myospheroid
The proventriculus is a multiply folded muscular organ of the foregut formed from a simple epithelial tube, whose function is grinding and masticating food. Coordinated cell movements are critical for tissue and organ morphogenesis in animal development. Drosophila genes hedgehog and wingless, which encode signaling molecules, and the gene myospheroid, which encodes a beta subunit of the integrins, are required for epithelial morphogenesis during proventriculus development. In contrast, this morphogenetic process is suppressed by the decapentaplegic gene (Pankratz, 1995).
Apterous plays a role controlling patterns of gene expression in the developing wing disc. The PS1 and PS2 integrins are normally expressed in primarily dorsal-specific and ventral-specific patterns, respectively. Ectopic expression of apterous induces ectopic ventral expression of alphaPS1 mRNA and PS1 integrin while loss of apterous can induce the ectopic dorsal expression of PS2 integrin. Thus, apterous plays a selector-like role both in terms of the control of lineage restrictions and the regulation of downstream gene expression (Blair, 1994).
There is a genetic interaction between blistered/BSRF and integrin genes inflated and myospheroid suggesting that blistered/DSRF might regulate integrin expression. There is an increased frequency and severity of blisters in progeny when mutant blistered males are crossed to females carrying if or mys. For the most part blistered/integrin combinations do not affect venation even when the blisters are very large (Fristrom, 1994).
The Drosophila PS1 and PS2 integrins are required to maintain the connection between the dorsal and ventral wing epithelia. aPS subunits are inappropriately expressed during early pupariation via the Blistermaker chromosome (containing a PS2 gene driven by the wing pouch enhancer trap, 684). Inappropriate expression of aPS2 results in the separation of epithelia, causing a wing blister. Two lines of evidence indicate that this apparent loss-of-function phenotype is not a dominant negative effect, but is due to inappropriate expression of functional integrins: (1) wing blisters are not generated efficiently by misexpression of loss-of-function aPS2 subunits with mutations that inhibit ligand binding, and (2) gain-of-function, hyperactivated mutant aPS2 proteins cause blistering at expression levels well below those required by wild-type proteins. A genetic screen was carried out for dominant suppressors of Blistermaker induced wing blisters. Suppression was induced by null alleles of a gene named moleskin, which encodes the protein DIM-7. DIM-7, a Drosophila homolog of vertebrate importin-7, has been shown to bind the SHP-2 tyrosine phosphatase homolog Corkscrew and to be important in the nuclear translocation of activated D-ERK (Rolled). Consistent with this latter finding, homozygous mutant clones of moleskin fail to grow in the wing. Genetic tests suggest that the moleskin suppression of wing blisters is not directly related to inhibition of D-ERK nuclear import (Baker, 2002).
The ß-importin family of proteins is principally linked with nuclear import of protein cargos. However, recently other functions have been associated with members of the importin superfamily. For example, importin-ß, in some cases with importin-a, functions in vertebrates to sequester microtubule polymerization factors early in mitosis. Mitotic microtubule formation can be triggered by the release of the polymerizaion regulators by RanGTP, just as RanGTP binding to importin-ß leads to release of cargos inside the nucleus. DIM-7 protein can be detected immunologically at the cell cortex, both in early Drosophila embryos and in S2 cells in culture. It thus seems reasonable to consider a more direct connection between the peripheral DIM-7 and integrin regulation. Additionally, it appears that a mutation in corkscrew, the Drosophila SHP-2 homolog, can also suppress Blistermaker and that Corkscrew protein binds directly to DIM-7. Although Corkscrew has been implicated primarily in signaling events downstream of receptor tyrosine kinases, vertebrate SHP-2 has been implicated in signaling via a host of growth factor receptors, cytokines, hormones, and antigens. Most relevant to this study, SHP-2, often in association with the membrane glycoproteins PECAM-1 or SHPS-1, has been shown to be involved in many integrin-dependent signaling events and also to be important in regulating integrin-mediated cell adhesion, spreading, or migration. While SHP-2 is a cytoplasmic tyrosine phosphatase, some experiments suggest that it can serve as a scaffolding protein at or near the plasma membrane. For example, a Corkscrew protein mutated in the phosphatase domain retains significant wild-type activity in situ, and this activity is increased if the protein is targeted to the plasma membrane (Baker, 2002).
It is likely therefore that cell surface receptors mediate a localized Corkscrew/SHP-2 activation of cortical DIM-7. This active DIM-7, in combination with associated factors such as D-ERK, could then function more directly in integrin regulation. A more direct connection between DIM-7 and integrin function is also consistent with the fact that moleskin mutations were especially common among the suppressors isolated in the screen. A key question for future work, therefore, will be defining the subcellular location at which DIM-7 functions with respect to integrin-related phenotypes (Baker, 2002).
Recently, evidence has begun to appear that integrin engagement with the ECM can regulate nuclear import of regulatory molecules. For example, there is an association between aLß2 and the c-Jun coactivator JAB1; this connection is suggested to regulate the nuclear localization of JAB1. More directly relevant to these results, ERK nuclear translocation in fibroblasts is dependent on an integrin-mediated event, also involving the actin cytoskeleton. Also, primary mouse embryo fibroblasts with a ß1 integrin cytoplasmic mutant show reduced nuclear translocation of phosphorylated ERK. Regardless of the importance of nuclear transport in Blistermaker suppression, the genetic data indicate a functional connection between integrins and a specific importin-ß that can transport activated ERK and suggest another potential molecular mechanism whereby integrin and growth factor signals can be integrated by the cell (Baker, 2002).
Tensin is an actin-binding protein that is localized in focal adhesions. At
focal adhesion sites, tensin participates in the protein complex that
establishes transmembrane linkage between the extracellular matrix and
cytoskeletal actin filaments. Even though there have been many studies on
tensin as an adaptor protein, the role of tensin during development has not
yet been clearly elucidated. Thus, this study was designed to dissect the
developmental role of tensin by isolating Drosophila tensin mutants
and characterizing its role in wing development. The Drosophila
tensin loss-of-function mutations results in the formation of blisters in the
wings, that are due to a defective wing unfolding process. Interestingly,
by1 -- the mutant allele of the gene blistery
(by) -- also shows a blistered wing phenotype, but fails to complement
the wing blister phenotype of the Drosophila tensin mutants, and
contains Y62N/T163R point mutations in Drosophila tensin coding
sequences. These results demonstrate that by encodes
Drosophila Tensin protein and that the Drosophila tensin
mutants are alleles of by. Using a genetic approach, it has been demonstrated that Tensin interacts with integrin and also with the components
of the JNK signaling pathway during wing development; overexpression of
by in wing imaginal discs significantly increases JNK activity and
induces apoptotic cell death. Besides the defects in wing cell adhesion process, another distinct mutant phenotype was observed in the by mutants; they lay rounded eggs due to defective oocyte elongation during oogenesis. Collectively, these data suggest that Tensin
relays signals from the extracellular matrix to the cytoskeleton through
interaction with integrin, and through the modulation of the JNK signal
transduction pathway during Drosophila wing development (Lee, 2003).
Drosophila wing development after pupariation (AP) consists of two
distinct stages: prepupal and pupal wing morphogenesis. Pupal wing morphogenesis is further divided into three stages: separation
(11-12 hours AP) of the ventral cell layer from the dorsal layer,
re-apposition of the inter-vein cells (21-36 hours AP) and re-separation (60
hours AP) of the two cell layers. Shortly after eclosion, wings expand and
unfold by an influx of hemolymph. PS integrins
are required for the attachment of the two wing surfaces during pupal wing
re-apposition and for the maintenance of the wing bilayer (Lee, 2003).
To determine the detailed roles of tensin during wing morphogenesis, the pupal wings of the by2 flies were examined. No
differences were observed in the attachment of two wing surfaces and in the integrin localization between wild type and by2 wings during both prepupal apposition (4-6 hours
AP) and pupal reapposition stages (30-36 hours AP) (Lee, 2003).
Because the pupal wing development was not disturbed in the
by2 mutants, the expansion and
unfolding processes of adult wings in the by2 mutants was investigated. After eclosion, the by2 flies display folded wings similar to the controls. Then, a
sudden and rapid influx of hemolymph induces the unfolding of folded wings in
the by2 mutants. However, as soon as
the wings of by2 flies unfold, fluid-filled blisters
began to appear at the distal part of the wings, and the boundary of
the blisters expand to a certain extent. After the fluid
dries, the wing blisters are fixed in place. Taken together, although the dorsal and ventral layers of a wing can be
brought into close association during apposition and re-apposition processes
in the by2 flies, the link between them may not be strong
enough to resist the hydrostatic pressures during the wing unfolding
process (Lee, 2003).
The functional significance of each domain of tensin in normal
wing development was examined. UAS lines overexpressing either
full-length tensin protein or various deletion mutant forms of tensin were generated. Unlike DeltaN and DeltaC, overexpression of DeltaPTB by MS1096-GAL4 driver completely rescues the blistered wing phenotype of by2. These data suggest
that both the N-terminal region and the SH2 domain of tensin are required for
proper attachment of two wing surfaces (Lee, 2003).
Since mammalian tensin is known to participate in the integrin signaling, whether tensin genetically interacts with integrin was examined. As expected,
the blistered wing phenotype become more severe in the
if3; by2/+ mutants
and extremely severe in the double homozygotic mutants for if3 and by2, compared with if3 homozygotes or by2 heterozygotes. In addition, the rate of flies showing blistered wings in the total population greatly increases in the double mutants (Lee, 2003).
