The function of PS integrins during Drosophila embryogenesis

The Drosophila position-specific (PS) antigens are homologous to the vertebrate fibronectin receptor family, or integrins. A Drosophila gene required for embryonic morphogenesis, l(1)myospheroid, codes for a product homologous to the beta subunit of the vertebrate integrins. l(1)myospheroid mutants die during embryogenesis. We show here that they lack the beta subunit of the PS antigens. In the absence of the beta subunit in mutant embryos, the PS alpha subunits are not expressed on the cell surface. We conclude that the l(1)myospheroid phenotype represents the lack-of-function phenotype for these Drosophila integrins. 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.     

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.     

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.     

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.     

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.     

Fibronectin receptor functions in embryonic cells deficient in alpha 5 beta 1 integrin can be replaced by alpha V integrins.

alpha 5 beta 1 integrin mediates cell adhesion to extracellular matrix by interacting with fibronectin (FN). Mouse lines carrying null mutations in genes encoding either the alpha 5 integrin subunit or FN have been generated previously. Both mutations are embryonic lethal with overlapping defects, but the defects of alpha 5-null embryos are less severe. Primary embryonic cells lacking alpha 5 beta 1 are able to adhere to FN, form focal contacts, migrate on FN, and assemble FN matrix. These results suggest the involvement of (an)other FN receptors(s). In this study, we examined functions of alpha 4 beta 1 and alpha V integrins in embryonic cells lacking alpha 5 beta 1. Our analysis of cells lacking both alpha 4 beta 1 and alpha 5 beta 1 showed that alpha 4 beta 1 is also not required for these FN-dependent functions. Using alpha V-specific blocking reagents, we showed that alpha V integrins are required for alpha 5-null cells, but not wild-type cells, to adhere and spread on FN. Our data also showed that, although the expression levels of alpha V integrins on the wild-type and alpha 5-null cells are similar, there is an increase in recruitment of alpha V integrins into focal contacts in alpha 5-null cells plated on FN, indicating that alpha V integrins can compensate functionally for the loss of alpha 5 beta 1 in focal contacts of alpha 5-null cells. Finally, our data suggested possible roles for alpha V integrins in replacing the role of alpha 5 beta 1 in FN matrix assembly in vitro and in FN-dependent embryonic functions in vivo.     

Genetic analysis of olfC demonstrates a role for the position-specific integrins in the olfactory system of Drosophila melanogaster

Genetic analysis of olfC provides evidence for a role for integrins in the development and/or function of the olfactory system of Drosophila. The olfC gene was identified on the basis of mutations that result in specific defects in behavioural responses to acetate esters, and has been mapped to the cytogenetic interval 7D1;D5–6 on the X chromosome. The myospheroid (mys) gene maps to this region and encodes a β subunit of integrins. Integrins are αβ heterodimers which are present on the cell surface and have been implicated in a variety of signalling roles. Mutations in mys fail to complement the olfactory deficits of olfC mutants. These defects can be rescued by misexpression of the mys ⁺ gene under control of a hsp70 promoter. Mutations that affect the α subunit of the position-specific integrin PS2 show a dominant interaction with olfC. These results suggest that olfC is allelic to mys and functions together with α_PS2 integrins in the olfactory pathway in Drosophila. Received: 3 November 1999 / Accepted: 17 January 2000     

Integrins modulate Sog activity in the Drosophila wing

Morphogenesis of the Drosophila wing depends on a series of cell-cell and cell-extracellular matrix interactions. During pupal wing development, two secreted proteins, encoded by the short gastrulation (sog) and decapentaplegic (dpp) genes, vie to position wing veins in the center of broad provein territories. Expression of the Bmp4 homolog dpp in vein cells is counteracted by expression of the Bmp antagonist sog in intervein cells, which results in the formation of straight veins of precise width. We screened for genetic interactions between sog and genes encoding a variety of extracellular components and uncovered interactions between sog and myospheroid (mys), multiple edematous wing (mew) and scab (scb), which encode betaPS, alphaPS1 and alphaPS3 integrin subunits, respectively. Clonal analysis reveals that integrin mutations affect the trajectory of veins inside the provein domain and/or their width and that misexpression of sog can alter the behavior of cells in such clones. In addition, we show that a low molecular weight form of Sog protein binds to alphaPS1betaPS. We find that Sog can diffuse from its intervein site of production into adjacent provein domains, but only on the dorsal surface of the wing, where Sog interacts functionally with integrins. Finally, we show that Sog diffusion into provein regions and the reticular pattern of extracellular Sog distribution in wild-type wings requires mys and mew function. We propose that integrins act by binding and possibly regulating the activity/availability of different forms of Sog during pupal development through an adhesion independent mechanism.     

