an Enhancer Piracy sog
line (sogEP) background, as below.
sogEP lines drive transgenic sog expression in
vein primordia.
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Eggs were collected for 24 hours and aged for 48 hours before the heat shock in order to generate predominantly small clones (<100 cells). First instar larvae were heat shocked for 15 minutes at 37°C. Unmarked clones generated with the same mysXB87 FRT line produced similar phenotypes. Twenty-seven dorsal clones, four ventral clones, and four dorsal and ventral clones were analyzed in detail.
mew clones were generated using the allele
mewM6 and scored using the bristle and trichome marker
forked (f36a). Clones were generated in a
wild-type background or in a sogEP background as indicated
below:
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Eggs were collected and aged as described above. First instar larvae were heat shocked for three 1 hour intervals at 37°C, with 30 minute recovery periods in between. Twenty-seven dorsal clones, seven ventral clones, and five dorsal and ventral clones were analyzed in detail.
scb clones were generated using the allele
scb1 and scored using the bristle and trichome marker
pawn (pwn). The scb1 allele was
recombined with FRT 42D and clones were induced in animals homozygous for FRT
42D and heterozygous for scb chromosomes. Clones were generated in a
wild-type background or in an sogEP background as
indicated below:
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Eggs were collected and aged as described above. First instar larvae were heat shocked for three 1 hour intervals at 37°C, separated by 45 minute recovery periods. Eighteen dorsal clones, nine ventral clones, and five dorsal and ventral clones were analyzed in detail.
In situ hybridization and immunohistochemistry
In situ hybridization was performed using digoxigenin-labeled antisense RNA
probes and visualized as a blue alkaline phosphatase precipitate
(O'Neill and Bier, 1994).
Immunohistochemistry was performed as described by Sturtevant et al.
(Sturtevant et al., 1993
). Sog
protein was detected using polyclonal 8B as primary antibody (1:500)
(Srinivasan et al., 2002
),
anti-rabbit HRP as secondary antibody (1:2000, Jackson Laboratories), and
visualized using the rhodamine TSA kit (NEB). For Sog and Integrin double
labels, Sog protein was detected with anti-8B antiserum as above, CF.6G11
monoclonal antiserum was used for ßPS integrin (1:500) and DK.1A4
monoclonal antiserum was used for
PS1 (1:500)
(Brower et al., 1984
), and
detected with secondary anti-mouse Alexa 488 (Molecular Probes). Images were
analyzed either on a Zeiss Axiovert 135, collected digitally with Axiocam or
on a LSM 510 Meta Zeiss Confocal Microscope.
Immunoprecipitation and immunoblotting
Co-immunoprecipitation was based on procedures described by Brower
(Brower, 1984), with minor
modifications. Wings of pupae taken 20-24 hours after puparium formation (APF)
were rapidly dissected from the pupal cases, homogenized with a pestle in ice
cold lysis buffer (10 mM Tris pH 8.1/75 mM NaCl/0.5 mM MgCl2/0.5 mM
CaCl2/0.25% NP40/0.25% BSA/0.01% NaN3/1 mM PMSF and
protease inhibitor cocktail Complete, Boehringer Mannheim), and left
for 30 minutes on ice with occasional rocking. After brief centrifugation (10
minutes at 10,000 g) to pellet non-homogenized tissue,
supernatants were incubated overnight at 4°C with protein A Sepharose
bound integrin antibodies. Unbound supernatants where collected as `unbound'
sample and 4x SDS sample buffer was added. Beads were washed twice with
lysis buffer and stripped of bound proteins by two rounds of acid elution with
200 mM glycine (pH 3.0) generating `bound 1' and `bound 2' samples to which
4xSDS sample buffer was added. All samples were boiled before running in
10% SDS-PAGE gels and transferred by electroblotting to nitrocellulose
membranes. Membranes were blocked in Tris/NaCl/0.3% BSA 0.1% Tween 20 and
incubated in primary antibody (anti-Sog 8A at 1:500 dilution) followed by
incubation in HRP-conjugated anti-rabbit secondary antibody (Sigma, at 1:5000
dilution) and developed using Supersignal (Pierce) according to manufacturer's
instructions. For detection of co-immunoprecipitated integrins used as
control, membranes were stripped of antibodies using 200 mM glycine (10
minutes at room temperature), incubated in biotin-conjugated Concanavalin A,
treated in Vectastain AB system and visualized by chemiluminescence as above.
