Requirements for integrins during Drosophila development

The common beta subunit of the PS antigens of Drosophila is homologous with vertebrate integrins and is encoded by the lethal(1)myospheroid gene. We have generated flies mosaic for wild-type and mutant alleles of lethal(1)myospheroid using adult gynandromorphs and radiation-induced somatic crossing over. The defects observed in the gynandromorphs demonstrate widespread requirements for PS integrins during development especially in ventrally derived structures, which also show strong expression of PS beta integrin. Smaller lethal(1)myospheroid clones induced during larval development result in blister and vein defects in the wings and aberrant development of photoreceptor cells, demonstrating roles for PS integrins during development of both wings and eyes. PS integrins are required for the close apposition of the dorsal and ventral wing epithelia and for the proper arrangement of photoreceptor cells. However, many other adhesive and morphogenetic processes proceed normally in the absence of integrins containing the beta subunit encoded by lethal(1)myospheroid.     

Integrins modulate the Egfr signaling pathway to regulate tendon cell differentiation in the Drosophila embryo

Changes in the extracellular matrix (ECM) govern the differentiation of many cell types during embryogenesis. Integrins are cell matrix receptors that play a major role in cell-ECM adhesion and in transmitting signals from the ECM inside the cell to regulate gene expression. In this paper, it is shown that the PS integrins are required at the muscle attachment sites of the Drosophila embryo to regulate tendon cell differentiation. The analysis of the requirements of the individual alpha subunits, alphaPS1 and alphaPS2, demonstrates that both PS1 and PS2 integrins are involved in this process. In the absence of PS integrin function, the expression of tendon cell-specific genes such as stripe and beta1 tubulin is not maintained. In addition, embryos lacking the PS integrins also exhibit reduced levels of activated MAPK. This reduction is probably due to a downregulation of the Epidermal Growth Factor receptor (Egfr) pathway, since an activated form of the Egfr can rescue the phenotype of embryos mutant for the PS integrins. Furthermore, the levels of the Egfr ligand Vein at the muscle attachment sites are reduced in PS mutant embryos. Altogether, these results lead to a model in which integrin-mediated adhesion plays a role in regulating tendon cell differentiation by modulating the activity of the Egfr pathway at the level of its ligand Vein.     

A role for integrin in the formation of sarcomeric cytoarchitecture

We propose that 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. We found that integrin alpha PS2 beta PS accumulated at contact regions of Drosophila embryo cells cultured in D-22 medium on Drosophila laminin. Myotubes formed, but subsequent addition of serum or fibronectin was needed for sarcomere formation: integrin and actin became concentrated at Z-bands; myosin and actin occurred between the Z-bands. This change failed to occur in the multinucleate myotubes derived from integrin beta PS null myospheroid mutants. In normal embryos/early larvae, integrin was located at Z-bands and at muscle insertions. Myogenesis and Z-bands were defective in myospheroid embryos. Attachment, spreading, and growth of myoblasts and neurons on the laminin substrate utilized different binding proteins and were independent of integrin.     

The function of PS integrins in Drosophila wing morphogenesis.

Integrins are found on many cell types during the development of most organisms. In Drosophila their functions can be analysed genetically. An analysis of lethal mutations in a PS integrin gene showed that the integrins were required for muscle attachment and for certain cell sheet migrations during embryogenesis. In this paper we use viable mutations in integrin component genes to look at integrin function in the later stages of development of one adult structure, the wing. We show that two known viable mutations, one which has its primary effect on the fly's escape response, the other on wing morphogenesis, are mutations in the beta and PS2alpha subunits, respectively, of the PS integrins. The mutation non-jumper (mys(mj42)) in the beta subunit leads to wasting of the thoracic jump muscles. Flies in which the dosage of this allele is reduced (and no wildtype copy is present) show defects also in wing morphogenesis. The two surfaces of the wing fail to connect properly, resulting in 'blistering' of the wing and the formation of extra crossveins. The mutation in the gene for the PS2alpha integrin subunit, inflated, also leads to a failure in wing surface apposition and consequent wing blistering. When the two mutations are combined, the mutant phenotype is greatly enhanced. Thus, one of the roles of the PS integrins in late Drosophila development is to ensure the correct apposition and patterning of the wing epithelia.     

