Download Integrins and cell differentiation

2607
Development 127, 2607-2615 (2000)
Printed in Great Britain © The Company of Biologists Limited 2000
DEV7803
Integrins modulate the Egfr signaling pathway to regulate tendon cell
differentiation in the Drosophila embryo
Maria D. Martin-Bermudo
Department of Anatomy, Cambridge University, Downing Street, Cambridge CB2 3DY, UK
e-mail: [email protected]
Accepted 11 April; published on WWW 23 May 2000
SUMMARY
Changes in the extracellular matrix (ECM) govern the
differentiation of many cell types during embryogenesis.
Integrins are cell matrix receptors that play a major role
in cell-ECM adhesion and in transmitting signals from the
ECM inside the cell to regulate gene expression. In this
paper, it is shown that the PS integrins are required at the
muscle attachment sites of the Drosophila embryo to
regulate tendon cell differentiation. The analysis of the
requirements of the individual α subunits, αPS1 and αPS2,
demonstrates that both PS1 and PS2 integrins are involved
in this process. In the absence of PS integrin function, the
expression of tendon cell-specific genes such as stripe and
β1 tubulin is not maintained. In addition, embryos lacking
the PS integrins also exhibit reduced levels of activated
INTRODUCTION
How do cells in different tissues interact? Cells can interact
with each other either directly or via the extracellular matrix
(ECM). The ECM is composed of a variety of proteins and
polysaccharides in a fibrous meshwork, closely associated with
the cell surface. The matrix can become calcified to make
bones or teeth, or can form basement membranes and tendons.
In addition to its structural role, the ECM also acts to regulate
the differentiation of many cell types (reviewed by Adams and
Watt, 1993; Lin and Bissell, 1993).
Most of the interactions between cells and the ECM are
mediated by integrins. Integrins are transmembrane receptors
composed of an α and a β subunit. They extend across the
membrane, recognizing and binding to ECM proteins outside
the cell and to cytoskeletal elements inside. Integrins therefore
act as bridges, connecting a cell to its ECM substratum or to
another cell. There are two possible outcomes of these ECMcell interactions mediated by integrins. On the one hand they
can result merely in adhesion of the cell to the ECM.
Alternatively, or in addition, they can trigger a signaling
cascade inside the cell transmitting the effect of ligand binding
across the membrane (reviwed by Giancotti and Ruoslahti,
1999). There are at least two ways by which integrins can
contribute to ECM signaling. First, they can act passively by
attaching the cell to the ECM, making it possible for other
MAPK. This reduction is probably due to a downregulation
of the Epidermal Growth Factor receptor (Egfr) pathway,
since an activated form of the Egfr can rescue the
phenotype of embryos mutant for the PS integrins.
Furthermore, the levels of the Egfr ligand Vein at the
muscle attachment sites are reduced in PS mutant embryos.
Altogether, these results lead to a model in which integrinmediated adhesion plays a role in regulating tendon cell
differentiation by modulating the activity of the Egfr
pathway at the level of its ligand Vein.
Key words: Tendon cell, Integrin, Differentiation, Egfr, Cell
signaling
signals from the ECM to be transmitted into the cell. Second,
they could themselves act as signaling receptors. Since
integrins do not have any enzymatic activity they are thought
to transmit signals through interactions of their cytoplasmic
tails with proteins in the cytoplasm. Integrin binding to the
ECM may activate two signaling pathways, mitogen-activated
protein kinase (MAPK) and Jun amino (N)-terminal kinase
(JNK) (Chen et al., 1994; Schlaepfer et al., 1994; Miyamoto et
al., 1995; Zhu and Assoian, 1995). MAPK and related kinases
then translocate to the nucleus to regulate gene expression
(Yurochko et al., 1992; Wary et al., 1996). This integrindependent regulation of gene expression may provide a way
for the ECM to act upon cell diferentiation.
The ECM can also regulate cell differentiation by triggering
rapid and localized changes in the activity of growth factors
and their receptors, the receptor tyrosine kinases (RTKs)
(Adams and Watt, 1993; Taipale and Keskioja, 1997). The
ECM exerts its control on growth factor activity in different
ways. (1) Oligomerisation – several molecules of growth
factors can bind to a single ECM component, such as heparan
sulfate, and, as oligomers, induce transphosphorylation of their
receptors (Schelessinger et al., 1993). (2) Proteolysis – heparan
sulfate can affect growth factors by protecting it from
proteolysis. (3) Endocytosis – binding of growth factors to the
ECM could prevent endocytosis and degradation of the ligandreceptor complexes (Flaumenhaft and Rifkin, 1992).
2608 M. D. Martin-Bermudo
There is increasing evidence suggesting that the signaling
pathways mediated by integrin-dependent adhesion and growth
factors, do not act in isolation, but interact to regulate cell
differentiation. This synergy between the two pathways can be
explained by a direct mechanism whereby integrins bind and
activate growth factor receptors (Sundberg and Rubin, 1996;
Schneller et al., 1997; Moro et al., 1998; Munger et al., 1999).