In mammalian cells, tensin has been implicated in signal transduction
related to cell adhesion such as Src, JNK and PI3K. To examine
the role of tensin in the signaling processes related to wing development, the in vivo interaction between tensin and signaling molecules
including rl/Erk, Src, JNK and PI3K was investigated. Interestingly, it was found that the JNK signaling pathway is tightly correlated with tensin in the wing development, while other signaling molecules including rl/Erk do not show any interactions with tensin. Homozygous by2 mutants with heterozygotic mutations of the JNK signaling components bsk1 or hep1 (the
loss-of-function mutants for Drosophila JNK and MKK7,
respectively) display a highly severe blistered wing phenotype, compared with
either homozygous by2, heterozygous
bsk1 or heterozygous hep1 mutants. Notably, the rate of flies, which show Class II blistered wings,
increases from 46.5% to 70% for these double mutants compared with homozygous
by2 mutants, and about 15% of these flies had multiple
blisters in their wings. Furthermore, the double homozygotic mutants for
by2 and hep1 die at pharate adult
stage. The lethality of these double mutants may be due to an impairment of essential in vivo interactions between tensin and the JNK signaling pathway in Drosophila (Lee, 2003).
Next, whether overexpressed by also interacts with the components of the JNK signaling pathway was tested. Overexpression of by using MS1096-GAL4 driver turns the adult wings into a convex shape with a smaller overall size, and this phenotype becomes more severe when two copies of the by gene are overexpressed. Simultaneous overexpression of bsk or hep with by results in a severely curled wing phenotype, which is fully penetrant, whereas overexpression of bsk or hep alone by MS1096-GAL4 driver did not induce any detectable phenotypes in the wing. Collectively, these data suggest that tensin activity is highly related to the JNK signaling pathway during wing development in Drosophila (Lee, 2003).
To further confirm the genetic interaction between tensin and the JNK
pathway, the effect of tensin on JNK activity in vivo was tested. The extent of JNK phosphorylation was tested using anti-phosphospecific JNK antibody in
the by overexpression line and the by2 mutants.
As expected, JNK phosphorylation is dramatically increased in the wing
imaginal discs overexpressing by; this
directly demonstrates increased JNK activity by by. On the contrary,
JNK phosphorylation in the imaginal discs of the by2
mutants is reduced compared with the control (Lee, 2003).
Moreover, the reduced size and the convex wing phenotype observed in the
wings overexpressing by can be most easily
explained by apoptosis in the wings. Since the induction of apoptosis by the JNK signaling is well established, it was expected that the wing phenotype induced by by overexpression might be due to apoptosis. To confirm the
by-induced apoptosis in vivo, Acridine Orange staining
of the relevant wing imaginal discs was carried out. As expected, the overexpression of by dramatically increases apoptotic cell death compared with the control (Lee, 2003).
Thus, tensin genetically interacts with the components of the JNK signaling pathway, and regulates JNK activity during wing development. The supporting evidence for the engagement of tensin in the JNK signaling pathway comes from a recent report that transfected mammalian tensin activates JNK signaling in HEK 293T cells (Katz, 2000). Interestingly, in mammalian cells, JNK is also activated via adaptor proteins p130 CAS and Crk which receive a signal from the FAK/Src tyrosine kinase complex in the cell adhesion sites when cells attach to the ECM. Since tensin is a possible substrate for FAK, and p130 CAS is able to interact with the C terminus of tensin, it is highly possible that tensin is involved in this signaling cascade and mediates signals from integrin and FAK to the JNK signaling pathway (Lee, 2003).
During development, morphogenesis involves migration and changes in the shape of epithelial sheets, both of which require coordination of cell adhesion. Thus, while modulation of integrin-mediated adhesion to the ECM regulates epithelial motility, cell-cell adhesion via cadherins controls the remodelling of epithelial sheets. The Drosophila wing epithelium was used to demonstrate that cell-ECM interactions mediated by integrins also regulate the changes in cell shape that underly epithelial morphogenesis. Integrins control the transitions from columnar to cuboidal cell shape underlying wing formation, and eliminating the ECM has the same effect on cell shape as inhibiting integrin function. Furthermore, lack of integrin activity also induces detachment of the basal lamina and failure to assemble the basal matrix. Hence, it is proposed that integrins control epithelial cell shape by mediating adherence of these cells to the ECM. Finally, it was shown that the ECM has an instructive rather than a structural role, because inhibition of Raf reverses the cell shape changes caused by perturbing integrins (Dominguez-Gimenez, 2007).
The generation of form in early animal development involves key cellular process such as epithelial morphogenesis. The reorganisation of cell shape is commonly associated with epithelial morphogenesis, which requires a precise and coordinated remodelling of the cytoskeleton and the adhesive properties of cells. In view of the two predominant cell adhesion systems, there is considerable evidence indicating that interactions between cells and the ECM modulate the shape of cells in culture. This study used the Drosophila wing to show that the regulation of cell shape by integrins also plays an important role during epithelial organ morphogenesis. Furthermore, this integrin function is shown to rely on interactions with a matrix whose assembly also depends on integrin activity. Finally, evidence is provided that the Raf kinase may act as a putative intracellular regulator of this integrin activity (Dominguez-Gimenez, 2007).
A chimera was formed in which the integrin PSβ subunit cytoplasmic domain was fused to the extracellular and transmembrane domains of mutant forms of the Torso receptor tyrosine kinase. These chimeras localise to the sites of endogenous integrins. There, they can act both as activated integrins and as dominant negatives. Whereas on one hand, they can substitute for the endogenous integrin and regulate integrin target genes in the Drosophila midgut they can, on the other hand, also inhibit cell adhesion, matrix assembly and cell migration mediated by the endogenous integrins. Since the key feature of this chimera appears to be the dimerisation of the PSβ cytoplasmic tail, it is henceforth referred to as diβ (Dominguez-Gimenez, 2007).
Integrins are thought to perform two distinct functions in the wing: an early regulatory one in which integrins signal to make cells competent for re-apposition and a later, more traditional one, where integrins mediate adhesion. This study has unravelled a new early function for integrins in maintaining the columnar cell shape of wing epithelial cells. It is proposed that maintenance of this columnar shape is necessary to achieve proper contact and recognition of cells on opposing surfaces during folding. Thus, interfering with this activity results in cells adopting a cuboidal shape, which prevents them from establishing appropriate dorso-ventral connections. These early connections are probably necessary for re-apposition and final adhesion between the dorsal and ventral epithelia. Indeed, disruption of integrin function by diβ overexpression during the initial apposition period results in the formation of wing blisters in the adult (Dominguez-Gimenez, 2007).
The simplest hypothesis as to how integrins maintain the columnar shape of cells is that they keep the wing disc cells firmly attached to the basal matrix. However, recent evidence supports the idea that integrins also play a role in mediating adhesion between the lateral surfaces of cells during the process of dorsal closure in the embryo. In the wing, integrins seem to be distributed basolaterally when cells are in close contact, such as during apposition and adhesion. By contrast, integrins are absent form the lateral cell surfaces and become restricted to basal junctions when cells diminish their basal contact, i.e. during the expansion period. It therefore seems reasonable to propose that, integrins can maintain the columnar shape by mediating basolateral contact between adjacent cells (Dominguez-Gimenez, 2007).
Cell culture studies have shown that integrins and members of the Rho family of GTPases function in a coordinated manner to regulate the morphological changes that accompany cell spreading and migration upon binding to the ECM. In the Drosophila wing, the Rho GTPase Dcdc42 localises predominantly to the basal and apical regions of epithelial columnar cells. Furthermore, expression of a dominant-negative form of Dcdc42 results in a shortening of epithelial cells in the third instar larvae and produces a wing blister in the adult. Therefore, it is possible that integrins and Rho GTPases also interact to regulate the changes in cell shape underlying epithelial morphogenesis during development (Dominguez-Gimenez, 2007).
Maintenance of the columnar state through integrins is also required for folding along the wing margin. During normal wing morphogenesis this is mainly accomplished by local changes in cell shape of the wing margin cells, involving a reduction in height. This ensures that folding only occurs at the middle of the wing disc, thereby allowing an alignment match between dorsal and ventral cells. This study shows that, when integrin function is disrupted in most of the wing pouch, folding does not always occur along the wing margin, probably because all cells now adopt a similar shape. In fact, wing margin cells cannot be distinguished morphologically from the rest, although they do maintain their identity. This results in a mismatch between dorsal and ventral cells that might also be important for later differentiation processes. In summary, this study has show that cell-ECM interactions mediated by integrins are required for the temporal and, most likely, the spatial regulation of the changes in cell shape that accompany the folding of epithelial sheets during organogenesis (Dominguez-Gimenez, 2007).
It still remains unclear to what extent signalling contributes to the activity of integrins during development. One of the main problems is how to distinguish between direct integrin signalling and the indirect effects caused by a lack of integrin adhesion. In Drosophila, the differentiation of some but not all cell types depends directly on integrin signalling downstream of diβ. The results presented here support the idea that the regulation of cell morphology by integrins depends more on integrin adhesion to the ECM than on signalling. Indeed, the diβ chimera does not prevent the changes in cell shape by interfering with integrin activity. However, the possibility that the signal pathway activated by diβ does not fully mimic integrin signalling cannot be ruled out. In fact, it has been demonstrated in muscle that the chimeric diβ integrin is not capable of recruiting certain proteins associated with sites of integrin activity, such as ILK, paxillin, PINCH and tensin (Tanentzapf, 2006a). Hence, it remains possible that the regulation of cell shape requires integrin signals that are only triggered when a complete integrin complex is assembled (Dominguez-Gimenez, 2007).
Additional evidence has been generated to support the idea that the interactions between integrins and the matrix affect cell shape. The elimination of ECM components by overexpressing metalloproteinases provoked changes in cell morphology that strongly resemble those observed when integrin activity is disrupted. Moreover, overexpression of metalloproteinases does not affect the normal distribution of endogenous integrins, which can still cluster in focal-adhesion like structures. Hence, integrins alone are insufficient to regulate changes in cell shape but, rather, they must interact with ECM components. This is in agreement with findings that, in most cases, a threshold of both clustering and binding to integrins must be reached before fully functional focal adhesion complexes are formed (Dominguez-Gimenez, 2007).
The interactions of cells with the ECM have long been proposed to involve 'dynamic reciprocity', whereby a cell response to its ECM affects the composition of the new matrix it secretes, which in turn alters the ensuing response of the cell. This study shows that disrupting integrin function leads to changes in the basal matrix containing laminin. As such, it seems reasonable to consider a model by which the main function of integrins in regulating cell shape during wing development is the correct assembly and/or attachment to the ECM. An organised ECM can then in turn modulate the activity of the integrins themselves and/or other receptors to regulate cell morphology (Dominguez-Gimenez, 2007).