The Drosophila PS2 antigen is an invertebrate integrin that, like the fibronectin receptor, becomes localized to muscle attachments

We establish that the position-specific antigen 2 (PS2), a Drosophila cell surface glycoprotein complex, is an invertebrate member of the vertebrate fibronectin receptor (integrin) family. New monoclonal antibodies show that in Drosophila embryos and larvae PS2 alpha subunits have a size of ca. 140 kd. Analysis of cDNA and genomic clones revealed that the canonical PS2 alpha subunit contains 1394 amino acids and has extensive homology to the heavy and light chains of integrin alpha subunits. The distribution of the PS2 antigen is regulated at the level of PS2 alpha subunit mRNA. In early Drosophila development the protein is restricted to mesoderm and appears to be involved in muscle attachment. We suggest that PS2, like vertebrate fibronectin receptors, mediates changes in cell shape and cell-extracellular matrix adhesion by binding to a basement membrane protein.     

Characterization of Mutant Alleles of Myospheroid, the Gene Encoding the β Subunit of the Drosophila PS Integrins

This paper presents the characterization of nine alleles of myospheroid, which encodes the beta PS subunit of the Drosophila PS integrins. On Southern blots, the mysXB87, mysXN101 and mysXR04 genes yield restriction digest patterns similar to that seen for wild-type chromosomes, however the mys1 and mysXG43 genes contain detectable deletions. mys1, mysXB87 and mysXG43 make little or no stable protein product, and genetically behave as strong lethal alleles. For the mysXN101 mutation, protein product is seen on immunoblots and a reduced amount of beta PS protein is seen at muscle attachment sites of embryos; this mutant protein retains some wild-type function, as revealed by complementation tests with weak alleles. Protein is also seen on immunoblots from mysXR04 embryos, and this allele behaves as an antimorph, being more deleterious in some crosses than the complete deficiency for the locus. mysts2 and mysnj42 are typically lethal in various combinations with other alleles at high temperatures only, but even at high physiological temperatures, neither appears to eliminate gene function completely. The complementation behaviors of mysts1 and mysts3 are quite unusual and suggest that these mutations involve regulatory phenomena. For mysts3, the data are most easily explained by postulating transvection effects at the locus. The results for mysts1 are less straightforward, but point to the possibility of a chromosome pairing-dependent negative interaction.     

Drosophila PS integrins recognize vertebrate vitronectin and function as cell-substratum adhesion receptors in vitro.

Using the Drosophila cell line MLDmBG-1, a monoclonal antibody aBG-1 that can inhibit not only cell clumping but also cell spreading was generated. This antibody immunoprecipitates a complex of molecules consisting of a major 120 x 10(3) Mr and other components. To characterize the 120 x 10(3) Mr component, we purified it, generated antibodies to it, and cloned its cDNA. Sequencing of this cDNA suggests that the 120 x 10(3) Mr molecule is identical to PS beta, a beta chain of Drosophila integrins. The other components immunoprecipitated included two alpha chains of Drosophila integrins, PS1 alpha and PS2 alpha, as revealed using specific antibodies to these molecules. These suggest that aBG-1 recognizes the PS beta associated with PS1 alpha or PS2 alpha. However, immunostaining of embryos and larvae with aBG-1 showed that the staining pattern is similar to that for PS2 alpha but not for PS beta, suggesting that the antibody preferentially recognizes the PS beta associated with particular alpha chains in situ. We then attempted to characterize the ligands for these integrin complexes, using culture dishes coated with various vertebrate matrix proteins. These cells spread very well on dishes coated with vitronectin and, to a lesser extent, on those with fibronectin. This spreading was partially inhibited by aBG-1, but not by other control antibodies or RGD peptides. The cell attachment to these substrata was not affected by the antibody. The cells also can attach to dishes coated with laminin but without spreading, and this attachment was not inhibited by aBG-1. Furthermore, they do not attach to dishes coated with collagen type I, type IV, and fibrinogen. These results indicate that Drosophila PS integrins can recognize vertebrate vitronectin, and also fibronectin with a weaker affinity, at sites other than RGD sequences, and thus can function in cell-substratum adhesion.     