In addition, integrin activity can be modulated by growth factors
at the level of regulation of integrin transcription (for review see
Sastry and Horwitz, 1996). In an indirect mechanism, on the
other hand, integrins and growth factor pathways could cooperate by activating common downstream molecules, such as
MAPK and integrin-linked kinase (ILK) (Chen et al., 1994;
Schlaepfer et al., 1994; Zhu and Assoian, 1995; Dedhar et al.,
1999). In another indirect mechanism, the adhesion sites formed
upon integrin clustering can serve as recruitment points that
bring together structural and signaling proteins thus enhancing
their ability to interact with the right partner, and therefore to
be activated (Miyamoto et al., 1996). And finally, integrins can
induce changes in the ECM that, as mentioned before, can lead
to changes in the activity of the growth factors (Brakebush et
al., 1997; Taipale and Keskioja, 1997).
The muscle attachment sites of the Drosophila embryo are
an ideal system for the analysis of the interactions between
integrins and growth factors. Half way through embryogenesis
the somatic muscles of the embryo form and attach to the basal
surface of the tendon cells, specialized epidermal cells, via the
tendon matrix (Tepass and Hartenstein, 1994; Prokop et al.,
1998). The differentiation of epidermal cells into tendon cells
takes place in two steps. The initial one is independent of
muscle insertion and it is induced by the activity of stripe,
which encodes for an early growth response (EGR)-like
transcription factor expressed in the putative tendon cells (Lee
et al., 1995). The next step depends on muscle insertion and
maintains the expression of stripe, and two other tendon cellspecific markers, groovin and β1 tubulin (Buttgereit, 1996;
Yarnitzky et al., 1997). This step requires the activation of the
epidermal growth factor receptor, Egfr, by one of its ligands,
Vein. Vein, produced only by the muscles, is secreted and
localized at the muscle-tendon junction sites. There, it activates
the Egfr on the tendon cells, leading to the transcription of
tendon cell-specific genes (Yarnitzky et al., 1997).
At these same muscle attachment sites, Position Specific
(PS) integrins are deployed; PS1 (αPS1βPS) is expressed at the
basal surface of the tendon cells, and PS2 (αPS2βPS) localizes
at the ends of the muscles where they attach to those cells
(Bogaert et al., 1987; Leptin et al., 1989). These PS integrins
are required for the maintenance of both muscle attachments
and β1 tubulin gene expression, since mutations in the gene
encoding the βPS subunit, myospheroid (mys), result in the
detachment of muscles from their tendon cells and in the decay
of β1 tubulin expression in the tendon cells (Wright, 1960;
Leptin et al., 1989; Buttgereit, 1996). Thus, like the Egfr,
integrins seem to be required for the second step of tendon cell
differentiation.
The results presented here show that the PS integrins control
the late differentiation of tendon cells. The integrin and Egfr
pathways are found to act interdependently to regulate tendon
cell gene expression. Thus, the tendon cells provide an ‘in vivo’
system to study interactions between growth factors and
adhesion pathways. The various mechanisms that are thought
to operate in cell cultures in vitro are here tested ‘in vivo’ and
demonstrate that integrin-mediated adhesion modulates the
Egfr pathway. Furthermore, evidence is presented for a model
in which integrins modulate the Egfr pathway by regulating
presentation and/or accumulation of its ligand Vein.
MATERIALS AND METHODS
Drosophila strains
The integrin mutant alleles used in this study are the null allele ifB4
(Brown, 1994), the null allele mewm6 (Brower et al., 1995) and the
null allele mysXG43 (Bunch et al., 1992). The GAL4 enhancer trap
lines used are twist-GAL4 and 24B, both expressed in the mesoderm
(Brand and Perrimon, 1993; Greig and Akam, 1993), and 69B,
expressed in the ectoderm from stage 12 onwards (Brand and
Perrimon, 1993). The following UAS construct used in this study
UAS-EgfrDN, UAS-λEgfr, UAS-vein, UAS-spitz, UAS-αPS12.1, UASαPS3.1 and UAS-torsoD/βcyt, have been described previously
(Schweitzer et al., 1995; Martin-Bermudo and Brown, 1996; O’Keefe
et al., 1997; Queenan et al., 1997; Yarnitzky et al., 1997; MartinBermudo and Brown, 1999).
Generation of germline clones
In order to examine the role of PS integrins during the differentiation
of muscle attachment cells embryos lacking both maternal and zygotic
mys function were generated by making germline clones for mysXG43.
This was achieved using the FLP-recombinase system and the
dominant female sterile mutation ovoD1 (Chou and Perrimon, 1992).