Overexpression of chimeras containing the cytoplasmic domains of the β1 or β3 subunits reduces integrin affinity. By contrast, chimeras containing a mutated β3 cytoplasmic domain with defective inside-out signalling, reduce the ability of the β3 cytoplasmic domain to block activation. These results suggest that there are limiting factors that bind to the cytoplasmic domains of integrins and which regulate ligand binding affinity. Modulation of these factors could be a way of regulating integrin activity. This study shows that diβ recruits the cytoplasmic protein Talin, opening the possibility that diβ exerts its dominant-negative effect by competing for Talin. However, this does not seem to be the case because overexpression of Talin does not rescue the diβ phenotype, contradicting data from CHO cells showing that competition for Talin underlies the trans-dominant inhibition exerted by isolated β tails. Nevertheless, complementation has been demonstrated between mutations in different motifs of the βPS cytoplasmic domain that eliminate the dominant-negative activity of diβ. This suggests that, if the dominant negative activity of diβ were due to competition for cytoplasmic components, this would involve the recruitment of at least two cytoplasmic proteins (Dominguez-Gimenez, 2007).
Alternatively, diβ could initiate a signalling cascade leading to the activation of a Raf-dependent integrin-suppressing pathway. Integrin-mediated adhesion to the ECM can trigger clustering and increase tyrosine phosphorylation of a number of intracellular proteins, including focal adhesion kinase (FAK), Raf, Ras, and MAPKs. However, it has been shown that activation of MAPK suppresses high-affinity ligand binding in integrins. Thus, a model has been proposed in which MAPK regulates integrin function through a negative feedback loop. This study shows that a dominant-negative form of Raf suppresses the capacity of diβ to inhibit integrin function. Furthermore, it is demonstrated that overexpression of diβ enhances Raf activity. Therefore, it is proposed that the trans-dominant inhibition exerted by diβ could result from the activation of a Raf-dependent signal transduction pathway that inhibits or modifies integrin-ECM interactions (Dominguez-Gimenez, 2007).
The negative regulation exerted by the Raf pathway could be part of a negative feedback loop that regulates integrin function during normal development. If this were the case, the expression of RafDN would be expected to constitutively activate integrin signalling and, therefore. provoke changes in cell morphology. But, none of these effects have been demonstrated upon expression of RafDN in the wing disc. However, since integrins can activate other pathways that are Raf independent, affecting one of these pathways might not produce a dramatic effect because the other pathways may compensate this deficiency. In this context, the results suggest that the chimeric diβ integrin is not able to activate intracellular signals other than those associated to the Raf pathway – probably be due to the failure of diβ to assemble a complete integrin complex (Dominguez-Gimenez, 2007).
The regulation of cell shape through cell-ECM interactions has been shown to have a dramatic influence on cell proliferation, patterning, differentiation, cell migration, cell branching and matrix production during development. This study shows that these interactions also play a crucial role in regulating the changes in cell shape that drive epithelial morphogenesis underlying the formation of organs and tissues. The molecular mechanisms by which these cell-ECM interactions influence the cytoskeleton and regulate cell shape during morphogenesis must now be identified. The easily detectable wing blister phenotype caused by expression of diβ in the wing provides a foundation to screen for mutations in genes required to modulate these integrin-ECM interactions (Dominguez-Gimenez, 2007).
scab was initially described in a study of mutations that affect the pattern of the larval cuticle. The defect in dorsal closure that describes the effects of scab mutation is similar to that seen in myospheroid mutant embryos. Myospheroid (also known as beta PS) is the dimerization partner of two previously characterized alpha integrins: alphaPS1 (Multiple edematous wings) and alphaPS2 (Inflated). Dorsal closure defect is not seen in null mutations of these two alpha integrins, indicating that some other alpha integrin must team up with betaPS during dorsal closure.
In a search for the presumed missing integrin, attention was focussed on a 90kDa band associated with immunoprecipitates of Myospheroid, resulting from the application of an anti-betaPS antiserum. This 90 kDa protein forms a non-covalent, divalent cation-dependent complex with Myospheroid. The 90 kDa protein binds well to both lentil lectin and Concanavilin A beads, suggesting that it is a glycoprotein. The protein was purified by immunoprecipitation, lecitin binding, elution and SDS gel electrophoresis and subjected to tryptic digestion; the resulting peptides were then sequenced. Degenerate primers based on the amino acid sequences were used to identify the cDNA coding for the 90 kDa protein. The sequence revealed an alpha integrin subunit that has been designated alphaPS3. Thus, the cloning of scab, revealing as it does the gene coding for the missing integrin, completes a picture of integrin activity with the discovery of a third alpha integrin partnering Myospheroid (Stark, 1997).
Scab RNA is localized to tissues undergoing invagination, tissue movement and morphogenesis: for example, salivary gland, trachea, midgut, dorsal vessel, midline of the ventral nerve cord, amnioserosa and the amnioproctodeal invagination. AlphaPS3 DNA localizes to the chromosomal vicinity of scab (scb), previously identified by a failure of dorsal closure. Embryos homozygous for the 119 allele of scb have no detectable alphaPS3 RNA. The 1035 allele of scb contains a P element inserted just 5' of the coding region for the shorter of the gene's two transcripts. Mutations in the scb locus exhibit additional defects corresponding to sites of alphaPS3 transcription, including abnormal salivary glands, mislocalization of the pericardial cells and interrupted trachea. Removal of both maternal and zygotic betaPS produces similar defects, indicating that these two integrin subunits associate in vivo and function in the movement and morphogenesis of tissues during development in Drosophila. Phenotypic similarities suggest that laminin A is a potential ligand for this integrin, at least in some tissues (Stark, 1997).
Tiggrin, a novel Drosophila extracellular matrix protein contains the potential integrin recognition sequence Arg-Gly-Asp (RGD) and 16 repeats of a novel 73-77 amino acid motif. The tiggrin gene is expressed by embryonic hemocytes and fat body cells. Tiggrin protein is detected in matrices, especially at muscle attachment sites that also strongly express integrins. Tiggrin may help to mediate integrin-dependent adhesion at embryonic muscle insertions. Tiggrin-coated surfaces support primary embryo cell culture and provide excellent substrates for alphaPS2 betaPS integrin-mediated cell spreading. Soluble RGD-peptides, able to act as ligands for integrins, inhibit this cell spreading (Fogerty, 1994).
PS1 (alphaPS1 betaPS) integrin is a laminin receptor. Both PS1 and PS2 integrins promote cell spreading on two different Drosophila extracellular matrix molecules, Laminin and Tiggrin, respectively. The differing ligand specificities of these two integrins, combined with data on the in vivo expression patterns of the integrins and their ligands, leads to a model for the structure of integrin-dependent attachments in the pupal wings and embryonic muscles of Drosophila (Gotwals, 1994a).
Two new potential ligands of the Drosophila PS2 integrins have been characterized by functional interaction in cell culture. These potential ligands are a new Drosophila laminin alpha2 chain encoded by the wing blister locus and Tenascin-major, an extracellular protein known to be involved in embryonic pattern formation. As with previously identified PS2 ligands, both contain RGD sequences, and RGD-containing fragments of these two proteins (DLAM-RGD and TENM-RGD) can support PS2 integrin-mediated cell spreading. In all cases, this spreading is inhibited specifically by short RGD-containing peptides. As previously found for the PS2 ligand Tiggrin (and the Tiggrin fragment TIG-RGD), TENM-RGD induces maximal spreading of cells expressing integrin containing the alphaPS2C splice variant. This is in contrast to DLAM-RGD, which is the first Drosophila polypeptide shown to interact preferentially with cells expressing the alphaPS2 m8 splice variant. The betaPS integrin subunit also varies in the presumed ligand binding region as a result of alternative splicing. For TIG-RGD and TENM-RGD, the beta splice variant has little effect, but for DLAM-RGD, maximal cell spreading is supported only by the betaPS4A form of the protein. Thus, the diversity in PS2 integrins due to splicing variations, in combination with diversity of matrix ligands, can greatly enhance the functional complexity of PS2-ligand interactions in the developing animal. The data also suggest that the splice variants may alter regions of the subunits that are directly involved in ligand interactions, and this is discussed with respect to models of integrin structure (Graner, 1998).
Curiously, the ten-m gene is expressed in an embryonic pair-rule pattern, and ten-m mutants display pair-rule patterning defects. Since the protein influences expression of downstream genes, it must communicate its presence to the cell nucleus. However, it does not appear that integrin signal transduction is important in early embryonic segmentation. PS integrins are not strongly expressed at this time, and, more importantly, mutations in integrin subunit genes do not cause segmentation phenotypes (Graner, 1998 and references).
Ten-m is later localized (among other places) at muscle attachment sites, where integrins are known to accumulate. This localization of Ten-m in vivo, as well as the demonstration of TENM-RGD interactions with PS2 integrins in vitro, suggests that Ten-m may function with PS2 integrins in muscle attachment. Interestingly, the heparan sulfate-containing protein D-syndecan also localizes to muscle attachments, and Ten-m contains a consensus heparin-binding sequence near the RGD, suggesting the potential of a Ten-m-syndecan-integrin complex. Syndecan proteoglycans recently have been shown to be important in signal transduction in focal adhesions in vertebrate cells (Graner, 1998 and references).
The available data, although very suggestive, do not demonstrate unequivocally that Ten-m serves as an integrin ligand at muscle attachment sites. However, other potential PS2 ligands, such as Tiggrin, also accumulate at muscle attachment sites, and genetic studies of tiggrin suggest considerable functional redundancy among the extracellular matrix components there. Because of this redundancy, a direct genetic demonstration of a role for Ten-m in muscle attachment may require simultaneous disruption of multiple genes encoding matrix proteins, and the early embryonic phenotype of ten-m mutants will further complicate such an analysis. One potential approach might be to demonstrate a dominant genetic effect of ten-m mutations in a background that has been sensitized for loss of function phenotypes by viable mutations in other genes that encode proteins important for muscle attachment or other integrin-dependent processes (Graner, 1998).
