myospheroid

DEVELOPMENTAL BIOLOGY

Embryonic

In wild-type embryos, PS antigens are found at the interface between mesoderm and ectoderm, and later mainly at the attachment sites of muscles to the epidermis and gut. Together these results indicate that during embryogenesis, Drosophila integrins are used to attach mesoderm to ectoderm, and are required for the proper assembly of the extracellular matrix and for muscle attachment (Leptin, 1989).

Time-lapse videomicroscopy was used to examine living embryos integrins. Roles for these molecules have been demonstrated as early as gastrulation [Images]. Abnormalities in mutant embryos include: separation and twisting of the embryonic germband, abnormal shape and migration of midgut primordia, irregular visceral mesoderm, detachment of amnioserosa cells, rupture of the cuticle along the dorsal midline, lack of midgut constriction, and detachment of somatic muscles. These observations suggest multiple roles for integrins in the adhesion of cells and in the formation, organization, and migration of embryonic tissues. The complete loss of both alpha subunits does not produce all of the phenotypes observed in embryos lacking betaPS. This suggests that alphaPS1 betaPS and alphaPS2 betaPS are not required in all embryonic processes utilizing integrins (Roote, 1995).

Integrins help to coordinate the differentiation of the internal, sarcomeric cytoarchitecture of a muscle fiber with its immediate environment and are essential for correct integration of muscle cells into tissue. Integrin alphaPS2 betaPS accumulates at contact regions of Drosophila cultured embryo cells. Myotubes form, but subsequent addition of serum or fibronectin is needed for sarcomere formation: Integrin and Actin become concentrated at Z-bands; Myosin and Actin are positioned between the Z-bands. This change fails to occur myotubes derived from myospheroid null myospheroid mutants (Volk, 1990).

The role of integrins was examined in the formation of the cell junctions that connect muscles to epidermis (muscle attachments) and muscles to neurons (neuromuscular junctions). At the ultrastructural level two types of muscle attachments can be distinguished: direct and indirect. At the direct muscle attachments, single muscles (such as the transverse muscles) attach to epidermal cells directly such that the hemiadherens junctions (HAJs) in opposing cells are separated by only 30-40nm, with a thin line of extracellular electron-dense material in between. These closed paired HAJs are referred to as connecting HAJs. Indirect muscle attachments occur at the segmental border, where the ends of multiple muscles attach at the same epidermal site, and contain extensive extracellular matrix consisting of fuzzy electron dense fibers, separated by up to several micrometers. This is referred to as tendon matrix because, like the vertebrate tendons, it is an extracellular matrix used to attach the muscles. Since HAJs at indirect muscle attachments are not closely paired but connected to the tendon matrix, they are referred to as tendon HAJs. Both types of muscle attachments have a common molecular basis: both contain PS integrins; both are sites were large secreted proteins Tiggrin and Masquerade accumulate; the intracellular appearance of connecting HAJs and tendon HAJs looks similar; connecting HAJs and tendon HAJs can appear together at the same site; they both appear to arise from short connecting HAJs; and both HAJs are separated from the extracellular electron dense matrix by a translucent gap of a few nanometers (Prokop, 1998).

Muscle attachments and neuromuscular junctions were examined ultrastructurally in single or double mutant Drosophila embryos lacking PS1 integrin (alphaPS1betaPS), PS2 integrin (alphaPS2betaPS), and/or their potential extracellular ligand Laminin A. At the muscle attachments PS integrins are essential for the adhesion of hemiadherens junctions to extracellular matrix, but not for their intracellular link to the cytoskeleton. The intracellular electron-dense material of connecting HAJs and tendon HAJs connects to microfilaments in the muscles, and to microtubules in the epidermis. The epidermal microtubules are anchored at the other end to apical focal HAJs that connect to the cuticle (Prokop, 1998).

The PS2 integrin is only expressed in the muscles, but it is essential for the adhesion of muscle and epidermal HAJs to electron dense extracellular matrix. PS2 integrin is also required for adhesion of muscle HAJs to a less electron dense form of extracellular matrix, the basement membrane. The PS1 integrin is expressed in epidermal cells and can mediate adhesion of the epidermal HAJs to the basement membrane. The ligands involved in adhesion mediated by both PS integrins seem distinct because adhesion mediated by PS1 appears to require the extracellular matrix component Laminin A, while adhesion mediated by PS2 integrin does not (Prokop, 1998).

At neuromuscular junctions (NMJs) the formation of functional synapses occurs normally in embryos lacking PS integrins and/or Laminin A, but the extent of contact between neuronal and muscle surfaces is altered significantly in embryos lacking laminin A. It is suggested that neuromuscular contact does not require laminin A directly at its point of contact, but requires basement membrane adhesion to the general muscle surface, and this form of adhesion is completely abolished in the absence of Laminin A. In contrast, loss of PS integrin function causes the boutons to make a more extensive contact with the muscle surface. Since no PS integrins are found at neuromuscular contacts it seems likely that the boutons can adhere to more muscle area because the muscle surfaces are more relaxed (allowing them to bend around the bouton) in the severely detached muscles of embryos lacking both PS integrins functions. Adhesion molecules expressed at Drosophila NMJs, like Fasciclin II, Fasciclin III or Connectin, are unlikely to mediate adhesion at the mature embryonic NMJ because they either fade during stage 16 or show no phenotype when mutated. Instead, mutant analysis reveals the existence of yet unknown embryonic adhesion factors downstream of mef2 regulation. Such factors might include laminin receptors that promote adhesion, or other receptors that displace the basement synaptic cell junction. Identification of mef2-dependent receptors might be aided by the use of lamA mutation as a sensitized background (Prokop, 1998).

Heterodimeric cell surface receptor integrin is widely expressed in the nervous system, but its specific role during axon development has not been directly tested in vivo. The Drosophila nervous system expresses low levels of positron-specific (PS) integrin subunits alphaPS1, alphaPS2, and betaPS during embryonic axogenesis. Furthermore, certain subsets of neurons express higher levels of integrin mRNAs than do the rest (Hoang, 1998).

