Early development of the thoracic discs ofDrosophila

Summary We estimate the number of blastoderm cells which generate the thoracic imaginal discs ofDrosophila. At hatching the wing disc is twice the size of the haltere disc, but the results suggest that both discs develop from a similar number of blastoderm cells. Two homeotic mutations, which transform the haltere into wing, affect embryonic growth but not the primordial number. All the segmental primordia may be of similar size and each may be similarly subdivided into a larger anterior, and a smaller posterior polyclone.     

Chemical Patterns, Compartments and a Binary Epigenetic Code in Drosophila

I propose a model which accounts for the geometries and sequencein which compartmental boundary lines arise on the differentimaginal discs, and on the blastoderm of Drosophila melanogaster;and propose that successive lines are recorded by differentbinary switches, to create a binary epigenetic code word specifyingeach disc, and disc compartment. I suppose a biochemical systemundergoing reaction and diffusion acts throughout development.As an imaginal disc grows, a succession of differently shapedchemical concentration patterns form at a discrete set of discsizes. I suppose a specific concentration of one chemical isa threshold. Concentrations above or below threshold switchcells to one or another of two commitments. Then the line acrossthe imaginal disc with the threshold concentration is a predictedcompartmental boundary. The sequence and geometries of suchlines predict the compartmental boundaries seen on the wingdisc, the other discs, and on the blastoderm stage egg. Thecompartmental lines on the wing disc suggest that a terminalcompartment is specified by a combination of binary names recordinga sequence of binary commitments: anterior, not posterior; dorsal,not ventral; wing, not thorax; proximal, not distal. Each combinationcomprises a binary epigenetic code word. Recently I constructedan independent model for transdetermination in Drosophila whichproposed a similar binary epigenetic code for the differentdiscs. The clone restriction lines predicted on the blastodermby my transdetermination model, the chemical pattern model,and analogy with the wing disc, are nearly identical. Severalare already confirmed. The resultant binary code scheme correctlypredicts many relative transdetermination frequencies and accountssimply for the action of most homeotic mutants as genes whichalter a single switch state in one or more discs.     

Localized ultraviolet laser microbeam irradiation of early Drosophila embryos: fate maps based on location and frequency of adult defects.

Drosophila embryos were locally irradiated with a 257-nm laser microbeam during blastoderm and germ band stages. Depending on stage and beam diameter (10–30 μm), from 0 to 45 nuclei were exposed to the uv radiation. The doses used, 5 or 10 erg, did not eliminate nuclei or cells at once, but up to 50% of the adult survivors from irradiated eggs carried defects in the thorax. These were scored with reference to the imaginal discs from which the affected structures derive. For each thoracic disc a “target center” was calculated as the weighted mean value of all beam locations affecting the respective adult derivatives. The target centers for the germ band stage map within the respective germ band segments. The pattern of target centers for the blastoderm stage is comparable to the thoracic region of published fate maps, and the distances between adjacent leg centers (approximately three cell diameters) agree with recent evidence based on mosaic flies. We discuss the question whether the target centers mark the position of the respective disc progenitor cells at the stages of irradiation and conclude that these positions are rendered rather correctly at least with reference to the longitudinal egg axis.     

A study of germinal mosaicism inDrosophila melanogaster

Summary Three-hundred and twenty fertile,pal-induced Y-chromosome mosaic males and females were obtained. Fractional analysis of the sons of 55 somatically mosaic flies that were also germinally mosaic tentatively suggests that the number of functional primordial germ cells inDrosophila melanogaster is variable and that it is seldom greater than 24. From the observed 0.17 frequency of germinal mosaicism it was estimated that the average number of pole cells at the end of blastoderm formation is 45. At present, the germ cells afford the only opportunity to compare genetic estimates of the number of blastoderm or primordial cells with available histological counts. The good agreement between them suggests that both the fractional and the mosaic frequency methods for estimating primordial or blastoderm cell numbers of various larval and imaginal anatomical structures provide reasonably close approximations of the actual values.     

Developmental analysis of Drosophila position-specific antigens

The distributions of three position-specific (PS) antigens have been examined in different Drosophila tissues and at various developmental times, using both immunofluorescence and affinity purification procedures. In the imaginal discs the PS antigens show nonuniform and nonhomologous distributions, and the expression of the antigens in a particular disc region can vary during development. In general, PS antigen expression appears to correlate with morphogenetic events in the disc epithelia, suggesting that the antigens are involved in cell-cell recognition and/or adhesion processes. PS antigens are also found in many other tissues, and in embryos as early as the cellular blastoderm stage. Affinity-purified PS antigens from different tissues or stages appear to be similar, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results are discussed in relation to Drosophila developmental events, with particular regard to the dorsoventral cell lineage restriction in the wing disc.     