The 8A anti-Sog antiserum was raised against a small peptide fragment that
included CR1 and the first part of the stem and the antibody recognizes a
sog construct on western blots that contains the stem but not
CR1.
Antibodies were covalently attached to protein A Sepharose beads by
incubating them with beads overnight at 4°C. After three washes in cold
PBS and two washes in 2 M sodium borate (pH 9.0), antibodies were crosslinked
to the beads by addition of 5 mg/ml DMP. Beads were incubated for 30 minutes
at room temperature, washed in 0.2 M triethanolamine pH 8.0, and incubated for
2 hours. After equilibrating in PBS, beads were stored at 4°C with 0.01%
sodium azide. Beads were washed in PBS before use. Antibodies used were:
CF.6G11 (monoclonal for ßPS integrin); DK.1A4 (monoclonal for PS1)
(Brower et al., 1984
); CF.2C7
monoclonal (Wilcox et al.,
1981
) for
PS2; and polyclonal
vol
(volado, also known as scab) for
PS3
(Grotewiel et al., 1998
).
Mounting fly wings
Wings from adult flies were dissected in isopropanol and mounted in Canada
Balsam mounting medium.
Microsequencing
N-terminal microsequencing was used to ensure the identity of the protein
band co-immunoprecipitated with PS1 integrin antibodies. Immobilon
instead of Nitrocellulose membranes were used, and BSA was omitted in the
homogenization buffer. Protein bands were transferred from SDS-PAGE onto
Immobilon membranes, followed by N-terminal microsequencing by Edman
degradation. Microsequencing revealed contaminating proteins in the band
recognized by the Sog antibody; however, we were able to detect the sequence
GV(X)EGR(X)H(XX)L(XX)EE(X). A Blast search for short sequences aligning to
this sequence found that it aligns to the N-terminal region of the Sog protein
(GVTEGRRHAPLMFEES).
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RESULTS |
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We also observed genetic interactions between integrins and other Bmp
signaling components. For example, the hypomorphic ß-integrin allele
mysnj42 suppresses the thickened vein phenotype of the
tkv1 allele of the Dpp receptor thick veins
(Fig. 1G,H;
Table 1). Similarly, decreasing
the level of scb (in scb1
tkv1/tkv1 flies) suppresses the
tkv1 phenotype (not shown). In addition, dpp and
gbb alleles enhance sogEP phenotypes as do
reduced levels of tld and tok, which encode highly related
metalloproteases (Table 1).
This latter observation is interesting in light of the fact that tld
and tok can collaborate to either degrade
(Marques et al., 1997) or
process Sog into more broadly active Bmp inhibitory forms in embryos and pupae
(Yu et al., 2000
).
Ectopic sog expression alters the behavior of clones lacking
ßPS and PS1 integrins
Because decreasing the dose of mys enhanced
sogEP phenotypes, we examined the effect of complete loss
of mys function in a sogEP7 background by
producing mys-null clones. Large mys- clones
generated by FLP-FRT-mediated recombination frequently induce blisters due to
non-apposition of the dorsal and ventral surfaces of the wing. In small
mys- clones, however, blisters are not observed and veins
appear normal although the two wing surfaces remain unapposed within the
center of the clone (Brabant et al.,
1996). When similar small mys- clones are
generated in a sogEP7 background, a different phenotype is
observed in which veins become ill-defined and broadened (e.g. four or five
cells across compared with two or three cells in diameter in wild type)
wherever the clones cross or abut longitudinal veins or the posterior
crossvein (Fig. 2), and is
observed in clones consisting of as few as 20 cells. The ability of
mys- clones to induce vein broadening non-autonomously in
neighboring cells occurs only at very short range as clones displaced by three
or more cell diameters from veins have a wild-type phenotype. Dorsal and
ventral mys- clones can induce non-apposition of the wing
surfaces in both wild-type and sogEP7 backgrounds,
consistent with the fact that ßPS integrin is expressed on both surfaces
of the wing during larval and pupal development
(Brabant et al., 1996
;
Brower et al., 1995a
). Vein
broadening in an sogEP7 background, however, is observed
only in dorsal clones, indicating that this phenotype is not simply a
secondary consequence of an adhesion defect. The restriction of
mys- vein phenotypes to the dorsal surface also suggests
that there is a dorsally expressed
-integrin, which acts in conjunction
with ß-integrin during vein development.