The expression pattern of alphaPS1, alphaPS2, and betaPS subunits were examined using in situ hybridization and immunocytochemistry in the embryonic nervous system. All three PS subunit mRNAs are expressed widely in the nervous system. Their expression levels during hours 9-18 of embryogenesis are notably low compared with that in the other tissues that have been studied previously, such as muscles and apodemes. alphaPS1 mRNA is detected widely in the CNS at a steady level during hours 13-18. During this period, axogenesis occurs within the CNS as well as in the periphery. At the ventral midline, cells appear to accumulate slightly higher levels of alphaPS1, as compared with most other cells in the CNS. The alphaPS2 mRNA expression pattern differs somewhat from that of alphaPS1. Specific clusters of cells, one near the midline and a bilateral pair at mediolateral sites, express alphaPS2 at relatively high levels in each segment of the CNS. The alphaPS2 expression in the CNS peaks during hours 9-15 of embryogenesis. The betaPS mRNA expression pattern partially overlaps with those of alphaPS1 and alphaPS2 mRNA in the CNS, with one prominent cluster of cells expressing relatively high levels at the ventral midline. betaPS mRNA expression in the CNS persists through hours 9-18. These in situ hybridization data suggest that the embryonic CNS expresses both PS1 (alphaPS1/betaPS heterodimer) and PS2 (alphaPS2/betaPS heterodimer) integrins during the period of axogenesis. Consistent with the mRNA data, immunocytochemistry shows that the betaPS protein subunit is unambiguously revealed on neuronal cell surfaces during hours 16-18. The major axon fascicles within the CNS, including the longitudinal connectives and the anterior and posterior nerve tracts as well as the cell surfaces of at least some identified neurons, are labeled with the betaPS antibodies. Unfortunately, both alphaPS1 and alphaPS2 subunits are below the threshold of detection with available antibodies. However, the detection of the betaPS protein subunit on the neuronal cell surfaces suggests that the alphaPS subunits are likely forming functional heterodimers with the betaPS subunit in those cells. These observations with in situ hybridization and immunocytochemistry have led to the conclusion that, similar to vertebrate neurons, many Drosophila neurons express relatively low levels of integrin on the neuronal surface during axogenesis (Hoang, 1998).

Null mutations in either the alphaPS1 or alphaPS2 subunit gene cause widespread axon pathfinding errors that can be rescued by supplying the wild-type integrin subunit to the mutant nervous system. In contrast, misexpressing either the alphaPS1 or alphaPS2 integrin subunit in all neurons leads to no obvious axon pathfinding errors. In the CNS, the longitudinal connective normally contains three prominent axon fascicles that are easily visualized by mAb 1D4. Axons in the alphaPS1 or alphaPS2 mutants appear somewhat wiggly or partially disconnected (Hoang, 1998).

In the PNS, one can visualize specific groups of motoneuron axons with higher cellular resolution than is possible in the CNS. Null mutations in alphaPS1 and alphaPS2 subunit genes both result in similarly widespread axon pathfinding defects for all five known motoneuron groups, despite apparently normal muscle development. The axon defects observed can be interpreted as a consequence of failing to turn at choice points and/or invading into neighboring muscle fields. These defects are most frequently detected in the SNb group. It is important to note that the axons in these loss-of-function mutants can extend as far as in wild type and sometimes beyond their normal stopping points. This suggests that integrin is unlikely to serve simply as a clutch-constituting molecule, on which growth cones depend for the adequate traction needed to extend forward. Another point is that each axon group selects a range of alternative pathways without obvious preferences. It is therefore likely that the loss of integrin leads to losses in the responsiveness of an axon to a large array, rather than a small specific set, of guidance cues. Finally, in general, loss of PS2 integrin (alphaPS2 null mutation) leads to higher axon guidance errors than does loss of PS1 integrin (alphaPS1 null mutation). Future analysis is needed to determine specific contributions of these two forms of integrin. On the basis of the current data, it is suggested that both the laminin-binding PS1 and RGD-dependent PS2 integrins are necessary for accurate axon guidance. It is proposed that integrin serves as part of a molecular network that cooperatively guarantees accurate axon guidance (Hoang, 1998).

Analysis of Talin protein distribution during embryogenesis shows that it is maternally deposited and is evenly distributed in the cytoplasm following cellularization. Talin becomes progressively concentrated at the membrane, first detected in the migrating primordial midgut cells and then at muscle attachment sites, where the muscles and epidermal cells are linked via integrins. Talin protein shows only a hint of the strong pattern of mRNA expressed in the nervous system. Consistent with the low level of protein in the nervous system, zygotic mutant rhea embryos do not have any defects in the structure of the nervous system, as assayed with glial and neuronal cell markers. The subcellular distribution of talin at the muscle attachment sites was examined by immunoelectron microscopy (IEM). Talin is found within submembranous electron-dense material associated with the hemiadherens junctions at muscle attachment sites. Most sites of Talin concentration at membranes correspond to sites of integrin concentration, and colocalization of the two proteins is seen at the edge of the epidermis, during dorsal closure, and at muscle attachments. No PS integrin staining lacking colocalized Talin was observed. However, Talin is concentrated at the membrane of some cells lacking integrin. This is clearest in the gonadal mesoderm, where recruitment of Talin to the cortex of the gonadal mesoderm cells occurs as the gonad condenses. To test whether recruitment of Talin to sites of integrin expression requires integrins, Talin distribution was examined in embryos lacking PS integrins. Embryos lacking the ßPS subunit show a loss of Talin concentration at muscle attachment sites. In embryos that lack the mesodermally expressed alphaPS2 integrin subunit, but that still contain epidermal PS1 (alphaPS1ßPS) integrin, Talin is lost from muscle ends but still concentrated at the basal ends of the attaching epidermal cells, the tendon cells. These results show that talin is recruited from the cytoplasm by integrins in both cell layers of the muscle attachment site (Brown, 2002).