Migration of the Drosophila primordial midgut cells requires coordination of diverse PS integrin functions.

Cell migration during embryogenesis involves two populations of cells: the migrating cells and the underlying cells that provide the substratum for migration. The formation of the Drosophila larval midgut involves the migration of the primordial midgut cells along a visceral mesoderm substratum. We show that integrin adhesion receptors are required in both populations of cells for normal rates of migration. In the absence of the PS integrins, the visceral mesoderm is disorganised, the primordial midgut cells do not display their normal motile appearance and their migration is delayed by 2 hours. Removing PS integrin function from the visceral mesoderm alone results in visceral mesoderm disorganization, but only causes a modest delay in migration and does not affect the appearance of the migrating cells. Removing PS integrin function from the migrating cells causes as severe a delay in migration as the complete loss of PS integrin function. The functions of PS1 and PS2 are specific in the two tissues, endoderm and mesoderm, since they cannot substitute for each other. In addition there is a partial redundancy in the function of the two PS integrins expressed in the endoderm, PS1 (alphaPS1betaPS) and PS3 (alphaPS3betaPS), since loss of just one alpha subunit in the midgut results in either a modest delay (alphaPS1) or no effect (alphaPS3). We have also examined the roles of small GTPases in promoting migration of the primordial midgut cells. We find that dominant negative (N17) versions of Rac and Cdc42 cause a very similar defect in migration as loss of integrins, while those of Rho and Ras have no effect. Thus integrins are involved in mediating migration by creating an optimal substratum for adhesion, adhering to that substratum and possibly by activating Rac and Cdc42.     

A role for PS integrins in morphological growth and synaptic function at the postembryonic neuromuscular junction of Drosophila

A family of three position-specific (PS) integrins are expressed at the Drosophila neuromuscular junction (NMJ): a beta subunit ((betaPS), expressed in both presynaptic and postsynaptic membranes, and two alpha subunits (alphaPS1, alphaPS2), expressed at least in the postsynaptic membrane. PS integrins appear at postembryonic NMJs coincident with the onset of rapid morphological growth and terminal type-specific differentiation, and are restricted to type I synaptic boutons, which mediate fast, excitatory glutamatergic transmission. We show that two distinctive hypomorphic mutant alleles of the beta subunit gene myospheroid (mys(b9) and mys(ts1)), differentially affect betaPS protein expression at the synapse to produce distinctive alterations in NMJ branching, bouton formation, synaptic architecture and the specificity of synapse formation on target cells. The mys(b9) mutation alters betaPS localization to cause a striking reduction in NMJ branching, bouton size/number and the formation of aberrant 'mini-boutons', which may represent a developmentally arrested state. The mys(ts1) mutation strongly reduces betaPS expression to cause the opposite phenotype of excessive synaptic sprouting and morphological growth. NMJ function in these mutant conditions is altered in line with the severity of the morphological aberrations. Consistent with these mutant phenotypes, transgenic overexpression of the betaPS protein with a heat-shock construct or tissue-specific GAL4 drivers causes a reduction in synaptic branching and bouton number. We conclude that betaPS integrin at the postembryonic NMJ is a critical determinant of morphological growth and synaptic specificity. These data provide the first genetic evidence for a functional role of integrins at the postembryonic synapse.     

