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Molecular, phenotypic, and expression analysis of vein, a gene required for growth of the Drosophila wing disc.

vein1 (vn1) mutants lack portions of longitudinal wing vein 4 and the anterior crossvein. Stronger alleles, originally called defective dorsal discs, show vn is also required for the growth of the wing and haltere discs, as mutants for these alleles have tiny dorsal discs. vn functions nonautonomously and encodes a secreted EGF-like protein. Here we characterize the role of vn in the imaginal wing disc by describing the expression pattern and correlating this pattern with vn mutant phenotypes and the requirement for vn. vn is expressed in wing discs in a complex and dynamic pattern. In larval wing discs vn is first expressed in the presumptive notum and later in the wing-pouch and hinge regions. There is a striking localization of vn transcripts to intervein regions which begins with a stripe of expression straddling the AP boundary in late larval discs and develops in all intervein regions after puparium formation. We isolated new vn mutations including nulls and hypomorphs. Hypomorphic vn alleles revealed region-specific requirements for vn within the wing disc. We mapped lesions caused by 10 vn mutations and defined a minimum size of 48 kb for the gene. The phenotype and expression analyses show vn has an early role in global proliferation of the wing disc and specific roles in the development of the notum, hinge, longitudinal vein 4, and all intervein regions. The role of vn in the EGF receptor signaling pathway is discussed.

Alleles↗

An analysis of muscle weight variations in the wing and leg of Sturnus vulgaris.

Dry weights of 18 wing and 23 leg muscles from both sides of 15 adult male starlings (Sturnus vulgaris) were determined. By means of factor analysis it is calculated which part of the variance in a muscle's weight can be accounted for by common factors and which part by the characteristics of the muscle itself (residual variance). Wing and leg muscles are analysed separately. From the wing muscle analysis one can draw the following inferences: 1. Three hierarchically ordered factors represent 66%, 8% and 6% of the total variance in the wing muscles. The first common factor will most likely reflect the size variation of the starlings. 2. It appeared advisable to perform rotation of the original factor solution because the second and third factor are bipolar. 3. The three rotated factors can not be interpreted as embryological sources of weight variations and are not related to the segmental or peripheral innervation of the muscles, but generally correspond with three groups of actions that the wing muscles perform. 4. Muscles correlated with the first rotated factor generally move the humerus, while muscles that move the forearm and (parts of) the hand are correlated with the third and second rotated factor, respectively. From the leg muscle analysis it follows that: 1. Five hierarchically ordered factors represent 57%, 13%, 7%, 6% and 4% of the total variance in the leg muscles. Body size (factor 1) plays a more important role in the wing than it does in the leg. 2. It was advisable to rotate the original factor solution since four bipolar factors are present. 3. As in the wing, residual variance shows no particular relation with the mean weight of a muscle. 4. As in the wing, the five rotated factors can only be related to five groups of muscle actions. 5. Muscles correlated with the five rotated factor cause protraction, adduction or inward rotation of the leg or combinations of these movements, whereas muscles correlated with the fourth rotated factor can perform the antagonistic actions. Muscles related to the third rotated factor flex the tarsometatarsus of flex more digits simultaneously, while muscles related to the second rotated factor flex a single digit or extend the tarsometatarus. The fifth rotated factor is a peculiar one since only the mm. peronei are correlated with it. The possibility to apply factor analysis also to experimental stimulated hypertrophy studies and to interspecific muscle weight analyses is discussed.

Animals↗

The polarity of axon growth in the wings of Drosophila melanogaster.

We have analyzed the growth of axons in the wings of the mutants Hairy wing and hairy of Drosophila melanogaster. These mutants produce many supernumerary bristle organs and sensilla campaniformia, whose axons grow between the two wing epithelia and can be visualized in both pupal and adult stages. The sensory axons of wild-type animals follow two paths in the wing, within longitudinal veins L1 and L3, and always grow with a distal to proximal polarity. In the mutants, all axons following these two paths likewise grow with correct polarity. Axons elsewhere in the wing, however, are found to grow in many different directions, including from proximal to distal and hence directly away from the central nervous system. A variety of patterns of axon growth and fasciculation are seen in different individuals. Only if the supernumerary axons encounter the two normal paths do they reliably grow toward the base of the wing. We conclude that these two paths provide polarity information for axon growth, information which is either not used or not available elsewhere in the wing in spite of the obvious morphological polarization of every epithelial cell. The time course of neural differentiation suggests that the normal sensory cells of mutant wings, which grow axons relatively early, may be the source of polarity information for the later-differentiating supernumerary cells.

