The vestigial locus of Drosophila melanogaster is involved in resistance to inhibitors of dTMP synthesis

The vestigal (vg) gene encodes a nuclear protein which plays a major role in the formation of the wing of Drosophila. Resistance or sensitivity to aminopterin, an inhibitor of the dihydrofolate reductase enzyme in D. melanogaster, seems to be associated with a specific alteration in vg gene function. Wild-type and vg mutant strains selected for growth on increasing concentrations of aminopterin display changes in physiological and biochemical parameters such as viability on normal and aminopterin-containing media, duration of development, wing phenotype, dihydrofolate reductase activity, and cross-resistance to fluorodeoxyuridine (FUdR) and to methotrexate. Our results indicate that the mechanisms of resistance differ in the wild-type and mutant strains. The vg ^83b27 mutant, in which the major part of intron 2 of the vg gene is deleted, is associated with a high rate of resistance to FUdR, an inhibitor of thymidylate synthetase. Moreover, vg ^83b27/vg ^BGheterozygotes, which are wild type when grown on normal medium, display a strong vg phenotype when grown on aminopterin. Our results indicate a role for the vestigial locus in mediating resistance to inhibitors of dTMP synthesis.     

The vestigial locus of Drosophila melanogaster is involved in resistance to inhibitors of dTMP synthesis

The vestigal (vg) gene encodes a nuclear protein which plays a major role in the formation of the wing of Drosophila. Resistance or sensitivity to aminopterin, an inhibitor of the dihydrofolate reductase enzyme in D. melanogaster, seems to be associated with a specific alteration in vg gene function. Wild-type and vg mutant strains selected for growth on increasing concentrations of aminopterin display changes in physiological and biochemical parameters such as viability on normal and aminopterin-containing media, duration of development, wing phenotype, dihydrofolate reductase activity, and cross-resistance to fluorodeoxyuridine (FUdR) and to methotrexate. Our results indicate that the mechanisms of resistance differ in the wild-type and mutant strains. The vg ^83b27 mutant, in which the major part of intron 2 of the vg gene is deleted, is associated with a high rate of resistance to FUdR, an inhibitor of thymidylate synthetase. Moreover, vg ^83b27/vg ^BGheterozygotes, which are wild type when grown on normal medium, display a strong vg phenotype when grown on aminopterin. Our results indicate a role for the vestigial locus in mediating resistance to inhibitors of dTMP synthesis.     

Vestigial mutants of Drosophila melanogaster live better in the presence of aminopterin: increased level of dihydrofolate reductase in a mutant

Summary Vestigial (vg) mutants of Drosophila melanogaster are characterized by atrophied wings. In this paper we show that: (1) aminopterin an inhibitor of dihydrofolate reductase (DHFR) and fluorodeoxyuridine (FUdR), an inhibitor of thymidylate synthetase induce nicks in the wings of wild-type flies and phenocopies of the vg mutant phenotype when vg/+ and vg ^B/+ flies are reared on these substances (vg^B is a deficiency of the vg locus). Only thymidine and thymidylate can rescue the flies from the effect of aminopterin. We propose that the vg phenotype is due to a decrease in the dTMP pool in the wings. (2) Mutant vg strains yield more offspring on medium containing aminopterin than on normal medium. The resistance of vg larvae to the inhibitor seems specific to the gene. This is the first case of aminopterin resistance in living eucaryotes. In contrast sensitivity of the vg larvae to FUdR is observed. (3) An increase in the activity and amount of DHFR is observed in mutant strains as compared with the wild-type flies.Our data suggest that the vg ⁺ gene is a regulatory gene acting on the DHFR gene or a structural gene involved in the same metabolic pathway.     