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.     

Cellular basis of the dynamic behavior of the imaginal thoracic discs during Drosophila metamorphosis

The eversion, migration, spreading, and fusion of the thoracic imaginal discs during metamorphosis of Drosophila are described using timed whole-mount preparations and several molecular markers. The leading edge of the migrating disc epithelia consists of two groups of cells, stalk cells (S cells) and specialized imaginal cells (I cells), that both express the gene puckered. With this and other markers, opening of the stalk, eversion of the discs, migration of the leading edges, and fusion of the imaginal epithelia can be visualized in detail. Fusion is initiated by S cells that migrate over the larval epithelium and constitute a bridge between two imaginal epithelia. S cells are subsequently lost and imaginal fusion is mediated by the I cells that remain at the site of fusion. The possible cellular basis of this process is discussed. Fusion along the dorsal midline of the notum from the mesothoracic wing discs occurs earlier than that of the prothoracic and metathoracic discs, which remain in a lateral position. For a relatively long period (30 h) the mesothoracic epithelium becomes attached to the head and abdomen, causing a temporary local discontinuity of the order of segments. Later the pro- and metathoracic discs intercalate between head and mesothorax and between abdomen and mesothorax, respectively, to reestablish the normal order.     

A fine-structure gynandromorph fate map of the Drosophila head

A gynandromorph fate map of the head of D. melanogaster was produced using 28 landmarks derived from one imaginal disc. An examination of the meaning of fine-structure mapping discloses that the sturt value observed between one pair of landmarks within a disc may approximate the relative physical distance of their progenitor cells at blastoderm, but for another pair of landmarks (assuming no directed cell movements), the sturt value may simply reflect their close geographic location at the time the cells are specified for their particular differentiation, a time much later in development when most cell division within the disc has come to an end. The formation of early developmental compartments has little effect on fate-map distances. Our analysis of the data suggests there are approximately ten cells present at the blastoderm stage that are head progenitors. Each blastoderm cell is likely to be the progenitor of a particular array of landmarks, but there is overlap between arrays from different blastoderm cells.     

Isolation and characterization of X-linked lethal mutants affecting differentiation of the imaginal discs in Drosophila melanogaster

Summary Twenty-seven late larval or early pupal lethal mutations were isolated for the X-chromosome, some of which showed structural and/or functional deficiencies of the imaginal discs. The mutants were grouped according to the size and morphology of their discs as follows: 1. discs normal: 18 mutants. 2. discs small: 2 mutants. 3. discs degenerate: 4 mutants. 4. discless: 1 mutant. 5. discs heterogeneous: 2 mutants. Preliminary characterization of the mutants included a study of disc morphology, puparium formation and pupal molt, in vivo and in vitro evagination of the imaginal discs, autonomy of the mutation in the disc tissue (differentiation after transplantation and gynander mosaicism test). Possible relations between disc morphology and the former characteristics are discussed.     

Genetic analysis of transdetermination in Drosophila. I. The effects of varying growth parameters using a temperature-sensitive mutation

The heat-sensitive mutation of Drosophila melanogaster l(3)c4(3)hs1, causes mutant larvae raised at a restrictive temperature to have abnormally large wing discs. The large size of these discs is a disc-autonomous property and results from an increase in the number rather than the size of wing disc cells. We have used wing discs from this mutant to further investigate properties of transdetermination which had previously been investigated with nonmutant discs. Transdetermination can occur in nonmutant discs when the proliferative phase of imaginal disc development is extended by wounding discs and culturing them in vivo. The results indicate that additional proliferation in the absence of wounding does not lead to transdetermination. There is a correlation between the extent of growth of a cultured disc and the probability that it will undergo transdetermination. The results suggest that this correlation does not depend on a differential rate of cell division. Finally, the results indicate that the cells which give rise to transdetermination are at an equivalent developmental stage no later than that characteristic of eye-antenna disc cells before the third larval instar.     