Virgin females of the genotype y w mysXG43 FRT101 were crossed with
males that were w ovoD1 FRT101; FLPF38. Larvae of this cross were
heat shocked twice at 37°C for 2 hours. The larvae were allowed to
recover for 2 hours at room temperature between the heat shocks. The
female progeny, y w mysXG43 FRT101/w ovoD1 FRT101 were then
crossed to males carrying an enhancer trap insertion in the stripe locus
which expresses nuclear β-galactosidase in a pattern identical to the
late expression the stripe gene (a gift from Bob Holmgren,
Northwestern University, Evanston, IL). Embryos were then either
stained with anti-β-galactosidase to follow stripe expression, or they
were hybridized with a probe against the β1 tubulin gene.
To create embryos that lack the βPS subunit and express the λEgfr,
TorsoD/βcyt, or Vein, in the epidermis, I first eliminated both maternal
and zygotic βPS function using the same strategy. In this case,
females of the genotype y w mysXG43 FRT101; UAS-λEgfr (or UASTorsoD/βcyt, or UAS-vein) were used to generate the germline clones.
The female progeny y w mysXG43 FRT101/w ovoD1 FRT101; UAS-λEgfr
(or UAS-TorsoD/βcyt)/FLPF38 were then crossed to 69B to drive
expression of activated Egfr (or TorsoD/βcyt, or Vein) in the ectoderm.
To express Vein in the mesoderm of embryos that completely lack
βPS function the same procedure was followed, but this time using
females of the genotype y w mysXG43 FRT101; UAS-vein and the
progeny crossed to twist-GAL4+24B.
Antibody staining and in situ hibridization
Whole-mount staining of embryos was performed using standard
procedures. The primary antibodies used were: rabbit anti-βgalactosidase (Cappel Laboratories, Malvern, PA), rat anti-Vein
(Yarnitzky et al., 1997), monoclonal anti-MAP Kinase activated
(Sigma), monoclonal anti-myc tag 9E10 (Oncogene Research
Products). A biotin-labelled secondary antibody, followed by the
Vectastain Elite ABC Kit (Vectorlabs) enhancement to stain the
embryos, or streptavidin-rhodamine for immunofluorescence was used.
Stained embryos were photographed either with a Spot digital camera
on a Zeiss Axiophot microscope, or directly from the MRC1024
Confocal microscope. The digital images were assembled with Adobe
Photoshop 5.0, and labelled in Freehand 8.0 on a Macintosh G3.
Integrins and cell differentiation 2609
Fig. 1. PS integrins regulate
tendon cell-specific gene
expression. A-F, Lateral views
of stage 16 embryos, in this and
all subsequent figures anterior is
to the left and dorsal to the top.
(A) Wild-type embryos have
been stained with an antibody
against the βPS subunit that
shows accumulation of the PS integrins at the tendon cells and at the end of the muscles. Arrowheads point to the ends of the ventral muscles
where they attach to tendon cells located at the segment border, and arrows point to the attachment of the lateral muscles. Tendon cells are
visualized by in situ hybridization with a β1 tubulin antisense probe (in blue, B-D), or using an enhancer trap insertion in the stripe gene and
staining the embryos with an anti-β-gal antibody (in brown E-G). In the absence of the βPS subunit the levels of β1 tubulin (C) or stripe (F) are
significantly reduced as compared to wild type embryos of a similar stage (B and E respectively). This reduction is comparable to that observed
in embryos expressing a dominant negative form of the Egfr, UAS-DN-Egfr, under the control of the epidermal GAL4 line 69B (compare D
with C, and G with F). Asteriks mark chordotonal organs.
Fig. 2. Both PS1 and PS2 integrins are required to regulate tendon
cell differentiation. Tendon cells of embryos lacking the αPS1
subunit (B) show no changes in the levels of β1 tubulin gene
expression when compared to wild-type embryos (A). On the
contrary, absence of the αPS2 subunit leads to reduced expression of
β1 tubulin, but this reduction is less strong than that observed in βPS
mutant embryos (compare C with Fig. 1C). When both αPS1 and
αPS2 subunits (D) are removed, we observe a stronger reduction in
β1 tubulin levels, similar to the reduction observed in embryos that
lack the βPS subunit. Arrowheads and arrows point to the tendon
cells for ventral and lateral muscles respectively.
RESULTS
Fig. 3. The Egfr pathway does not regulate integrin expression in the
tendon cells. The epidermal GAL4 line 69B has been used to express
either an activated form of the Egfr, UAS-λEgfr (A,B), or a dominant
negative form, UAS-DN-Egfr (C,D), and expression of integrins has
been monitored using an anti-βPS antibody (B,D). Ectopic
expression of λEgfr cannot induce ectopic βPS expression (B),
despite it being able to ectopically activate β1 tubulin expression (A).
Note that there are more cells expressing β1 tubulin (arrowheads)
between the segment borders (arrows). Similarly, expression of the
dominant negative form of the Egfr does not change integrin
expression in the tendon cells (D), despite its ability to cause a
reduction in the levels of β1 tubulin expression (C).
PS integrins are required for tendon cell
differentiation
Once they are formed, the somatic muscles of the Drosophila
embryo attach, via the PS integrins, to specific tendon cells in
the epidermis, located at each the end of the developed muscle.
