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.     

Overexpression of transglutaminase in the Drosophila wing imaginal disc induced an extra wing crossvein phenotype

To determine the roles of Drosophila transglutaminase-A (dTG-A), we examined a phenotype induced through ectopic expression of dTG-A. Overexpression of dTG-A in the wing imaginal disc induced an extra wing crossvein phenotype. This phenotype was suppressed by crossing with epidermal growth factor receptor (Egfr) signaling pathway mutant flies. These results indicate that this phenotype, induced by dTG-A, is related to enhancement of the Egfr signaling pathway.     

Metalloproteinases in Drosophila to humans that are central players in developmental processes

Many secreted proteins are synthesized as precursors with propeptides that must be cleaved to yield the mature functional form of the molecule. In addition, various growth factors occur in extracellular latent complexes with protein antagonists and are activated upon cleavage of such antagonists. Research in the separate fields of embryonic patterning and extracellular matrix formation has identified members of the BMP1/Tolloid-like family of metalloproteinases as key players in these types of biosynthetic processing events in species ranging from Drosophila to humans.     

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

Abstract.— 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.     

Drawing lines in the Drosophila wing: initiation of wing vein development

It has been proposed that wing veins in Drosophila form at boundaries between discrete sectors of cells that subdivide the anterior-posterior axis of the developing wing primordium. Recently, analysis of events underlying initiation of vein formation suggests that there is a general developmental mechanism for drawing lines between adjacent domains of cells, which is referred to as 'for-export-only-signaling'. In this model, cells in one domain produce a short range signal to which they cannot respond. As a consequence of this constraint, cells lying in a narrow line immediately outside the signal-producing domain are the only cells that can respond to the signal by activating expression of vein-promoting genes.     

Mutations in the catalytic loop HRD motif alter the activity and function of Drosophila Src64

The catalytic loop HRD motif is found in most protein kinases and these amino acids are predicted to perform functions in catalysis, transition to, and stabilization of the active conformation of the kinase domain. We have identified mutations in a Drosophila src gene, src64, that alter the three HRD amino acids. We have analyzed the mutants for both biochemical activity and biological function during development. Mutation of the aspartate to asparagine eliminates biological function in cytoskeletal processes and severely reduces fertility, supporting the amino acid's critical role in enzymatic activity. The arginine to cysteine mutation has little to no effect on kinase activity or cytoskeletal reorganization, suggesting that the HRD arginine may not be critical for coordinating phosphotyrosine in the active conformation. The histidine to leucine mutant retains some kinase activity and biological function, suggesting that this amino acid may have a biochemical function in the active kinase that is independent of its side chain hydrogen bonding interactions in the active site. We also describe the phenotypic effects of other mutations in the SH2 and tyrosine kinase domains of src64, and we compare them to the phenotypic effects of the src64 null allele.     

Lack of response to artificial selection on developmental stability of partial wing shape components in Drosophila melanogaster

Developmental stability, the ability of organisms to buffer their developmental processes against developmental noise is often evaluated with fluctuating asymmetry (FA). Natural genetic variation in FA has been investigated using Drosophila wings as a model system and the recent estimation of the heritability of wing shape FA was as large as 20 %. Because natural genetic variation in wing shape FA was found to localize in a partial component of the wings, heritable variation in specific parts of the wings might be responsible for FA estimation based on the whole wing shape. In this study, we quantified the shape of three partial components of the wings, and estimated the heritability of the wing shape FA based on artificial selections. As a result, FA values for the partial wing shape components did not respond to artificial selections and the heritability scores estimated were very small. These results indicate that natural additive genetic variation in FA of partial wing components was very small compared with that in a complex wing trait.     

CDC42 and Rac1 control different actin-dependent processes in the Drosophila wing disc epithelium

Cdc42 and Rac1 are members of the rho family of small guanosinetriphosphatases and are required for a diverse set of cytoskeleton-membrane interactions in different cell types. Here we show that these two proteins contribute differently to the organization of epithelial cells in the Drosophila wing imaginal disc. Drac1 is required to assemble actin at adherens junctions. Failure of adherens junction actin assembly in Drac1 dominant-negative mutants is associated with increased cell death. Dcdc42, on the other hand, is required for processes that involve polarized cell shape changes during both pupal and larval development. In the third larval instar, Dcdc42 is required for apico-basal epithelial elongation. Whereas normal wing disc epithelial cells increase in height more than twofold during the third instar, cells that express a dominant-negative version of Dcdc42 remain short and are abnormally shaped. Dcdc42 localizes to both apical and basal regions of the cell during these events, and mediates elongation, at least in part, by effecting a reorganization of the basal actin cytoskeleton. These observations suggest that a common cdc42-based mechanism may govern polarized cell shape changes in a wide variety of cell types.     