Cell-autonomous requirement for beta1 integrin in endothelial cell adhesion, migration and survival during angiogenesis in mice

beta1 integrin (encoded by Itgb1) is established as a regulator of angiogenesis based upon the phenotypes of complete knockouts of beta1 heterodimer partners or ligands and upon antibody inhibition studies in mice. Its direct function in endothelial cells (ECs) in vivo has not been determined because Itgb1(-/-) embryos die before vascular development. Excision of Itgb1 from ECs and a subset of hematopoietic cells, using Tie2-Cre, resulted in abnormal vascular development by embryonic day (e) 8.5 and lethality by e10.5. Tie1-Cre mediated a more restricted excision of Itgb1 from ECs and hematopoietic cells and resulted in embryonic lethal vascular defects by e11.5. Capillaries of the yolk sacs were disorganized, and the endothelium of major blood vessels and of the heart was frequently discontinuous in mutant embryos. We also found similar vascular morphogenesis defects characterized by EC disorganization in embryonic explants and isolated ECs. Itgb1-null ECs were deficient in adhesion and migration in a ligand-specific fashion, with impaired responses to laminin and collagens, but not to fibronectin. Deletion of Itgb1 reduced EC survival, but did not affect proliferation. Our findings demonstrate that beta1 integrin is essential for EC adhesion, migration and survival during angiogenesis, and further validate that therapies targeting beta1 integrins may effectively impair neovascularization.     

Mouse primordial germ cells lacking beta1 integrins enter the germline but fail to migrate normally to the gonads

Primordial germ cells are the founder cells of the gametes. They are set aside at the initial stages of gastrulation in mammals, become embedded in the hind-gut endoderm, then actively migrate to the sites of gonad formation. The molecular basis of this migration is poorly understood. Here we sought to determine if members of the integrin family of cell surface receptors are required for primordial germ cell migration, as integrins have been implicated in the migration of several other motile cell types. We have established a line of mice which express green fluorescent protein in germline cells that has enabled us to efficiently purify primordial germ cells at different stages by flow cytometry. We have catalogued the spectrum of integrin subunit expression by primordial germ cells during and after migration, using flow cytometry, immunocytochemistry and RT-PCR. Through analysis of integrin beta1(-/-)-->wild-type chimeras, we show that embryonic cells lacking beta1 integrins can enter the germline. However, integrin beta1(-/-) primordial germ cells do not colonize the gonad efficiently. Embryos with targeted deletion of integrin subunit alpha3, alpha6, or alphaV show no major defects in primordial germ cell migration. These results demonstrate a role for beta1-containing integrins in the development of the germline, although an equivalent role for * integrin subunit(s) has yet to be established.     

Roles for beta(pat-3) integrins in development and function of Caenorhabditis elegans muscles and gonads

Heterodimeric integrin receptors for extracellular matrix (ECM) play vital roles in bidirectional signaling during tissue development, organization, remodeling, and repair. The beta integrin subunit cytoplasmic domain is essential for transmission of many of these signals and overexpression of an unpaired beta tail in cultured cells inhibits endogenous integrins. Unlike vertebrates, which have at least nine beta subunit genes, the nematode Caenorhabditis elegans expresses only one beta subunit (betapat-3), and a null mutation in this gene causes embryonic lethality. To determine the functions of integrins during larval development and in adult tissues, we have taken a dominant negative approach by expression of an HA-betatail transgene composed of a hemagglutinin (HA) epitope tag extracellular domain connected to the betapat-3 transmembrane and cytoplasmic domains. Expression of this transgene in muscle and gonad, major sites of integrin expression, caused a variety of phenotypes dependent on the level of transgene expression. Abnormalities in body wall and sex muscles led to uncoordinated movement and egg-laying defects. Significant anomalies in migration and pathfinding were caused by tissue-specific expression of HA-betatail in the distal tip cells (DTC), the cells that direct gonad morphogenesis. A pat-3 gene with Tyr to Phe mutations in the cytoplasmic domain was able to rescue pat-3 null animals but also showed DTC migration defects. These results show that betapat-3 plays important roles in post-embryonic organogenesis and tissue function.     