The PS integrins are expressed in both muscles and tendon
cells, αPS1βPS being expressed on the basal surfaces of the
tendon cells and αPS2βPS at the ends of the muscles where
2610 M. D. Martin-Bermudo
they attach to the tendon cells (Fig. 1A). Terminal
differentiation of the tendon cells depends on the secretion of
Vein by the muscle cells and the subsequent activation of the
Egfr pathway in the tendon cells (Yarnitzky et al., 1997). Egfr
function in the tendon cells is required for proper expression
of at least two genes, the β1 tubulin gene and the stripe gene.
Embryos mutant for Egfr, or embryos in which a dominant
negative form of Egfr has been ectopically expressed in the
epidermis, show a reduction in the levels of β1 tubulin and
stripe expression in the tendon cells (Yarnitzky et al., 1997;
Fig. 1D,G). Interestingly, it has been shown that embryos
mutant for the βPS subunit also show a strong reduction in the
levels of β1 tubulin expression (Buttgereit, 1996). To further
investigate whether integrin function is required for tendon cell
differentiation, and does not just down-regulate β1 tubulin gene
expression, the pattern of Stripe was also analysed in the
absence of integrins. To completely eliminate PS integrin
function, germline clones of a βPS null mutation were
produced to create embryos lacking both maternal and zygotic
βPS contributions (βPS−, see Materials and Methods). In the
absence of PS integrins a reduction in the levels of both β1
tubulin and stripe expression in the tendon cells was observed
(Fig. 1C,F), which is comparable to that found in embryos
lacking Egfr function (Fig. 1D,G). The expression of β1
tubulin in the chordotonal organs was used as an internal
control (asterisk in Fig. 1B-D). The conclusion from these
results is that integrins contribute, much like the Egfr pathway,
to regulate tendon cell differentiation.
Two different αPS integrins are present at the muscle-tendon
cell sites, αPS1 is expressed in the epidermis, and αPS2 is
expressed in the muscles. How do they each act to control
differentiation of the tendon cells?
Integrin function to mediate tendon cell
differentiation is required in both the muscles and
the epidermis
By examining the phenotype of embryos mutant in the single
α subunits we can investigate the action of the PS integrins in
the two tissues. Embryos lacking the PS1 integrin show levels
of β1 tubulin expression indistinguishable from wild type,
suggesting that PS1 integrin function in the epidermis is not
required for tendon cell differentiation (Fig. 2B). In contrast,
loss of PS2 integrin causes a decrease in the levels of β1 tubulin
(Fig. 2C). However, this decrease is not as strong as the one
observed in embryos that lack both maternal and zygotic
contribution of the βPS subunit (compare Fig. 2C with 1C).
There are at least two alternative explanations for this
difference: (1) there is another α subunit required for tendon
cell differentiation, or (2) αPS1 has a function that is rescued
by the presence of αPS2. To distinguish between these two
possibilities, β1 tubulin gene expression was examined in
embryos mutant for both αPS1 and αPS2. Tendon cells from
embryos that lack both α subunits show a reduction in the
levels of β1 tubulin comparable to those observed in βPS−
embryos (compare Fig. 2D with 1C). These results indicate that
another alpha is not functioning, and that removing αPS2 from
the muscles has a non-autonomous effect on the tendon cells.
Therefore, both the integrin and the Egfr pathways are
required for proper tendon cell differentiation. The next step
was to test whether the different signaling pathways induced
by integrins and by Egfr are connected or act independently.
The PS integrins regulate MAPK activation and
interact with the Egfr pathway to induce tendon cell
differentiation
There are a number of potential mechanisms for synergy
between integrins and the Egfr pathway to control gene
expression. One possibility is that growth factors act first to
regulate PS integrin expression in the tendon cells. Indeed such
regulation by growth factors has been observed for both
integrins and cadherins in cell cultures (Sastry and Horwitz,
1996). To test for this possibility, we have analysed the
expression of βPS in embryos that ectopically express (1) an
activated form of the Egfr or (2) a dominant negative form of
the Egfr, in the tendon cells and other epidermal cells. If the
Egfr pathway positively regulates integrin expression, one
would expect, in the first case, ectopic βPS expression in the
epidermal cells, and, in the second case, down regulation of
βPS expression in the tendon cells. Activation of the Egfr
pathway in the ectoderm, using UAS-λEgfr; GAL4-69B (see
Materials and Methods), does not lead to ectopic βPS
expression (Fig. 3B). As a control for activation of the Egfr
pathway we observe ectopic β1 tubulin expression in the
epidermal cells (Fig. 3A). Reciprocally, expression of a
dominant negative form of the Egfr suppresses β1 tubulin
expression at the muscle attachment sites (Fig. 3C) but does
not affect integrin expression (Fig. 3D). These results suggest
that control of tendon cell differentiation by the Egfr pathway
is not mediated through regulation of PS integrin expression.