Animals↗

Independent development of homologous pattern elements in the wing patterns of butterflies.

Rank correlation analyses demonstrate that the degree of color pattern development in each wing cell of Cercyonis pegala (Satyridae) and Smyrna blomfildia (Nymphalidae) is either weakly or not at all correlated with that in other wing cells. There is much greater individual variability in pattern development in different wing cells than there is in homologous wing cells on opposite wings. This finding indicates that differences in pattern development in adjacent wing cells are not due to developmental noise, but are programmed, and that pattern development in each wing cell is in large measure independent of that in other wing cells.

Animals↗

The ability of the chick wing bud to regulate positional disparity along the anterior-posterior axis.

When wedges of wing bud tissue are added to a host wing bud so there is positional disparity between graft and host, skeletal duplications result (L. E. Iten and D. J. Murphy 1980) Dev Biol. 75, 373-385. The polarity of the duplications is predictable by the polar coordinate model, leading to the interpretation that the positional disparity caused the duplications. To determine whether positional disparity alone causes duplications, without the complication of added tissue, we rotated wedges of ectoderm and mesoderm around the proximodistal axis within the wing bud. Wedges measuring 200-800 micron along the distal edge were rotated 180 degrees at stages 20-22, reversing the anteroposterior and dorsoventral axes relative to the bud. This caused positional disparity, similar to that achieved by Iten and Murphy (1980), without the addition of tissue. We found that rotations involving no polarizing zone tissue produced normal wings or wings lacking some distal parts, as did rotations of tissue lying entirely within the polarizing zone. However, when polarizing zone mesoderm was displaced, so that polarizing and nonpolarizing tissues were juxtaposed, a majority of the operations produced polarized skeletal duplications. Our data demonstrate that positional disparity alone does not cause skeletal duplications in the chick wing bud, unless polarizing zone tissue is displaced. Further, these data demonstrate that the chick wing bud can regulate to form a normal wing skeleton in the face of large positional disparity, provided that the polarizing zone is not moved. Finally, our results may be explained by the action of the proposed polarizing morphogen on the displaced cells causing repolarization.

Animals↗

fringe, a Boundary-specific signaling molecule, mediates interactions between dorsal and ventral cells during Drosophila wing development.

Wing formation in Drosophila requires interactions between dorsal and ventral cells. We describe a new gene, fringe, which is expressed in dorsal cells and encodes for a novel protein that is predicted to be secreted. Wing margin formation and distal wing outgrowth can be induced by the juxtaposition of cells with and without fringe expression, whether at the normal wing margin, at the boundaries of fringe mutant clones in the dorsal wing, or at sites of fringe misexpression in the ventral wing. By contrast, both loss of fringe expression and uniform fringe expression cause wing loss. These observations suggest that fringe encodes a boundary-specific cell-signaling molecule that is responsible for dorsal-ventral cell interactions during wing development.

Amino Acid Sequence↗

Morphogenesis of Drosophila pupal wings in vitro.

We have developed an in vitro culture system which supports the differentiation of Drosophila pupal wings. Cultured wings develop marginal bristles and wing veins, and wing cells form a single prehair at their distal vertex at the appropriate developmental stages. We have tested two molecules with well defined activities to determine the usefulness of this system for applying pharmacological approaches to wing differentiation. Cycloheximide (CY) is a small molecule which inhibits protein synthesis. We found that 50 nM CY rapidly blocks all stages of wing differentiation without lowering cell viability. Chitinase is an enzyme which cleaves chitin polymers and is involved in normal cuticle apolysis. Chitinase applied prior to 28 h apf caused a contraction of the wing without affecting the general wing pattern. We have detected connections between the epithelium and pupal cuticle that are presumably targets of chitinase and are necessary for maintaining normal tissue shape during morphogenesis. Later in development exposure to chitinase caused a loss of normal prehair and bristle polarity, and high doses resulted in a severe disruption of the actin cytoskeleton.

Acetylglucosaminidase↗

Fourier analysis of wing beat signals: assessing the effects of genetic alterations of flight muscle structure in Diptera.

