The ultrastructure of the developing leg ofDrosophila melanogaster

Summary The ultrastructure of the imaginal discs ofDrosophila melanogaster was compared with that of other chitogenous tissues with different developmental capacities, namely, embryonic, larval, pupal and adult epidermis. Attention was paid to features which might be correlated with specific morphogenetic activities. Previous morphological studies of imaginal discs of Diptera were analyzed in detail and a somewhat revised view of imaginal disc structure emerged. The results reveal that the imaginal discs ofD. melanogaster consist of three types of cells: cells of the single layered disc epithelium, adepithelial cells and nerves. Four types of specialized junctions connect the cells of the disc epithelium: zonulae adhaerens, septate desmosomes, gap junctions and cytoplasmic bridges. The junctions are discussed in relation to their possible roles in adhesion and intercellular communication. It was concluded that gap junctions may be a more likely site for the intercellular communication involved in pattern formation than septate desmosomes. Evidence is presented that adepithelial cells are the precursors of imaginal muscles and that some cell lines (atelotypic) are in fact lines of adepithelial cells which can differentiate into muscle.Specific imaginal discs can be easily recognized by their overall morphology, i.e. patterns of folds. However, no ultrastructural features were found which we could correlate with the state of determination of the cells. Most differences in the ultrastructure of different discs at several developmental stages were attributable to different phases of cuticle secretion. The cells of the imaginal disc epithelium are packed with ribosomes but very little rough ER. The amount of rough ER increases rapidly at puparium formation. Cuticulin is recognizable 4–6 hours after puparium formation. Six hours after puparium formation, the cells of the disc epithelium are secreting the epicuticle of the pupa. As the imaginal disc of a leg everts from a folded sac to the tubular pupal leg, the cells of the disc epithelium change from tall columnar to cuboidal. A loss of microtubules in the long axis of the columnar cells accompanies this change. Prepupal morphogenesis of the leg appears to be caused by the change in cell shape. Evidence is presented which is incompatible with previous explanations of the mechanism of eversion of imaginal discs.There is some turnover of the cells of the disc epithelium as evidenced by autophagy and the occasional heterophagy of a dead neighbor. However this does not appear to be an important factor in the morphogenesis of discs. Plant peroxidase which was used as a tracer of proteins in the blood was taken up from the hemolymph by the disc epithelium. Imaginal disc cells contain many lipid droplets which coalesce and are replaced by glycogen during the prepupal period.

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Zusammenfassung Die Feinstrukturen der Imaginalscheiben, der embryonalen, larvalen, pupalen und adulten Epidermis, alles chitinbildende Gewebe, wurden untersucht und miteinander verglichen. Besondere Aufmerksamkeit legten wir auf ultrastrukturelle Merkmale, die mit spezifischen morphogenetischen Vorgängen korreliert sein können. Frühere Untersuchungen über die Morphologie der Imaginalscheiben bei Dipteren wurden kritisch analysiert und führten mit unseren Resultaten zu einem etwas veränderten Bild der Scheibenstruktur. Die Imaginalscheiben vonDrosophila melanogaster bestehen aus drei Zelltypen: Zellen des einschichtigen Epithels, adepitheliale Zellen und Nerven. Die Epithelzellen weisen vier spezialisierte Zellverbindungen auf: zonulae adherens, septate desmosomes, gap junctions und zytoplasmatische Brücken. Die Funktion dieser Zellverbindungen wird im Zusammenhang mit der Zelladhäsion und Zellkommunikation diskutiert. Es scheint, daß während der Musterbildung, die gap junctions, eher als die septate desmosomes, die Orte der Zellkommunikation sind. Wir haben gezeigt, daß adepitheliale Zellen Vorläufer der imaginalen Muskeln sind. Einige atelotypische Linien, die sich als Kulturen adepithelialer Zellen erwiesen, differenzieren Muskeln.Die Imaginalscheiben können leicht an ihrer Gesamtmorphologie, d.h. an ihrem Faltenmuster erkannt werden. Ultrastrukturelle Merkmale wurden jedoch nicht beobachtet, die mit dem Determinationszustand der Zelle korrelierbar wären. Während der Entwicklung sind die meisten Unterschiede in der Feinstruktur auf verschiedene Phasen der Kutikulasekretion zurückzuführen. Die Epithelzellen der Imaginalscheiben zeigen viele Ribosomen, besitzen aber nur sehr wenig endoplasmatisches Reticulum. Dieses nimmt erst bei der Pupariumbildung stark zu. 4–6 Std nach Puparisierung ist Kutikulin nachweisbar und nach 6 Std scheiden die Epithelzellen die Epikutikula aus. Während sich die Beinscheibe vom gefalteten Sack zum röhrenförmigen Bein ausstülpt, werden die länglichen Epithelzellen kubisch. Gleichzeitig mit dieser Formänderung verschwinden die Microtubuli in der Längsachse der Zellen. Die Morphogenese des Beines im Vorpuppenstadium scheint auf eine Änderung der Zellform zu beruhen. Früher beschriebene Erklärungen für den Mechanismus der Ausstülpung sind mit unseren Beobachtungen nicht vereinbar. Autophagozytose und gelegentlich Heterophagozytose einer toten Nachbarzelle konnten in den Epithelzellen nachgewiesen werden. Dies scheint jedoch kein wesentlicher Faktor für die Morphogenese der Scheibe zu sein. Pflanzenperoxydase, als Tracer-Protein im Blut, wird vom Scheibenepithel aus der Hämolymphe aufgenommen. Scheibenzellen enthalten viele Lipidtröpfchen, die sich vereinigen und während des Vorpuppenstadiums durch Glycogen ersetzt werden.