A second possibility for the synergy between integrin and
the Egfr is that integrin function is required for the proper
activation of the Egfr pathway in the tendon cells. As in many
other organisms, activation of the Egfr pathway in Drosophila
leads to the activation of a kinase cascade that sequentially
involves Raf, MEK and MAPK (Perrimon, 1994). If integrins
are required for activation of the Egfr pathway, one might
expect the absence of integrins to result in lack of MAPK
activation. We have tested this possibility by making use of an
antibody that recognizes an activated form of MAPK. In wildtype embryos, activated MAPK is expressed at high levels at
the muscle attachment sites (Fig. 4A, arrow). In contrast,
embryos that lack the βPS subunit activated MAPK show
reduced expression in these cells (Fig. 4B, arrow),
demonstrating that integrins are required to activate MAPK.
Such requirement can be a direct consequence of integrinmediated adhesion to the ECM as shown previously (Howe et
al., 1998). In this case, integrin could function in parallel to the
Egfr pathway to achieve certain levels of MAPK activation
required for tendon cell differentiation. Alternatively, integrin
requirements for MAPK activation could reflect a regulation of
the Egfr pathway by the integrins. To distinguish between these
two possibilities we have asked whether an activated form of
the Egfr could rescue the reduced expression of β1 tubulin
present in embryos that lack the βPS subunit. As shown in Fig.
4D, activation of the Egfr pathway in the epidermis can rescue
β1 tubulin gene expression in βPS− mutant embryos (Fig. 4C).
Taken together, these results suggest that integrins regulate
tendon cell differentiation by modulating the activity of the
Egfr signaling pathway. However, activation of the Egfr could
lead to artificial activation of the MAPK, making it difficult to
completely rule out the possibility that integrins modulate the
Egfr pathway downstream of the Egfr, i.e. at the level of the
MAP Kinase.
Integrins and cell differentiation 2611
Integrins are required for the accumulation of Vein
at the muscle-tendon cell junctions
The PS integrins could modulate Egfr function at two levels:
(1) at the level of the receptor, with integrins perhaps
facilitating Egfr recruitment and activation; and/or (2) at the
level of the ligand, whereby integrins could be required for
proper assembly and/or stabilization of the ECM at the muscle
attachment sites, which in turn might contribute to the
localization and/or activation of Vein. In fact, ultrastructural
analysis has shown that in the absence of PS integrin function
the tendon matrix accumulates at the muscle attachment sites,
but detaches from both muscles and tendon cells (Prokop et al.,
1998). Furthermore, the basement membrane, a specialized
matrix in close apposition to cell surfaces, also detaches or fails
to assemble in the tendon cells of embryos lacking PS integrin
function. To further analyse how ECM assembly is affected in
the absence of PS integrins, embryos that lack both maternal
and zygotic βPS product were stained with an antibody against
the ECM protein Tiggrin (Fogerty et al., 1994). Since muscles
of βPS− embryos detach, muscle attachment sites were
localized using an enhancer trap line inserted in the stripe gene
(Fig. 5A,D). Although we can detect Tiggrin localized at the
muscle attachment in βPS− embryos, it is less concentrated and
not properly assembled compared to wild type (compare Fig.
5B with E). These results further demonstrate a role for the PS
integrins in either the stabilization of the tendon matrix or,
proper accumulation of tendon matrix components. To test
whether this function of the PS integrins has an effect on Vein
localization and/or accumulation, the pattern of Vein was
analyzed in βPS− mutant embryos. In wild type embryos Vein
localizes to the sites of contact between the muscles and the
epidermal attachment cells (Fig. 5C, arrow), as well as in some
cells along the central nervous system (Fig. 5C, arrowhead)
(Yarnitzky et al., 1997). Embryos that completely lack the βPS
subunit do not accumulate Vein at the muscle attachment sites
(Fig. 5F, arrow), although it is still present in the nervous
system (Fig. 5F, arrowhead). Integrins could be required for the
production of Vein or for its accumulation at the muscle-tendon
cell junctions. To distinguish between these two possibilities,
Vein was ectopically produced in the muscles or in the
epidermis of βPS− embryos to see if it rescued the decrease of
β1 tubulin in these mutant embryos. Expression of Vein in the
tendon cells of integrin mutant embryos, which creates an
autocrine system by which the tendon cell produces its Egfr
ligand locally, can rescue the phenotype of βPS− embryos,
while expression in the mesoderm cannot (data not shown).
Altogether, these results demonstrate that the PS integrins are
required for proper localization and/or accumulation of Vein at
the muscle-tendon cell junctions.