The integrin family of cell surface receptors mediates cell-substrate and cell-to-cell adhesion and transmits intracellular signals. In Drosophila there is good evidence for an adhesive role for integrins, but evidence for integrin signaling has remained elusive. Each integrin is an alphabeta heterodimer; the Drosophila betaPS subunit forms at least two integrins by association with different alpha subunits: alphaPS1betaPS (PS1) and alphaPS2betaPS (PS2). The complex pattern of PS2 integrin expression includes, but is more extensive than, the sites where PS2 has a known requirement. a comprehensive genetic analysis was carried out on the gene inflated, which encodes alphaPS2. Thirty-five new inflated alleles were isolated; 10 additional alleles were obtained from other investigators. The majority of alleles are amorphs (36/45) or hypomorphs (4/45), but five alleles that affect specific developmental processes were identified. Interallelic complementation between these alleles suggests that some alleles may affect distinct functional domains of the alphaPS2 protein, which specify particular interactions that promote adhesion or signaling. One new allele reveals that the PS2 integrin is required for the development of the adult halteres and legs as well as the wing (Bloor, 1998).
An examination of the phenotypes of the new classes of inflated alleles demonstrates that the inflated gene has separate functions in the somatic musculature vs. the gut and nerve cord. The class II allele, if SEF, is an embryonic lethal yet the phenotype is surprisingly mild: the nerve cord is fully condensed, midgut morphogenesis occurs normally, and the vast majority of muscles remain attached to the epidermis. The somatic muscles in if SEF mutant embryos, however, do exhibit a defect in the contractile ultrastructure. Examination by polarized light shows little evidence of sarcomeric structure in these muscles; staining for filamentous actin with rhodamine-phalloidin reveals that the f-actin fails to become properly organized. Embryos were examined at earlier times during stage 17 with polarized light. The appearance of striations in if SEF mutant embryos was not observed, suggesting that the PS2 integrin is required for the formation of muscle sarcomeric structure rather than for its maintainance. The strong waves of muscle contraction that normally accompany hatching from the vitelline membrane and chorion are not observed in these mutants, although some residual muscle function is present, since weak muscle contractions occur if the mutant animal is poked with a needle. Therefore, the if SEF mutant appears to be unable to form normal contractile somatic muscles. When if SEF mutant embryos were stained with the PS2hc/2 monoclonal antibody, staining was detected; however, wild-type staining could be detected with a polyclonal antisera directed against the C-terminal 15 amino acids of the PS2 subunit. This suggests that this mutant alters the conformation of the PS2 integrin (and the PS2hc/2 epitope) rather than its expression (Bloor, 1998).
A significant fraction of embryos mutant for the class III inflated alleles hatch to first instar larvae. Approximately one-fifth of the mutant individuals carrying the class III if C2B allele hatch: these larvae slowly become less motile and die over the next 48 hr. if C2B mutant embryos that fail to hatch have been examined by polarized light and rhodamine-phalloidin staining. The muscles remain attached and have normal sarcomeric structure. In contrast, the midgut fails to elongate and only two fat gastric caecae are formed. Staining of the visceral muscles in the mutant midguts shows that there is some detachment of the visceral muscle layer, but that the sarcomeric structure is not perturbed. Hatched if C2B larvae possess the same phenotypic characteristics as their unhatched counterparts, that is, wild-type muscles and abnormal midguts. It seems likely that the larval lethality is a result of their inability to feed, altough it is unknown why some of the mutant embryos fail to hatch. The lethal class III embryos also have defective nerve cord condensation. When these alleles are placed over Df(1)rif, the gut and nerve cord phenotypes are enhanced, and rare muscle detachments are observed (Bloor, 1998).
The if V2 allele causes a substantial reduction in the expression of the PS2 subunit in the third instar wing imaginal disc and is semilethal with a very strong adult phenotype. There is some larval lethality (but no embryonic lethality), to judge by the reduced numbers of if V2 pupae and adults relative to their siblings. The majority of the mutant individuals die while eclosing: they get their head and legs out but then become stuck. This is likely due to the inflated wings sticking to the pupal case. A few mutant individuals do successfully eclose and have severe adult abnormalities, although they are viable and fertile. The two layers of the wing blade are completely separated and the wings appear as hemolymph filled balloons. The hemolymph often becomes dried and blackened within the wing. In addition to this extreme version of the wing blister phenotype previously observed for inflated, two novel phenotypes are observed in this mutant. The halteres are distorted, appearing longer and less rounded than in wild type and have a rougher surface. The legs are also misshapen, with a kink in the femur, particularly in the second and third legs (Bloor, 1998).
Using a Drosophila cell line, a monoclonal antibody that inhibits not only cell clumping but also cell spreading has been generated. This antibody immunoprecipitates a complex of proteins identical to PS beta and other proteins. The antibody preferentially recognizes the PS beta associated with particular alpha chains in situ. The cells spread very well on dishes coated with vitronectin and, to a lesser extent, on those with fibronectin. The cells also can attach to dishes coated with laminin but without spreading; this attachment was not inhibited by antibody (Hirano, 1991).
The closest mammalian equivalents of Drosophila alphaPS1 are alpha3, alpha6 and alpha7, while the closest mammalian equivalents of alphaPS2 are alpha11b, alpha8, alphaV and alpha5. The ability of different Drosophila integrin alpha subunits to substitute for one another during embryonic development was tested. Two alpha subunits, which form heterodimers with the same betaPS subunit, are expressed in complementary tissues in the Drosophila embryo, with alphaPS1 expressed in the epidermis and endoderm, and alphaPS2 expressed in the mesoderm. As a result the two integrin heterodimers are present on opposite surfaces at sites of interaction between the mesoderm and the other cell layers, where they are required for normal development. Using the GAL4 system, the embryonic lethality of an alphaPS2 null mutation was rescued with a UAS-alphaPS2 transgene, but only partially with a UAS-alphaPS1 gene, as evidenced by the partial rescue of both muscle and midgut phenotypes. Similarly the embryonic/first instar larval lethality of an alphaPS1 null mutation gene was rescued with UAS-alphaPS1, but only partially with UAS-alphaPS2. Each UAS-alpha gene, when it contains the cytoplasmic domain from the other alpha subunit, maintains an equivalent ability to rescue its own mutation and cannot fully rescue a mutation in the other alpha. It is concluded that the two alpha subunits are not equivalent and have distinct functions that reside in the extracellular domains (Martin-Bermudo, 1997).
Integrin cell surface receptors are ideally suited to coordinate cellular differentiation and tissue assembly during embryogenesis because they can
mediate both signaling and adhesion. The identification of two genes, Mt and 258,
that require integrin function for their normal expression in Drosophila midgut endodermal cells has shown that integrins regulate gene expression in the intact developing embryo. The relative roles of integrin
adhesion versus signaling in the regulation of these integrin target genes was determined. Integrin-mediated adhesion is not required between the
endodermal cells and the surrounding visceral mesoderm for integrin target gene expression. In addition, a chimeric protein that lacks
integrin-adhesive function, but maintains the ability to signal (TorsoD), can substitute for the endogenous integrin and regulate integrin target genes. This
chimera consists of an oligomeric extracellular domain fused to the integrin betaPS subunit cytoplasmic domain; a control monomeric
extracellular domain fusion does not alter integrin target gene expression. Therefore, oligomerization of the 47-amino-acid betaPS intracellular
domain is sufficient to initiate a signaling pathway that regulates gene expression in the developing embryo (Martin-Bermudo, 1999a).
Integrin regulation of gene expression does not require specific
alpha subunit function. Whereas the betaPS cytoplasmic domain alone can mimic PS1
integrin signaling when fused to TorsoD, in the intact
integrin the alpha subunit will be required for interaction with the
extracellular ligands to promote clustering and may also play a role
inside the cell in the signaling pathway. To
test whether specific alpha subunits are required for signaling by PS
integrin heterodimers, the consequences of switching alpha subunits were examined in the endodermal cells.
alphaPS2 is not able to substitute for alphaPS1
function in the midgut when assayed by larval lethality. Expression of
UAS-alphaPS1 with the GAL4 driver can
substitute for endogenous alphaPS1 function to repress the
target gene 258 . Next to be tested were two chimeric alpha subunits, in
which the cytoplasmic domains were swapped between
alphaPS1 and alphaPS2, were tested, as well at the normal alphaPS2 subunit. It was found that all three can substitute
for alphaPS1 and repress 258 expression. This shows that the alpha subunits do not provide specificity
to this signaling event. In addition, it shows that the PS2 integrin is
able to interact with enough ligands to become clustered and initiate a
signaling pathway, even when it is expressed in an ectopic location. These results suggest that dimerization of the PS
subunit intracellular domain is sufficient to initiate a signaling pathway that can upregulate and
downregulate gene expression. This shows that whereas integrin ligand binding is used for adhesion to
the extracellular matrix, as signaling receptors, the integrins are formally equivalent to growth factor
receptors, in that their ability to mediate adhesion is not required for integrins to regulate gene
expression. Thus, these results have confirmed the importance of integrins in providing a vital link
between cell adhesion during morphogenesis and cellular differentiation (Martin-Bermudo, 1999a).
The assembly of an organism requires the interaction between different layers of cells, in many cases via an extracellular matrix. In the developing Drosophila larva, muscles attach in an integrin-dependent manner to the epidermis, via a specialized extracellular matrix called tendon matrix. Tiggrin, a tendon matrix integrin ligand, is primarily synthesized by cells distant to the muscle attachment sites, yet it accumulates specifically at these sites. Previous work has shown that the PS integrins are not required for tiggrin localization, suggesting that there is redundancy among tiggrin receptors. This was examined by testing whether the PS2 integrin can recruit Tiggrin to ectopic locations within the Drosophila embryo. It was found that neither the wild type nor modified forms of the PS2 integrin, which have higher affinity for Tiggrin, can recruit Tiggrin to new cellular contexts. Next, the fate was genetically manipulated of the muscles and the epidermal muscle attachment cells; this demonstrated that muscles have the primary role in recruiting Tiggrin to the tendon matrix and that cell-cell contact is necessary for this recruitment. Thus it is proposed that the inherent polarity of the muscle cells leads to a molecular specialization of their ends, and interactions between the ends produces an integrin-independent Tiggrin receptor. Thus, interaction between cells generates an extracellular environment capable of nucleating extracellular matrix assembly (Martin-Bermudo, 2000b).