Morphogenesis in the absence of integrins: mutation of both Drosophila beta subunits prevents midgut migration

Two integrin beta subunits are encoded in the Drosophila genome. The betaPS subunit is widely expressed and heterodimers containing this subunit are required for many developmental processes. The second betasubunit, betanu, is a divergent integrin expressed primarily in the midgut endoderm. To elucidate its function, we generated null mutations in the gene encoding betanu. We find that betanu is not required for viability or fertility, and overall the mutant flies are normal in appearance. However, we could observe betanu function in the absence of betaPS. Consistent with its expression, removal of betanu only enhanced the phenotype of betaPS in the developing midgut. In embryos lacking the zygotic contribution of betaPS, loss of betanu resulted in enhanced separation between the midgut and the surrounding visceral mesoderm. In the absence of both maternal and zygotic betaPS, a delay in midgut migration was observed, but removing betanu as well blocked migration completely. These results demonstrate that the second beta subunit can partially compensate for loss of betaPS integrins, and that integrins are essential for migration of the primordial midgut cells. The two beta subunits mediate midgut migration by distinct mechanisms: one that requires talin and one that does not. Other examples of developmental cell migration, such as that of the primordial germ cells, occurred normally in the absence of integrins. Having generated the tools to eliminate integrin function completely, we confirm that Drosophila integrins do not control proliferation as they do in mammals, and have identified alphaPS3 as a heterodimeric partner for betanu.     

Drosophila position-specific antigens resemble the vertebrate fibronectin-receptor family.

The Drosophila position-specific (PS) antigens are a family of cell surface glycoprotein complexes thought to be involved in morphogenesis. Their overall structures and biochemical properties are similar to those of a group of vertebrate receptors, including those for fibronectin, fibrinogen and vitronectin, and also the leukocyte antigens Mac-1, LFA-1 and p150,95 and the VLA family of cell surface antigens. The N-terminal sequences of the alpha subunits of some of these molecules are homologous to the N-terminus of a PS antigen component. The Drosophila PS antigens thus appear to be homologous to these vertebrate receptors.     

WNT5 interacts with the Ryk receptors doughnut and derailed to mediate muscle attachment site selection in Drosophila melanogaster

In recent years a number of the genes that regulate muscle formation and maintenance in higher organisms have been identified. Studies employing invertebrate and vertebrate model organisms have revealed that many of the genes required for early mesoderm specification are highly conserved throughout evolution. Less is known about the molecules that mediate the steps subsequent to myogenesis, e. g. myotube guidance and attachment to tendon cells. We use the stereotypic pattern of the Drosophila embryonic body wall musculature in genetic approaches to identify novel factors required for muscle attachment site selection. Here, we show that Wnt5 is needed in this process. The lateral transverse muscles frequently overshoot their target attachment sites and stably attach at novel epidermal sites in Wnt5 mutant embryos. Restoration of WNT5 expression in either the muscle or the tendon cell rescues the mutant phenotype. Surprisingly, the novel attachment sites in Wnt5 mutants frequently do not express the Stripe (SR) protein which has been shown to be required for terminal tendon cell differentiation. A muscle bypass phenotype was previously reported for embryos lacking the WNT5 receptor Derailed (DRL). drl and Wnt5 mutant embryos also exhibit axon path finding errors. DRL belongs to the conserved Ryk receptor tyrosine kinase family which includes two other Drosophila orthologs, the Doughnut on 2 (DNT) and Derailed-2 (DRL-2) proteins. We generated a mutant allele of dnt and find that dnt, but not Drl-2, mutant embryos also show a muscle bypass phenotype. Genetic interaction experiments indicate that drl and dnt act together, likely as WNT5 receptors, to control muscle attachment site selection. These results extend previous findings that at least some of the molecular pathways that guide axons towards their targets are also employed for guidance of muscle fibers to their appropriate attachment sites.     