     

Chromosomal effects on peptidase activities inDrosophila melanogaster

The peptidase system inDrosophila melanogaster (dipeptidase-A, -B, and -C and leucine aminopeptidases G and P) was used as a model to study the effects of modifier genes on activity of enzymes with similar functions. A screen of X, second, and third chromosome substitution isogenic lines revealed the presence of activity modifiers for peptidases on all three chromosomes. Correlation analyses indicated that covariation between some of the peptidase activities is independent of genetic background, while others are associated with variable second chromosomes. Chromosome-specific effects onK _m ,V _max, and specific activity of partially purified peptidases were also detected. Moreover, a repeatable technique using anion-exchange column chromatography allowed the characterization of possibly two putative peptidic enzymes, glycyl-l-isoleucine-ase andl-leucyl-l-proline-ase, whose kinetic properties differ from the dipeptidases and the leucine aminopeptidases. These findings confirm the existence of activity modifiers for peptidases, much like other enzymes inDrosophila melanogaster. These studies were supported by grants from the National Institutes of Health (GM42-115-01A1), the Whitaker Foundation of the Research Corporation (C-2560), and the National Science Foundation (USE 8951018) to Kazuo Hiraizumi.     

Sensitive periods for abnormal patterning on a leg segment inDrosophila melanogaster

Summary The development of a leg segment of the fruitflyDrosophila melanogaster was analyzed in order to determine whether the orderliness of the segment's bristle pattern originates via waves of cellular interactions, such as those that organize the retina. Fly development was perturbed at specific times by either teratogenic agents (gamma rays, heat shock, or the drug mitomycin C) or temperature-sensitive mutations (l(1)63, l(1) Notch^ts1, orl(1) shibire ^ts1 ), and the resulting abnormalities (e.g., missing or extra structures) were mapped within the pattern area. If bristles develop in a linear sequence across the pattern, then they should show sensitivity to perturbations in the same order, and wavefronts of cuticular defects should result. Contrary to this prediction, the maps reveal no evidence for any directional waves of sensitivity. Nevertheless, other clues were uncovered as to the nature and timing of patterning events. Chemosensory bristles show earlier sensitivities than mechanosensory bristles, and longer bristles precede shorter ones. The types and sequence of cuticular abnormalities imply the following stages of bristle pattern development: (1) scattered inception of bristle mother cells, each surrounded by an inhibitory field, (2) alignment of the mother cells into rows, (3) differential mitoses, (4) assignment of cuticular fates to the mitotic progeny, (5) polytenization of the bristle cells, (6) fine-tuning adjustments in bristle spacing, and (7) signalling from bristle cells to adjacent epidermal cells, inducing them to form bracts.     

The effects of tandem duplications on crossing over inDrosophila melanogaster

Each of three tandem duplications,Bar, Beadex ^r49k andDp(I)z-w, when homozygous increases crossing over in their environs in excess of the genetic length of the duplication.Detailed crossing over studies withDp(I)z-w showed that in the duplication homozygotes interference is reduced and when combined with heterologous autosomal inversions, double crossovers occurring in less than 10 map units are readily recovered.These results are interpreted in terms of the concept of effective pairing and suggest that tandem duplications increase crossing over by increasing effective pairing.     

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.     

Cell clones and pattern formation: Genetic eye mosaics inDrosophila melanogaster

Summary Genetic eye mosaics ofDrosophila melanogaster have been studied by means of anatomical techniques. Using different cell markers it was found that the ommatidia at the boundaries between phenotypes are composed of cells belonging to different clones. Therefore, the formation of an individual ommatidium does not obey a mechanism based on a common clonal origin of its constituent elements. A statistical analysis of mosaic ommatidia shows that there is a significant tendency for the receptor cellsR2-R5 on the one hand and the receptor cellsR1, R6 andR7 on the other to belong to the same cell clone. The implications of these findings are discussed.     