This paper uses the muscle attachment sites and the
integrin ligand Tiggrin as a model system to study the
mechanisms that regulate the spatial and temporal assembly of
ECM during embryogenesis. How the
extracellular matrix protein Tiggrin comes to be tightly
localized at the interface between the specialized epidermal
tendon cells and the ends of the muscles at the muscle
attachment sites was examined. Whether one Tiggrin cell surface
receptor, the PS2 integrin, is able to localize Tiggrin to new sites
within the embryo was tested -- it is not. Then an examination was performed of what cells are required for the localization of Tiggrin; muscles are required, while the tendon cells are not.
Unexpectedly it was found that the localization of Tiggrin to the
end of a muscle requires contact between the muscle and
another cell (Martin-Bermudo, 2000b).
The requirement for integrins in the assembly of ECM in
vivo is clearly variable. In amphibian embryos, the
accumulation of fibronectin fibrils is blocked by antibodies
against the integrins. However, genetic
elimination of integrin function has more modest effects. The
initial assembly of extracellular matrices appears normal in
mouse embryos lacking a variety of integrin subunits, although
the matrices formed may be less stable. Ultrastructural
analysis shows that in the absence of PS integrin function the tendon
matrix still accumulates at the muscle attachment sites,
although it is separated from the cell surfaces. This is further supported by light microscopic findings
showing that the tendon matrix protein Tiggrin accumulates
correctly in embryos lacking PS integrins. In addition, the PS2 integrin
is not only not necessary for Tiggrin localization but is also not
sufficient. Therefore, a mechanism for the assembly of
extracellular matrix at the muscle attachment sites has to be
integrin-independent (Martin-Bermudo, 2000b).
This work has posed two key questions that will have to be
resolved in order to understand the mechanism of Tiggrin
localization: why does it require cell-cell contact, and why do
some sites of cell-cell contact recruit Tiggrin, while others do
not? The latter point implies that something must be special
about the ends of the muscles; when they make cell-cell
contacts they recruit Tiggrin, while other cell-cell contacts, for
example between the lateral surfaces of the muscles, do not.
This difference reflects the inherent polarity present within the
developing muscles, which has been revealed by two separate
experiments. When the rat transmembrane protein CD2 is
expressed in Drosophila muscles it is uniformly distributed on
membrane, but when its cytoplasmic tail is replaced with that
from the ßPS subunit, then the chimeric protein is localized to
the ends of the muscles. This demonstrates that the ßPS cytoplasmic tail is recognized inside the cell and localized to the ends of the muscles. By examination of the localization of kinesin-ß-galactosidase
fusion proteins, it has been shown that the muscles contain a
bipolar arrangement of microtubules, with the plus ends at the
termini of the muscles. Thus the muscles
clearly have an internal polarity that is able to localize
molecules specifically to the ends of the muscles, and it is
proposed that this is the first step leading to the localization of
Tiggrin (Martin-Bermudo, 2000b).
There are a variety of possible models to explain why cell-cell
contact is required for Tiggrin localization and three will
be outlined here. In the first model, the inherent
polarity of the muscles leads to the localization of a
transmembrane Tiggrin receptor at the ends of the muscle.
However, this receptor is not active until the muscle cells have
made contact with another cell, such as the end of the
equivalent muscle in the next segment. Following this cell
contact-dependent interaction, Tiggrin can bind to the receptor,
and later in development binds more strongly to other proteins
in the extracellular matrix, possibly by becoming crosslinked
to them, so that when the muscle detaches in the PS2 integrin
mutant, Tiggrin remains with the extracellular matrix.
In a second model, polarization of the cells results
in the specific transport of vesicles containing transmembrane
receptors and or extracellular matrix components to the ends
of the muscles. Then, the fusion of these vesicles with the
plasma membrane, which releases the contents, requires cell-cell
contact. This could be achieved by the interaction of a
transmembrane receptor with a ligand on the apposing cell,
which triggers an intracellular pathway leading to vesicle
fusion. In this model, since the vesicles are localized, the
receptor that triggers fusion does not have to be. One of the
proteins in the vesicle is not freely diffusible (for example by
being tethered to the membrane) and
contains a binding site for Tiggrin, thus recruiting Tiggrin to the
tendon matrix. Such polarized discharge of matrix components
has been described in diverse vertebrate cells, although it has
not been shown to require cell-cell contact (Martin-Bermudo, 2000b).
In these models, focus was placed on the muscles, since the
localization of Tiggrin requires only the muscles. However,
both models may also be applicable to the localization by
tendon cells of ECM proteins involved in tendon cell attachment to the tendon
matrix, such as the proteins affected by the rhea
mutation. The main difference between the cells on the two
sides of muscle attachments is that the muscles make cell-cell
contacts with each other and with the tendon cells, while the
tendon cells only make contact with the muscles. One of the
attractive aspects to the vectorial discharge model is that the
epidermal cells and muscles could secrete components that
crosslink together when they interact in the extracellular space,
forming a stable matrix. This would be similar to basement
membrane assembly at the interface between the epidermis and
the dermis, where laminin and its interacting partner nidogen
are expressed in different layers. Such
interaction between components secreted by the two layers
may be important for the formation of a functional tendon
matrix, as suggested by the rhea phenotype, but is not required for Tiggrin localization (Martin-Bermudo, 2000b).
The two models described above involve cell contact-dependent
activation of Tiggrin receptors, but it is difficult to
rule out the third model, where cell-cell contact has
a more mechanical role and is only required to reduce Tiggrin
diffusion. For example the inherent polarity could produce a
Tiggrin receptor at the ends of the muscles that is fully active
prior to cell-cell contact. The interaction of Tiggrin with this
receptor could be short-lived, so that it comes off again and
diffuses away. The cell-cell interaction would serve to make an
enclosed space or 'basket', where the resulting concentration
of Tiggrin would reach a high enough concentration to assemble
into an insoluble matrix. However, the assembly of the tendon
matrix clearly differs from the assembly of basement
membranes and fibronectin fibrils, which can form on a cell
surface that faces the extracellular fluid. Of course the actual
mechanism of tendon matrix localization could easily involve
all of these possible mechanisms (Martin-Bermudo, 2000b).
In summary, a combination of cell polarity and cellular
interaction result in the assembly of the tendon matrix in the
right place. These results have shown that in the developing
embryo cell-cell contact is necessary and may be sufficient for
the formation of a localized matrix, and have allowed the
formulation of models that are consistent with diverse experimental
results. Further characterization of the components of the
tendon matrix and the transmembrane receptors on the cells
should allow a determination of the mechanism of cell contact
dependent localization (Martin-Bermudo, 2000b).
The genetic locus that encodes Talin is rhea. The first two alleles, rhea1 and rhea2, were isolated in the wing blister screen (Prout, 1997). Two other alleles, rhea17 and rhea3, were isolated as mutations that dominantly enhance weak integrin mutations. The rhea1 and rhea2 alleles were mapped to 66D5-6. By locally hopping a P element in this region, which is not allelic to rhea, l(3)S1760, rhea79a was generated. The P element in this strain is inserted in the same position as l(3)S1760, within the coding region of the Drosophila ortholog of ergic-53, but is deleted for the ergic-53 coding region, leading to the initial suggestion that rhea encodes ergic-53. However, recombinational analysis placed rhea1 0.08 map units (25-50 kb) from the l(3)S1760 P element. The gene adjacent to ergic-53 was revealed to be Talin by the genome sequence. It was then found that Talin protein is reduced in rhea mutant embryos and imaginal disc clones and that Talin mRNA is absent in rhea79a (Brown, 2002).
To confirm that rhea is the Talin gene, the Talin-coding region was sequenced from rhea1 and rhea2. For each allele a small deletion was found that produces a frameshift in the Talin-coding sequence. For rhea1 the frameshift occurs after amino acid 1139, and the new reading frame terminates after two amino acids in the wrong frame. For rhea2 the frameshift occurs after amino acid 1279 and terminates after 31 out of frame amino acids. Inverse PCR was used to identify the proximal insertion site of the P element in rhea79a, which was found to be 1931 bp downstream of Talin, showing that the rhea79a deficiency deletes three genes, ergic-53, Talin, and CG6638. Each rhea allele has an aberration in the Talin coding sequence. A mutant deficient for Ergic53 complements rhea1, rhea2, and rhea17. Therefore, it is concluded that rhea encodes Talin (Brown, 2002).
Three aspects of the Talin mutant phenotype have been described (Prout, 1997). Clones of rhea/rhea cells in the wing do not attach to the other cell layer of the wing, causing a wing blister. The two initial alleles and two recently identified ones (rhea3 and rhea17) dominantly enhanced the wing blister phenotype of hypomorphic alleles of integrin genes (Prout, 1997). Finally, rhea mutant embryos have a detachment of the epidermis from the muscles, although the muscles remain attached end to end. The embryonic phenotype of Talin mutants was examined by EM, with particular attention to tendon cells. In wild-type embryos, tendon cells are spanned by microtubules that link basal hemiadherens junctions to tonofibrils that insert into the apical exoskeleton, thereby transferring the force of muscle contraction to the exoskeleton. In rhea/rhea cells, microtubules extended from apical tonofibrils toward the basal membrane, but mature basal attachment sites fail to form. Structural features of normal attachment sites, such as extensive folding of basal membranes and linkage of microtubules to the inner surface of basal membranes, are not generally present in rhea tendon cells. Also, in these rhea mutant tendon cells, microtubules are abnormally oriented, in some cases running parallel, rather than perpendicular, to the exoskeleton. Loss of Talin results in reduction of electron-dense material from the cytoplasmic face of hemiadherens junctions at muscle attachment sites. This suggests that Talin and/or the proteins it recruits make a significant contribution to this dense material (Brown, 2002).