A method for determining and analyzing the wing beat frequency in Diptera is presented. This method uses an optical tachometer to measure Diptera wing movement during flight. The resulting signal from the optical measurement is analyzed using a Fast Fourier Transform (FFT) technique, and the dominant frequency peak in the Fourier spectrum is selected as the wing beat frequency. Also described is a method for determining quantitatively the degree of variability of the wing beat frequency about the dominant frequency. This method is based on determination of a quantity called the Hindex, which is derived using data from the FFT analysis. Calculation of the H index allows computer-based selection of the most suitable segment of recorded data for determination of the representative wing beat frequency. Experimental data suggest that the H index can also prove useful in examining wing beat frequency variability in Diptera whose flight muscle structure has been genetically altered. Examples from Drosophila indirect flight muscle studies as well as examples of artificial data are presented to illustrate the method. This method fulfills a need for a standardized method for determining wing beat frequencies and examining wing beat frequency variability in insects whose flight muscles have been altered by protein engineering methods.

Animals↗

A chick wingless mutation causes abnormality in maintenance of Fgf8 expression in the wing apical ridge, resulting in loss of the dorsoventral boundary.

We analyzed a Japanese chick wingless mutant (Jwg) to know a molecular mechanism underlying wing development. We observed expression patterns of eleven marker genes to characterize the mutant. Expressions of dorsoventral (DV) and mesenchymal marker genes were intact in nascent Jwg limb buds. However, expression of Fgf8, a marker gene for the apical ectodermal ridge (AER), was delayed and shortly disappeared in the wing regressing AER. Later on, ventral expression of dorsal marker genes of Wnt7a and Lmx1 indicated that the wing bud without the AER became bi-dorsal. In addition, the posterior mesoderm became defective, as deduced from the impaired expression patterns of Sonic hedgehog (Shh), Msx1, and Prx1. We attempted to rescue a wing by implanting Fgf8-expressing cells into the Jwg wing bud. We found that FGF8 can rescue outgrowth of the wing bud by maintaining Shh expression. Thus, the Jwg gene seems to be involved in maintenance of the Fgf8 expression in the wing bud. Further, it is suggested that the AER is required for maintenance of the DV boundary and the polarizing activity of the established wing bud.

Animals↗

Serrate and wingless cooperate to induce vestigial gene expression and wing formation in Drosophila.

BACKGROUND: The appendages of insects, like the limbs of vertebrates, grow out of the body wall after the establishment of a proximo-distal axis among a group of primordial cells. In Drosophila, the wing develops in the limbless larva from one of the imaginal discs of the thorax, which give rise to the adult epidermis. The earliest identified requirement in wing development is for the induction of vestigial (vg) gene expression at the interface between ventral cells and dorsal cells of the wing disc. It has been proposed that this event requires two reciprocal signals--one from the dorsal to the ventral cells and the other from the ventral to the dorsal cells--which trigger vg expression at the presumptive wing margin and hence initiate the development of the wing tissue. RESULTS: We have identified four genes--Serrate (Ser), wingless (wg), Notch and Suppressor of Hairless (Su(H))--whose activity is required during the second and early third larval instars for the expression of vg. Analysis of the functions and patterns of expression of these genes at the time of the inductive event indicates that the Ser protein acts as a dorsal signal, and the Wg protein as a ventral signal for the induction of vg expression. Furthermore, the expression of both Ser and Wg is sufficient to trigger ectopic wing development in the wing disc and leg discs. The product of the Notch gene, which encodes a receptor, is also required for this event and we suggest that its role is to integrate the inputs of Ser and Wg. CONCLUSIONS: We show that the induction of vg, which initiates wing development in Drosophila, requires the combined activities of Ser, wg and Notch. Based on the patterns of expression and requirements for Ser and wg in this process, we propose that Ser is a dorsal signal and that Wg is a ventral signal, and that their combination at the dorso-ventral interface activates the Notch receptor and leads to vg expression.

Animals↗

Effect of heat shock, pretreatment and hsp70 copy number on wing development in Drosophila melanogaster.

