Roles for scalloped and vestigial in regulating cell affinity and interactions between the wing blade and the wing hinge

The scalloped and vestigial genes are both required for the formation of the Drosophila wing, and recent studies have indicated that they can function as a heterodimeric complex to regulate the expression of downstream target genes. We have analyzed the consequences of complete loss of scalloped function, ectopic expression of scalloped, and ectopic expression of vestigial on the development of the Drosophila wing imaginal disc. Clones of cells mutant for a strong allele of scalloped fail to proliferate within the wing pouch, but grow normally in the wing hinge and notum. Cells overexpressing scalloped fail to proliferate in both notal and wing-blade regions of the disc, and this overexpression induces apoptotic cell death. Clones of cells overexpressing vestigial grow smaller or larger than control clones, depending upon their distance from the dorsal-ventral compartment boundary. These studies highlight the importance of correct scalloped and vestigial expression levels to normal wing development. Our studies of vestigial-overexpressing clones also reveal two further aspects of wing development. First, in the hinge region vestigial exerts both a local inhibition and a long-range induction of wingless expression. These and other observations imply that vestigial-expressing cells in the wing blade organize the development of surrounding wing-hinge cells. Second, clones of cells overexpressing vestigial exhibit altered cell affinities. Our analysis of these clones, together with studies of scalloped mutant clones, implies that scalloped- and vestigial-dependent cell adhesion contributes to separation of the wing blade from the wing hinge and to a gradient of cell affinities along the dorsal-ventral axis of the wing.     

Developmental effects of modifiers of the vg mutant in Drosophila melanogaster

An analysis of the modifiers affecting the expression of the vg gene was performed. We selected for weak and strong expression of the vg mutant in F2 segregating populations obtained by crossing a vestigial stock with an Oregon laboratory stock (O) and with a wild strain (B) captured near Bologna, Italy. The selection for enlarged wings was more effective in the vg B population where wild wings appeared from the 10th generation. The assay of the three major chromosomes showed that the modifiers are located on chromosomes 2 and 3. The mutant imaginal disc cell death phenotype is evident in vg/vg strains that have a wild-type wing phenotype. It is suggested that the selected modifiers do not prevent cell death but induce regenerative growth.     

Genetic requirements of vestigial in the regulation of Drosophila wing development

The gene vestigial has been proposed to act as a master gene because of its supposed capacity to initiate and drive wing development. We show that the ectopic expression of vestigial only induces ectopic outgrowths with wing cuticular differentiation and wing blade gene expression patterns in specific developmental and genetic contexts. In the process of transformation, wingless seems to be an essential but insufficient co-factor of vestigial. vestigial ectopic expression alone or vestigial plus wingless co-expression in clones differentiate 'mixed' cuticular patterns (they contain wing blade trichomes and chaetae characteristic of the endogenous surrounding tissue) and express wing blade genes only in patches of cells within the clones. In addition, we have found that these clones, in the wing imaginal disc, may cause autonomous as well as non-autonomous cuticular transformations and wing blade gene expression patterns. These non-autonomous effects in surrounding cells result from recruitment or 'inductive assimilation' of vestigial or wingless-vestigial overexpressing cells.     

Growth and patterning are evolutionarily dissociated in the vestigial wing discs of workers of the red imported fire ant, Solenopsis invicta

Over the last decade, it has become clear that organismal form is largely determined by developmental and evolutionary changes in the growth and pattern formation of tissues. Yet, there is little known about how these two integrated processes respond to environmental cues or how they evolve relative to one another. Here, we present the discovery of vestigial wing imaginal discs in worker larvae of the red imported fire ant, Solenopsis invicta. These vestigial wing discs are present in all worker larvae, which is uncommon for a species with a large worker size distribution. Furthermore, the growth trajectory of these vestigial discs is distinct from all of the ant species examined to date because they grow at a rate slower than the leg discs. We predicted that the growth trajectory of the vestigial wing discs would be mirrored by evolutionary changes in their patterning. We tested this prediction by examining the expression of three patterning genes, extradenticle, ultrabithorax, and engrailed, known to underlie the wing polyphenism in ants. Surprisingly, the expression patterns of these three genes in the vestigial wing discs was the same as those found in ant species with different worker size distributions and wing disc growth than fire ants. We conclude that growth and patterning are evolutionarily dissociated in the vestigial wing discs of S. invicta because patterning in these discs is conserved, whereas their growth trajectories are not. The evolutionary dissociation of growth and patterning may be an important feature of gene networks that underlie polyphenic traits.     