Cell degeneration and elimination in the imaginal wing disc,caused by the mutationsvestigial andUltravestigial ofDrosophila melanogaster

Summary The mutationsvestigial (vg; recessive) andUltravestigial (vg ^U; dominant) ofDrosophila melanogaster give rise to identical mutant adult phenotypes in which much of the cases this results from cell death in the presumptive wing margin of the wing disc in the third larval instar, but the process of cell degeneration is quite different in the two mutants. Invg cell death occurs continuously throughout the third larval instar, while invg ^U it occurs only in the early third instar. Cells fragment and some of the fragments condense, becoming electron dense (apoptosis). Both condensed and ultrastructurally normal cell fragments are extruded to the basal side of thevg disc epithelium. They accumulate under the basal lamina in the wing pouch area until they are phagocytosed by blood cells entering the wing pouch during the six hours following pupariation. Fragments are not extruded from thevg ^U epithelium but are apparently phagocytosed by neighboring epithelial cells. The basal lamina undergoes mophological changes following pupariation and is phagocytosed by blood cells in both wild-type andvestigial, but investigial the degenerated cell fragments are also engulfed by the same blood cells.     

The effect of temperature and genetic background on the phenotypic expression of severalVestigial strains ofDrosophila melanogaster

The eightvestigial strains,vg, vg-co-iso,vg;se-co-iso,vg-ms-1-co-iso,vg-ms-1; 3-co-iso,vg-ms-co-iso,vg-ms;se andvg-ms;se-co-iso showed various temperature responses. The five strains of thevg-ms group showed a greater response to temperature than did the three strains of thevg group. This tendency became more pronounced the higher the temperature was. The difference in temperature response between thevg-ms-1; 3-co-iso andvg-ms-co-iso strains indicates that the phenotypic expression ofvestigial is influenced by a modifier or modifiers located on the second chromosome of the Oregon(iso) strain. It was found that the X chromosome of the Oregon(iso) strain showed a slight modifier action in females but not in males of thevg-ms mutant.These gene in thevg-ms;se strain seemed to enhance the sensitivity to heat, and to inhibit the emergence of adult flies from pupal cases at 30°C when combined with thevg-ms gene, but no interaction was seen between these gene and thevg gene. From the results of this experiment, it is assumed that thevg-ms mutant either has a new recessive allele of thevg gene, or a modifier gene(s) closely linked tovg.This work forms part of a thesis for the doctorate of Kyoto University.     

Control of phenotypic variability in selected populations of Drosophila melanogaster

Summary In order to collect information on the mechanism determining the observed increase of wing length phenotypic variability in selected Drosophila lines, reciprocal crosses were made between +/vg and vg/vg flies within each selected population.The results obtained suggest that in our lines the increased phenotypic variability may be produced by maternally inherited agents whose activity seems to be changed according to which of the two alleles, vg or vg ⁺, is present at the vestigial locus.     

Hypoxanthine-guanine-phosphoribosyl-transferase (HGPRT) activity in the vestigial mutant of Drosophila melanogaster: Effect of inhibitors of the purine pathway

In cultured cells established from Drosophila melanogaster embryos, and in wild type files, no hypoxanthineguaninephosphoribosyltransferase (HGPRT) could be detected. In extracts of vestigial mutants and in vg/+ flies, HGPRT and 5nucleotidase activities were detected without treatment by charcoal. A considerable diminution of HGPRT for vg flies reared at 30°C was detected, but none for the vg ^np stock. A slight resistance of vg flies to azaserine and 6-mercaptopurine was observed, along with a sensitivity to increasing concentrations of 8-azaguanine. These results suggest that HGPRT is active in vivo in vestigial mutants and that the nucleotide purine pool of such flies is perturbed.     