Isolation of temperature sensitive mutations blocking clone development inDrosophila melanogaster,and the effects of a temperature sensitive cell lethal mutation on pattern formation in imaginal discs

Summary A method of isolating temperature-sensitive (ts) mutations blocking clone development, based on the analysis of twin spots produced by X-ray induced somatic recombination is reported. From this screen 10 ts mutations were recovered which caused an absence of the lethal-bearing clone at the restrictive temperature. Eight of these mutations were analyzed. Seven proved to be autonomous ts cell lethals and one was an autonomous ts mutation which reversibly affected cell division and growth of imaginal disc cells and growth of larval cells. The effects on development of one of the cell lethal mutations,l(1)ts-504, are described. Heat pulses (29°C) 24–72 hrs long caused a high frequency (up to 90%) of morphologically abnormal animals. The abnormalities observed were of two major kinds: deficiencies and duplications of imaginal disc derivatives. In addition, alterations of tarsal segmentations occurred. Heat pulses to larvae also delayed pupariation and eclosion by as much as four days. In general, longer pulses led to a greater delay in pupariation and eclosion and a higher frequency of deficiencies and duplications than shorter pulses. Exposure to restrictive temperature early in larval development delayed pupariation and resulted in mostly normal animals; exposure during the second and early third larval instar also delayed pupariation and led to a high frequency of duplications; exposure later in larval life, i.e. mid and late third larval instar, caused no delay in pupariation but led to a high frequency of deficiencies. These results can be explained by the occurrence of areas of cell death, which can be seen in the imaginal discs of larvae exposed to restrictive temperature by staining with trypan blue. This conclusion is further supported by the observation in gynandromorphs of duplications of female nonmutant tissue. These results are discussed in relation to current theories of pattern formation.     

Selection and temperature effects on extra dorsocentral bristles inDrosophila melanogaster

Starting from base populations which showed a tendency to form supernumerary dorsocentral bristles, selection for high numbers of dorsocentrals was carried out. In all, 9 lines were selected 6 to 14 generations. Selection proved to be effective in all but two lines. Selection for reduced numbers in one of the lines also proved effective.Mean values of over 10 were reached in the females of certain high lines. Average counts in males were always lower than those of females. This sex difference is not a consequence of the difference in size between the sexes.The base populations were of mutant stock origin, and some of them segregated for mutant genes which proved to be correlated with extrabristle phenotype. Supernumerary bristles were not distributed in the same manner in all of the selection lines, nor was the reaction of phenotypic expression to temperature the same in various lines.     

Arrangement of bristles as a function of bristle number on a leg segment inDrosophila melanogaster

Summary The arrangement of bristles on a leg segment of the fruitflyDrosophila melanogaster was studied in various mutants that have abnormal numbers of bristles on this segment. Eighteen mutations at six different genetic loci were analyzed, plus five double or triple mutant combinations. Recessive mutations at theachaete-scute locus were found to affect distinct groups of bristles:achaete mutations remove mechanosensory bristles, whereasscute mutations remove mainly chemosensory bristles. Mechanosensory bristles remain uniformly spaced along the longitudinal axis unless their number decreases below a certain threshold, suggesting that spacing is controlled by cell interactions that cannot function when bristle cells are too far apart. Above a certain threshold, bristle spacing and alignment both become irregular, perhaps due to excessive force from these same interactions. Chemosensory bristles occupy definite positions that are virtually unaffected by removal of individual bristles from the array. Extra chemosensory bristles develop only near the six normal sites. At two of the six sites the multiple bristles tend to exhibit uniform longitudinal spacing — a property confined to mechanosensory bristles in wild-type flies. To explain the various mutant phenotypes the following scheme is proposed, with different mutations directly or indirectly affecting each step: (1) spots and stripes are demarcated within the pattern area, (2) one bristle cell normally arises within each spot, multiple bristle cells within each stripe, (3) incipient bristle cells inhibit neighboring cells from becoming bristle cells, and (4) the bristle cells within each stripe become aligned to form rows and then repel one another to generate uniform spacing.     

