Pyrimidine auxotrophy and the complementation map of the Rudimentary locus of Drosophila melanogaster

Summary The complementation pattern of twelve rudimentary mutations has been analyzed at two different levels. When analyzed on the basis of complementation for a wing abnormality the mutations can be divided into three groups, each of which is believed to affect the activity of one of the first three enzymes of pyrimidine synthesis (Norby, 1973; Jarry and Falk, 1974; Rawls and Fristrom, 1975). However, when the mutants are analyzed for complementation on the basis of a second phenotype, pyrimidine auxotrophy, the distinction between two of these three groups is not evident. The disparity in the two patterns probably reflects a different threshold of gene activity required for the detection of an auxotrophic phenotype as compared to that at which a wing abnormality is detectable.The biochemical basis of these results is interpreted in light of recent data suggesting that at least the first two enzymes of pyrimidine synthesis are contained within a single multifunctional protein complex (Soderholm et al., 1975).     

Repair of a genetically-caused defect in oogenesis in Drosophila melanogaster by transplantation of cytoplasm from wild-type eggs and by injection of pyrimidine nucleosides

Eggs produced by homozygous mutant rudimentary (r⁹, 154.5) females of Drosophila melanogaster die during embryogenesis, apparently because the mutant female fails to incorporate certain substances into the egg during oogenesis. These eggs can be rescued by injecting them at the preblastoderm stage with wild-type nuclei and cytoplasm or wild-type cytoplasm alone from unfertilized eggs. Some preblastoderm eggs injected with 1.5% of egg volume of cytoplasm from unfertilized wild-type eggs were able to complete both embryonic and postembryonic development and emerged as adults, whereas not a single uninjected control egg was able to complete embryonic development. The eggs of rudimentary mothers can also be rescued by injecting each egg at the blastoderm stage with 0.01 μg of pyrimidine nucleosides. The results demonstrate that a pyrimidine deficiency is the cause of abortive embryogenesis, and confirm the previous finding that the rudimentary mutation leads to pyrimidine auxotrophy.     

A genetic and dietary study of the physiology of pyrimidine synthesis in Drosophila melanogaster

A combination of genetic and dietary manipulations have been utilized to investigate the physiology of pyrimidine metabolism throughout the Drosophila life cycle. We present evidence that the dietary sources of pyrimidines ingested at the larval stage are sufficient for all subsequent stages of the life cycle, except the process of oögenesis in adult females where a maternal supply of endogenously synthesized pyrimidines is normally required. Deprivation of dietary and de novo synthesis does not affect adult longevity; indicating that with the exception of oögenesis, all normal functions are carried out by recycling pyrimidines produced or ingested at the larval stage.In an enzymatic analysis, we have determined that Drosophila does not adjust for pyrimidine dietary deficiencies by significantly altering the level of synthesis of two tested enzymes encoded by the rudimentary locus, even though the pyrimidine deficiency is a rate limiting step in the completion of development.     

Role of the <Emphasis Type="Italic">porcupine</Emphasis> gene in the development of the wing imaginal disk of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

A search for the genes interacting with the Merlin tumor suppressor gene revealed a Merlin-porcupine interaction during wing morphogenesis. Ectopic expression of the porcupine gene in the wing imaginal disk reduced the adult wing, while addition of an UAS construct with a full-length or truncated copy of the Merlin gene partly restored the wing phenotype. The highest restoration level was observed upon adding the fragments coding for the C end of the Merlin protein. In addition, the porcupine gene was shown to mediate the wingless gene autoregulation, which occurs at two ontogenetic stages, segmentation during embryo development and determination of the wg expression band at the boundary between the dorsal and ventral compartments of the wing imaginal disk.     

Control of pupal commitment in the imaginal disks of Precis coenia (Lepidoptera: Nymphalidae)