The role of DE-cadherin during cellularization, germ layer formation and early neurogenesis in the Drosophila embryo

The Drosophila E-cadherin homolog, DE-cadherin, is expressed and required in all epithelial tissues throughout embryogenesis. Due to a strong maternal component of DE-cadherin, its early function during embryogenesis has remained elusive. The expression of a dominant negative DE-cadherin construct (UAS-DE-cad(ex)) using maternally active driver lines allowed us to analyze the requirements for DE-cadherin during this early phase of development. Maternally expressed DE-cad(ex) result in phenotype with variable expressivity. Most severely affected embryos have abnormalities in epithelialization of the blastoderm, resulting in loss of the blastodermal cells' apico-basal polarity and monolayered structure. Another phenotypic class forms a rather normal blastoderm, but shows abnormalities in proliferation and morphogenetic movements during gastrulation and neurulation. Mitosis of the mesoderm occurs prematurely before invagination, and proliferation in the ectoderm, normally a highly ordered process, occurs in a random pattern. Mitotic spindles of ectodermal cells, normally aligned horizontally, frequently occurred vertically or at an oblique angle. This finding further supports recent findings indicating that, in the wild-type ectoderm, the zonula adherens is required for the horizontal orientation of mitotic spindles. Proliferation defects in DE-cad(ex)-expressing embryos are accompanied by the loss of epithelial structure of ectoderm and neuroectoderm. These germ layers form irregular double or triple layers of rounded cells that lack zonula adherens. In the multilayered neuroectoderm, epidermal precursors, neuroblasts and ganglion mother cells occurred intermingled, attesting to the pivotal role of DE-cadherin in delamination and polarized division of neuroblasts. By contrast, the overall number and spacing of neuroblasts was grossly normal, indicating that DE-cadherin-mediated adhesion is less important for cell-cell interaction controlling the ratio of epidermal vs. neural progenitors.     

Hindgut visceral mesoderm requires an ectodermal template for normal development in Drosophila

During Drosophila embryogenesis, the development of the midgut endoderm depends on interactions with the overlying visceral mesoderm. Here we show that the development of the hindgut also depends on cellular interactions, in this case between the inner ectoderm and outer visceral mesoderm. In this section of the gut, the ectoderm is essential for the proper specification and differentiation of the mesoderm, whereas the mesoderm is not required for the normal development of the ectoderm. Wingless and the fibroblast growth factor receptor Heartless act over sequential but interdependent phases of hindgut visceral mesoderm development. Wingless is required to establish the primordium and to enhance Heartless expression. Later, Heartless is required to promote the proper differentiation of the hindgut visceral mesoderm itself.     

PS integrins and laminins: key regulators of cell migration during Drosophila embryogenesis

During embryonic development, there are numerous cases where organ or tissue formation depends upon the migration of primordial cells. In the Drosophila embryo, the visceral mesoderm (vm) acts as a substrate for the migration of several cell populations of epithelial origin, including the endoderm, the trachea and the salivary glands. These migratory processes require both integrins and laminins. The current model is that αPS1βPS (PS1) and/or αPS3βPS (PS3) integrins are required in migrating cells, whereas αPS2βPS (PS2) integrin is required in the vm, where it performs an as yet unidentified function. Here, we show that PS1 integrins are also required for the migration over the vm of cells of mesodermal origin, the caudal visceral mesoderm (CVM). These results support a model in which PS1 might have evolved to acquire the migratory function of integrins, irrespective of the origin of the tissue. This integrin function is highly specific and its specificity resides mainly in the extracellular domain. In addition, we have identified the Laminin α1,2 trimer, as the key extracellular matrix (ECM) component regulating CVM migration. Furthermore, we show that, as it is the case in vertebrates, integrins, and specifically PS2, contributes to CVM movement by participating in the correct assembly of the ECM that serves as tracks for migration.     

Expression of nicotinic acetylcholine receptors in aneural Xenopus embryos

During gastrulation in vertebrate embryos, the mesoderm moves inward and under the ectoderm and these two cell layers subsequently differentiate in close proximity to each other, providing an opportunity for the exchange of inductive signals. This study examines whether the activation of muscle nicotinic acetylcholine receptor (AChR) genes and the subsequent expression of receptors in Xenopus myotomal muscle are dependent on interaction between the ectoderm and the mesoderm, or their derivatives, after the onset of gastrulation. We eliminated such interaction by inducing total exogastrulation of Xenopus embryos. During exogastrulation, the mesoderm moves away from the ectoderm, and the nervous system fails to develop. Single channel recordings from the myotomal muscle of exogastrulated embryos revealed the presence of two major classes of AChRs, which could be distinguished on the basis of channel conductance. The current amplitudes, conductances, reversal potentials, and open times of these channels closely resembled those reported for the two major classes of AChR channels normally expressed in vivo. We conclude that interaction between ectoderm and mesoderm following the onset of gastrulation is not required for the future expression of the major classes of AChRs in myotomal muscle.     