Naturally occurring heat shock (HS) during pupation induces abnormal wing development in Drosophila; we examined factors affecting the severity of this induction. The proportion of HS-surviving adults with abnormal wings varied with HS duration and intensity, and with the pupal age or stage at HS administration. Pretreatment (PT), mild hyperthermia delivered before HS, usually protected development against HS. Gradual heating resembling natural thermal regimes also protected wing development against thermal disruption. Because of the roles of the wings in flight and courtship and in view of natural thermal regimes that Drosophila experience, both HS-induction of wing abnormalities and its abatement by PT may have marked effects on Drosophila fitness in nature. Because PT is associated with expression of heat-inducible molecular chaperones such as Hsp70 in Drosophila, we compared thermal disruption of wing development among hsp70 mutants as well as among strains naturally varying in Hsp70 levels. Contrary to expectations, lines or strains with increased Hsp70 levels were no more resistant to HS-disruption of wing development than counterparts with lower Hsp70 levels. In fact, wing development was more resistant to HS in hsp70 deletion strains than control strains. We suggest that, while high Hsp70 levels may aid cells in surviving hyperthermia, high levels may also overly stimulate or inhibit numerous signalling pathways involved in cell proliferation, maturation and programmed death, resulting in developmental failure.

Age Factors↗

Proximo-distal specification in the wing disc of Drosophila by the nubbin gene.

Mutations in the nubbin (nub) gene have a phenotype consisting of a severe wing size reduction and pattern alterations, such as transformations of distal elements into proximal ones. nub expression is restricted to the wing pouch cells in wing discs since early larval development. These effects are also observed in genetic mosaics where cell proliferation is reduced in all wing blade regions autonomously, and transformation into proximal elements is observed in distal clones. Clones located in the proximal region of the wing blade cause in addition nonautonomous reduction of the whole wing. Cell lineage experiments in a nub mutant background show that clones respect neither the anterior-posterior nor the dorsal-ventral boundary but that the selector genes have been correctly expressed since early larval development. The phenotypes of nub el and nub dpp genetic combinations are synergistic and the overexpression of dpp in clones in nub wings does not result in overproliferation of the surrounding wild-type cells. We discuss the role of nub in the wing's proximo-distal axis and in the formation of compartment boundaries.

Animals↗

The function of resilin in beetle wings.

This account shows the distribution of elastic elements in hind wings in the scarabaeid Pachnoda marginata and coccinellid Coccinella septempunctata (both Coleoptera). Occurrence of resilin, a rubber-like protein, in some mobile joints together with data on wing unfolding and flight kinematics suggest that resilin in the beetle wing has multiple functions. First, the distribution pattern of resilin in the wing correlates with the particular folding pattern of the wing. Second, our data show that resilin occurs at the places where extra elasticity is needed, for example in wing folds, to prevent material damage during repeated folding and unfolding. Third, resilin provides the wing with elasticity in order to be deformable by aerodynamic forces. This may result in elastic energy storage in the wing.

Animals↗

On aerodynamic modelling of an insect-like flapping wing in hover for micro air vehicles.

This theoretical paper discusses recent advances in the fluid dynamics of insect and micro air vehicle (MAV) flight and considers theoretical analyses necessary for their future development. The main purpose is to propose a new conceptual framework and, within this framework, two analytic approaches to aerodynamic modelling of an insect-like flapping wing in hover in the context of MAVs. The motion involved is periodic and is composed of two half-cycles (downstroke and upstroke) which, in hover, are mirror images of each other. The downstroke begins with the wing in the uppermost and rearmost position and then sweeps forward while pitching up and plunging down. At the end of the half-cycle, the wing flips, so that the leading edge points backwards and the wing's lower surface becomes its upper side. The upstroke then follows by mirroring the downstroke kinematics and executing them in the opposite direction. Phenomenologically, the interpretation of the flow dynamics involved, and adopted here, is based on recent experimental evidence obtained by biologists from insect flight and related mechanical models. It is assumed that the flow is incompressible, has low Reynolds number and is laminar, and that two factors dominate: (i) forces generated by the bound leading-edge vortex, which models flow separation; and (ii) forces due to the attached part of the flow generated by the periodic pitching, plunging and sweeping. The first of these resembles the analogous phenomenon observed on sharp-edged delta wings and is treated as such. The second contribution is similar to the unsteady aerodynamics of attached flow on helicopter rotor blades and is interpreted accordingly. Theoretically, the fluid dynamic description is based on: (i) the superposition of the unsteady contributions of wing pitching, plunging and sweeping; and (ii) adding corrections due to the bound leading-edge vortex and wake distortion. Viscosity is accounted for indirectly by imposing the Kutta condition on the trailing edge and including the influence of the vortical structure on the leading edge. Mathematically, two analytic approaches are proposed. The first derives all the quantities of interest from the notion of circulation and leads to tractable integral equations. This is an application of the von Kármán-Sears unsteady wing theory and its nonlinear extensions due to McCune and Tavares; the latter can account for the bound leading-edge vortex and wake distortion. The second approach uses the velocity potential as the central concept and leads to relatively simple ordinary differential equations. It is a combination of two techniques: (i) unsteady aerodynamic modelling of attached flow on helicopter rotor blades; and (ii) Polhamus's leading-edge suction analogy. The first of these involves both frequency-domain (Theodorsen style) and time-domain (indicial) methods, including the effects of wing sweeping and returning wake. The second is a nonlinear correction accounting for the bound leading-edge vortex. Connections of the proposed framework with control engineering and aeroelasticity are pointed out.

