Imaginal discs regulate developmental timing in Drosophila melanogaster

The regulation of body size in animals involves mechanisms that terminate growth. In holometabolous insects growth ends at the onset of metamorphosis and is contingent on their reaching a critical size in the final larval instar. Despite the importance of critical size in regulating final body size, the developmental mechanisms regulating critical size are poorly understood. Here we demonstrate that the developing adult organs, called imaginal discs, are a regulator of critical size in larval Drosophila. We show that damage to, or slow growth of, the imaginal discs is sufficient to retard metamorphosis both by increasing critical size and extending the period between attainment of critical size and metamorphosis. Nevertheless, larvae with damaged and slow growing discs metamorphose at the same size as wild-type larvae. In contrast, complete removal of all imaginal tissue has no effect on critical size. These data indicate that both attainment of critical size and the timely onset of metamorphosis are regulated by the imaginal discs in Drosophila, and suggest that the termination of growth is coordinated among growing tissues to ensure that all organs attain a characteristic final size.     

What are and what are not imaginal discs: reevaluation of some basic concepts (Insecta, Holometabola).

Some general aspects of the concept of imaginal discs in the Holometabola are reevaluated. Their monolayer character and continuity with the surrounding epidermis are confirmed. Studies on the imaginal discs of the silkworm (Bombyx mori) and data from the literature show that the discs and their peripodial cells produce cuticle during larval life, as well as at metamorphosis. In B. mori it is demonstrated that adult and larval antennae are produced by the same cells or their progeny. The results also suggest that segments of the typically three-segmented larval antenna of Holometabola are not scape, pedicel, and one-segmented flagellum; at least segments 2 and 3 are of flagellar origin. Based on these and some additional facts it is argued that: (1) No larval organs are "replaced" at metamorphosis, but strict "sequential homology" is always maintained. (2) Imaginal discs are not undifferentiated structures destined to form the adult after larval breakdown, cannot be unambiguously defined, and do not represent qualitatively different epidermal structures. Classical imaginal discs (invaginated and present also in pre-final larval instars) arose several times independently and were not present in the larvae of ancestral Holometabola. (3) Since the disc cells are not undifferentiated and "embryonic" (if these words have a defined meaning at all), it is unreasonable to expect that the processes taking place in discs at metamorphosis would differ fundamentally from those occurring in other diploid metamorphosing epidermal cells.     

Larva-adult relationships in an ancestral dipteran: A re-examination of sensillar pathways across the antenna and leg anlagen of Chaoborus crystallinus (DeGeer, 1776; Chaoboridae)

 In one of his classical studies on insect metamorphosis, Weismann compared the imaginal anlagen of the ancestral phantom midge, Chaoborus, with those of advanced brachycerans. We have expanded his findings on the relationships between larval and imaginal organs using electron microscopy and cobalt backfilling of the antenna and leg anlagen and the axonal trajectories of corresponding larval sensilla. We show that both primordia are confluent with the larval antennae and ”leg” sensilla (an ancestral Keilin organ), respectively. These fully developed larval organs represent the distal tips of the imaginal anlagen rather than separate cell clusters. The axons of the larval antenna and leg sensilla project across the corresponding anlagen to their target neuromeres within the central nervous system (CNS). Within the discs, nerves composed of these larval axons, developing afferent fibres and efferences ascending from the CNS are found. Both the structure of the primordia and the axonal trajectories thus relate the situation found in advanced brachycerans with that seen in more ancestral insects. In addition, the larval antennae, legs, wings and even the eyes possess very similar afferent pioneer trajectories supporting the idea that the described pattern is generally used in the ontogeny of sensory systems. Received: 30 June 1998 / Accepted: 27 September 1998     

Expression profiling of Drosophila imaginal discs

Background  

In the Drosophila larva, imaginal discs are programmed to produce adult structures at metamorphosis. Although their fate is precisely determined, these organs remain largely undifferentiated in the larva. To identify genes that establish and express the different states of determination in discs and larval tissues, we used DNA microarrays to analyze mRNAs isolated from single imaginal discs.     