Drosophila talin and integrin genes are required for maintenance of tracheal terminal branches and luminal organization

Epithelial tubes that compose many organs are typically long lasting, except under specific developmental and physiological conditions when network remodeling occurs. Although there has been progress elucidating mechanisms of tube formation, little is known of the mechanisms that maintain tubes and destabilize them during network remodeling. Here, we describe Drosophila tendrils mutations that compromise maintenance of tracheal terminal branches, fine gauge tubes formed by tracheal terminal cells that ramify on and adhere tightly to tissues in order to supply them with oxygen. Homozygous tendrils terminal cell clones have fewer terminal branches than normal but individual branches contain multiple convoluted lumens. The phenotype arises late in development: terminal branches bud and form lumens normally early in development, but during larval life lumens become convoluted and mature branches degenerate. Their lumens, however, are retained in the remaining branches, resulting in the distinctive multi-lumen phenotype. Mapping and molecular studies demonstrate that tendrils is allelic to rhea, which encodes Drosophila talin, a large cytoskeletal protein that links integrins to the cytoskeleton. Terminal cells mutant for myospheroid, the major Drosophila beta-integrin, or doubly mutant for multiple edematous wings and inflated alpha-integrins, also show the tendrils phenotype, and localization of myospheroid beta-integrin protein is disrupted in tendrils mutant terminal cells. The results provide evidence that integrin-talin adhesion complexes are necessary to maintain tracheal terminal branches and luminal organization. Similar complexes may stabilize other tubular networks and may be targeted for inactivation during network remodeling events.     

Expression of chicken integrin beta 1 subunit in rat PC12 cells

We transfected rat pheochromocytoma (PC12) cells with a cDNA encoding chicken integrin beta 1 subunit. The chicken integrin beta 1 subunit produced in stable transfectants associated with two major alpha subunits of rat integrins to form interspecific chimeric receptors. These receptors mediated cell spreading and initial neurite outgrowth on laminin as did corresponding endogenous integrins, although they were slightly less effective in inducing cell adhesion to laminin. These results indicate that chicken integrin beta 1 may functionally substitute for beta 1 subunit of rat integrins in PC12 cells. Apparently, the structure of the integrin beta 1 subunit is highly conserved in the evolution of these species.     

Identification of integrin beta subunit mutations that alter heterodimer function in situ

We conducted a genetic screen for mutations in myospheroid, the gene encoding the Drosophila betaPS integrin subunit, and identified point mutants in all of the structural domains of the protein. Surprisingly, we find that mutations in very strongly conserved residues will often allow sufficient integrin function to support the development of adult animals, including mutations in the ADMIDAS site and in a cytoplasmic NPXY motif. Many mutations in the I-like domain reduce integrin expression specifically when betaPS is combined with activating alphaPS2 cytoplasmic mutations, indicating that integrins in the extended conformation are unstable relative to the inactive, bent heterodimers. Interestingly, the screen has identified alleles that show gain-of-function characteristics in cell culture, but have negative effects on animal development or viability. This is illustrated by the allele mys(b58); available structural models suggest that the molecular lesion of mys(b58), V409>D, should promote the "open" conformation of the beta subunit I-like domain. This expectation is supported by the finding that alphaPS2betaPS (V409>D) promotes adhesion and spreading of S2 cells more effectively than does wild-type alphaPS2betaPS, even when betaPS is paired with alphaPS2 containing activating cytoplasmic mutations. Finally, comparisons with the sequence of human beta8 suggest that evolution has targeted the "mys(b58)" residue as a means of affecting integrin activity.     