The final overall shape of the salivary gland and its position within the developing embryo arise as a consequence of both its intrinsic properties and its interactions with surrounding tissues. This study focuses on the role of directed cell migration in shaping and positioning the Drosophila salivary gland. The salivary gland turns and migrates along the visceral mesoderm (VM) to become properly oriented with respect to the overall embryo. Salivary gland posterior migration requires the activities of genes that position the visceral mesoderm precursors, such as heartless, thickveins, and tinman, but does not require a differentiated visceral mesoderm. A role for integrin function in salivary gland migration is demonstrated. Although the mutations affecting salivary gland motility and directional migration cause defects in the final positioning of the salivary gland, most do not affect the length or diameter of the salivary gland tube. These findings suggest that salivary tube dimensions may be an intrinsic property of salivary gland cells (Bradley, 2003).

In htl, tkv, and tinman, the residual fragments of VM express Fas3, have a VM-like structure, and are able to direct salivary gland migration if present along its migratory path. Thus, the residual structures appeared to be differentiated VM with wild-type properties. To determine whether salivary gland migration requires a differentiated VM, embryos with mutations in the VM-specific gene biniou (bin) were examined. In bin mutant embryos, VM precursors segregate from dorsal mesoderm and move internally where they coalesce into the typical VM band; however, all tested VM-specific genes, including Fas3, fail to be expressed in bin mutants. Thus, an intact structure formed from VM precursors is present in bin mutants, but the VM precursor cells fail to express markers indicative of differentiation from a general mesodermal cell into a VM-specific cell. The salivary glands in bin mutants had no defects in turning or posterior migration, suggesting that guidance of salivary gland posterior migration by the VM requires neither the terminal differentiation of the precursors nor the function of any VM gene whose expression is bin-dependent (Bradley, 2003).

The VM forms a contiguous structure that may physically block salivary cells from further dorsal movement, thereby causing the cells to move posteriorly, in the path of least resistance. Alternatively or additionally, there may be a bin-independent factor (or factors) that guides salivary gland migration in a more instructive way, perhaps via a secreted signal or a transmembrane guidance molecule. If the mesodermal cue were informational, a signaling pathway functioning within salivary gland cells would have to be involved. A screen of several candidate pathways revealed that mutations disrupting the FGFR1-, FGFR2-, EGF-, DPP-, JNK-, or Wg-signaling pathway did not have phenotypes consistent with a role in the salivary cells for their migration. Thus, focus was placed on molecules known to have a more direct role in migration, specifically the integrin family of cell adhesion molecules, which are heterodimers of two transmembrane proteins, an alpha and a ß subunit. In Drosophila, each of the five identified alpha subunits (alpha_PS1-5) is thought to dimerize with the ß_PS subunit encoded by the myospheroid (mys) gene. The alpha subunit of alpha_PS2ß_PS (PS2) integrin is expressed in all mesodermal cells beginning at a very early stage, suggesting that PS2 integrin is likely to be present in the VM precursor cells prior to bin-dependent differentiation. Indeed, alpha_PS2 RNA expression was observed in the mesoderm of bin^R22 homozygotes. In embryos mutant for inflated (if), the gene encoding the alpha_PS2 subunit, migration of two tissues along the VM is affected, the endoderm and the tracheal visceral branch. Thus, the PS2 integrin is required to make the VM a suitable substrate for the migration of at least two distinct cell populations (Bradley, 2003).

Whether PS2 integrin is required for salivary gland migration was examined by staining if mutant embryos for several salivary gland proteins. In if homozygotes, salivary cells appear to invaginate normally. The first group of salivary cells to be internalized reaches the approximate level of the wild-type turning point but fails to migrate. During subsequent stages, the remaining if salivary cells continue to internalize, but the distal tip remains at the approximate VM turning point, and the tube is often slightly bent. By late stages, if salivary tubes are frequently folded in half with the distal tips oriented anteriorly. The apparent lack of salivary gland migration in if mutants is distinct from the mismigration phenotypes in htl, hbr, tkv, and tin mutants (Bradley, 2003).

Analysis of PINCH function in Drosophila demonstrates its requirement in integrin-dependent cellular processes

Integrins play a crucial role in cell motility, cell proliferation and cell survival. The evolutionarily conserved LIM protein PINCH is postulated to act as part of an integrin-dependent signaling complex. The molecular architecture of PINCH (Particularly Interesting New Cysteine-Histidine rich protein), which consists exclusively of multiple LIM domains suggests that it may function as a platform for the docking and/or productive juxtaposition of proteins involved in integrin signaling. In order to evaluate the role of PINCH in integrin-mediated cellular events, function of PINCH in Drosophila melanogaster was directly tested in vivo. The steamer duck (stck) alleles, that were first identified in a screen for potential integrin effectors, represent mutations in Drosophila pinch. stck mutants die during embryogenesis, revealing a key role for PINCH in development. Muscle cells within embryos that have compromised PINCH function display disturbed actin organization and cell-substratum adhesion. Mutation of stck also causes failure of integrin-dependent epithelial cell adhesion in the wing. Consistent with the idea that PINCH could contribute to integrin function, PINCH protein colocalizes with ßPS integrin at sites of actin filament anchorage in both muscle and wing epithelial cells. Furthermore, it is shown that integrins are required for proper localization of PINCH at the myotendinous junction. Integrin-linked kinase (Ilk), is also essential for integrin function. Drosophila PINCH and Ilk are complexed in vivo and are coincident at the integrin-rich muscle-attachment sites in embryonic muscle. Interestingly, Ilk localizes appropriately in stck mutant embryos, therefore the phenotypes exhibited by the stck mutants are not attributable to mislocalization of Ilk. These results provide direct genetic evidence that PINCH is essential for Drosophila development and is required for integrin-dependent cell adhesion (Clark, 2003).

The genetic analysis of PINCH function has led to four main conclusions: (1) Drosophila PINCH is encoded by the stck locus and is essential for embryonic development and maintenance of tissue architecture; (2) PINCH is necessary for stable actin-membrane anchorage in muscle and contributes to integrin-dependent adhesion in muscle cells and epithelial cells; (3) integrins are required for the stable association of PINCH with muscle-attachment sites; and (4) the lethal stck mutant phenotype cannot be attributed to mislocalization of the PINCH-binding partner, Ilk, whose recruitment to muscle-attachment sites appears normal in stck mutant embryos (Clark, 2003).