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PS3 is expressed dorsally in the pupal wing and regulates vein
formation
It has not yet been reported whether the -integrin scb is
expressed in the wing or functions during wing development. Because
scb alleles interacted genetically with sogEP7,
we examined the pattern of scb expression during larval and pupal
wing development by in situ hybridization. No scb expression was
detected during larval and prepupal stages (data not shown); however, a
dynamic pattern of scb expression emerges in 20-30 hour pupal wings
(Fig. 4A,B). scb
expression, which is restricted to the dorsal surface of the wing at all
times, is initiated most intensely in intervein regions in the vicinity of L2
and L3 and then rapidly expands to encompass all intervein cells.
Subsequently, scb expression is also observed within the provein
domain, initially at high levels and then tapering off after 25 hours of pupal
development (Fig. 4B,F).
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Integrins regulate the distribution of Sog protein in the pupal
wing
It has been observed that sog mRNA is confined to intervein cells
during pupal development (Yu et al.,
1996) (Fig. 4E). As
Sog protein diffuses during early embryonic development
(Srinivasan et al., 2002
),
however, we wondered whether Sog might also travel from its intervein site of
production into the provein region during pupal development. We stained pupal
wings with the 8B anti-Sog antiserum
(Srinivasan et al., 2002
) and
observed a dynamic pattern of Sog protein distribution, which includes vein
competent domains as well as intervein cells
(Fig. 7). Anti-Sog staining is
initially patchy (around 20 hours apf), stronger on the dorsal surface and
mostly restricted to intervein cells (Fig.
7A). Shortly thereafter (22-26 hours apf), Sog staining spreads
into provein cells on the dorsal surface of the wing, at which point it is
excluded only from the most central vein-proper cells
(Fig. 7B,E). On the
corresponding ventral surface, however, Sog staining remains excluded from the
entire provein region throughout pupal development (i.e. up to 34 hours apf)
(Fig. 7F,H). Between 26 and 30
hours apf, Sog staining fills all the provein domains on the dorsal surface,
with increased levels of staining observed at the provein/intervein border
(Fig. 7C,G). At 30 hours apf,
Sog staining diminishes overall and becomes restricted to intervein cells and
hemocytes running in the middle of the vein
(Fig. 7D). As sog mRNA
is detectable only in intervein cells during the examined pupal period, we
conclude that Sog protein must be delivered to cells within the provein
territory on the dorsal surface by some form of passive diffusion or active
transport.
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DISCUSSION |
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Integrins regulate Sog distribution in the pupal wing
Consistent with Sog interacting genetically with integrins to alter the
course of veins on the dorsal surface of the wing, we found that the
PS1 and ßPS-integrins are required for the diffusion or transport
of Sog from dorsal intervein cells where sog mRNA is expressed into
adjacent provein regions. As
PS1 binds Sog, this physical interaction
may contribute to regulating the distribution of Sog. The 8B anti-Sog
antiserum, which recognizes Sog protein in intervein cells and inside the
provein domain, detects an epitope located near the second cystein repeat
(CR2). Consequently, Sog fragments that diffuse or that are delivered to
provein cells must be either full length, which weakly binds to
PS1 in
co-immunoprecipitation experiments, or fragments that contain CR2. The
truncated Supersog-like fragment that binds strongly to
PS1 in
coimmunoprecipitation experiments should not be recognized by the 8B
antiserum. Therefore, integrins may differentially regulate the distribution
of Sog fragments on the dorsal surface of the pupal wing, restraining the
movement of broad spectrum Bmp inhibitory Sog fragments (such as Supersog-like
molecules) and allowing or mediating transport of other fragments to provein
cells, such as full-length Sog, which also has a vein inhibitory function.
Unfortunately, it is not possible currently to examine the diffusion of
Supersog-like fragments directly because the 8A antiserum is not suitable for
staining pupal wings. These findings suggest that integrins regulate the
delivery or diffusion of active Sog protein from intervein cells into the vein
competent domain. In contrast to the dorsally restricted functions of
integrins required for vein development, the previously analyzed adhesive
functions of integrins depends on subunits functioning on both surfaces of the
wing.