The morphostatic actions of juvenile hormone

The maintenance of "status quo" in larvae by juvenile hormone (JH) involves both the programming of ecdysteroid-dependent synthesis during the molt and the suppression of morphogenetic growth during the intermolt. The latter morphostatic action does not require ecdysteroids, and has been studied in the formation of imaginal discs in Manduca sexta. Preultimate larval instars have both invaginated discs and imaginal primordia, both of which grow isomorphically with the larva. In the last instar, the young discs/primordia initiate the morphogenesis and patterning that results in a mature disc. JH suppresses both the initiation and progression of the signaling that transforms immature discs or primordia into a fully patterned imaginal disc. This transformation normally occurs in the context of the rapid growth of the last larval stage, and nutrient-dependent factors appear to be able to override the JH suppression. The morphostatic action of JH may have been important for the evolution of the larval stage. Studies on embryos of basal, hemimetabolous insects show that their premature exposure to JH can truncate patterning programs and cause precocious tissue maturation, factors essential for organizing a novel larval form. This suppression of embryonic patterning then results in embryonic fields that remain dormant as long as JH is present. These are the primordia that can transform into imaginal discs once JH disappears in preparation for metamorphosis.     

Analysis of morphogenetic movements in the development of the notum anlage of Drosophila melanogaster

Summary A comparison of the morphogenetic maps of the notum anlage of Drosophila melanogaster derived from the gynandromorph data and mosaics induced by somatic crossing-over during the first instar larval stage revealed that practically no major morphogenetic movements occur in the development of the anlage between the blastoderm and first instar larval stages and the adult stage. By comparing the morphogenetic map derived from gynandromorphs and the fate map derived from data on the transplantation of fragments of the mature wing imaginal disc, it was observed that no major morphogenetic movements occur in the notum anlage between the stages of the allocation of the disc and the mature disc. The results are consistent with the observations of other authors concerning the larval development of eye-antenna, wing and leg discs.     

Monoclonal antibody probes discriminate early and late mutant defects in development of the Drosophila retina

Many mutations in Drosophila melanogaster affect the morphology of the adult compound eye. However, the times at which the phenotypes first become manifest in development are, in most cases, unknown; they can occur at any of a series of stages. Among mutants in which eyes appear externally similar, the developmental stage of onset of each defect may be quite different. Pattern formation in the compound eye begins during the late third larval instar in the eye imaginal disc, when a wave of morphogenesis crosses the disc from posterior to anterior. As this wave crosses the disc, there appears in its wake an array of photoreceptor neuron clusters and accessory cells that will comprise the adult ommatidia. Eye discs from 20 abnormal-eye mutants were analyzed using monoclonal antibodies that highlight various aspects of the developing array, to observe the stage at which each anomaly becomes evident. Some mutations apparently affect precursor cells, others the setting up of the pattern, others maintenance of the pattern, and still others later morphogenetic events.     

Defective gap-junctional communication associated with imaginal disc overgrowth and degeneration caused by mutations of the dco gene in Drosophila

The lethal(3)discs overgrown (dco) locus of Drosophila melanogaster, located on the third chromosome at cytogenetic position 100A5,6-100B1,2, is necessary for normal development and growth control in the imaginal discs of the larva. Three recessive lethal alleles (dco2, dco3, and dco18) in heteroallelic combinations and one allele (dco3) when homozygous cause the imaginal discs to continue to grow beyond the normal disc-intrinsic limit during an extended larval period. Some degeneration also occurs in the overgrowing discs. The discs overgrow even when transplanted early in their development into wild-type hosts, whereas normal discs stop growth at about the normal final size under such conditions, indicating that the overgrowth is a disc-autonomous effect of the mutations. During overgrowth the imaginal discs retain their single-layered epithelial structure except near regions of degeneration, and they differentiate into disc-appropriate but abnormal adult structures when transplanted into wild-type larval hosts. When the mutant larvae are reared under certain conditions a small percentage develop to the pharate adult stage, and these animals show a characteristic syndrome of abnormalities including swollen leg segments with many extra bristles, small or missing eyes, duplicated antennae and palpi, and separated vesicles of cuticle. A fourth recessive lethal allele (dcole88), when homozygous or in heteroallelic combination with the overgrowth alleles, causes the imaginal discs to degenerate, producing a "discless" phenotype. Gap junction-mediated communication was assayed by observing the intercellular transfer of injected fluorescein complexon (dye coupling). Dye coupling in the imaginal discs of the dco genotypes that cause overgrowth was dramatically reduced at 4 days after egg laying (AEL) compared with wild-type controls. Coupling was more normal although still significantly reduced at 7-8 and 12-14 days AEL. In c43hs1, another disc overgrowth mutant, the imaginal disc cells also showed very reduced dye coupling at 4 days and incomplete coupling at 9 days. In contrast, discs from wild-type larvae, two other imaginal disc overgrowth mutants, and a cell death mutant showed extensive dye coupling at all stages tested. Electron microscopic morphometry revealed a reduction in gap-junction length per unit lateral plasma membrane length in dco3/dco18 and c43hs1 wing discs, although not in dco2/dco3, compared with wild-type wing discs. The results suggest that gap-junctional cell communication may be involved in the cell interactions that limit cell proliferation in vivo.     