Regulative Properties of Wing Discs from the Vestigial Mutant of Drosophila melanogaster

The vestigial (vg) mutant of Drosophila melanogaster shows reduced wing size and lacks margin structures from the wing blade. The expressivity is temperature-sensitive, more structures being formed at 29°C than at 25°C. There is cell death in the third instar wing disc which to some extent parallels the fate map locations of the structures absent in the adult.
Vestigial wing discs are unable to regenerate margin structures even when given extra time for growth by culturing them in an adult abdomen before metamorphosis. If the region of cell death is excised from the disc before culture, there is still no regeneration of margin structures, indicating that the dead cells do not physically prevent regulation. Furthermore, by metamorphosing young vg wing discs, it was discovered that cells never acquire competence to make margin during wing disc development. Experiments mixing fragments of vg wing disc with non- vg wing disc fragments of ebony multiple wing hairs (e mwh) genotype showed that the vg cells interacted with the e mwh cells and wing blade was intercalated of both genotypes. However, structures such as wing margin, and alar lobe, usually affected in vg wings, were always made from e mwh cells and not from vg cells. Analysis of mutants which are unable to differentiate particular cell types may help us to understand the mechanism of pattern establishment in developing imaginal discs.     

Truncated products of the vestigial proliferation gene induce apoptosis.

The vestigial (vg) gene in D. melanogaster, whose mutant phenotype is characterized by wing atrophy, encodes a novel nuclear protein involved in cell proliferation. The original vg mutant (vgBG) displays massive apoptosis in the wing imaginal disc. Here we tested the hypothesis that the vg mutant phenotype could be due: (i) to lack of cell proliferation in null mutants due to the absence of the Vg product and, (ii) to apoptosis in vgBG and other mutants due to the presence of a major Vg truncated product. In agreement with our hypothesis no cell death was observed in null vg mutants, and the anticell death baculovirus P35 product is unable to rescue the mutant phenotype caused by absence of the Vg product. In addition, expression of the antiproliferative gene dacapo, the homolog of p21, induces a mutant wing phenotype without inducing cell death. In contrast the wing phenotype of the original vg mutant could be reproduced by the ectopic expression of the reaper cell death gene when expressed by vg regulatory sequences. In agreement with the hypothesis, the classic vg mutant spontaneously displays an increase in reaper expression in the wing disc and its phenotype can be partially rescued by the P35 product. Finally, we showed that ectopic expression of a truncated Vg product is able on its own to induce ectopic cell death and reaper expression. Our results shed new light on the function of the vg gene, in particular, they suggest that the normal and truncated products affect vg target genes in different ways.     

Mutants of the bithorax complex and determinative states in the thorax of Drosophila melanogaster.

Homoeotic mutations of the bithorax complex cause segmental transformations. The genes in which these mutations occur are good candidates for genes that are involved in determination. The determination system in imaginal discs must have at least two functions. One is a cell heredity function that is responsible for maintaining the determined state during growth and development. A second is the expression of the determined state (e.g., different imaginal discs have different morphologies). The homoeotic mutations of the bithorax complex could be affecting either of these two functions. I have found that when posterior haltere disc cells, that are transformed by the mutation postbithorax so that they form wing cuticle in situ, regenerate anterior structures, these structures are anterior wing. This is the same result as that seen when wild-type posterior-wing disc cells regenerate anterior structures. On the other hand, when anterior haltere disc cells transformed by the mutation bithorax³, so that they produce wing cuticle in situ, regenerate, they produce posterior haltere structures. This is unlike wild-type anterior-wing disc cells, which regenerate posterior-wing structures. From these results, I conclude that bithorax³ affects the expression of the determined state and postbithorax affects the cell heredity of determination.     