Control and function of terminal gap gene activity in the posterior pole region of the Drosophila embryo.

We have studied the genetic requirement for the normal expression of the terminal gap genes huckebein (hkb) and tailless (tll) and their possible function in the posterior pole region of the Drosophila embryo. At the early blastoderm stage, both genes are expressed in largely coextensive expression domains. Our results show that in the posterior region of the embryo both the activation and the control of the spatial limits of tll and hkb expression are critically dependent on torso (tor) activity, which is thought to be a crucial component of a cellular signal transduction pathway provided by the terminal maternal system. Furthermore, the spatial control of hkb and tll expression does not require mutual interactions among each other, nor does it require regulatory input from other gap genes which are essential for the establishment of segmentation in the trunk region of the embryo ("central gap genes"). Therefore, the terminal gap genes have unique regulatory features which are distinct from the central gap genes. In the absence of terminal gap gene activities, as in hkb and tll mutant embryos, the expression domains of the central gap genes expand posteriorly, indicating that the terminal gap gene activities prevent central gap gene expression in the posterior pole region of the wildtype embryo. This, in turn, suggests that the terminal gap gene activities prevent metamerization by repression of central gap genes, thereby distinguishing the segmented trunk from the nonsegmented tail region of the embryo.     

Quantitative developmental genetic analysis reveals that the ancestral dipteran wing vein prepattern is conserved in Drosophila melanogaster

Quantitative complementation tests provide a quick test of the hypothesis that a particular gene contributes to segregating phenotypic variation. A set of wild-type alleles is assayed for variation in their ability to complement the degree of dominance of the quantitative effect of a loss of function allele. Analysis of 15 loci known to be involved in wing patterning in Drosophila melanogaster suggests that the genes decapentaplegic, thickveins, EGFR, argos and hedgehog, each of which are involved in secreted growth factor signaling, may contribute to wing shape variation. The phenotype of one deficiency, Df(2R)Px2, which removes blistered/Plexate, is also highly sensitive to the wild-type genetic background and at intermediate expressivity reveals six ectopic veins. These form in the same locations as a projection of the ancestral pattern of dipteran wing veins onto the D. melanogaster wing. This atavistic phenotype indicates that the wing vein prepatterning mechanism can be conserved in highly derived species, and implies that homoplasic venation patterns may be produced by derepression of vein primordia. Received: 13 March 2000 / Accepted: 13 August 2000     

Dpp signaling activity requires Pentagone to scale with tissue size in the growing Drosophila wing imaginal disc

The wing of the fruit fly, Drosophila melanogaster, with its simple, two-dimensional structure, is a model organ well suited for a systems biology approach. The wing arises from an epithelial sac referred to as the wing imaginal disc, which undergoes a phase of massive growth and concomitant patterning during larval stages. The Decapentaplegic (Dpp) morphogen plays a central role in wing formation with its ability to co-coordinately regulate patterning and growth. Here, we asked whether the Dpp signaling activity scales, i.e. expands proportionally, with the growing wing imaginal disc. Using new methods for spatial and temporal quantification of Dpp activity and its scaling properties, we found that the Dpp response scales with the size of the growing tissue. Notably, scaling is not perfect at all positions in the field and the scaling of target gene domains is ensured specifically where they define vein positions. We also found that the target gene domains are not defined at constant concentration thresholds of the downstream Dpp activity gradients P-Mad and Brinker. Most interestingly, Pentagone, an important secreted feedback regulator of the pathway, plays a central role in scaling and acts as an expander of the Dpp gradient during disc growth.     

An anterior/posterior communication compartment border in engrailed wing discs: possible implications for Drosophila pattern formation

Our previous studies have suggested that all the known lineage compartment borders in the wing imaginal disc of Drosophila are coincident with boundaries of reduced gap junctional communication (communication compartment borders). Since engrailed discs have a disrupted anterior/posterior (A/P) lineage border (G. Morata and P. A. Lawrence, 1975, Nature (London) 255, 614-617), it was of great interest to determine if their A/P communication restriction boundary is similarly disrupted. Examination of gap-junction-mediated exchange of small fluorescent molecules between cells in the engrailed wing disc revealed a boundary of restricted communication that appeared to be identical to the wild-type A/P communication restriction boundary. This result suggests that lineage compartments are not required for the formation of A/P communication restrictions. Furthermore, we suggest that perhaps communication compartments are the domains within which information is provided for specifying the formation of lineage compartments.     