These zygotic rhea mutant embryos still have some maternally deposited Talin. To analyze the phenotype resulting from the complete absence of Talin, the maternal contribution was removed by generating germline clones. Half of these rhea/rhea eggs receive a wild-type paternal allele, and the zygotic expression of Talin rescues the absence of maternal Talin in some, but not all, embryos. The number of hatching embryos varied from 33%–41%, rather than the expected 50% (depending on the allelic combination). Viable fertile adults developed from hatched embryos. Thus, maternal deposition of Talin protein is important, but not essential, for normal development (Brown, 2002).
Embryos lacking both maternal and zygotic talin have a stronger phenotype than those lacking either maternal or zygotic product, the most prominent features of which are a failure in germband retraction and strong muscle detachment. This phenotype is very similar to that of embryos lacking both maternal and zygotic ßPS. The similarities between the two phenotypes suggest that talin is essential for integrin function. Close examination of the muscle phenotype provides insight into the role of Talin in integrin-mediated adhesion. PS2 (alphaPS2ßPS) integrin localizes normally, demonstrating that integrins reach the cell surface and localize to the ends of muscles in the absence of talin. In detached muscles, actin staining is separate from PS2 integrin staining. This demonstrates that integrin is able to bind to the ECM, since, if it could not, it would be expected to remain on the surface of the detached muscle. Thus, a separation is seen between integrins and actin, not between integrins and the ECM, suggesting that the primary role of talin is to link integrins to the cytoskeleton, and not to stimulate their ligand binding. Talin does not appear to be required for condensation of the gonad, since this occurs in some mutant embryos. Condensation does fail in some embryos, but this could be a secondary effect caused by other morphogenetic defects. By examining different rhea alleles, it has been confirmed that this represents the null Talin phenotype. The phenotypes of mutant embryos from germline clones of rhea1 and rhea2 are indistinguishable, as are those of rhea79a, which deletes rhea and two flanking genes. Other data suggest, however, that the rhea1 and rhea2 mutations are weak dominant negatives. For example, producing rhea2/rhea79A embryos from maternal germline clones of rhea2 causes 65% (n = 153) failure of germ band retraction, while those of the rhea79A deficiency caused only 48% failure (Brown, 2002).
Further insight into the role of Talin was gained by looking at Talin and integrins in the imaginal disc epithelia. Just prior to pupariation it has been found that Talin and integrins colocalize into focal adhesion-like structures at the basal surface of the wing imaginal disc. Making clones of cells mutant for rhea results in loss of the staining of these structures with the Talin antibody. The clusters of Talin also fail to form in clones of cells lacking the ßPS integrin subunit. Clustering of integrins into these focal adhesion-like structures requires Talin function, as it does not occur in clones of cells lacking Talin. Therefore, clustering of integrins requires Talin, and clustering of Talin requires integrins. Loss of Talin does not grossly impair the rate of proliferation of the imaginal disc cells, since mutant clones are of a similar size to the wild-type twin spots. In addition, loss of Talin does not alter overall levels of integrin synthesis. Combining these results with those from the embryo suggests that Talin's role may be to promote integrin clustering, which, in turn, allows the establishment of a strong connection with the cytoskeleton (Brown, 2002).
The involvement of Talin in the transmission of integrin signals regulating gene expression was also examined. In Drosophila one signaling assay uses the enhancer trap 258, which is expressed in the midgut and fails to be repressed in the absence of PS1 integrin. The expression of this integrin target gene was examined in the absence of Talin. In embryos lacking maternal and zygotic Talin, midgut development was too disrupted to assay 258 expression. In the absence of the zygotic Talin, the midgut shows the characteristic phenotype of an integrin mutation: the gastric caeca fail to split from two initial evaginations into four slender tubes, the midgut does not elongate into a slender tube, and the proventriculus does not form properly. Despite these morphological defects, the 258 gene was repressed. The same result was obtained in more than 30 mutant midguts. While the possiblility cannot be ruled out that the small amount of maternal Talin left is sufficient for integrin signaling, but not for integrin adhesion, these results suggest that Talin is not required for integrin signaling to the nucleus (Brown, 2002).
A ligand-mimetic antibody Fab fragment specific for Drosophila alphaPS2ßPS integrins was developed to probe the ligand binding affinities of these invertebrate receptors. TWOW-1 was constructed by inserting a fragment of the extracellular matrix protein Tiggrin into the H-CDR3 of the alphavß3 ligand-mimetic antibody WOW-1. The specificity of alphaPS2ßPS binding to TWOW-1 was demonstrated by numerous tests used for other integrin-ligand interactions. Binding was decreased in the presence of EDTA or RGD peptides and by mutation of the TWOW-1 RGD sequence or the ßPS metal ion-dependent adhesion site (MIDAS) motif. TWOW-1 binding was increased by mutations in the alphaPS2 membrane-proximal cytoplasmic GFFNR sequence or by exposure to Mn2+. Although Mn2+ is sometimes assumed to promote maximal integrin activity, TWOW-1 binding in Mn2+ could be increased further by the alphaPS2 GFFNR --> GFANA mutation. A mutation in the ßPS I domain (ßPS-b58; V409D) greatly increased ligand binding affinity, explaining the increased cell spreading mediated by alphaPS2ßPS-b58. Further mutagenesis of this residue suggested that Val-409 normally stabilizes the closed head conformation. Mutations that potentially reduce interaction of the integrin ß subunit plexin-semaphorin-integrin (PSI) and stalk domains have been shown to have activating properties. Complete deletion of the ßPS PSI domain enhanced TWOW-1 binding. Moreover the PSI domain is dispensable for at least some other integrin functions because ßPS-DeltaPSI displayed an enhanced ability to mediate cell spreading. These studies establish a means to evaluate mechanisms and consequences of integrin affinity modulation in a tractable model genetic system (Bunch, 2006).
Integrins are the primary family of receptors that connects cells to the extracellular matrix (ECM). The cytoplasmic tails of integrin subunits associate with multiple intracellular components, which mediate both signaling functions and ECM-cytoskeleton connections. Cells can regulate the functions of integrins at least in part by inducing conformational changes in integrin structure that alter the affinity of the integrin heterodimer for ECM ligands. Similarly integrin binding to ligands can lead to outside-in signal propagation, which can regulate cellular behaviors such as growth, differentiation, and survival (Bunch, 2006)
The integrin heterodimer is composed of alpha and ß subunits that are nonhomologous to one another but strongly conserved structurally across the animal kingdom. A long history of studies with conformation-sensitive antibodies indicates that integrins undergo large and concerted conformational changes as a result of cellular activation, and recent studies have begun to provide details of this structural switching. Inactive integrins are likely to be bent in the middle so that the headpiece faces in toward the membrane-proximal part of the extracellular stalks. As a result of cellular activation, the integrin adopts an extended conformation with the headpiece facing away from the cell in optimal position to engage ECM proteins. The headpiece also can adopt two states, termed the open or closed conformations, corresponding to high and low affinity states for ligand binding. Binding of substrate ligands traps the integrin in the open conformation, and this shift in the equilibrium may then trigger outside-in signaling in many cases involving integrin clustering. This view of integrin dynamics is almost certainly simplified with other intermediate states probable (for example, bent integrins can also bind ligand, but overall it can provide a consistent explanation of the structure-function relationships between cellular regulation and signaling (Bunch, 2006).
Invertebrates such as Drosophila provide a system for sophisticated genetic studies of integrin function that are not practical with vertebrates. Genetic studies with flies can also be complimented with cell biology experiments in cell culture, and this system has become especially attractive with the simplicity of RNA interference methods in Drosophila. However, one persistent drawback to studies of integrin structure and function in invertebrates has been a lack of probes for measuring affinity states of integrins directly. Even in cell culture, Drosophila experiments have relied on indirect assays such as cell adhesion or spreading to quantitate integrin activity. Although these methods have generally provided a consistent set of data, a tool for measuring ligand affinity directly could permit much more reliable and rapid assays that can complement genetic analyses of integrin function in Drosophila (Bunch, 2006).
This study describes the generation of TWOW-1, a novel monovalent antibody Fab fragment that functions as a ligand-mimetic affinity sensor for alphaPS2ßPS. Using this probe, it is shown that previously described mutants in both the alphaPS2 and ßPS subunits lead to changes in affinity of the fly integrins. Finally it is demonstrated that complete removal of the ß subunit N-terminal PSI domain causes an increase in ligand affinity (Bunch, 2006).
Organogenesis of the somatic musculature in Drosophila is directed by the precise adhesion between migrating myotubes and their corresponding ectodermally derived tendon cells. Whereas the PS integrins mediate the adhesion between these two cell types, their extracellular matrix (ECM) ligands have been only partially characterized. This study shows that the ECM protein Thrombospondin (Tsp), produced by tendon cells, is essential for the formation of the integrin-mediated myotendinous junction. Tsp expression is induced by the tendon-specific transcription factor Stripe, and accumulates at the myotendinous junction following the association between the muscle and the tendon cell. In tsp mutant embryos, migrating somatic muscles fail to attach to tendon cells and often form hemiadherens junctions with their neighboring muscle cells, resulting in nonfunctional somatic musculature. Talin accumulation at the cytoplasmic faces of the muscles and tendons is greatly reduced, implicating Tsp as a potential integrin ligand. Consistently, purified Tsp C-terminal domain polypeptide mediates spreading of PS2 integrin-expressing S2 cells in a KGD- and PS2-integrin-dependent manner. A model is proposed in which the myotendinous junction is formed by the specific association of Tsp with multiple muscle-specific PS2 integrin receptors and a subsequent consolidation of the junction by enhanced tendon-specific production of Tsp secreted into the junctional space (Subramanian, 2007).