When final (5th) instar larvae of Precis coenia were treated with the juvenile hormone analog (JHA) methoprene, they underwent a supernumerary larval molt, except for certain regions of their imaginal disks, which deposited a normal pupal cuticle. Evidently those regions had already become irreversibly committed to pupal development at the time JHA was applied. By applying JHA at successively later times in the instar, the progression of pupal commitment could be studied. Pupal commitment in the proboscis, antenna, eye, leg and wing imaginal disks occurred in disk-specific patterns. In each imaginal disk there were distinct initiation sites where pupal commitment began during the first few hours of the final larval instar, and from which commitment spread across the remainder of the disk over a 2- to 3-day period. The initiation sites were not always located in homologous regions of the various disks. As a rule, pupal commitment also spread from imaginal disk tissue to surrounding epidermal tissue. The regions of pupal commitment in all disks except those of the wings, coincided with the regions of growth of the disk. Only portions of the disk that had undergone cell division and growth underwent pupal commitment. Shortening the growth period did not prevent pupal commitment in the wing imaginal disk, indicating that, in this disk at least, a normal number of cell divisions was not crucial in reprogramming of disk cells for pupal cuticle synthesis. The apparent growth spurt of imaginal disks that occurs during the last part of the final larval instar is merely the final stage of normal and constant exponential growth. Juvenile hormone (JH) and ecdysteroids appeared to play little role in the regulation of normal imaginal disk growth. Instead, growth of the disks may be under intrinsic control. Interestingly, even though endogenous fluctuation in JH titers do not affect imaginal disk growth, exogenous JHA proved able to inhibit both pupal commitment, cell movement, and growth of the disks during the last larval instar. This function of JH could be important under certain adverse conditions, such as when metamorphosis is delayed in favor of a supernumerary larval molt.     

Chitin synthesis in Spodoptera frugiperda wing imaginal discs. III: Role of the peripodial membrane

We determined the contribution of the peripodial membrane to chitin synthesis in cultured wing imaginal discs of Spodoptera frugiperda. This was accomplished by examining chitin synthesis in vitro in intact imaginal discs, in the peripodial membrane, and in imaginal discs in which the peripodial membrane had been injured. Chitin synthesis in peripodial membrane-deprived imaginal discs, peripodial membrane injured imaginal discs, and peripodial membrane fragments was assessed by measuring incorporation of [¹⁴C]GlcNAc after treatment with 20-hydroxyecdysone in tissue culture. Removing or injuring the peripodial membrane resulted in a marked decrease in ecdysteroid-dependent chitin synthesis in these wing discs compared with intact wing discs. In addition, a break in the ecdysteroid treatment of 4 h reduced chitin synthesis in the wing discs substantially. These biochemical experiments were supplemented with ultrastructural and immunocytochemical approaches. A wheat germ agglutinin colloidal gold complex was used to visualize the presence of chitin synthesized by wing discs including the peripodial membrane. These experiments confirmed the importance of an intact peripodial membrane for optimal production of cuticle by the wing pouch. Our results demonstrate that for opti-ma1 production of chitin in tissue culture, wing discs must be treated with 20-hydroxyecdysone for an uninterrupted period of 48 h, and the peripodial membrane of these imaginal discs must be present and uninjured. © 1995 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Control of growth and differentiation of the wing imaginal disk of Precis coenia (Lepidoptera: Nymphalidae)

During the last larval instar, the wing imaginal disks of Precis coenia grow continuously. The rate of disk growth is not disk-autonomous but closely matches the rate of somatic growth of the larva, so that the size of the disks is a function of the size of the body, irrespective of the growth rate of the larva. When larvae are starved, their wing disks cease growth within 4 h, which indicates the existence of an efficient coupling mechanism between the growth of the soma and growth of the imaginal disks. Disk growth is inhibited by juvenile hormone in a dose-dependent manner. In the presence of the hormone the wing disks stop growing even while the larva continues to grow normally. During the last larval instar the wing imaginal disks also undergo a complex differentiation, consisting of the development of the lacunae and tracheation that define the future adult wing venation system. In normally growing larvae, differentiation of the wing disk is tightly correlated with wing size. Differentiation and size can be dissociated by starvation. If larvae are starved at any time after differentiation has begun, differentiation continues at a normal rate, even though the wing disk does not grow. Differentiation does not begin spontaneously in larvae that are starved before differentiation has begun. These findings indicate that the initiation of differentiation and its continuation are controlled independently. Juvenile hormone inhibits differentiation in a dose-dependent manner. Upon treatment with juvenile hormone, the stage of differentiation becomes fixed. These findings indicate that continued differentiation of the wing disk can only occur in the absence of juvenile hormone. Although the circulating level of juvenile hormone may be elevated during starvation, it is unlikely that this elevation is responsible for the observed effect of starvation on growth and differentiation of the disk.     

The time during which beta-ecdysone is required for the differentiation in vitro and in situ of wing imaginal discs of Drosophila melanogaster.