Dual function of the Drosophila Alk1/Alk2 ortholog Saxophone shapes the Bmp activity gradient in the wing imaginal disc

Wing patterning in Drosophila requires a Bmp activity gradient created by two Bmp ligands, Gbb and Dpp, and two Bmp type I receptors, Sax and Tkv. Gbb provides long-range signaling, while Dpp signals preferentially to cells near its source along the anteroposterior (AP) boundary of the wing disc. How each receptor contributes to the signaling activity of each ligand is not well understood. Here, we show that while Tkv mediates signals from both Dpp and Gbb, Sax exhibits a novel function for a Bmp type I receptor: the ability to both promote and antagonize signaling. Given its high affinity for Gbb, this dual function of Sax impacts the function of Gbb in the Bmp activity gradient more profoundly than does Dpp. We propose that this dual function of Sax is dependent on its receptor partner. When complexed with Tkv, Sax facilitates Bmp signaling, but when alone, Sax fails to signal effectively and sequesters Gbb. Overall, our model proposes that the balance between antagonizing and promoting Bmp signaling varies across the wing pouch, modulating the level and effective range, and, thus, shaping the Bmp activity gradient. This previously unknown mechanism for modulating ligand availability and range raises important questions regarding the function of vertebrate Sax orthologs.     

Cubitus interruptus acts to specify naked cuticle in the trunk of Drosophila embryos.

One function of the Wingless signaling pathway is to determine the naked, cuticle cell fate choice in the trunk epidermis of Drosophila larvae. The zinc finger protein Teashirt binds to the transactivator domain of Armadillo to modulate Wingless signaling output in the embryonic trunk and contributes to the naked cell fate choice. The Hedgehog pathway is also necessary for the correct specification of larval epidermal cell fate, which signals via the zinc finger protein, Cubitus interruptus. Here, we show that Cubitus interruptus also has a Wingless-independent function, which is required for the specification of the naked cell fate; previously, it had been assumed that Ci induces naked cuticle exclusively by regulation of wg. Wg and Hh signaling pathways may be acting combinatorially in the same, or individually in different, cells for this process, by regulating common sets of target genes. First, the loss of the naked cuticular phenotype in embryos lacking cubitus interruptus activity is very similar to that induced by a late loss of Wingless function. Second, overexpression of Cubitus interruptus causes the suppression of denticles (as Wingless does) in absence of Wingless activity in the anterior trunk. Using epistasis experiments, we conclude that different combinations of the three proteins Teashirt, Cubitus interruptus, and Armadillo are employed for the specification of naked cuticle at distinct positions both along the antero-posterior axis and within individual trunk segments. Finally, biochemical approaches suggest the existence of protein complexes consisting of Teashirt, Cubitus interruptus, and Armadillo.     

The determination of aldehyde oxidase activity patterns in the wing discs of Drosophila melanogaster

Summary The pattern of aldehyde oxidase (AO) activity was determined in wing discs of Drosophila melanogaster larvae homozygous for the mutants apt ⁷³ⁿ, Beaded, and vestigial (vg) in order to determine if reduction in field size in the pouch could be related to alterations of the wild-type AO pattern, as suggested by the Kauffman (1978) hypothesis. The pattern in wild-type discs was resolved into six areas for comparison with mutant discs. vg discs developed at 25° C showed restriction of the pattern into a small area on the anterior side of the disc, and comparison of vg and wild-type prepupal wings allowed positive identification of the AO pattern elements which remained. AO patterns in vg wing discs grown at 27°, 29°, and 31° C were progressively more complete and similar to wild-type, reflecting the reduction in cell death in discs grown at higher temperatures. These results show that cell loss during the third instar in vg development at 25° C is responsible for the alteration of the AO pattern, rather than field size reduction, and that determination of the pattern must take place much earlier than the time of its first appearance during the third larval instar, and before cell death in vg discs begins. Thus mutants acting at earlier stages will be necessary for further tests of the Kauffman hypothesis.