Integration of complex larval chemosensory organs into the adult nervous system of Drosophila

The sense organs of adult Drosophila, and holometabolous insects in general, derive essentially from imaginal discs and hence are adult specific. Experimental evidence presented here, however, suggests a different developmental design for the three largely gustatory sense organs located along the pharynx. In a comprehensive cellular analysis, we show that the posteriormost of the three organs derives directly from a similar larval organ and that the two other organs arise by splitting of a second larval organ. Interestingly, these two larval organs persist despite extensive reorganization of the pharynx. Thus, most of the neurons of the three adult organs are surviving larval neurons. However, the anterior organ includes some sensilla that are generated during pupal stages. Also, we observe apoptosis in a third larval pharyngeal organ. Hence, our experimental data show for the first time the integration of complex, fully differentiated larval sense organs into the nervous system of the adult fly and demonstrate the embryonic origin of their neurons. Moreover, they identify metamorphosis of this sensory system as a complex process involving neuronal persistence, generation of additional neurons and neuronal death. Our conclusions are based on combined analysis of reporter expression from P[GAL4] driver lines, horseradish peroxidase injections into blastoderm stage embryos, cell labeling via heat-shock-induced flip-out in the embryo, bromodeoxyuridine birth dating and staining for programmed cell death. They challenge the general view that sense organs are replaced during metamorphosis.     

Development Capacities of Mixed Normal and Neoplastic 1(3)gl Imaginal Discs and Neuroblast Tissue in Drosophila hydei

The pleiotropic mutant lethal(3)giant larvae [l(3)gl] of Drosophila hydei exhibits among other anatomical defects, hypertrophy of the larval brain and imaginal discs. Both hypertrophic tissues when transplanted into wild-type female flies behave as fast growing and lethal neoplasms. Implanted into mature wild-type larvae they fail to metamorphose. When l(3)gl neoplastic brain tissue or imaginal discs were mixed with normal imaginal discs, cultured in vivo in the abdomen of adult females and transplanted into mature wild-type larvae, the following results were obtained. The invasive l(3)gl brain neoplasm, while fatal for adult hosts, had no effect on the metamorphosis of normal imaginal disc tissue. On the other hand, the noninvasive l(3)gl imaginal disc neoplasms when mixed with normal imaginal disc tissue inhibited its development and metamorphosis in the wild-type host. This inhibitory effect was not observed when the tissues were injected as separate implants into the same host.     

Invasive cell behavior during Drosophila imaginal disc eversion is mediated by the JNK signaling cascade

Drosophila imaginal discs are monolayered epithelial invaginations that grow during larval stages and evert at metamorphosis to assemble the adult exoskeleton. They consist of columnar cells, forming the imaginal epithelium, as well as squamous cells, which constitute the peripodial epithelium and stalk (PS). Here, we uncover a new morphogenetic/cellular mechanism for disc eversion. We show that imaginal discs evert by apposing their peripodial side to the larval epidermis and through the invasion of the larval epidermis by PS cells, which undergo a pseudo-epithelial-mesenchymal transition (PEMT). As a consequence, the PS/larval bilayer is perforated and the imaginal epithelia protrude, a process reminiscent of other developmental events, such as epithelial perforation in chordates. When eversion is completed, PS cells localize to the leading front, heading disc expansion. We found that the JNK pathway is necessary for PS/larval cells apposition, the PEMT, and the motile activity of leading front cells.     

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.     

Metamorphosis of imaginal discs of Drosophila melanogaster

Summary Imaginal discs ofDrosophila melanogaster larvae, 24–53 hrs after oviposition, were transplanted into mature immobile larval hosts. The transplants did not respond to the hormonal stimuli of metamorphosis, but instead completed their larval development. When reinjected into mature larval hosts, they now differentiated the full set of their presumptive imaginal structures. The process of acquiring competence for metamorphosis appears to be independent of the hormonal conditions.Supported by a credit of the Swiss National Foundation granted to Prof. Dr. E. Hadorn. I thank Dr. R. Nöthiger for his valuable criticism during this investigation.     

Genesis of the reproductive system of mosquitoes II. Male of Aedes stimulans (walker)