Integrin signaling versus adhesion
PS integrins have been traditionally presented as mediators of
adhesion between different cell layers (for review see Brown,
1993). However, there is increasing evidence demonstrating
that integrins can also act as signaling receptors. Therefore,
there are two different ways that integrins could regulate the
Egfr pathway. The first is direct signaling in which integrins
themselves initiate a signaling event that regulates the Egfr
pathway. In the second, indirect signaling, integrin-dependent
cell adhesion modulates the signaling pathway initiated by the
Egfr. To test whether it is integrin adhesion or signaling that is
required for normal tendon cell differentiation, we made used
of a chimeric integrin, TorsoD/βcyt, which has been shown to
lack the integrin-adhesive function, but can signal to regulate
gene expression, at least in the midgut (Martin-Bermudo and
Brown, 1999). We expressed the chimeric protein in the
muscles of embryos lacking the βPS subunit. using a
combination of two GAL4 lines, twist and 24B (Fig. 6A, see
Materials and Methods). The pattern of expression of the β1
tubulin gene in these mutant embryos is indistinguishable from
that found in βPS− embryos (compare Fig. 6B with 1C).
Expression of the chimera in the epidermis of βPS− mutant
embryos using the 69B GAL4 line (Fig. 6C) was also unable
to rescue tendon cell-specific gene expression (Fig. 6D). These
results show that the signaling pathway initiated by the
clustering of the βPS cytoplasmic domain is not sufficient to
regulate tendon cell differentiation.
PS integrins are not sufficient to induce tendon cell
fate
We have shown that integrins act in cooperation with the Egfr
pathway for late muscle attachment cell differentiation.
However, the Egfr pathway can also induce de novo expression
of tendon cell markers when ectopically expressed in the
epidermis (Yarnitzky et al., 1997). To investigate whether
integrin function is also able to induce tendon cell fate, we have
expressed the PS1 integrin throughout the epidermis, using
69B-GAL4 driver in conjunction with UAS-αPS1; UAS-βPS.
We monitored the expression of the β1 tubulin gene as an
indication of tendon cell fate. Since the transport of integrins
to the surface requires both subunits, we confirmed the
successful expression of the heterodimer by examining the
localization of the βPS subunit (Fig. 7A). When we express the
PS1 heterodimer throughout the epidermis, we found that it is
unable to ectopically activate β1 tubulin gene expression (Fig.
7B). One explanation for the failure of ectopic PS1 integrin to
activate β1 tubulin expression is that the ectopic integrin is not
active, since ligand binding precedes integrin activation and the
PS1 ligand might not be present at the ectopic places. To
overcome this problem we made use of two different ligandindependent activated forms of integrins. We have used the
chimera TorsoD/βcyt, which, as mentioned above, behaves as
an activated integrin that can signal independently of adhesion
(Martin-Bermudo and Brown, 1999). In addition, we have used
an integrin that carries a deletion of the cytoplasmic domain of
the α subunit, which has been shown to behave as
constitutively active (O’Toole et al., 1994; Martin-Bermudo et
al., 1998). Expression of either UAS-TorsoD/βcyt (data not
shown) or UAS-αPS1∆cyt; UAS-βPS (Fig. 7C) in the
epidermis, using the 69B driver, is still unable to activate
ectopic tendon cell genes such as β1 tubulin (Fig. 7D).
Altogether, these results suggest that integrins are not sufficient
for the specification of tendon cells, but that integrins are
required for their differentiation.
DISCUSSION
This paper focuses on the differentiation of tendon cells as a
model system to study how growth factors and integrins
interact to produce a specific cellular response. It first shows
that integrins are required in both muscles and tendon cells to
2612 M. D. Martin-Bermudo
Fig. 4. Integrins modulate the Egfr
pathway by regulating MAPK
activation. Horizontal views of stage 16
embryos are shown in A and B, and
dorsolateral views in C and D. Wild
type (A) and βPS– mutant embryos (B)
are stained with an antibody that
recognizes an activated form of MAPK.
While activated MAPK is expressed in
the tendon cells of wild type embryos
(arrow in A), this expression is reduced
in βPS– mutant embryos (arrow in B).
To determine whether reduction in
activated MAPK is a consequence of
downregulation of the Egfr pathway,
we expressed the activated form of Egfr
in embryos lacking the βPS subunit. The decrease in the levels of β1 tubulin expression that occurs in βPS– mutant embryos (C) is suppressed
when these embryos carry the 69B-GAL4 and UAS-λEgfr (D).
Fig. 5. Integrins control proper
accumulation of Vein at the
muscle attachment sites via
regulation of matrix assembly.
The tendon cells are visualized
using a stripe enhancer trap and
stained with anti-βgalactosidase antibody (A,D,
red). We examined assembly of
the matrix at the tendon cells
using an antibody against the
PS2 integrin ligand Tiggrin, a
component of the tendon matrix
(B and E, green). In wild-type
embryos Tiggrin is localized at
the ends of the muscles (B)
where they contact the tendon
cells (A). In embryos that lack
the βPS subunit there is a
reduction in the levels of stripe
in the tendon cells (D), and the tendon matrix appears disorganized (E). In wild-type embryos Vein is found at the muscle attachment sites
(arrow in C) as well as in some cells of the CNS (arrowhead in C). However, in the absence of the βPS subunit, Vein expression at the muscle
attachment sites is significantly reduced (arrow in F), while expression in the CNS remains unaltered (arrowhead in F). Lateral views of
embryos are shown in A, B, D and E, and horizontal views in C and F.