Studies in biochemical genetics in Drosophila

The metabolism of the imaginal discs of wild type, miniature, vestigial, and four-jointed varieties of Drosophila was investigated using the Cartesian diver ultramicrorespirometer. Wild type and vestigial wing disc respiration is inhibited by cyanide and azide and thus is mediated by an iron or copper porphyrin system, presumable cytochrome-cytochrome oxidase. Respiration is also inhibited by certain hydroxynaphthoquinones, believed to inactivate some enzyme between cytochromes b and c. The respiration of the vestigial and miniature wing discs is increased to normal by the addition of ascorbic acid and to a lesser extent by p-phenylenediamine and hydroquinone, hence the cytochrome oxidase and cytochrome c systems of vestigial and miniature wing discs are normal and the effects of these genes are on enzymes below cytochrome c in the respiratory chain. The respiratory enzymes of the developing imaginal discs of insects are similar to those of a wide variety of cells from bacteria to mammals. The correlation of these biochemical findings with embryological studies of the discs is discussed.     

Compartments and the control of growth in the Drosophila wing imaginal disc

The mechanisms that control organ growth are among the least known in development. This is particularly the case for the process in which growth is arrested once final size is reached. We have studied this problem in the wing disc of Drosophila, the developmental and growth parameters of which are well known. We have devised a method to generate entire fast-growing Minute(+) (M(+)) discs or compartments in slow developing Minute/+ (M/+) larvae. Under these conditions, a M(+) wing disc gains at least 20 hours of additional development time. Yet it grows to the same size of Minute/+ discs developing in M/+ larvae. We have also generated wing discs in which all the cells in either the anterior (A) or the posterior (P) compartment are transformed from M/+ to M(+). We find that the difference in the cell division rate of their cells is reflected in autonomous differences in the developmental progression of these compartments: each grows at its own rate and manifests autonomous regulation in the expression of the developmental genes wingless and vestigial. In spite of these differences, ;mosaic' discs comprising fast and slow compartments differentiate into adult wings of the correct size and shape. Our results demonstrate that imaginal discs possess an autonomous mechanism with which to arrest growth in anterior and posterior compartments, which behave as independent developmental units. We propose that this mechanism does not act by preventing cell divisions, but by lengthening the division cycle.     

Cellular degeneration in the production of some mutant phenotypes in Drosophila melanogaster

Summary A number of mutants of Drosophila melanogaster are characterized by the absence of structures present in the wild type. Imaginal discs from the wing mutants vestigial, apterous-Xasta, Beadex and cut and from the eye mutants Bar, eyeless and lozenge were examined by light and electron microscopy. In all these mutants, with the exception of lozenge, clear evidence of degeneration was found. The onset and duration of degeneration and the number and distribution of dying cells were specific characteristics of each mutant. In most cases the degenerate areas of the disc could be correlated with the missing parts of the adult wing or eye. In contrast, in wild type wing and eye discs and in wing discs from the mutant miniature, which has a wing reduced in size but fully formed, extensive cell death was not observed.The ultrastructural features of the degenerating areas weresimilar in all the mutants studied. Conspicuous aspects of the cytolytic process included condensation and fragmentation of the dying cells followed by phagocytosis of the cell fragments by neighboring disc cells.The results indicate that localized cell death during development is a widespread occurrence among Drosophila mutants which exhibit structural deficiències.     