The imaginal male of mosquitoes bears a combination of organs and appendages that make it morphologically distinctive. Its reproductive organs produce sperm cells, convey and extrude them, provide accessory fluids, and insure copulation and insemination. In Aedes stimulans (Walker) these organs are derived from one of the two sets of primordia provided by the embryo. The second set of primordia is capable of producing the feminine reproductive system under unusual circumstances. Testes are derived from two compact ovoid masses of cells suspended in the hemocoel of abdominal segment 6. Each enlarges slowly throughout larval instars 1–3 and elongates very rapidly late in instar 4. Specialization of the cellular mass into sperm cells proceeds forward from the caudal end early in pupal life. From the beginning, a sheath of nutritive cells or fatbody encases each gonad, and no tracheation of the mass is evident although one small trachea sends branches to the encasing fatbody late in larval life. The efferent canal from each testis is derived from a tenuous filament extending caudally from each gonad to the venter of segment 9 and a small cluster of cells in the wall of the hemocoel on the ental surface of imaginal disc 9. Early in pupal life the filaments become the tubular vasa efferentia. The caudal clusters are primordial terminal parts of the lateral tract that become vasa deferentia, seminal vesicles and associated accessory glands. The ejaculatory canal comes from a short pouch derived from the median genital plate of disc 9. All external parts except the paraprocts are products of disc 9. The bilateral buds begin to proliferate in larval instar 4 and become the basistyles, dististyles and claspettes of the gonapophyses during pupal life. The phallosome is derived from the median genital plate. Primordia of a possible feminine reproductive system and cerci remain undifferentiated and disappear early in pupal life in the normal course of events. Primordia that were recognizable include those of ovaries, parts of lateral oviducts, median genital tract and cerci.     

Mutations at the fat locus interfere with cell proliferation control and epithelial morphogenesis in Drosophila

Lethal mutations at the fat locus in Drosophila cause imaginal discs to continue to grow by cell proliferation far beyond their normal final size. During a greatly extended larval period, the overgrowing imaginal discs develop additional folds and lobes, but retain a single-layered epithelial structure. In the wing disc, the additional lobes originate in the proximal fold area, and in the extra tissue the cells are less columnar than normal. Mutant disc cells lack zonulae adherents as well as associated microtubules and microfilaments, and they show an abnormal distribution and reduced density of gap junctions. The effect on growth is disc-autonomous as shown by transplantation experiments. The overgrown imaginal discs retain the ability to differentiate adult cuticular structures, as shown by metamorphosis of discs after transplantation into wild-type larval hosts and by the ability of some mutant animals to develop to the pharate adult stage. The structures differentiated by mutant discs show many abnormalities including ingrowths, outgrowths, separated cuticular vesicles, and areas of reversed bristle polarity; some of these abnormalities suggest that the mutations interfere with cell adhesion as well as the control of cell proliferation. The fat locus is located in cytogenetic interval 24D5.6-7, and 18 alleles are known including spontaneous, chemically induced, X-ray-induced, and dysgenic mutations.     

Visualization of proprioceptors in Drosophila larvae and pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs). Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis. The development of larval ChOs during embryogenesis has been studied extensively. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system. Multiple ChOs have been described in the adult fly. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy. In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary. Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.     

Origin and development of the dorso-ventral flight muscles in Chironomus (Diptera; Nematocera)

The origin and development of the dorso-ventral flight muscles (DVM) was studied by light and electron microscopy in Chironomus (Diptera; Nematocera). Chironomus was chosen because unlike Drosophila, its flight muscles develop during the last larval instar, before the lytic process of metamorphosis. Ten fibrillar DVM were shown to develop from a larval muscle associated with myoblasts. This muscle is connected to the imaginal leg discso that its cavity communicates with the adepithelial cells present in the disc; but no migration of myoblasts seems to take place from the imaginal leg disc towards the larval muscle or vice versa. At the beginning of the last larval instar, the myoblasts were always present together with the nerves in the larval muscle. In addition, large larval muscle cells incorporated to the imaginal discs were observed to border on the area occupied by adepithelial cells, and are probably involved in the formation of 4 other fibrillar DVM with adepithelial cells. Three factors seem to determine the number of DVM fibres: the initial number of larval fibres in the Anlage, the fusions of myoblasts with these larval fibres and the number of motor axons in the Anlage. The extrapolation of these observations to Drosophila, a higher dipteran, is discussed.     

Lipid accumulation in liver, spleen, lungs and kidneys of miniature-pigs after chloroquine treatment.

Chronic chloroquine treatment of type-Göttingen miniature-pigs induced lipid accumulation in the liver, spleen, lungs and kidneys. The lipid analyses showed marked quantitative and qualitative differences between the organs. In the liver the lipids affected most were cholesteryl esters and glucosylceramides, which were increased at the most 20 times. Cholesterol and ganglioside concentrations were also increased, though less markedly. The concentration of acidic phospholipids was slightly increased but that of the neutral phospholipids was unaffected. There was a considerable inter-individual variation in the lipid changes. Spleen and lung showed significant increases of all the major lipids. Glucosylceramide was increased more than the other lipids, namely 6-fold in the spleen and 10-fold in the lung. The concentration of acidic phospholipids as well as that of gangliosides was increased by 50% in the spleen and by 100% in the lung. The organ affected least was the kidney, in which only the glycolipids, both acidic and neutral, were significantly increased. Common to all the organs of the chloroquine-treated pigs was the large increase of glucosylceramide, ganglioside CM2 and bis(monoacylglyceryl)phosphate. The ganglioside increase affected all the individual gangliosides and, except for the increased proportion of ganglioside GM2, there were not remarkable changes in the ganglioside pattern in any of the organs.     