Integrins modulate Sog activity in the wing
There are several possible mechanisms by which interaction with integrins
could modulate Sog activity in pupal wings. It has been previously shown that
elevated sog expression results in vein truncation, while
misexpression of dpp induces ectopic veins, indicating that
sog restricts vein formation by opposing Bmp signals emanating from
the center of the vein (Yu et al.,
1996). One possibility is that such a Bmp inhibitory form(s) of
Sog must interact with integrins in order to diffuse or be transported into
provein domains (on the dorsal surface of the wing only). This hypothesis
would be consistent with the finding that veins appear to be attracted to
integrin- clones. Such a vein repulsive form(s) of Sog would
presumably act as a Bmp antagonist.
According to the simple model in which integrins are essential for
delivering a Bmp inhibitory form of Sog to provein cells, one would expect
that integrin- and sog- clones would generate
similar phenotypes in which veins deviated towards the mutant clones and/or
broadened within them. However, sog- clones induce
meandering of veins (Yu et al.,
1996), which show only a weak tendency to track along the outside
of sog- clones (B.N. and E.B., unpublished), in contrast
to integrin- clones, which bend or widen veins in a more dramatic
fashion. One possible explanation for the differences between the
sog- and integrin- phenotypes is that there are
several different endogenous forms of Sog in pupal wings
(Yu et al., 2000
), which might
exert opposing activities. If multiple Sog fragments exert effects on vein
development, some providing repulsive and others attractive activities on vein
formation, the differences between the behaviors of sog- and
integrin- clones could be explained by a repulsive (Bmp inhibitory) form(s) of
Sog selectively requiring an interaction with integrins. The possibility that
a positive Bmp-promoting activity of Sog might also be present that acts as a
vein attractant has precedent in that a positive Sog activity has been
proposed to explain a requirement for Sog in activating expression of the Dpp
target gene race in early embryos
(Ashe and Levine, 1999
).
Structure/function studies of Sog have also revealed a potential Bmp promoting
form of Sog, which is longer than Supersog forms (K. Yu and E.B.,
unpublished). According to this model, altering the balance between repulsive
and attractive Sog activities would generate different vein phenotypes. In the
total absence of sog, both repulsive and attractive activities would
be lost, generating a mild meandering vein phenotype in which neither
attraction nor repulsion clearly dominated, as is observed in
sog- clones (Yu et
al., 1996
). If an interaction with integrins were required only
for production or delivery of Bmp inhibitory forms of Sog into the vein, then
integrin- clones, which still contain the Bmp-activating forms of
Sog, could exert a net attractive influence on veins, leading to more
pronounced deviation of veins toward the clones. This hypothesis is consistent
with vein phenotypes we have observed associated with integrin-
clones that cross over veins or run along both sides of the vein, such as
narrowing, bending and wandering of veins which are similar to phenotypes
observed in correspondingly located sog- clones. The
existence of different Sog fragments bearing opposing activities would also
explain the different phenotypes we obtain upon ectopic Sog expression in some
sogEP lines (Yu et al.,
1996
), such as sporadic ectopic vein material between L3 and L2
and meandering L2 veins in addition to vein loss in other areas.
Another possible explanation for the differences between the
sog- and integrin- phenotypes is that integrins
may regulate the activity of extracellular signals in addition to Sog. One
hint of such an activity is that when a scb- clone falls
within the provein area, the vein splits around the border of the clone in a
cell autonomous fashion. As this later phenotype is enhanced by ectopic
sog expression in veins (e.g. in a sogEP
background), PS3 may normally promote Bmp signaling within the vein.
Although the identity of such potential targets is unknown, candidates would
include Bmps (e.g. Dpp or Gbb) or Bmp receptors. Further analysis will be
needed to explain the basis for the different behaviors of
sog- and integrin- clones, as well as the
variations observed in different integrin- clones.
In summary, we propose that Sog fragments with differential activities may
regulate vein formation. The vein bending phenotype observed in the absence of
PS1 would result from a remaining attractive Sog activity that
outweighs the activity of a repulsive form of Sog, which can no longer be
delivered from intervein cells (Fig.
9). As ßPS integrin forms heterodimers with both
PS1
and
PS3 (Brower et al.,
1984
; Stark et al.,
1997
) mys would be expected to be required for the
activity of both
PS chains. Consistent with this expectation, the
phenotype of mys- clones (i.e. broad poorly defined veins)
resembles a hybrid of those observed for mew- and
scb- clones.
|
Although we have not directly addressed whether integrins regulate Sog
endocytosis in this current study, the altered distribution of Sog within
integrin- clones is suggestive of such a role. Reticular Sog
staining, which outlines the cell perimeter is lost in integrin-
clones on the dorsal surface, leaving only a punctate intracellular staining.