Animals↗

Direct and correlated responses to artificial selection on developmental time and wing length in Drosophila buzzatii.

Developmental time and body size are two positively correlated traits closely related to fitness in many organisms including Drosophila. Previous work suggested that these two traits are involved in a trade-off that may result from a negative genetic correlation between their effects on pre-adult and adult fitness. Here, we examine the evolution of developmental time and body size (indexed by wing length) under artificial selection applied to one or both traits in replicated D. buzzatii populations. Directional changes in both developmental time and wing length indicate the presence of substantial additive genetic variance for both traits. The strongest response to selection for fast development was found in lines selected simultaneously to reduce both developmental time and wing length, probably as an expected consequence of a synergistic effect of indirect selection. When selection was applied in the direction opposite to the putative genetic correlation, that is, large wing length but fast development, no responses were observed for developmental time. Lines selected to reduce both wing length and developmental time diverged slightly faster from the control than lines selected to increase wing length and reduce developmental time. However, wing length did not diverge from the control in lines selected only for fast development. These results suggest a complex genetic basis of the correlation between developmental time and wing length, but are generally consistent with the hypothesis that both traits are related in a trade-off. However, we found that this trade-off may disappear under uncrowded conditions, with fast-developing lines exhibiting a higher pre-adult viability than other lines when tested at high larval density.

Analysis of Variance↗

The evolution and development of dipteran wing veins: a systematic approach.

In this review, we use the wing veins of dipteran insects as potential models for understanding the evolution of development. We briefly discuss previous work in this field and examine the genetic complexity of wing formation, discussing the genes involved in wing formation and their roles in Drosophila wing development and vein formation. Furthermore, patterns of wing vein formation, addition, and reduction are discussed as they occur throughout the Diptera. Using the phyletic phenocopy paradigm, we draw attention to many wing vein morphologies that phenocopy various wing mutants in Drosophila melanogaster. The systematic issues of the nature of characters, homology, and the role of modern developmental approaches to evolutionary studies, which has recently become important, can be addressed from the perspective of the wing. We argue that further developmental evolutionary studies, and the interpretation of data therefrom, must be conducted within the context of a well-supported phylogeny of the organisms under study.

Animals↗

blistery encodes Drosophila tensin protein and interacts with integrin and the JNK signaling pathway during wing development.

Tensin is an actin-binding protein that is localized in focal adhesions. At focal adhesion sites, tensin participates in the protein complex that establishes transmembrane linkage between the extracellular matrix and cytoskeletal actin filaments. Even though there have been many studies on tensin as an adaptor protein, the role of tensin during development has not yet been clearly elucidated. Thus, this study was designed to dissect the developmental role of tensin by isolating Drosophila tensin mutants and characterizing its role in wing development. The Drosophila tensin loss-of-function mutations resulted in the formation of blisters in the wings, which was due to a defective wing unfolding process. Interestingly, by(1)-the mutant allele of the gene blistery (by)-also showed a blistered wing phenotype, but failed to complement the wing blister phenotype of the Drosophila tensin mutants, and contains Y62N/T163R point mutations in Drosophila tensin coding sequences. These results demonstrate that by encodes Drosophila tensin protein and that the Drosophila tensin mutants are alleles of by. Using a genetic approach, we have demonstrated that tensin interacts with integrin and also with the components of the JNK signaling pathway during wing development; overexpression of by in wing imaginal discs significantly increased JNK activity and induced apoptotic cell death. Collectively, our data suggest that tensin relays signals from the extracellular matrix to the cytoskeleton through interaction with integrin, and through the modulation of the JNK signal transduction pathway during Drosophila wing development.

Animals↗

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.

Animals↗