Evolutionary origin of the insect wing via integration of two developmental modules

SUMMARY Insect wing is a key evolutionary innovation for insect radiation, but its origins and intermediate forms are absent from the fossil record. To understand the ancestral state of the wing, expression of three key regulatory genes in insect wing development, wingless (wg), vestigial (vg), and apterous (ap) was studied in two basal insects, mayfly and bristletail. These basal insects develop dorsal limb branches, tracheal gill and stylus, respectively, that have been considered candidates for wing origin. Here we show that wg and vg are expressed in primordia for tracheal gill and stylus. Those primordia are all located in the lateral body region marked by down‐regulation of early segmental wg stripes, but differ in their dorsal–ventral position, indicating their positions drifted within the lateral body region. On the other hand, ap expression was detected in terga of mayfly and bristletail. Notably, the extensive outgrowth of the paranotal lobe of apterygote bristletail developed from the border of ap‐expressing tergal margin, and also expressed wg and vg. The data suggest that two regulatory modules involving wg–vg are present in apterygote insects: one associated with lateral body region and induces stick‐like dorsal limb branches, the other associated with the boundary of dorsal and lateral body regions and the flat outgrowth of their interface. A combinatorial model is proposed in which dorsal limb branch was incorporated into dorsal–lateral boundary and acquired flat limb morphology through integration of the two wg–vg modules, allowing rapid evolution of the wing.     

Dihydrofolate reductase activity and resistance to aminopterin in various species of Drosophila

Summary The aim of our work was to compare the mechanisms of resistance to aminopterin, inhibitor of the dihydrofolate reductase enzyme, between different Drosophila species and those described for cultured cells. Moreover we compared the systematic species divisions based on morphological traits and those based on a molecular approach. For this purpose, the effect of aminopterin on viability and wing phenotype was studied in different Drosophila species. Dihydrofolate reductase was measured in adult flies. We found an important dihydrofolate reductase activity in the melanogaster sub-group compared to the other species studies. Wing effect was observed only in this sub-group. The effects of aminopterin on the wing phenotype were very similar to the phenotype of rudimentary mutants. Both deplete the pyrimidine pool and it has been shown by the studies of the structural genes of the nucleotide pyrimidine pathway that the wing tissue is very sensitive to every pertubation of this metabolism.The D. ananassae species was found to be fully resistant at the concentrations of the inhibitor tested. No or very little dihydrofolate reductase activity was detected. The binding of the enzyme to the inhibitor was comparable to that found in the Oregon strain of D. melanogaster. The purine and pyrimidine salvage pathways were investigated and the D. ananassae species displayed an important thymidine kinase activity. The D. ananassae flies were sensitive on Sang medium compared to the Oregon flies but were able to use exogenous bases or nucleosides more efficiently. Therefore the mechanism of resistance to aminopterin in Drosophila may be different from those described for methotrexate in mammalian cultured cells, as indicated by the results obtained for D. ananassae.     

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.     

Mutants of Saccharomyces cerevisiae That Incorporate Deoxythymidine-5′-Monophosphate Into Deoxyribonucleic Acid In Vivo

Spontaneous mutants of Saccharomyces cerevisiae able to incorporate deoxythymidine-5′-monophosphate (dTMP) into deoxyribonucleic acid (DNA) have been selected based on their ability to grow in the presence of aminopterin and sulfanilamide if dTMP is present. Essentially all mutants (called tup) selected in this way required dTMP for growth in the presence of the two drugs, but none required dTMP in the absence of the drugs. Neither thymine nor thymidine would satisfy this requirement. Equimolar amounts of ³²P- and ³H-base-labeled dTMP were incorporated by the mutants into alkali-stable, deoxyribonuclease-sensitive material. In the presence of aminopterin and sulfanilamide, this incorporation was sufficient to account for a substantial proportion of the thymine residues in the cellular DNA, whereas in the absence of the drugs only about 40% as much of the thymine residues originated from the medium. Of 29 mutants examined, all were recessive and 17 showed 2:2 segregation in crosses with a wild-type strain. The lesions in these mutants fell into four complementation groups: one (tup1) occurs on chromosome III; another (tup3) is on chromosome II; and a third (tup4) was centromere linked. Strains of the genotype α tup1 mated with lower than normal efficiency with a strains, but with higher than normal efficiency with α strains. Strains of genotype a/α tup1/tup1 failed to sporulate, whereas homozygous diploids for tup2, tup3, or tup4 sporulated normally, as did a/α tup1/+ strains.     