This mis-localization of Sog implicates integrins in internalizing and/or
trafficking of Sog to the cell surface. Because appropriately located
integrin- clones also block the accumulation of Sog in adjacent
provein domains, the observed defects in Sog distribution between the surface
and the cytoplasm may underlie the failure to deliver Sog to vein competent
cells. The endocytic pathway could promote the transport of Sog to provein
cells by a mechanism similar to that proposed to be involved in the transport
of Dpp along the AP axis during larval stages
(Entchev et al., 2000;
Teleman and Cohen, 2000
).
Alternatively, endocytosis could function to limit Sog diffusion as is the
case during embryogenesis (Srinivasan et
al., 2002
). According to this latter scenario, integrins would
normally prevent or reduce Sog endocytosis because integrins are necessary for
delivery of Sog to provein cells. Integrins have been shown to play a direct
role in endocytosis of viral particles and in mediating membrane traffic
through the endocytic cycle (de Curtis,
2001
; Triantafilou et al.,
2001
). Indirect mechanisms for integrin-mediated endocytosis may
also exist that would not involve endocytosis of the integrin receptor itself,
but of other components that regulate Sog trafficking. Further analysis will
be necessary to investigate whether Drosophila integrins regulate
delivery of Sog to endocytic vesicles or transport of Sog through the
endocytic pathway to adjacent cells.
Do integrins regulate other pathways required for vein
development?
The modulatory effect of integrins on Sog activity described in this paper
are likely to be mediated by dpp and/or gbb signaling
because existing evidence indicates that Sog is a dedicated modulator of Bmp
signaling. In addition, the phenocritical period for mys and
sog interaction coincides with that for interaction between
sog and dpp (Yu et al.,
1996). On the one hand, we cannot exclude the existence of an
additional role of integrins in regulating vein formation through another
pathway, such as the Egf and Notch pathways, which have been shown to exert
important roles on vein development (de
Celis et al., 1997
; de Celis
and Garcia-Bellido, 1994
;
Garcia-Bellido and de Celis,
1992
; Guichard et al.,
1999
; Huppert et al.,
1997
; Martin-Blanco et al.,
1999
; Sturtevant and Bier,
1995
). On the other hand, the integrin- clonal
phenotypes described in this manuscript are observed only on the dorsal
surface and all known components of the Egfr pathway promote vein development
on both surfaces of the wing
(Diaz-Benjumea and Garcia-Bellido,
1990
; Diaz-Benjumea and Hafen,
1994
; Guichard et al.,
1999
).
We also found that mysnj42 and scb1
suppress the thickened vein phenotype of tkv1 mutants,
which raises the possibility of a direct interaction between integrins and a
Bmp receptor involved in wing vein development. The vein splitting and vein
thickening scb- clonal phenotypes are reminiscent of
tkv mutant phenotypes, which derive from a positive requirement for
Bmp signaling for vein formation inside the vein competent domain and a
negative ligand titrating function that limits the range of Bmp diffusion into
the intervein territory adjacent to the provein domain
(de Celis, 1997). The fact
that scb is expressed in both vein and intervein territories is
consistent with a dual action of scb. Additional experiments will be
necessary to investigate whether scb plays a direct role in
modulating Bmp receptor activity.
Interactions with the extracellular matrix may help shape morphogen
gradients
Diffusion of putative growth factors and the shaping of their activity
gradients have been the focus of intense interest since Allan Turing
formulated the concept of morphogens
(Turing, 1952). Recently,
several groups have described mechanisms to explain how soluble factors can
create morphogen gradients. These include degradative proteolysis and a
retrieval role for endocytosis in creating the early embryonic Sog gradient
(Srinivasan et al., 2002
),
regulated endocytosis of wingless
(Strigini and Cohen, 2000
),
extracellular transport of Wg in membrane bound argosomes
(Greco et al., 2001
), planar
transcytosis [as is required for Dpp movement in the wing imaginal disc
(Entchev et al., 2000
;
Teleman and Cohen, 2000
)], and
the formation of thin cell extensions (cytonemes) that deliver Dpp over
several rows of cells (Ramirez-Weber and
Kornberg, 1999
). Protein-protein interactions in the extracellular
milieu, such as those described here, may also be capable of modulating the
magnitude and spatial pattern of Bmp activity, working independently or in
conjunction with other mechanisms.
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ACKNOWLEDGMENTS |
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REFERENCES |
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