The time during which β-ecdysone is required for the apolysis and imaginal differentiation of wing discs of Drosophila both in vitro and in situ has been examined, and it is concluded that β-ecdysone is required as a sustained stimulus rather than as a trigger for differentiation. These results are compared with the requirement for β-ecdysone for the puffing of salivary gland polytene chromosomes during the prepupal stage (Richards, G. P., 1976, Develop. Biol.48, 191–195). It is suggested that imaginal discs and larval salivary glands require different exposures to β-ecdysone to fulfill their developmental commitments and that the drop in β-ecdysone titer during the early prepupal stage, which is necessary for the subsequent puffing of the polytene chromosomes, plays little or no part in imaginal disc differentiation.     

Spalt major controls the development of the notum and of wing hinge primordia of the Drosophila melanogaster wing imaginal disc

The Drosophila wing and the dorsal thorax develop from primordia within the wing imaginal disc. Here we show that spalt major (salm) is expressed within the presumptive dorsal body wall primordium early in wing disc development to specify notum and wing hinge tissue. Upon ectopic salm expression, dorsally located second leg disc cells develop notum and wing hinge tissue instead of sternopleural tissue. Similarly, by salm over-expression within the wing disc, wing blade formation is suppressed and a mirror-image duplication of the notum and wing hinge is formed. In large dorsal clones, which lack salm and its neighboring paralogue spalt related (salr), the cells of the notum primordium do not grow; these dorsal cells are not specified as notum, hence no notum outgrowth develops. These results suggest that the zinc finger factors encoded by the salm/salr complex play important roles in defining cells of the early wing disc as dorsal body wall cells, which develop into a large dorsal body wall territory and form mesonotum and some wing hinge tissue, and in delimiting the wing primordium. We also find that salm activity is down-regulated by its own product and by that of the Pax gene eyegone.     

Studies on the female-sterile mutant rudimentary of Drosophila melanogaster,: I. An analysis of the rudimentary wing phenotype

The rudimentary wing phenotype was examined in detail, using six different alleles of rudimentary, and a number of points about the genesis of the r phenotype were made. (1) All of the r alleles in which the wings are defective produce wings in which the area of individual hair cells is reduced. The more severely affected the allele, the greater is the reduction in wing cell area. This reduction in area is probably uniform throughout the wing rather than localized to specific wing regions. (2) The total number of cells per wing is also greatly reduced in phenotypically r wings. As with cell area, the more severely affected the allele, the greater the reduction in cell number. However, the reduction in cell number is not uniform throughout the wing. In the less severely affected alleles, the cell number reduction is much greater in those regions of the wing which are drastically altered in shape (truncated), while those wing regions which show only slight size reductions but no overall shape changes have near normal numbers of cells. In the most deformed wings, there is a reduction in cell number throughout the wing, but again those regions with are severely truncated are the most drastically reduced in cell number. Measurements of the amount of chitin per wing indicated that the three most severely affected alleles had as much or more chitin than the wild type. It is suggested that overproduction of chitin in these alleles prevents normal expansion of the wing cells, thus increasing the severity of the wing defect. Finally, the validity and limitations of a quantitative measure of the r phenotype were defined. This measure was utilized to demonstrate a clear-cut effect of nutrition on the expression of the r phenotype.     

Clonal analysis of the undifferentiated wing disk of Drosophila

Using a new cell marker, we have examined the early clonal restrictions in wing imaginal disks from late third instar larvae of Drosophila. Large clones do not significantly alter the gross structure of the disks, allowing us to map the clones relative to morphological landmarks. Clones in the posterior region of the disks behave in a similar way to clones in the adult cuticle; that is, they appear to be restricted to a defined compartment, and the presumptive anterior/posterior compartment border defined by these clones is located in a similar place in every disk. In contrast, clones in the anterior region of the wing disks often cross into the region normally occupied by the posterior compartment and, especially near the margins of the disk, show no common posterior boundary. We believe that the anterior clones are “pushing” the anterior/posterior compartment border, and that this pushing is related to the growth advantage of the marked cells, which are Minute⁺ in a Minute background. Finally, we find that clones do not cross between the adepithelial cells, which contribute to the adult musculature, and the disk epithelium.     