The Effect of the Genotype on the Expressiveness of Character vestigial and Polyteny of Giant Chromosomes in Drosophila melanogaster Meig.

The effects of the Bar (B) andwhite (w) mutations on the expressiveness of the character vestigial (vg) and the degree of polyteny of salivary gland giant chromosomes were studied in Drosophila melanogaster.Either mutation changed both the expressiveness of vestigial and the degree of chromosome polyteny. A negative association between the vg expressiveness and the degree of chromosome polyteny was revealed and proved to be stronger in females than in males. The parameters under study were shown to differ between females and males.     

Spatiotemporally modulated Vestigial gradient by Wingless signaling adaptively regulates cell division for precise wing size control

In animal development, the growth of a tissue or organ is timely arrested when it reaches the stereotyped correct size. How this is robustly controlled remains poorly understood. The prevalent viewpoint, which is that morphogen gradients, due to their organizing roles in development, are directly responsible for growth arrest, cannot explain a number of observations. Recent findings from studies of the Drosophila wing have revealed that the interpretation of the Wingless gradient requires signaling-induced self-inhibition and that cell proliferation is controlled by graded vestigial expression. These findings highlight a growth control mechanism that involves Wingless regulated vestigial expression, but a question is whether they can quantitatively explain the observed preciseness and robustness of wing size control. Quantitative and systematic investigation into Wingless signaling using a mathematical model has elucidated two points. First, negative regulation of the Vestigial gradient by Wingless signaling makes vestigial expression precise and robust. Second, weak Wingless signaling in a primarily small wing pouch causes a short and steep Vestigial gradient, which stimulates more cell divisions and leads to a significant expansion of the wing pouch; however, strong Wingless signaling in a primarily large wing pouch causes a long and smooth Vestigial gradient, which stimulates fewer cell divisions and results in a slight expansion of the wing pouch. These results substantially decipher an inherent mechanism of tissue and organ size control. Our model explains, and is supported by, a number of experimental observations.     

The effects of chemicals on the phenotypic expression of aVestigial mutant inDrosophila melanogaster

Several kinds of chemicals were tested to determine their effects on the wings of avestigial mutant inDrosophila melanogaster. Two isogenic strains were used, viz.vg-ms andB; vg-ms, both co-isogenic with the Oregon-isogenic strain. Ammonium lactate was found to have a conspicuous effect in enlarging thevestigial wings. At an appropriate concentration of this agent the wings of thevg-ms- andB;vg-ms-co-isogenic strains were restored almost to wild-type wing at 27°C. This fact indicates that the wings of thevg-ms mutant can be restored to wild type not only by temperature but also by certain chemicals. Ammonium lactate was effective conspicuously on bothBar andvestigial-ms mutants. However, there was found an interaction betweenBar andvestigial-ms when these genes were combined. The manifestation ofB is enhanced byvg-ms, especially under the the condition of feeding in medium containing the chemical, and the manifestation ofvg-ms is reduced byB.This work forms part of a thesis for the doctorate of Kyoto University.     

Integration of differentiation signals during indirect flight muscle formation by a novel enhancer of Drosophila vestigial gene

The gene vestigial (vg) plays a key role in indirect flight muscle (IFM) development. We show here that vg is controlled by the Notch anti-myogenic signaling pathway in myoblasts and is regulated by a novel 822 bp enhancer during IFM differentiation. Interestingly, this muscle enhancer is activated in developing fibers and in a small number of myoblasts before the fusion of myoblasts with the developing muscle fibers. Moreover, we show that this enhancer is activated by Drosophila Myocyte enhancing factor 2 (MEF2), Scalloped (SD) and VG but repressed by Twist, demonstrating a sensitivity to differentiation in vivo. In vitro experiments reveal that SD can directly bind this enhancer and MEF2 can physically interact with both SD and TWI. Cumulatively, our data reveal the interplay between different myogenic factors responsible for the expression of an enhancer activated during muscle differentiation.