The costae presenting in high-temperature-induced <Emphasis Type="Italic">vestigial</Emphasis> wings of <Emphasis Type="Italic">Drosophila</Emphasis>: implications for anterior wing margin formation

It has long been noted that high temperature produces great variation in wing forms of the vestigial mutant of Drosophila. Most of the wings have defects in the wing blade and partially formed wing margin, which are the result of autonomous cell death in the presumptive wing blade or costal region of the wing disc. The vestigial gene (vg) and the interaction of Vg protein with other gene products are well understood. With this biochemical knowledge, reinvestigations of the high-temperature-induced vestigial wings and the elucidation of the molecular mechanism underlying the large-scale variation of the wing forms may provide insight into further understanding of development of the wing of Drosophila. As a first step of such explorations, I examined high-temperature-induced (29°C) vestigial wings. In the first part of this paper, I provide evidences to show that the proximal and distal costae in these wings exhibit regular and continuous variation, which suggests different developmental processes for the proximal and distal costal sections. Judging by the costae presenting in the anterior wing margin, I propose that the proximal and distal costal sections are independent growth units. The genes that regulate formation of the distal costal section also strongly affect proliferation of cells nearby; however, the same phenomenon has not been found in the proximal costal section. The distal costal section seems to be an extension of the radius vein. vestigial, one of the most intensely researched temperature-sensitive mutations, is a good candidate for the study of marginal vein formation. In the second part of the paper, I regroup the wing forms of these wings, chiefly by comparison of venation among these wings, and try to elucidate the variation of the wing forms according to the results of previous work and the conclusions reached in the first part of this paper, and provide clues for further researches.     

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.     

Differential division rates and size control in the wing disc

We have generated wing disc compartments that contain marked fast growing M+ clones surrounded by slow dividing M/+ cells. Under these conditions the interactions between fast and slow dividing cells at the clone borders frequently lead to cell competition. However, our assay suppressing apoptosis indicates that cell competition plays no major role in size control. We argue that cells within a compartment proliferate according to their genotype independently of each other and that their contribution to the final structure will depend solely on their proliferation rate. This model is supported by a computer simulation that predicts values similar to those found experimentally. Our results on the growth of M+ clones within compartments and on the expression of developmental genes like vestigial and wingless suggest the existence of a non-cell autonomous mechanism that functions at the level of the entire cell population. It measures the population size in each moment, determines the corresponding expression levels of developmental genes and establishes the time to arrest growth.     

Correlations between spatiotemporal changes in gene expression and apoptosis underlie wing polyphenism in the ant Pheidole morrisi

Wing polyphenism, which is the ability of a single genome to produce winged and wingless castes in a colony in response to environmental cues, evolved just once and is a universal feature of ants. The gene network underlying wing polyphenism, however, is conserved in the winged castes of different ant species, but is interrupted at different points in the network in the wingless castes of these species. We previously constructed a mathematical model, which predicts that a key gene brinker (brk) mediates the development and evolution of these different "interruption points" in wingless castes of different ant species. According to this model, brk is upregulated throughout the vestigial wing discs of wingless ant castes to reduce growth and induce apoptosis. Here, we tested these predictions by examining the expression of brk, as well as three other genes up- and downstream of brk-decapentaplegic (dpp), spalt (sal), and engrailed (en)-in the winged reproductive and wingless soldier castes in the ant Pheidole morrisi. We show that expression of these genes is conserved in the wing disc of winged castes. Surprisingly, however, we found that brk expression is absent throughout development of the vestigial soldier forewing disc. This absence is correlated with abnormal growth of the soldier forewing disc as revealed by En expression and morphometric analyses. We also discovered that dpp and sal expression change dynamically during the transition from larval-to-prepupal development, and is spatiotemporally correlated with the induction of apoptosis in soldier forewing disc. Our results suggest that, contrary to our predictions, brk may not be a key gene in the network for suppressing wings in soldiers, and its absence may function to disrupt the normal growth of the soldier forewing disc. Furthermore, the dynamic changes in network interruptions we discovered may be important for the induction of apoptosis, and may be a general feature of gene networks that underlie polyphenism.