The development of functional musculature depends on the correct encounter
and adhesion of muscles with their corresponding tendon cells. In
Drosophila the hemiadherens junctions, formed on both sides of the
myotendinous junction, mediate the adhesion between muscles and their
corresponding tendon cells. The muscle-specific integrin heterodimer αPS2ßPS
accumulates at the muscle counterpart of this junction, and binds to its
specific extracellular matrix (ECM) ligand Tiggrin.
Correspondingly, the tendon-specific integrin heterodimer αPS1ßPS
accumulates at the tendon counterpart of the myotendinous junction, and is
thought to associate with the laminin ligand. Both
hemiadherens junctions on each cell type exhibit a symmetrical distribution,
raising the possibility that, although each cell utilizes a distinct integrin
heterodimer, the formation of the myotendinous junction is coordinated between
the two cell types. In the absence of the common ßPS subunit Myospheroid, muscles
initially interact with tendon cells; however, following muscle contraction
the muscles detach from the tendon cells and round up (the
myospheroid phenotype). Notably, lack of the muscle-specific αPS2
subunit similarly leads to muscle detachment; by contrast,
however, lack of the tendon-specific αPS1 (e.g., in the mew
mutant embryos) does not lead to muscle detachment.
mew mutant embryos hatch, suggesting that occupation of the
muscle-specific αPS2ßPS by its ligand may be sufficient for the
formation of embryonic myotendinous junctions. The αPS1 belongs to the
laminin-binding type α family of receptors and binds to laminin.
Drosophila laminin may consist of ß1 and ß2 subunits and
either of two laminin α subunits. The
αPS1 is thought to associate with laminin containing the LanA
subunit (also known as α3,5), which when deleted does not exhibit
significant muscle-tendon attachment defects. By
contrast, lack of the laminin α1,2 (wing blister), which associates with the
αPS2ßPS (Graner, 1998), results in a mild muscle-detachment phenotype (e.g.,
wing blister mutants) , pointing to the crucial function of the
muscle-specific PS2 in the formation of the myotendinous junction (Subramanian, 2007).
Tiggrin, a Drosophila-specific ECM component, has been shown to
associate with the muscle-specific αPS2ßPS integrin. However,
homozygous tiggrin mutant embryos do form muscle-tendon junctions and
the adult flies are only semilethal (Subramanian, 2007).
In addition to its role in the establishment of myotendinous junctions,
integrin-mediated adhesion is essential for several biological processes,
including dorsal closure, visceral mesoderm development and the development of
the adult fly wing. Wing epithelial cells from the dorsal and ventral aspects
of the wing form specialized integrin-mediated adherens junctions required for
the development of the adult fly wing. At morphogenesis dorsal wing epithelial
cells expressing αPS1ßPS are brought together with ventral cells
that express αPS2ßPS. Adhesion between these two epithelial sheets
of cells is presumably mediated by specific ECM ligands. Although the
involvement of the laminin α1,2 (wing blister) has been
described, ligand specificity of each of the PS integrin receptors in this
context has yet to be elucidated (Subramanian, 2007).
Tendon cells are specified in the Drosophila ectoderm as a result
of the activity of the tendon-specific transcription factor Stripe. Embryos
mutant for stripe do not develop normal tendon cells.
Conversely, ectopic expression of Stripe leads to ectopic development of
tendon cells. Thrombospondin was recovered in a search for genes that are regulated by the tendon-specific transcription factor Stripe (Subramanian, 2007).
Thrombospondins (Tsps) are a family of extracellular matrix proteins that
mediate cell-cell and cell-matrix interactions by binding membrane receptors,
extracellular matrix proteins and cytokines (Adams, 2001; Lawler, 2000). In vertebrates there are five tsp genes expressed in various tissues, including the brain (TSP1 and Tsp2), bones (Tsp5) and tendons (Tsp4). Tsp1 and Tsp2 are
closely related trimeric proteins that share the same set of structural and
functional domains. Tsp4 and Tsp5 are pentameric and differ from Tsp1 and Tsp2
in their domain arrangement. All Tsps share a typical C-terminal domain (CTD)
that contains epidermal growth factor (EGF)-like repeats, and a Ca-binding
domain. The N-terminal domain contains additional conserved regions including
the laminin G-like domain (which is not present in Tsp5). Drosophila tsp is encoded by a single gene that is spliced into four variants, among which only one (TspA) contains the conserved CTD, which in addition to the EGF repeats and Ca-binding domains also includes a putative integrin-binding KGD motif. The N-terminal domain contains a conserved heparin-binding domain and putative integrin-binding motifs RGD and KGD. Drosophila Tsp is closest in structure to vertebrate Tsp-5/COMP,
which is expressed mainly in cartilage and certain other connective tissues
and has a role in chondrocyte attachment, differentiation and cartilage ECM
assembly (Subramanian, 2007).
A wide range of functions has been attributed to the different Tsps,
including a role in platelet aggregation, inflammatory response, regulation of
angiogenesis during wound healing, and tumor growth. Tsp1 and Tsp2 have been described as astrocyte-secreted components that promote synapse formation in the CNS (Subramanian, 2007).
The large isoform of Drosophila Tsp has been shown to form
pentamers and exhibits heparin-binding activity. Its major sites of expression
in the embryo are the muscle attachment sites, and also the precursors of the
longitudinal visceral muscles. In larval stages it is expressed in wing imaginal discs (Subramanian, 2007).
This study reports that Drosophila Tsp is a key ECM component that is
required for muscle-specific adhesion to tendon cells. In tsp mutant
embryos muscles fail to attach to tendon cells, and often aggregate and form
ectopic integrin-mediated junctions with neighboring muscles. This leads to
nonfunctional somatic musculature and embryonic lethality. In the embryo, Tsp
is required for integrin-mediated adhesion as measured by Talin-specific
accumulation. Furthermore, Tsp can functionally bind to
αPS2ßPS-integrins; the purified CTD of Tsp mediates PS2
integrin-dependent cell spreading in a KGD- and PS2-dependent manner (Subramanian, 2007).
Taken together, these results suggest a model whereby Tsp produced by tendon
cells is required for muscle-specific adhesion to tendons by binding the
muscle-specific αPS2ßPS integrin receptors, and a subsequent
consolidation of the junction by enhanced tendon-specific production of Tsp
secreted into the junctional space (Subramanian, 2007).
It is suggested that the dynamics of myotendinous junction
formation involve the following sequential steps. (1) When the myotube is very
close to the tendon cell, Tsp secreted continuously from the tendon cell
associates with the muscle leading edge and binds to the muscle-specific
αPS2ßPS integrin receptors. Because Drosophila Tsp forms
pentamers, each pentamer potentially associates with several PS2 receptors,
leading to accumulation of αPS2ßPS receptors at the myotube leading
edge. This association triggers integrin-mediated adhesion and Talin
accumulation at the cytoplasmic tail of the PS2 integrin receptors. (2) Tsp
may bind to the tendon surfaces through an unknown ligand. (3) Stripe levels
in the tendon cell are elevated following the establishment of the
muscle-tendon junction, because of Vein-EGF receptor (EGFR) signaling.
Stripe induces the elevation of Tsp levels, creating a positive feedback loop
that encourages further secretion and accumulation of Tsp at the junction
site, strengthening the myotendinous junction (Subramanian, 2007).
The KGD site in the CTD of Tsp was shown to trigger PS2
integrin-dependent cell spreading. This sequence had been shown to bind
certain types of vertebrate integrin receptors
(Scarborough, 1993). The N-terminal domain of Tsp contains an additional KGD site, and an RGD site, both implicated in integrin-binding activity. These sites may also contribute
to the binding of the PS2 muscle-specific integrins. Therefore, each Tsp
pentamer contains multiple binding sites for PS2 integrin receptors, and thus
may induce receptor aggregation at the muscle leading edge. It remains to be
determined whether Tsp is capable of binding to PS1 integrins or other
receptors expressed by the tendon cell (Subramanian, 2007).
Whether Tsp functions as an integrin ligand in other tissues (e.g., midgut,
salivary gland, dorsal closure and the wing epithelium) is yet to be
elucidated. Phenotypic analysis of the tsp8R mutant
embryos did not reveal a major phenotype in the gut, CNS or dorsal closure.
Similarly, tsp8R mutant clones induced at the larvae stage
did not result with wing blisters as in integrin-induced clones. Although
mutants for the tsp8R allele did not show staining with
the anti-Tsp antibody, it is still possible that residual Tsp activity is
retained in the mutants because of the activity of the other TSP isoforms
(which were not affected by the deletion of the EP excision at the CTD). In
addition, maternal tsp transcripts were detected that may partially rescue the zygotic tsp phenotype in the early developmental stages (Subramanian, 2007).
An additional relevant ECM component at the myotendinous junction is
laminin. Laminin α1,2 (encoded by wing blister) is required for
the formation of the myotendinous junction (Martin, 1999). Laminin α1,2 contains an RGD sequence and also binds to the PS2 integrins (Graner, 1998),
demonstrating the crucial role of these receptors in the formation of the
myotendinous junctions. It is possible that laminin containing the laminin
α1,2 subunit associates with Tsp in the myotendinous junctional space.
Both laminin and Tsp carry a heparin-binding domain and it is possible that
they interact indirectly through a putative heparin-containing proteoglycan.
Because no changes in laminin distribution was detected following
overexpression of Tsp (using anti-laminin antibody), it is not thought that there
is any direct Tsp-laminin interaction. The heparan sulfate
glycoprotein Syndecan is produced by the muscle cells. In syndecan
mutant embryos the somatic muscle pattern is defective, a phenotype that is
attributed to an effect of Syndecan on Slit distribution and function.
However, Syndecan at the muscle cell membrane may contribute to a putative
indirect interaction between Tsp and laminin through its heparin-containing
domain. Such interaction may enhance the accumulation of ECM components such
as Tsp and laminin at the myotendinous junction. In support of this
hypothesis, vertebrate Tsp has been shown to bind Syndecan at its CTD
(Adams, 2004). However, syndecan homozygous mutant embryos do not exhibit
alterations in Tsp distribution, arguing against a central
role for Syndecan in Tsp distribution. Nevertheless, it remains possible that
another heparin domain-containing protein functions to promote Tsp and laminin
deposition at the myotendinous junction (Subramanian, 2007).