Genetic analyses of the roles of integrins in Drosophila have clearly highlighted the importance of integrins for adhesion and signaling in vivo. Drosophila PINCH is colocalized with integrins in both muscle and epithelial cells. Integrins retain the capacity to accumulate at muscle-attachment sites in stck mutants, illustrating that PINCH does not have an obligatory role in the proper processing and membrane targeting of integrins in vivo. The integrin staining in stck mutants does lack the high degree of order and lateral registration observed in wild-type embryos. In the Drosophila system, it is difficult to distinguish whether this modest disorganization simply reflects the underlying disturbance of the musculature or if it is revealing some contribution of PINCH to maintenance of spatially restricted integrin localization. In C. elegans embryos in which PINCH function is compromised by unc-97 mutation, both integrin and vinculin spread laterally beyond their normal zones of accumulation in dense plaques, suggesting a role for PINCH in clustering of adhesive junction components in this system (Clark, 2003).

Interestingly, PINCH depends on the presence of integrins for its stable accumulation at muscle-attachment sites. Several other proteins, including Talin, Ilk, Myosin II and Short stop colocalize with ßPS integrin at Drosophila muscle-attachment sites. These proteins display variable levels of dependence on integrins for their localization. Like Talin, a well-established integrin effector, PINCH depends on the presence of integrins for its concentration at muscle-attachment sites. The reliance of PINCH and Talin on integrins for their spatially restricted accumulation in muscle emphasizes their connection to the integrin receptors (Clark, 2003).

Integrins must establish links to both extracellular determinants and to intracellular cytoskeletal elements in order to support strong adhesion. Examination of the cellular defects in stck mutant muscle suggests that PINCH contributes to the stabilization of actin-membrane linkages at integrin-rich adhesion sites. In a stck mutant muscle cell, the actin filaments lose their linear organization and eventually accumulate in clumps at one end of the cell. These defects are interpreted to mean that a primary consequence of disturbed PINCH function is a destabilization of the linkage between the actin cytoskeleton and the muscle membrane; it appears that the actin-membrane attachments in stck mutants lack the mechanical strength to remain intact during cyclic muscle contraction. Because integrin functionality relies on the ability of the receptors to establish a transmembrane link between the cytoskeletal elements and the extracellular matrix, reduced substratum attachment strength and/or stability might also be expected to occur if membrane cytoskeletal linkages were compromised. Consistent with this prediction, loss of adhesion is evident in the stck^17-/- wing cell clones and, to some extent, in muscles of stck mutant embryos (Clark, 2003).

The molecular architecture of PINCH suggests that it may function as a platform for the docking and/or productive juxtaposition of protein partners. Ilk, a binding partner of PINCH, is thus a candidate to collaborate with PINCH in the stabilization of integrin-cytoskeletal linkages. Consistent with the view that PINCH and Ilk cooperate to promote stable actin anchorage at sites of integrin-mediated adhesion, the phenotypes that result from compromised function of either protein in Drosophila are very similar (Zervas, 2001; Clark, 2003). Moreover, PINCH and Ilk are colocalized in Drosophila embryos and are recovered in a protein complex isolated from embryos by immunoprecipitation. Drosophila PINCH also interacts directly with Ilk using two-hybrid methods. These results are consistent with findings for vertebrate PINCH and Ilk. PINCH and Ilk also colocalize at actin-membrane anchorage sites in C. elegans muscle, and elimination of either gene product was shown to produce a paralyzed at twofold stage (PAT) phenotype similar to that seen for ß-integrin mutants. Collectively, results in both invertebrate and vertebrate systems illustrate that the capacity to form a PINCH/Ilk complex has been conserved through evolution (Clark, 2003 and references therein).

Given the fact that Ilk and PINCH colocalize, co-precipitate and have similar loss of function phenotypes, it is possible that disturbed PINCH function could adversely affect Ilk localization and that such mislocalization might account for the stck mutant phenotype. To explore this possibility the localization of Ilk was examined in stck mutant embryos; Ilk was found to be unperturbed in its ability to accumulate at muscle-attachment sites, even when a dramatic lethal phenotype is evident in stck mutant embryos. As noted above, ßPS integrin also accumulates at muscle-attachment sites in stck mutant embryos. These findings illustrate that the proper localization of integrin and Ilk is not sufficient to stabilize actin membrane linkages at sites of integrin-dependent adhesion, and define PINCH as a critical component of the molecular machinery necessary for the tethering of actin to the integrin-rich membranes (Clark, 2003).

The demonstration that single ilk and stck mutants both display deficiencies in integrin-dependent processes illustrates that neither PINCH nor Ilk is sufficient on its own to support full integrin function. It is possible that PINCH acts as a positive regulator of Ilk function, either by modulating Ilk function by direct binding or by recruitment of an Ilk-modifying factor. Alternatively, Ilk may activate some PINCH function that is crucial for stabilization of actin-membrane linkages. Finally, a PINCH-Ilk protein complex may be a key component of the platform necessary for the recruitment of other proteins required to achieve stable actin-membrane associations. In this regard, it is of interest that PINCH and Ilk can be recovered in a complex with the Ilk-binding partner, CH-IlkBP, a calponin domain-containing protein related to Affixin and Actopaxin that could provide the link to actin filaments. Because the localization of Drosophila PINCH is dependent on integrins, the establishment of PINCH-Ilk complexes at muscle-attachment sites is not be supported in the absence of integrin function. This dependence of PINCH localization on integrins could provide a means to couple integrin adhesive function to its role in cytoskeletal anchorage (Clark, 2003).

In vertebrate cells, PINCH and Ilk appear to be mutually dependent on each other for their localization to integrin-rich focal adhesions (Zhang, 2002b). However, as noted above, despite their ability to interact with each other, PINCH and Ilk show distinct requirements for their recruitment to specific subcellular domains in Drosophila. In particular, it is shown that PINCH requires functional integrins for its localization to muscle-attachment sites, whereas it has previously been demonstrated that Drosophila Ilk fails to bind integrins directly and localizes normally in an integrin mutant. Rather than employing an association with integrins, Ilk may rely on a protein such as Paxillin for its targeting to integrin-rich sites. Although Drosophila PINCH requires integrins for its stable accumulation at muscle-attachment sites, there is no evidence that PINCH can associate directly with integrin cytoplasmic domains, therefore additional proteins probably act as a bridge (Clark, 2003 and references therein).