mediate tendon cell differentiation. It then investigates possible
mechanisms of interactions between the integrin and the Egfr
pathways. It shows that in the absence of integrins the levels
of activated MAPK are reduced, suggesting that a putative
point of convergence between the two pathways is at the level
of MAPK activation. The ability of an activated form of the
Egfr to rescue the lack of integrin function favours the
hypothesis that integrin function is required for the activation
of the Egfr pathway, over the hypothesis that both pathways
act independently on the common downstream molecule,
MAPK. This work also shows that while ectopic activation of
the Egfr pathway leads to ectopic β1 tubulin gene expression,
ectopic expression of activated integrins does not. This leads
to the proposal that activation of the Egfr pathway in the tendon
cells has two functions. The first one is integrin-independent
and is required for proper specification of tendon cells. The
second one is integrin-dependent and maintains tendon cellspecific gene expression.
Cell culture experiments have shown that integrins can
regulate activation of the Egfr pathway at the level of the
ligand, or at the level of the receptor. In the first case, integrins
could regulate ligand activity through modulation of the
composition and assembly of the ECM. There is increasing
evidence suggesting that the binding of growth factors to the
extracellular matrix is a major mechanism regulating growth
factor activity (Taipale and Keskioja, 1997). The largest group
of ECM proteins that interact with growth factors include the
heparan sulfates, which are the major components of the
Integrins and cell differentiation 2613
Fig. 6. The integrin pathway initiated by dimerization of the
cytoplasmic domain of the βPS subunit is not sufficient to regulate
tendon cell differentiation. We have used a combination of two
mesodermal GAL4 lines, tw+24B, and the epidermal GAL4 line 69B
to express the chimeric integrin TorsoD/βcyt respectively in the
muscles (A,B) or the epidermis (C,D), of embryos lacking the βPS
subunit. Expression of the chimera in either the mesoderm (A) or the
epidermis (C) fails to rescue the reduction in the levels of the β1
tubulin gene that occurs in the absence of the βPS subunit (B,D).
A-D, ventrolateral close up views of stage 16 embryos.
Fig. 7. Activation of the integrin pathway is not sufficient to induce
tendon cell fate. The GAL4 line 69B has been used to express either
a wild type, UAS-αPS1; UAS-βPS (A,B), or a ligand-independent
activated form, UAS-αPS1∆cyt; UAS-βPS (C,D) of the PS1 integrin
throughout the ectoderm. Embryos were stained with an anti-βPS
antibody to show that both wild-type (A) and activated (C) forms of
the PS1 integrin are expressed in the embryonic ectoderm. Ectopic
expression of any of the forms does not induce β1 tubulin gene
expression, as judged by hybridization with a β1 tubulin probe (B,D).
basement membrane (Hardingham and Fosang, 1992) – indeed
integrins contribute to the stabilization of the epidermal
basement membrane (DiPersio et al., 1997). Integrins can also
exert control on the Egfr pathway at the level of the receptor.
In this scenario, the adhesion sites formed upon integrin
activation, focal adhesions, can serve as recruitment points that
bring together structural and signaling proteins thus enhancing
their ability to interact with the right partner, and therefore to
be activated. Indeed, Miyamoto et al. (1996) have shown that
clustering of integrins results in co-clustering of epidermal
growth factor receptor molecules leading to receptor activation,
and enhanced EGF-dependent activation of MAPK (Miyamoto
et al., 1996). In another example, integrins can also enhance
the efficiency of signal transduction between the Egfr and
MAPK by promoting the recruitment and activation of Raf (Lin
et al., 1997; Renshaw et al., 1997).
The data presented here supports a model by which the PS
integrins regulate Egfr signaling pathway at the level of its
ligand Vein. This regulation involves the ability of the PS
integrins to organize the tendon matrix and the basement
membrane at the basal surface of muscles and tendon cells. In
fact, integrin function in regulating assembly of the ECM,
rather than integrin signaling, has been shown to be crucial in
keratinocyte differentiation (Bagutti et al., 1996). We have
shown here that in the absence of integrin function the levels
of Vein at the muscle attachment sites are decreased compared
to wild type. Therefore, we propose that integrins are required
for the proper assembly of the basement membrane and the
tendon matrix, which in turn regulates Vein activity. A role for
PS2 in matrix assembly is in agreement with results showing
a requirement for α3β1 integrin in mediating assembly of
basement membrane between the epidermis and the dermis in
mice (DiPersio et al., 1997). Furthermore, our results showing
that defects in tendon cell-specific gene expression are stronger
when we eliminate both integrins are consistent with data
showing that the failure in assembly of the matrix is more
severe in embryos lacking both PS1 and PS2 integrins than in
single mutants (Prokop et al., 1998). The basement membrane
and the tendon matrix could then regulate Vein activity in
different ways. (1) They could promote a higher affinity of Vein
for the Egfr. In fact, heparan sulfate has been reported to
promote high-affinity binding of the fibroblast growth factor 2
(FGF2) and hepatocyte growth factor (HGF) to their receptors
(Lyon et al., 1994; Faham et al., 1996). (2) They could also
direct the movement of Vein by limiting its diffusion. This
could be a mechanism for muscles to specifically transmit
signals to those epidermal cells that are in contact with the
same matrix, the tendon cells. (3) They could promote the
accumulation or clustering of Vein to specific levels required
for the activation of its receptor. And finally, (4) binding of
integrins to the ECM might either protect Vein from proteolysis
or lead to the production of proteolytic enzymes that release
Vein from the tendon matrix and activate it. Several of these
mechanisms could be operating at the same time. Thus,
organization and assembly of the tendon matrix via the PS
integrins would ensure the localized production and
concentration of an active ligand for the Egfr at the muscle
attachment sites.