Structural basis of integrin activation by talin

Regulation of integrin affinity (activation) is essential for metazoan development and for many pathological processes. Binding of the talin phosphotyrosine-binding (PTB) domain to integrin beta subunit cytoplasmic domains (tails) causes activation, whereas numerous other PTB-domain-containing proteins bind integrins without activating them. Here we define the structure of a complex between talin and the membrane-proximal integrin beta3 cytoplasmic domain and identify specific contacts between talin and the integrin tail required for activation. We used structure-based mutagenesis to engineer talin and beta3 variants that interact with comparable affinity to the wild-type proteins but inhibit integrin activation by competing with endogenous talin. These results reveal the structural basis of talin's unique ability to activate integrins, identify an interaction that could aid in the design of therapeutics to block integrin activation, and enable engineering of cells with defects in the activation of multiple classes of integrins.     

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.     

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.     

In vivo roles of integrins during leukocyte development and traffic: insights from the analysis of mice chimeric for alpha 5, alpha v, and alpha 4 integrins

Mice chimeric for integrins alpha(5), alpha(V), or alpha(4) were used to dissect the in vivo roles of these adhesion receptors during leukocyte development and traffic. No major defects were observed in the development of lymphocytes, monocytes, or granulocytes or in the traffic of lymphocytes to different lymphoid organs in the absence of alpha(5) or alpha(V) integrins. However, in agreement with previous reports, the absence of alpha(4) integrins produced major defects in development of lymphoid and myeloid lineages and a specific defect in homing of lymphocytes to Peyer's patches. In contrast, the alpha(4) integrin subunit is not essential for localization of T lymphocytes into intraepithelial and lamina propria compartments in the gut, whereas one of the partners of alpha(4), the beta(7) chain, has been shown to be essential. However, alpha(4)-deficient T lymphocytes cannot migrate properly during the inflammatory response induced by thioglycolate injection into the peritoneum. Finally, in vitro proliferation and activation of lymphocytes deficient for alpha(5), alpha(V), or alpha(4) integrins upon stimulation with different stimuli were similar to those seen in controls. These results show that integrins play distinct roles during in vivo leukocyte development and traffic.     

Integrins hold Drosophila together

The Drosophila position-specific (PS) integrins are members of the integrin family of cell surface receptors and are thought to be receptors for extracellular matrix components. Each PS integrin consists of an α subunit, α_PS1 or α_PS2, and a β_PS subunit. Mutations in the β_PS subunit and the α_PS2 subunit have been characterised and reveal that the PS integrins have an essential role in the adhesion of different cell layers to each other. The PS integrins are especially required for the function of the cell-matrix-cell junctions, where the muscles attach to the epidermis and where one surface of the developing wing adheres to the other. These junctions are similar to vertebrate focal adhesions and hemidesmosomes, which also contain integrins. Integrin-mediated cell to cell adhesion via the extracellular matrix provides a way for tissues to adhere to each other without intermingling of their cells.     

The alpha 1-alpha 6 subunits of integrins are characteristically expressed in distinct segments of developing and adult human nephron

We studied the distribution of the alpha 1-alpha 6 subunits of beta 1 integrins in developing and adult human kidney using a panel of mAbs in indirect immunofluorescence microscopy. Uninduced mesenchyme displayed a diffuse immunoreactivity for only the alpha 1 integrin subunit. At the S-shaped body stage of nephron development, several of the alpha subunits were characteristically expressed in distinct fetal nephron segments, and the pattern was retained also in the adult nephron. Thus, the alpha 1 subunit was characteristically expressed in mesangial and endothelial cells, the alpha 2 in glomerular endothelium and distal tubules, the alpha 3 in podocytes, Bowman's capsule, and distal tubules, and the alpha 6 subunit basally in all tubules, and only transiently in podocytes during development. Unlike the alpha 3 and alpha 6 subunits, the alpha 2 subunit displayed an overall cell surface distribution in distal tubules. It was also distinctly expressed in glomerular endothelia during glomerulogenesis. The beta 4 subunit was expressed only in fetal collecting ducts, and hence the alpha 6 subunit seems to be complexed with the beta 1 rather than beta 4 subunit in human kidney. Of the two fibronectin receptor alpha subunits, alpha 4 and alpha 5, only the latter was expressed, confined to endothelia of developing and adult blood vessels, suggesting that these receptor complexes play a minor role during nephrogenesis. The present results suggest that distinct integrins play a role during differentiation of specific nephron segments. They also indicate that alpha 3 beta 1 and alpha 6 beta 1 integrin complexes may function as basement membrane receptors in podocytes and tubular epithelial cells.