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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. 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