In addition, or alternatively, the PS integrins could be
required in the tendon cells to regulate Egfr function at the level
of the receptor. At the muscle attachment sites of the Drosophila
embryo, there are special cell junctions, called hemiadheren
junctions (HAJs), which form between the ends of the muscles
and the basal surface of the tendon cells in opposing pairs
(Tepass and Hartenstein, 1994; Prokop et al., 1998). HAJs are
2614 M. D. Martin-Bermudo
organized sites of membrane-cytoskeletal linkage which have
been proposed to recruit integrins (Prokop et al., 1998). It is
worth mentioning here that although PS2 has been shown to be
expressed only in the muscles, loss of PS2 integrin function
affects adhesion of both muscle and epidermal HAJs (Prokop
et al., 1998). This can explain why lack of PS2 alone leads to
a reduction in the expression of tendon cell-specific genes. At
this level for integrin modulation of the Egfr signaling, a first
step requires that epidermal HAJs act as recruitment centres for
the Egfr or other signalling molecules, in the same way as focal
adhesions. In this case, the detachment of the epidermal HAJs
from the matrix found in embryos lacking the integrins results
in the disorganization of these adhesion centres leading to a
failure to cluster the Egfr and/ or signalling molecules, and
therefore, to activate the Egfr pathway. In this scenario it is also
possible that integrins and the Egfr activate parallel pathways
needed to reach the threshold level of MAPK activation,
required for optimal transcription of tendon cell-specific genes.
This will be consistent with the results presented here where
over activation of the Egfr pathway can compensate for lack
integrin function. Thus, integrin-mediated cell adhesion might
produce a long-lasting activation of MAPK which cooperates
with the fast and short stimulation of MAPK normally induced
by activation of growth factors pathways.
Finally, experiments were performed to try to determine the
relative roles of integrin adhesion versus signaling in
modulating the Egfr pathway in the process of tendon cell
differentiation. One of the best characterized integrin signaling
events involves tyrosine phosphorylation of the focal adhesion
kinase, FAK. This pathway can be mimicked by clustering the
cytoplasmic domain of the βPS subunit (Akiyama et al., 1994;
Lukashev et al., 1994). It has been shown previously that
clustering of the cytoplasmic tail of the βPS subunit is
sufficient to initiate a signaling pathway that regulates gene
expression in the Drosophila midgut (Martin-Bermudo and
Brown, 1999). However, this signaling pathway is found to be
insufficient to regulate tendon cell differentiation in the
embryo. These results suggest that integrin-mediated adhesion,
rather than signaling, is required to regulate tendon cell
differentiation. Some experiments have shown that clustering
of the cytoplasmic domain of the β subunits does not fully
mimic integrin signaling, the α subunits are also important and,
in some cases sufficient. Wary et al (1996) have identified a
pathway from integrins to MAPK which is mediated by
interactions between the transmembrane and/or extracellular
domains of the α subunit and the adaptor protein Shc. The
pathway from integrins to MAPK is α subunit specific, being
α5 and αv, which belongs to the same family as the αPS2, the
α subunits that signal through Shc. Therefore, it still remains
possible that PS2 integrin requirements to regulate tendon cell
differentiation include a signaling function through Shc.
In summary, the results presented here show that PS integrin
function is required in both layers, muscles and epidermis, to
mediate Egfr regulation of tendon cell differentiation. They
support a model in which integrin function is placed outside of
the cell. In this case, integrin-mediated adhesion is required to
assemble basement membrane components which in turn
interact with Vein to influence its ability to activate the receptor.
In addition, in a more speculative model, integrin function can
also be confined to the cell membrane. In this case, PS1 and
PS2 mediate the formation of adhesion complexes which
contribute to the recruitment of the Egf receptor and/or its
downstream effectors, enhancing Vein activity.
I am grateful to D. Buttgereit, M. Freeman, B-Z. Shilo, M. Mlodzik,
T. Schupbach, and T. Volk, for providing reagents and fly stocks. I
thank N. Brown, A. Gonzalez-Reyes, A. Hidalgo and P. Lawrence for
helpful comments on the manuscript. This work was supported by a
Royal Society University Research Fellowship and Wellcome Trust
project grant 054613 to M. D. M. B.
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