Crk-associated substrate (Cas) signaling protein functions with integrins to specify axon guidance during development

Members of the Cas family of Src homology 3 (SH3)-domain-containing cytosolic signaling proteins are crucial regulators of actin cytoskeletal dynamics in non-neuronal cells; however, their neuronal functions are poorly understood. This study identified a Drosophila Cas (DCas, CG1212, p130CAS; not to be confused with the Drosophila genes CAS/CSE1 segregation protein and castor), found that Cas proteins are highly expressed in neurons and showed that DCas is required for correct axon guidance during development. Functional analyses reveal that Cas specifies axon guidance by regulating the degree of fasciculation among axons. These guidance defects are similar to those observed in integrin mutants, and genetic analysis shows that integrins function together with Cas to facilitate axonal defasciculation. These results strongly support Cas proteins working together with integrins in vivo to direct axon guidance events (Huang, 2007).

In mammals, Cas proteins function downstream of several different receptors in non-neuronal cells, including growth factor receptors, G-protein-coupled receptors, T-cell receptors, B-cell receptors and integrins. Interestingly, integrin receptor subunit mutations in Drosophila give rise to CNS and motor axon guidance defects that are strikingly similar to those observed in DCas mutants, suggesting that Cas might function together with integrin receptors to guide axons (Huang, 2007).

Drosophila integrins, like vertebrate integrins, are composed of an α-subunit and a ß-subunit. In Drosophila, there is one gene encoding a typical laminin-binding-type α-subunit (α1, called mew), one encoding an RGD-binding-type α-subunit (α2, called if), and a single ß-subunit gene (ß1, called mys) very similar to the prototype vertebrate ß1 receptor. To investigate the connection between integrin and Cas signaling, the role of integrin receptor function in embryonic motor axon pathfinding was revisited and it was found that integrin-null mutant embryos exhibit defects that are qualitatively and quantitatively similar to DCas mutants. Embryos harboring null alleles for either α1 (mew^M6) or α2 (if^K27E) integrin genes exhibited ISNb and SNa axon guidance defects very similar to those observed in DCas mutants, including increased fasciculation resulting in the absence of muscle innervation. CNS axon guidance defects in were also observed both α1 and α2 integrin mutants that were similar to those observed in DCas mutants (Huang, 2007).

To further address DCas involvement in integrin-mediated axon guidance, dominant genetic interactions were sought between DCas and integrin subunit LOF mutations. Such transheterozygous interactions provide genetic support for two proteins functioning together in the same signaling pathway. It was asked whether removal of a single copy of DCas dominantly enhances heterozygosity at the α1, α2 or ß1 integrin loci. It was found that in α1, α2, ß1 or DCas heterozygotes, motor axon trajectories were not significantly different from wild type. However, removal of a single copy of DCas together with a single copy of α1, α2 or ß1 integrin resulted in highly penetrant axon guidance defects, suggesting that these three genes function in the same signaling pathway. Importantly, the phenotypes resulting from dominant enhancement by DCas are indicative of increased fasciculation, similar to those observed in DCas, α1 or α2 integrin LOF embryos (Huang, 2007).

To further assess the role integrins play in DCas-mediated axon guidance, it was asked whether ß1integrin LOF mutants dominantly suppress DCas GOF motor axon guidance phenotypes. The Drosophila ß1 integrin (encoded by mys¹) is the predominant neuronal ß1 integrin and therefore likely to mediate most, if not all, nervous system integrin signaling. If integrins are indeed necessary for activating DCas signaling, removing a single copy of ß1 integrin should suppress DCas GOF phenotypes. When low levels of DCas were expressed in all neurons in an otherwise wild-type background, moderate guidance defects were observed involving axons of the ISNb, SNa and CNS third longitudinal. Removing a single copy of the ß1 integrin gene in this same neuronal DCas GOF genetic background significantly rescued axon guidance phenotypes resulting from DCas GOF. Importantly, these results also show that neuronal overexpression of DCas does not simply function in a dominant-negative fashion to block integrin, or other, signaling pathways (Huang, 2007).

Supporting a model for how integrins and Cas regulate axonal fasciculation and pathfinding is extensive work on neuronal integrin functions in vitro. Growing axons and migrating cells preferentially elongate on surfaces to which they adhere most strongly, including integrin ligands. How might adhesive interactions influence axonal guidance decisions in vivo? Neuronal growth cones tend to form extensive lamellae, which are indicative of strong adhesive interactions, when cultured on highly adhesive substrata containing integrin ligands. These adhesive interactions stabilize elongating nerve fibers by promoting filopodial extension and expansion of growth cone surfaces. Disruption of axon-substrate attachment in vitro with integrin function-blocking antibodies encourages axon-axon adhesive interactions (fasciculation) in place of axon-substrate adhesion. Furthermore, contact with integrin ligands can slow axon elongation, as axons encountering an increasing gradient of laminin peptide exhibit reduced velocity, but growth cone velocity returns to previous rates when axons turn down the gradient. This in vitro observation resembles in vivo situations in which growth cones slow at a choice point, exhibit increased morphological complexity and then extend along distinct pathways. Drosophila motor axon growth cones also exhibit similar changes in morphological complexity upon contacting different substrates in vivo, suggesting that similar processes function to generate motor axon trajectories. Different combinations of integrin ligands might be responsible for these effects. When vertebrate growth cones in vitro contact either the α1 or α2 integrin ligands, laminin and fibronectin respectively, they decelerate, pause and exhibit short-term growth arrest. Interestingly, in vivo observations show that DCas functions with both α1 (laminin-binding) and α2 [RGD (e.g., Tiggrin)-binding] integrins to mediate correct axon navigation by regulating motor axon fasciculation at choice points, suggesting that integrin/Cas-mediated spatial regulation of growth cone extension underlies correct navigation at these choice points (Huang, 2007 and references therein).