Towards Long Term Cultivation of Drosophila Wing Imaginal Discs In Vitro

The wing imaginal disc of Drosophila melanogaster is a prominent experimental system for research on control of cell growth, proliferation and death, as well as on pattern formation and morphogenesis during organogenesis. The precise genetic methodology applicable in this system has facilitated conceptual advances of fundamental importance for developmental biology. Experimental accessibility and versatility would gain further if long term development of wing imaginal discs could be studied also in vitro. For example, culture systems would allow live imaging with maximal temporal and spatial resolution. However, as clearly demonstrated here, standard culture methods result in a rapid cell proliferation arrest within hours of cultivation of dissected wing imaginal discs. Analysis with established markers for cells in S- and M phase, as well as with RGB cell cycle tracker, a novel reporter transgene, revealed that in vitro cultivation interferes with cell cycle progression throughout interphase and not just exclusively during G1. Moreover, quantification of EGFP expression from an inducible transgene revealed rapid adverse effects of disc culture on basic cellular functions beyond cell cycle progression. Disc transplantation experiments confirmed that these detrimental consequences do not reflect fatal damage of imaginal discs during isolation, arguing clearly for a medium insufficiency. Alternative culture media were evaluated, including hemolymph, which surrounds imaginal discs during growth in situ. But isolated larval hemolymph was found to be even less adequate than current culture media, presumably as a result of conversion processes during hemolymph isolation or disc culture. The significance of prominent growth-regulating pathways during disc culture was analyzed, as well as effects of insulin and disc co-culture with larval tissues as potential sources of endocrine factors. Based on our analyses, we developed a culture protocol that prolongs cell proliferation in cultured discs.     

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.     

Adult abnormalities resulting from a localized blastoderm defect in a maternal-effect mutant of Drosophila.

In the mutant mat(3)3 of Drosophila melanogaster, there is a temperature-sensitive maternal effect on blastoderm formation. When oogenesis occurs in homozygous mat(3)3 females at the fully restrictive temperature of 29°C, the embryonic progeny form a defective cellular blastoderm in which cells are either completely or partially missing from a posterior-dorsal region, and the embryos die before hatching. Transplantation tests for the presence in the embryos of primordial imaginal cells capable of developing into adult structures showed a relatively high yield of eye and antenna structures, an intermediate yield of labium structures, and low or zero yields of wing, haltere, and leg structures. These results are consistent with the fate mapping of the primordial imaginal cells by analysis of gynandromorph mosaics; the eye and antenna map in the fully cellular region of the mutant blastoderm, the labium near the border of the defective region, and the wing, haltere, and legs within the defective region. When oogenesis oocurs at a lower temperature, the lethal maternal effect in mat(3)3 is reversed, but there is a nonlethal effect on larval and adult progeny of the mat(3)3 females. Many of the adults are missing one or more cuticular structures, usually a leg, haltere, or abdominal segment, and many of the larvae are missing the corresponding imaginal discs from which the thoracic structures are derived. These selective effects on imaginal development appear to be caused by maternally induced blastoderm defects that are less extensive at the lower temperature of oogenesis.     

Multiple roles of the gene zinc finger homeodomain-2 in the development of the Drosophila wing

The gene zfh2 and its human homolog Atbf1 encode huge molecules with several homeo- and zinc finger domains. It has been reported that they play important roles in neural differentiation and promotion of apoptosis in several tissues of both humans and flies. In the Drosophila wing imaginal disc, Zfh2 is expressed in a dynamic pattern and previous results suggest that it is involved is proximal–distal patterning. In this report we go further in the analysis of the function of this gene in wing development, performing ectopic expression experiments and studying its effects in genes involved in wing development. Our results suggest that Zfh2 plays an important role controlling the expression of several wing genes and in the specification of those cellular properties that define the differences in cell proliferation between proximal and distal domains of the wing disc.     

Intercellular junctions during development and in tissue cultures ofDrosophila melanogaster: An electron-microscopic study

Summary The present investigation analyzes intercellular junctions in tissues with different developmental capacities. The distribution of junctions was studied inDrosophila embryos, in imaginal disks, and in cultures of disk cells that were no longer able to differentiate any specific pattern of the adult epidermis.The first junctions —primitive desmosomes andclose membrane appositions — already appear in blastoderm.Gap junctions are first detected in early gastrulae and later become more and more frequent.Zonulae adhaerentes are formed around 6 h after fertilization, whileseptate junctions appear in the ectoderm of 10-h-old embryos.Inwing disks of all stages studied (22–120 h), three types of junctions are found: zonulae adhaereentes, gap junctions, and septate junctions. Gap junctions, which are rare and small at 22 h, increase in number and size during larval development. The other types of junctions are found between all cells of a wing disk throughout development.All types of junctions that are found in normal wing disks are also present in theimaginal disk tissues cultured in vivo for some 15 years and in thevesicles of imaginal disk cells grown in embryonic primary cultures in vitro. However, gap junctions are smaller and in the vesicles less frequent than in wing disks of mature larvae.Thus gap junctions, which allow small molecules to pass between the cells they connect, are present in the early embryo, when the first developmental decisions take place, and in all imaginal disk tissues studied, irrespective of whether or not these are capable of forming normal patterns.