It is considered that the Stripe-dependent positive feedback that upregulates
tsp transcription contributes significantly to the establishment of
the myotendinous junction. Previous studies have shown that muscle-tendon
interactions form a signaling center, which is initiated by muscle-dependent
Vein secretion and accumulation at the myotendinous junction. Vein activates
the EGFR pathway in the tendon cell, leading to a significant elevation of the
transcription factor Stripe. This study shows that Stripe induces upregulation of Tsp.
Taking these results together, it is suggested that the initial formation of the
hemiadherens junction creates a self-auto-regulatory nucleation center, which
leads to additional deposition of Tsp and possibly other ECM components.
These, in turn, gradually strengthen the hemiadherence junction formed between
the muscle and the tendon cell (Subramanian, 2007).
Vertebrate Thrombospondins are essential for a variety of biological
activities, including cell adhesion, migration, angiogenesis, etc. This work
reveals an intriguing similarity between the role of Tsp in the formation of
the myotendinous junction and the role of vertebrate Tsp1 and Tsp2 in the
induction of synapses. It was shown that Tsp provided by oligodendrocytes is a
potent inducer of synapse formation on the dendrites of cultured neurons
(Christopherson, 2005). Although these synapses are not electrically active, the Tsp-induced synapses exhibit typical synaptic ultra-structures. The biogenesis of the myotendinous junction carries several similarities to the biogenesis of synapses, including the mutual crosstalk between the two cell types involved and the gradual
formation of the junction at both cell membranes involved (Subramanian, 2007).
In summary, this analysis of Tsp function reveals the molecular dynamics and
biogenesis of the myotendinous junction. A similar scenario may unfold during
Tsp-dependent synapse formation in the development of vertebrate embryos (Subramanian, 2007).
During Drosophila embryogenesis, the attachment of somatic muscles to epidermal tendon cells requires heterodimeric PS-integrin proteins (α- and β-subunits). The α-subunits are expressed complementarily, either tendon cell- or muscle-specific, whereas the β-integrin subunit is expressed in both tissues. Mutations of β-integrin cause a severe muscle detachment phenotype, whereas α-subunit mutations have weaker or only larval muscle detachment phenotypes. Furthermore, mutations of extracellular matrix (ECM) proteins known to act as integrin binding partners have comparatively weak effects only, suggesting the presence of additional integrin binding ECM proteins required for proper muscle attachment. This study reports that mutations in the Drosophila gene thrombospondin (tsp) cause embryonic muscle detachment. tsp is specifically expressed in both developing and mature epidermal tendon cells. Its initial expression in segment border cells, the tendon precursors, is under the control of hedgehog-dependent signaling, whereas tsp expression in differentiated tendon cells depends on the transcription factor encoded by stripe. In the absence of tsp activity, no aspect of muscle pattern is affected, nor is formation of the initial contact between muscle and tendon cells or muscle-to-muscle attachments. However, when muscle contractions occur during late embryogenesis, muscles detach from the tendon cells. The Tsp protein is localized to the tendon cell ECM where muscles attach. Genetic interaction studies indicate that Tsp specifically interacts with the αPS2 integrin and that this interaction is needed to withstand the forces of muscle contractions at the tendon cells (Chanana, 2007).
Attachment of muscles to tendon cells critically depends on integrin activity. The integrin-like muscle detachment phenotype of the tsp mutants as well as the co-localization of Tsp and integrin proteins strongly suggest that Tsp plays a decisive role in the integrin-mediated cell adhesion process of muscles and tendon cells. In fact, Tsp was shown to encode a pentameric glycosylated protein that is part of the ECM (Adams, 2003). Thus, it could indeed function as a direct binding partner of the PS-integrins (Chanana, 2007).
In order to establish such a functional link between tsp and PS-integrins by genetic means, double mutant embryos were generated that carry either a strong loss of function allele for the αPS1 subunit of integrin due to the mewM6 mutation or a hypomorphic allele for the αPS2 subunit due to the ifB2 mutation in combination with only one wild-type copy of tsp (tspΔ6 and tspΔ79) (Chanana, 2007).
In comparison with wild-type embryos mewM6 mutants bearing two copies of tsp develop a normal muscle pattern with muscle detachment in only few segments. mewM6 mutant embryos that have only one wild-type copy of tsp develop a weak muscle detachment phenotype affecting a small number of longitudinal muscles. In contrast, ifB2 mutants bearing two copies of tsp developed a mild detachment phenotype in several segments, which was strongly enhanced, both with respect to the extent of detachment and penetrance in embryos with only one remaining wild-type copy of tsp. In these embryos, longitudinal, ventral as well as dorsal muscles were found to be detached, a phenomenon not observed in tsp mutant embryos. Furthermore, in mewM6 mutants lacking both wild-type copies of tsp, only a mild enhancement of the tsp mutant phenotype was observed. In contrast, ifB2 lacking both wild-type copies of tsp develop dramatic muscle pattern defects beyond an additive effect of the two individual mutant phenotypes. The enhancement of the muscle detachment phenotype of if mutants by the removal of one copy of tsp and the dramatic enhancement that affects even the set of muscles that are not affected in each of the single if or tsp mutants establishes an essential role of Tsp in the αPS2-dependent muscle attachment process. In contrast, the weak effects of the reduction of the tsp dose in hemizygous mewM6 mutants makes a prominent role of Tsp in αPS1-dependent muscle attachment rather unlikely (Chanana, 2007).
Vertebrate Tsp is a glycosylated protein that forms oligomers and is capable of interacting with both calcium and heparin (Adams, 2001). Furthermore, it has been shown to directly interact with the extracellular part of integrin proteins (Lawler, 1988). This interaction depends on a highly conserved RGB motif, which is characteristic of integrin binding proteins of the ECM (Chanana, 2007).Vertebrate genomes code for up to five Tsps (Adams, 2001), whereas the Drosophila genome contains only a single tsp-coding sequence (Adams, 2003), which, however, encodes two Tsp variants which differ in their carboxyterminal regions. Previous biochemical studies on Drosophila Tsp showed that the protein is secreted and able to form a pentameric structure as suggested by the molecular weight of the secreted native complex (Adams, 2003). At the sequence level, the conserved Drosophila Tsp contains all functionally characterized domains including the critical RGD motif required for integrin binding. In contrast to vertebrate Tsp, the RGD motif in Drosophila Tsp is positioned in the aminoterminal region, close to two BBXB sequence motifs known to bind to heparin, instead of the third Tsp/COMP domain (Adams, 2003). In addition, a KGD motif is observed in the third Tsp/COMP domain of Drosophila Tsp which was shown to serve also as an interaction motif for integrins (Ruoslahti, 1996). Drosophila Tsp contains therefore two RGD/KGD motifs that would allow direct binding of PS2, the integrin heterodimer that was previously found to associate with Tig, an interaction that was shown to be dependent on the presence of the RGD motif. Furthermore, an RGD motif is also required to mediate the interaction of PS2 with the laminin α-chain Wb (Chanana, 2007).
Drosophila tsp is expressed in all ectodermal tendon precursor cells, strongly enriched in those positioned at the segment border of the embryo. Furthermore, tsp is expressed in all differentiated tendon cells after muscle contact. Therefore, tsp is expressed in all cells that have previously been identified by the expression of stripe. stripe encodes an EGR-type Zn-finger transcription factor that is required for tendon cell differentiation. Like stripe, the initial expression of tsp is controlled by Hedgehog signaling at the segment borders and requires stripe activity only during the later stages when the tendon cells are already differentiated. These results suggest that the genes stripe and tsp are activated in parallel by Hh-dependent Ci activity, and that stripe activity maintains the expression of tsp during the later stages when Ci activity has ceased (Chanana, 2007).
Tsp is secreted from epidermal tendon cells and accumulates at the tendon cell matrix, a specific ECM to which the muscles attach. The functional characterisation of the newly generated tsp alleles, which fail to express detectable amounts of tsp transcript, showed that Tsp is necessary for the proper anchoring of muscles at the tendons cells. As observed with mutants affecting the β subunit and the αPS2 subunit of integrin, mys and if, respectively, the muscles were found to detach from their epidermal attachment sites once muscle contraction occurs. Thus, tsp activity is not required for any aspect of muscle pattern formation and/or muscle guidance as well as proper adherence to tendon cells but plays an essential role in maintaining the interconnection between muscles and tendons cells once contraction occurs. Although the muscle detachments are less pronounced than in mys or if mutants, the detachment phenotype of tsp mutants is by far stronger than the corresponding phenotypes that are caused by the loss of other ECM proteins, such as Tig, Wb and LanA, known to be integrin interaction partners. The strong and specific enhancement of the detachment phenotype of mutants that carry a weak if allele, in response to the loss of one or both tsp wild-type alleles strongly suggests that Tsp functions as an essential ECM binding partner of αPS2 encoded by if. The specificity of the genetic interaction shown in this study is consistent with the finding that binding to αPS2 requires an RGD motif as has been found in Tig and Wb as well. Mutations of either tig or wb cause weak muscle detachment phenotypes as observed with tsp mutants, suggesting a redundant αPS2 integrin interaction system in which ECM binding is provided by different partners and that each of them is required for the proper anchoring of the muscles. This conclusion is consistent with the finding that tig, wb and tsp mutants display a weaker phenotype than the if loss of function mutants. Based on the specific expression of tsp in both tendon cell precursors and differentiated tendon cells, which differs from the multiple expression sites of tig and wb, together with the strong enhancement of the muscle detachment phenotype in if and tsp double mutants, it appears likely that tsp is the crucial interaction partner of the αPS2 integrin subunit to provide proper anchoring of muscles to tendon cells. This proposal, and the relative contribution of each of the by now three different αPS2 integrin subunit binding proteins, can be tested once double and triple mutant combinations for all the genes involved and biochemical test systems become available (Chanana, 2007).
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