The molecular mechanisms underlying integrin-mediated axon guidance remain to be completely defined. However, results derived from analysis of integrin/Cas signaling on cell migration shed light on how Cas and integrins might specify axonal defasciculation events in vivo. During cell migration, Cas proteins serve to establish linkage between migrating cells and the ECM. Cas plays an important role in regulating cytoskeletal organization, cell adhesion and force sensing, and fibroblasts isolated from p130Cas-null mutant mouse embryos exhibit disorganized and short actin filaments and decreased cell migration. In non-neuronal cells, Cas becomes phosphorylated in response to integrin engagement by many ECM components, including fibronectin and laminin (Defilippi, 2006). FAK and Src family kinases have been implicated in integrin-dependent phosphorylation of Cas. Interestingly, recent in vitro observations reveal that FAK signaling at sites of integrin-mediated adhesion controls axon pathfinding. Furthermore, pharmacological inhibitors of Src family kinases decrease the level of neuronal phosphorylated Cas in vitro, supporting a role for Src kinases in regulating Cas proteins in neurons. Finally, the activity of Rho-family small GTPases is also regulated by Cas interactions with the guanine nucleotide exchange factor Dock180 (Dock1) (Defilippi, 2006). Taken together, these links between Cas signaling components and cytoskeletal reorganization suggest that some of these signaling proteins might also influence axon guidance in vivo during development (Huang, 2007 and references therein).

The results demonstrate that integrin/Cas-mediated signaling is necessary but not sufficient for axonal defasciculation, revealing that integrin/Cas-mediated axon guidance must be integrated with other axon guidance signaling cascades to regulate axon defasciculation events during development. The identity of these other axon guidance pathways is not known. The attractive/permissive guidance cue Netrin binds to integrins, and functions with integrins in non-neuronal cells. The Netrin receptor Deleted in colorectal cancer (DCC) has been found to utilize the integrin effector FAK and recently p130Cas, to mediate Netrin-dependent attractive growth cone steering. Ephrins, best known for their role as repulsive axon guidance cues, also induce cell adhesion and actin cytoskeletal changes in fibroblasts in a p130Cas-dependent manner. Repulsive axon guidance cues may also regulate integrin/Cas-dependent axon guidance during development. The axonal repellent Slit genetically interacts with integrins and their ligands to guide commissural axons in Drosophila. Semaphorin and Ephrin-mediated repulsive effects on non-neuronal cells also appear to involve inhibition of integrin signaling events. Interestingly, a crucial component of semaphorin-dependent repulsive axon guidance, a member of the molecule interacting with Cas-L (MICAL) family, physically associates with Cas-L and preliminary data suggest that these interactions are functionally important for axon guidance. The observation that Cas functions with integrins to mediate axon guidance during development suggests new directions to better understand how integrin/Cas signaling modulates neuronal guidance through interactions with distinct axon guidance signaling pathways (Huang, 2007).

Larval

PS1 integrin is expressed primarily on the presumptive dorsal wing epithelium, and the PS2 integrin (alpha PS2 beta PS) is found almost exclusively on the ventral epithelium. Immediately after pupariation, the central wing pouch evaginates, folding along its center to appose the epithelia that will secrete the dorsal and ventral surfaces of the adult wing blade. Both of the PS integrins are required to maintain the close apposition of the dorsal and ventral wing epithelia during morphogenesis (Brower, 1989).

Wing disc ultrastructure is correlated with the distribution of the beta chain of integrin, laminin A, and filamentous actin for each key stage of pupal development. Integrin is present on the basal surface of intervein cells but not on vein cells whereas laminin A is absent from the basal surfaces of intervein cells but is present on vein cells. Laminin is not a ligand for integrin in this context. During apposition and adhesion stages integrin is broadly distributed over the basal and lateral surfaces of intervein cells but subsequently becomes localized to small basal foci. Basal adherens-type junctions are first evident during the adhesion stage and become closely associated with the cytoskeleton during the separation stage. Basal junction formation occurs in two discrete steps; intercellular connections are established first and junction/cytoskeletal connections are formed about 20 hours later (Fristrom, 1993). Drosophila serum response factor, otherwise known as Blistered is confined to intervein cells (Montagne, 1996).

A network of cell-cell contacts mediated by adherens junctions and cell-extracellular matrix contacts defines the architecture of the Drosophila ommatidium. This network is built incrementally; contacts established early in eye development typically persist into adulthood. Photoreceptor apical surfaces are involuted into the retinal epithelium and are subsequently elaborated to form the photosensitive rhabdomeres (see The Drosophila Adult Ommatidium: Illustration and explanation with Quicktime animation). Rhabdomeres become aligned to the ommatidial optical axis via their anchorage to the retinal floor at the cone cell plate, a specialized nexus of cell-cell and cell-extracellular matrix contacts. Several eye phenotypes trace their origin to the structural failure of the cone cell plate (Longley, 1995).

Oogenesis

Drosophila alpha-actinin in ovarian follicle cells is regulated by EGFR and Dpp signalling and required for cytoskeletal remodelling; alpha-actinin is involved in the assembly of a β-integrin-dependent adhesion site and Enabled localization

α-Actinin is an evolutionarily conserved actin filament crosslinking protein with functions in both muscle and non-muscle cells. In non-muscle cells, interactions between α-actinin and its many binding partners regulate cell adhesion and motility. In Drosophila, one non-muscle and two muscle-specific α-actinin isoforms are produced by alternative splicing of a single gene. In wild-type ovaries, α-actinin is ubiquitously expressed. The non-muscle α-actinin mutant Actn^Δ233, which is viable and fertile, lacks α-actinin expression in ovarian germline cells, while somatic follicle cells express α-actinin at late oogenesis. This latter population of α-actinin, termed FC-α-actinin, is shown to be absent from the dorsoanterior follicle cells, and evidence is presented that this is the result of a negative regulation by combined Epidermal growth factor receptor (EGFR) and Decapentaplegic signalling. Furthermore, EGFR signalling increases the F-actin bundling activity of ectopically expressed muscle-specific α-actinin. A novel morphogenetic event in the follicle cells is described that occurs during egg elongation. This event involves a transient repolarisation of the basal actin fibres and the assembly of a posterior β-integrin-dependent adhesion site accumulating α-actinin and Enabled. Clonal analysis using Actn null alleles demonstrated that although α-actinin is not necessary for actin fibre formation or maintenance, the cytoskeletal remodelling is perturbed, and Enabled does not localise in the posterior adhesion site. Nevertheless, epithelial morphogenesis proceeded normally. This work provides the first evidence that α-actinin is involved in the organisation of the cytoskeleton in a non-muscle tissue in Drosophila (Wahlström, 2006).