The occurrence of the transposable elementpogo inDrosophila melanogaster

We examined the genomic occurrence of the transposable elementpogo in over 120 strains ofDrosophila melanogaster, from around the world and from different eras. All had multiple copies of a 2.1 kilobase (kb)pogo element, and multiple copies of several size classes between 1.0 and 1.8 kb. There were differences between strains in intensities or presences of deletion-derivative size classes, suggesting current or recent mobility in the species. We were unable to find anypogo-hybridization in eight other species in the genus, in three subgenera, or in the relatedScaptomyza pallida. Thepogo element may be a ‘middle-aged’ element in the genome ofD. melanogaster, having entered the species since its divergence from its sibling species, but long before theP andhobo elements.     

Establishment of compartments in the developing leg imaginal discs ofDrosophila melanogaster

Summary By X-irradiation ofM/M ⁺ embryos and larvae to induce mitotic recombination, clones ofM ⁺/M⁺ genotype were obtained (Fig. 1). Since such cells grow faster than the surroundingM/M ⁺-cells they can fill large areas within the compartments of an imaginal disc.The present studies concentrated mainly on the three leg discs. Clones were induced by doses of 1000 r at ages ranging from 3±0.5 h after oviposition to 144 h.All clones induced later than the blastoderm stage were absolutely restricted to either the anterior or the posterior compartment of a disc. The border between the anterior and posterior compartment runs as a straight line along the entire leg and at the distal end separates the two claws (Figs. 5, 6, 7). A further subdivision of the anterior compartment is indicated by clones initiated in the second larval instar (Fig. 11). A parallel subdivision could not be detected in the posterior compartment. Irradiation in the early third instar led to clones which were restricted to single longitudinal bristle rows and leg segments. But no clear-cut compartment borders could be found; in particular a proximo-distal separation appears to be absent.Among the 318 clones induced at the blastoderm stage eleven extended from the wing into the second leg (Fig. 8), or from the haltere into the third leg.With the exception of 3 clones that apparently occupied the anterior as well as the posterior compartment of a wing or a leg, all clones remained confined to either the anterior or the posterior compartment.Frequently clones overlapped left and right forelegs (Fig. 9). Intersegmental overlaps were not observed.The results show that the earliest compartment borders appear in all thoracic discs. This suggests that compartmentalization is a fundamental process common to all discs.Supported bySchweizerischer Nationalfonds Gesuch Nr. 3.480-0.75     

Short- and long-term effects of environmental urea on fecundity inDrosophila melanogaster

Previous studies have shown that exposure to urea-supplemented food inhibited fecundity inDrosophila females, and that this inhibition was not expressed when females were given a choice between regular and urea-supplemented food as an oviposition substrate. We assayed fecundity, on both regular food and urea-supplemented food, at 5, 15 and 25 days post eclosion on females from ten laboratory populations ofDrosophila melanogaster. The females assayed came from one of two treatments; they were maintained as adults on either regular or urea-supplemented food. We found that exposure to urea-supplemented food inhibited fecundity, relative to the levels exhibited on regular food, regardless of whether the urea was present in the assay medium, or in the medium on which the flies were maintained over the course of the experiment, thereby suggesting that urea has both a long-term (possibly physiological) as well as a short-term (possibly behavioural) inhibitory effect on fecundity ofDrosophila females. We also tested and ruled out the hypothesis that prior yeasting could ameliorate the inhibitory effect of urea in the assay medium on fecundity, as this was a possible explanation of why flies given a choice between regular and urea-supplemented food did not exhibit a preference for regular food in a previous study.     

Aspects of amino acid requirements for in vitro evagination of imaginal leg discs inDrosophila melanogaster

Summary A method of staging late third instar larvae on the basis of salivary gland morphology is described. Using this technique, we investigated stage related amino acid requirements forDrosophila leg disc evagination in vitro. It was found that the requirement for glutamine lasted longer than that of proline. The staging technique should help in the detailed exploration of the late 3rd instar time period in order to bridge the gap between biochemistry and morphogenesis in the initiation of disc evagination.     

The obscure parts of the X chromosome inDrosophila melanogaster

Through the use of small atelic fragments derived from T (1; 4) A,B ^S the right arm of the X chromosome ofD. melanogaster has been demonstrated.The proximal portion of XR is a non-chromatic or ghost reversed repeat of the proximal portion of XL.Polytene X chromosomes show no indications of the limits of the heterochromatic blocks which characterize mitotic X chromosomes.The nuclear organizer is a subterminal portion of the polytene XR, whereas it is situated in XL in mitotic neuroblast cells.It is suggested that the non-chromatic XR may contain a ghost allele of the genebobbed, and of perhaps other adjacent genes.This investigation was supported in part by Contract No. AT (30-1)-2113 from the U.S. Atomic Energy Commission, and in part by Public Health Service Research Grant No. C-4362 from the National Institutes of Health.