Cloning and characterization of Dfak56, a homolog of focal adhesion kinase, in Drosophila melanogaster.

The focal adhesion kinase (FAK) protein-tyrosine kinase plays important roles in cell adhesion in vertebrates. Using polymerase chain reaction-based cloning strategy, we cloned a Drosophila gene that is homologous to the vertebrate FAK family of protein-tyrosine kinases. We designated this gene Dfak56 and characterized its gene product. The overall protein structure and deduced amino acid sequence of Dfak56 show significant similarity to those of FAK and PYK2. Dfak56 has in vitro autophosphorylation activity at tyrosine residues. Expression of the Dfak56 mRNA and the protein was observed in the central nervous system and the muscle-epidermis attachment site in the embryo, where Drosophila position-specific integrins are localized. The results suggest that like FAK in vertebrates, Dfak56 functions downstream of integrins. Dfak56 was tyrosine-phosphorylated upon integrin-dependent attachment of the cell to the extracellular matrix. We conclude that the Dfak56 tyrosine kinase is involved in integrin-mediated cell adhesion signaling and thus is a functional homolog of vertebrate FAK.     

Delayed behavioural aging and altered mortality in Drosophila beta integrin mutants

The genetic basis for aging is being intensely investigated in a variety of model systems. Much of the focus in Drosophila has been on the molecular-genetic determinants of lifespan, whereas the molecular-genetic basis for age-related functional declines has been less vigorously explored. We evaluated behavioural aging and lifespan in flies harbouring loss-of-function mutations in myospheroid, the gene that encodes betaPS, a beta integrin. Integrins are adhesion molecules that regulate a number of cellular processes and developmental events. Their role in aging, however, has received limited attention. We report here that age-related declines in locomotor activity are ameliorated and that mean lifespan is increased in myospheroid mutants. The delayed functional senescence and altered mortality in myospheroid flies are independent of changes in body size, reproduction or stress resistance. Our data indicate that functional senescence and age-dependent mortality are influenced by beta integrins in Drosophila.     

Characterization of Brachyury-downstream notochord genes in the Ciona intestinalis embryo

The notochord has two major roles during chordate embryogenesis, as a source of inductive signals for the patterning of neural tube and paraxial mesoderm and as a supportive organ of the larval tail. Despite the recent identification of mutations that affect the notochord development in vertebrate embryos, little is known about genes that are expressed in the differentiating notochord itself. In the urochordate ascidian Ciona intestinalis, Brachyury (Ci-Bra) plays a key role in notochord differentiation. In a previous study, we isolated cDNA clones for nearly 40 potential Ci-Bra target genes that are expressed in notochord cells (H. Takahashi et al., 1999, Genes Dev. 13, 1519-1523). Here we characterized 20 of them by determining the complete nucleotide sequences of the cDNAs. These genes encode a broad spectrum of divergent proteins associated with notochord formation and function. Two genes encode ascidian homologs of the Drosophila Prickle LIM domain proteins and another encodes the ERM protein, all 3 of which appear to be involved in the control of cytoskeletal architecture. In addition, genes for netrin, leprecan, cdc45, ATP:citrate lyase, ATP sulfurylase/APS kinase, protein tyrosine phosphatase, beta4-galactosyltransferase, fibrinogen-like protein, divergent tropomyosin-like proteins, and Drosophila Pellino-like protein were identified. The observation of the netrin gene expression in the notochord may provide the first molecular evidence that the ascidian notochord is a source of signals as in vertebrates. In addition, the present information should be used to identify nonchordate deuterostome tissues homologous to the notochord as well as genes which are expressed in the notochord cells of vertebrate embryos.