To understand how α-actinin is involved in the function of the follicle cells at late oogenesis, α-actinin localisation was studied in wild-type follicle cells at stages 10-14. For detection, used the monoclonal antibody MAC276, which recognises all three α-actinin isoforms was used, along with a staining protocol that does not allow simultaneous labelling of F-actin with phalloidin. The follicle cells are polarised with the apical side facing the germline and the basal side facing the epithelial sheath surrounding each string of developing egg chambers. At the time of egg chamber assembly, the basal surface of the follicle cells acquires a layer of stress fibre-like actin bundles, which is maintained throughout oogenesis. At stage 10A, α-actinin was localised at the cell cortex (not shown) and was especially abundant in the basal actin fibres. At stage 10B/11, the evenly stained actin fibres began to reorganise, and by stage 11, a distinct patch of α-actinin accumulation was detected in the posterior part of the cell. This pattern was seen in all main body follicle cells, i.e. ventral follicle cells and dorsal cells posterior to the dorsal appendages. In the dorsoanterior follicle cells, α-actinin was expressed at lower levels and showed less distinct localisation patterns. α-Actinin was also detected at the roof cell apices of the elongating dorsal appendages. At the end of stage 12, the basal α-actinin pattern in the main body follicle cells was reorganised again. The accumulation at the posterior end of the cell gradually dispersed, and at stage 13, α-actinin was concentrated at the lateral cell margins. The central actin fibres were less strongly labelled. By stage 14, when the basal actin fibres have disappeared, α-actinin displayed a cortical localisation (Wahlström, 2006).

The lateral stripes of α-actinin in the follicle cells at stage 13 correspond to the previously described adhesion sites shown to contain β-integrin and Ena. Integrins are transmembrane receptors for ligands in the extracellular matrix (ECM), and they mediate adhesion between the cell and the ECM. Ena is the sole Drosophila member of the conserved family of Ena/VASP proteins, which act as positive regulators of actin filament assembly. Co-localisation studies of α-actinin and Ena revealed a complete overlap in the basal cytoskeleton, including the posterior patch, during stages 11 and 12. At stage 13, there was also extensive co-localisation, although Ena appeared to be located closer to the cell margin than α-actinin. Thus, both α-actinin and Ena accumulate in a transient adhesion site-like structure that forms at the onset of egg elongation (Wahlström, 2006).

The basal stress fibres are aligned perpendicular to the A/P axis of the oocyte between stages 7 and 10, but then a phase of slight disorganisation occurs before the perpendicular alignment is reassumed by stage 13. The disorganised phase correlates with the relocalisation of α-actinin and Ena observed in the basal cytoskeleton. The remodelling could also be recognised by phalloidin-staining of the actin fibres, although they indeed appeared quite irregular in most cells. In several cells, they are polarised in the A/P direction, and they often also converge in a denser patch of F-actin, which overlaps with the posterior patch containing Ena and α-actinin. Thus, egg elongation involves an organised repolarisation of the basal actin fibres (Wahlström, 2006).

Analysis of the α-actinin expression pattern in the non-muscle mutant Actn^Δ233 revealed that at least two separate populations of α-actinin are present in the follicle cells. α-Actinin produced from an mRNA that is transcribed from the upstream promoter (NC-α-actinin) is ubiquitously expressed in the egg chamber. The second α-actinin population, FC-α-actinin, corresponds to α-actinin that is present in certain non-muscle cells of all examined non-muscle-specific α-actinin mutants. FC-α-actinin is most likely produced from an mRNA transcribed from the downstream promoter and may include both non-muscle α-actinin and adult muscle-specific α-actinin. However, an analysis using isoform-specific antibodies or a complete sequencing of the mRNAs expressed in the egg chamber will be required in order to clarify this issue. FC-α-actinin protein was expressed in the main body follicle cells starting from stage 10, but excluded from the dorsoanterior cells. The dorsoanterior cells are patterned by the EGFR and Dpp signalling pathways, and the results showed that these two pathways together downregulate FC-α-actinin expression, but not the expression of NC-α-actinin (Wahlström, 2006).

The dorsoanterior and main body follicle cells undergo very different morphogenetic changes. The dorsoanterior cells elongate in the apicobasal direction and migrate (Dorman, 2004), an event that did not seem to require α-actinin. In contrast, the main body follicle cells flatten and expand their surfaces. These events are expected to involve different sets of cytoskeletal regulators, of which very little is yet known. The formation of a dense layer of basal actin fibres in the main body follicle cells may include upregulation of proteins known to be involved in stress fibre formation, such as α-actinin. It has been shown that the dorsal midline cells upregulate basal E-cadherin and FasIII, indicating increased cell-cell adhesion. These cells also lose their basal actin fibres, which may explain why less α-actinin, i.e., only NC-α-actinin, is expressed in these cells (Wahlström, 2006).

Throughout oogenesis, the basal cytoskeleton is organised into actin fibres aligned in parallel. Variation has been noted in the actin fibre polarity at the late stages of oogenesis. These observations are extended by showing that the basal cytoskeleton undergoes an organised remodelling during the final stages of oogenesis. The rapid increase in oocyte volume during nurse cell dumping at stage 11 requires that the follicle cells expand their surfaces in order to maintain a coherent epithelium. This process involves a transient change in the polarity of the basal actin fibres, from a perpendicular to a parallel orientation relative to the A/P axis of the egg chamber, and the assembly of a transient structure that accumulates α-actinin and Ena. The association of this structure with an accumulation of β-integrin and its dependence on integrin adhesion demonstrate that the cytoskeletal reorganisation is linked to a remodelling of integrin-based adhesion sites. The fact that α-actinin and β-integrin did not show a strict co-localisation is in good agreement with studies on mammalian cells showing that integrin, but not α-actinin, is present in nascent adhesion sites termed focal complexes. α-Actinin accumulation in the adhesion site occurs later, as the focal complexes mature into focal adhesions (Zaidel-Bar, 2003). The signal that induces the remodelling of the basal cytoskeleton remains to be identified. An intriguing possibility is that the mechanical stress applied to the epithelium during nurse cell dumping is transduced into biochemical signals that result in the observed reorganisation. Two different mechanisms are known to mediate mechanotransduction: stretch-activated ion channels or conformational changes within cell-matrix adhesion sites (Wahlström, 2006).