Position-specific interaction between cells of the imaginal wing and haltere discs of Drosophila melanogaster.

When fragments of the imaginal wing disc from opposite ends of the disc are mixed prior to culture, intercalary regeneration occurs so that structures are produced which neither of the fragments would have produced if they had been cultured alone. I report here that fragments of the imaginal wing and haltere disc interact in a position-specific way. Mixing of homologous fragments does not result in regeneration, while mixing of fragments from opposite ends of the discs does. Thus the interaction of wing and haltere disc fragments shows the same positional specificity as the mixing of two wing fragments.     

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     

The Mechanism of Evagination of Imaginal Discs of Drosophila melanogaster

The evagination of imaginal leg discs to produce legs is a usefulmodel for studying morphogenesis. Evagination of imaginal legdiscs occurs in vitro in defined culture media in the presenceof the molting hormone ß-ecdysone. Evagination involveslimited, organized movement of imaginal disc cells. The movementappears to be a result of contractile activity, coordinatedwith the presence of appropriate structural and surface propertiesof disc cells. However, ecdysone does not produce its effectsdirectly, but acts through the genome to cause evagination.Evagination is a result then of increased synthesis of differentproteins, one of which is myosin. If the results on discs aregeneralizable they indicate that similar morphogenetic processesare the direct result of the readout of the specific geneticprograms.     

Misexpression screen in Drosophila melanogaster aiming to reveal novel factors involved in formation of body parts

To identify novel factors that lead a fly imaginal disc to adopt its developmental fate, we carried out a modular dominant misexpression screen in imaginal discs. We have identified two factors that appear to change the fate of the respective body structure and appear to lead to the transformation of a body part. In one mutant line, notum tissue, normally derived from wing imaginal tissue, formed close to the site of the sternopleural bristles, which are leg disc derivatives. In the other line, the arista is transformed into a tubular structure, resembling an abnormal leg. We found that ectopic expression of abrupt was responsible for this potential transformation of the arista.     

The role of nutrition in creation of the eye imaginal disc and initiation of metamorphosis in Manduca sexta

With the exception of the wing imaginal discs, the imaginal discs of Manduca sexta are not formed until early in the final larval instar. An early step in the development of these late-forming imaginal discs from the imaginal primordia appears to be an irreversible commitment to form pupal cuticle at the next molt. Similar to pupal commitment in other tissues at later stages, activation of broad expression is correlated with pupal commitment in the adult eye primordia. Feeding is required during the final larval instar for activation of broad expression in the eye primordia, and dietary sugar is the specific nutritional cue required. Dietary protein is also necessary during this time to initiate the proliferative program and growth of the eye imaginal disc. Although the hemolymph titer of juvenile hormone normally decreases to low levels early in the final larval instar, eye disc development begins even if the juvenile hormone titer is artificially maintained at high levels. Instead, creation of the late-forming imaginal discs in Manduca appears to be controlled by unidentified endocrine factors whose activation is regulated by the nutritional state of the animal.     

Intercellular adhesivity and pupal morphogenesis inDrosophila melanogaster

Summary Leg and wing imaginal discs of mature larvae ofDrosophila melanogaster when treated with 0.1% trypsin for 5–10 min underwent a change in shape that closely resembled normal pupal morphogenesis. Simultaneously, the cells of the disc epithelium changed in shape from tall columnar to cuboidal. Colcemid eliminated microtubules but was without effect on the shape of the imaginal discs or their cells. Tryptic digestion reduced non-junctional intercellular adhesivity but septate desmosomes and gap junctions remained intact.It is proposed that the structure of imaginal discs permits the packaging of the anlagen of the adult integument so that they can change shape and replace the larval structures in a brief period. Apparently most of the definitive form of the pupal leg is built into the disc and becomes visible within a few minutes as intercellular adhesivity is changed.