The longitudinal visceral musculature of Drosophila melanogaster persists through metamorphosis

The larval gut of Drosophila is coated with visceral muscles of mesodermal origin. In the midgut region this musculature comprises circular and longitudinal fibres. The complete visceral musculature is described to be removed during metamorphosis and to be replaced by a newly differentiated imaginal tissue resembling the morphology of the larval musculature. However, progenitors of this imaginal visceral musculature have never been detected prior to differentiation. Here I present results indicating that the longitudinal visceral musculature of the midgut completely persists through metamorphosis. Single cells expressing green fluorescent protein (GFP) as a marker were transplanted at the blastoderm stage. All clones contributing to the longitudinal visceral musculature detected in third instar larvae were recovered after metamorphosis in adult flies. Further evidence for the persistence of the larval visceral musculature was obtained from the P[Gal4] insertion line 5053A. It expresses GAL4 specifically in the longitudinal visceral muscles of the midgut of all developmental stages to the adult fly beginning at the end of embryogenesis. By using GFP as a reporter, it was possible to follow these cells through the entire metamorphosis. Although the muscles undergo dramatic morphological changes including the loss of their contractile system, no evidence for a replacement of the larval visceral musculature by imaginal precursor cells was detected.     

Larval legs of mulberry silkworm Bombyx mori are prototypes for the adult legs

Morphological diversity of leg appendages is one of the hallmarks of developmental evolution. Limbs in insects may develop either from their embryonic prototypes or from imaginal discs harbored inside the larva. Bombyx mori (B. mori), a Lepidopteran insect, develops adult wings from larval wing imaginal discs. However, it has been debated whether the adult legs of B. mori arise from imaginal discs or from the larval legs. Here we addressed how the larval legs relate to their adult counterparts. We present the morphological landmarks during early leg development. We used expression of developmental genes like Distalless and extradenticle to mark leg primordia. Finally, we employed classical excision approach to develop a fate map of the adult leg. Excision and ablation of thoracic legs along proximo-distal axis at various times during larval development resulted in the loss of corresponding adult leg segments. Our data suggest that B. mori legs develop from larval appendages rather than leg imaginal discs.     

Down-regulation of RpS21, a putative translation initiation factor interacting with P40, produces viable minute imagos and larval lethality with overgrown hematopoietic organs and imaginal discs

Down-regulation of the Drosophila ribosomal protein S21 gene (rpS21) causes a dominant weak Minute phenotype and recessively produces massive hyperplasia of the hematopoietic organs and moderate overgrowth of the imaginal discs during larval development. Here, we show that the S21 protein (RpS21) is bound to native 40S ribosomal subunits in a salt-labile association and is absent from polysomes, indicating that it acts as a translation initiation factor rather than as a core ribosomal protein. RpS21 can interact strongly with P40, a ribosomal peripheral protein encoded by the stubarista (sta) gene. Genetic studies reveal that P40 underexpression drastically enhances imaginal disc overgrowth in rpS21-deficient larvae, whereas viable combinations between rpS21 and sta affect the morphology of bristles, antennae, and aristae. These data demonstrate a strong interaction between components of the translation machinery and showed that their underexpression impairs the control of cell proliferation in both hematopoietic organs and imaginal discs.     

Taste organ growth and development in the direct developing frog Eleutherodactylus coqui (Lissamphibia: Eleutherodactylidae)

Previous research on amphibian taste organs concerned amphibians with a biphasic life history, that is, with larval period and metamorphosis. Direct developing frog species, such as Eleutherodactylus coqui, undergo a cryptic metamorphosis before hatching, and many larval‐specific features are vestigial or have been lost entirely from their ontogeny. Taste buds are present in larval stages of biphasically developing anurans and are replaced by taste discs during metamorphosis. One goal of this study was to characterize the ontogeny of taste buds and/or discs in E. coqui. The other goal was to examine correlations between body size and taste organ density and size in different regions of oral epithelium. The research reveals the presence of only one type of taste organ, characteristic of metamorphs of biphasic amphibians, namely taste disc. In addition, taste disc density and the area of the taste disc sensory zone change dramatically during growth.     

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.