The current view is that the parallel basal actin fibres shape the oocyte during egg elongation by preventing axial expansion. However, since the basal actin fibres repolarise during egg elongation, the current model does not adequately explain how the oocyte acquires its final shape. The fact that Ena accumulates in the posterior of the cell during egg elongation suggests that a mechanism involving localised actin polymerisation and directed cell growth may also contribute to shaping the oocyte. It has been reported that egg elongation is blocked by mutations in the genes encoding α-integrin, β-integrin, the adhesion site components talin or tensin, the receptor tyrosine phosphatase Dlar or the ECM component Laminin A. In the case of β-integrin and Dlar, it has been shown that the actin fibre polarity is disturbed (Bateman, 2001; Frydman, 2001), and this has been suggested to be the cause of the short egg phenotype. However, the data presented in this work give reason to speculate that defective adhesion between the follicle cells and the ECM might play a role as well (Wahlström, 2006).

To explore the function of α-actinin in the main body follicle cells, clones of cells lacking α-actinin were generated. This experiment unexpectedly revealed that α-actinin is not required for the formation or maintenance of the basal actin fibres. Previous studies, relying on the introduction of truncated α-actinin molecules into cultured mammalian cells, have suggested that α-actinin is important for stress fibre maintenance. Furthermore, examination of transformed cells expressing different levels of α-actinin showed that cells with low α-actinin levels had poorly developed stress fibres, an effect was not observe in this study. It is possible that the follicle cell basal actin fibres are not true contractile stress fibres and therefore do not depend on α-actinin. Alternatively, an alternative pathway for stress fibre assembly that is independent of α-actinin might be activated in the follicle cells following removal of α-actinin (Wahlström, 2006 and references therein).

The clonal analysis revealed that while α-actinin was not necessary for the lateral accumulation of Ena at stage 13, it was cell-autonomously required for the posterior localisation of Ena at stages 11 and 12. The reason for this could be that α-actinin is specifically required for recruiting Ena to the posterior adhesion site, perhaps by recruiting their common binding partner zyxin. Alternatively, the posterior adhesion site may not form at all. The latter possibility is supported by observations on mosaic stage 10B/11 egg chambers that are in the process of assembling the posterior adhesion site. While wild-type cells are in the process of translocating Ena towards the posterior, neighbouring cells lacking α-actinin still showed a lateral Ena pattern. At stage 12/13, the lateral adhesion sites are assembled earlier in the mutant cells than in the wild-type cells, perhaps because the mutant cells had not reorganised their cytoskeleton to the same extent as the wild-type cells had. Thus, these results clearly demonstrate that adhesion site remodelling is altered in the absence of α-actinin. However, in contrast to the cells lacking β-integrin, the Actn mutant cells appear to maintain their adhesion to the ECM, since they appear equally well spread as the wild-type cells at stage 13 (Wahlström, 2006).

The results are in agreement with the current view that vertebrate α-actinin is involved in adhesion site disassembly. This is a strictly regulated process that involves signalling by phosphoinositides, tyrosine phosphorylation and proteolytic cleavage of individual components. α-Actinin is one of the targets for these activities. Phosphorylation of α-actinin by focal adhesion kinase (FAK) reduces α-actinin’s affinity for F-actin and regulates the activity of FAK itself, PtdIns(3,4,5)-P₃ binding to α-actinin disrupts α-actinin binding to β-integrin and F-actin, and cleavage of α-actinin by calpain has been associated with cell shape changes in certain cell types. It has also been shown that α-actinin is essential for maintaining the link between the adhesion site and the stress fibre. This conclusion was reached based on an experiment showing that laser-mediated inactivation of α-actinin located in an adhesion site resulted in stress fibre detachment from the adhesion site. In the Drosophila follicle cells, α-actinin is clearly not required for actin fibre attachment. However, by the laser-mediated inactivation, α-actinin was removed from an adhesion site, whereas in the Actn null mutant follicle cells, the adhesion sites never contained α-actinin. Considering the large number of proteins that interact with α-actinin, it is expected that a signal targeted at α-actinin indirectly affects many other proteins and processes as well. An adhesion site lacking α-actinin may well be functional, but it may respond differently to various signals that induce adhesion site remodelling (Wahlström, 2006).

Interestingly, even very large clones of Actn mutant cells had no negative effects on egg morphology. This indicates that proper cytoskeletal remodelling and posterior localisation of Ena is not necessary for egg elongation. Apparently, the expansion of the main body follicle cells in the Actn mutant cells occurs by an alternative mechanism that is not dependent on α-actinin. This raises the question of whether the wild-type remodelling mechanism would become important under some specific conditions not prevailing in the laboratory. The impact of the environment on the development of mutant phenotypes has been well documented in the slime mould Dictyostelium discoideum. Lack of α-actinin results in only minor alterations in cellular functions and did not reduce viability. However, when the cells were grown under conditions resembling their natural habitat, specific developmental defects appeared (Wahlström, 2006).

This study contributes new data to the field of cytoskeletal dynamics in Drosophila follicle cells. A surprisingly complex regulation was undercovered underlaying α-actinin expression in the follicle cells. The basal cytoskeleton of the main body follicle cells undergoes an organised remodelling during egg elongation, and α-actinin is required in this process. This observation provides the first identified phenotype in a Drosophila non-muscle tissue lacking α-actinin. The fact that both loss of α-actinin and overexpression of α-actinin results in very distinct cellular phenotypes suggests that the follicular epithelium could serve as a very useful in vivo system for further studies on mechanisms that regulate α-actinin function and activity. Furthermore, the cytoskeletal remodelling may provide an easily accessible and genetically tractable model for studies on adhesion dynamics in vivo (Wahlström, 2006).

Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.