Homologies of positional information in thoracic imaginal discs ofDrosophila melanogaster

Summary The regulative behavior of fragments of the imaginal discs of the wing and first leg was studied when these fragments were combined with fragments of other thoracic imaginal discs. A fragment of the wing disc which does not normally regenerate when cultured could be stimulated to regenerate by combination with certain fragments of the haltere disc. When combined with a haltere disc fragment thought to be homologous by the criteria of morphology and the pattern of homoeotic transformation, such stimulated intercalary regeneration was not observed. Combinations of first and second leg disc fragments showed that a lateral first leg fragment could be stimulated to regenerate medial structures when combined with a medial second leg disc fragment but not when combined with a lateral second leg disc fragment. Combinations of wing and second leg disc fragments showed that one fragment of the second leg disc is capable of stimulating regeneration from a wing disc fragment while another second leg disc fragment fails to stimulate such regeneration. It is suggested that absence of intercalary regeneration in combinations of fragments of different thoracic imaginal discs is a result of homology or identity of the positional information residing in the cells of the fragments. The pattern of correspondence of positional information revealed by this analysis is consistant with the pattern of homology determined by morphological observation and by analysis of the positional specificity of homoeotic transformation among serially homologous appendages. The implications of the existence of homologous positional information in wing and second leg discs which share a common cell lineage early in development are discussed.     

The wing imaginal disc

The Drosophila wing imaginal disc is a tissue of undifferentiated cells that are precursors of the wing and most of the notum of the adult fly. The wing disc first forms during embryogenesis from a cluster of ∼30 cells located in the second thoracic segment, which invaginate to form a sac-like structure. They undergo extensive proliferation during larval stages to form a mature larval wing disc of ∼35,000 cells. During this time, distinct cell fates are assigned to different regions, and the wing disc develops a complex morphology. Finally, during pupal stages the wing disc undergoes morphogenetic processes and then differentiates to form the adult wing and notum. While the bulk of the wing disc comprises epithelial cells, it also includes neurons and glia, and is associated with tracheal cells and muscle precursor cells. The relative simplicity and accessibility of the wing disc, combined with the wealth of genetic tools available in Drosophila, have combined to make it a premier system for identifying genes and deciphering systems that play crucial roles in animal development. Studies in wing imaginal discs have made key contributions to many areas of biology, including tissue patterning, signal transduction, growth control, regeneration, planar cell polarity, morphogenesis, and tissue mechanics.     

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.     

Regeneration following duplication in imaginal wing disc fragments of Drosophila melanogaster

Fragments from the imaginal wing disc of Drosophila melanogaster were cultured in vivo for periods up to 28 days. One type of edge fragment first duplicated and then ceased to grow, but others often continued to grow following initial duplication and regenerated structures characteristic of other areas of the disc. After 28 days of culture, about 50% of fragments from the presumptive ventral hinge region of the disc grew extensively and produced regenerated as well as original structures. The regenerated structures in some implants were produced at the line of mirror-image symmetry. Regeneration was associated with fragment growth and in many cases was accompanied by loss of duplicate structures. Fragments which were only duplicated after the culture period could in some cases be stimulated to grow by additional culture in fresh hosts, but the results of coculturing two fragments in each host show that culture conditions alone do not control growth and regulation in the fragments. The large, normally regenerating fragment, complementary to the ventral fragment, did not appear to grow following regeneration and only occasionally produced supernumerary structures during prolonged culture. Intact wing discs cultured under similar conditions never produced supernumerary structures. Our results suggest that a duplicated pattern is less stable than a complete, regenerated pattern, which in turn is less stable than an intact disc. We propose that the growth of duplicated disc fragments is stimulated by polarity reversals present at lines of mirror-image symmetry.     

Compartments and distal outgrowth in theDrosophila imaginal wing disc

Summary Peripheral tissue of the imaginal wing disc gives rise to the proximal mesothoracic structures of the adult. Pieces of peripheral tissue, which have no regenerative capacity when cultured as intact fragments, are capable of distal outgrowth (regeneration) after dissociation and reaggregation. This ability depends on the region of the disc periphery from which the fragment is taken. Extensive distal outgrowth occurs in reaggreages of a fragment containing equal proportions of tissue from anterior and posterior developmental compartments. The extent of outgrowth decreases as the proportion of posterior tissue is reduced, so that a fragment containing only anterior tissue shows no regeneration after dissociation. Limited distal outgrowth occurs in reaggregates of a wholly posterior fragment, but the regenerative capacity is increased greatly when a small amount of anterior tissue is included. It is concluded that distal outgrowth in the wing disc requires an interaction between cells of the anterior and posterior compartments.     

Cell-free translation of Drosophila imaginal disc mRNA

Total RNA derived from the imaginal discs of Drosophila melanogaster was translated in vitro, and the polypeptide products electrophoresed on two-dimensional gels. In agreement with previously published examinations of imaginal disc protein synthesis and content, we can detect no reproducible differences in abundant mRNA populations between different disc types (foreleg and wing). Differences can be found, however, between imaginal discs and other tissues. We also present evidence for a nonuniformly distributed wing disc mRNA.     

Wound healing and regenerative response of fragments of the Drosophila wing imaginal disc cultured in vitro

Fragments of imaginal discs of the fruitfly Drosophila undergo growth and pattern regulation when cultured in vivo in adult female hosts for several days prior to metamorphosis in host larvae. Pattern regulation results in either regeneration of excised pattern elements or duplication of elements whose fate map positions lay within the fragment. Initial wound healing along the cut edge of a fragment is thought to be a crucial first step in the process of pattern regulation. We have examined the capacity for wound healing and pattern regulation of fragments (distal halves) of the wing disc cultured in vitro, using the culture system recently reported to support extensive growth and transdetermination of slightly wounded whole imaginal discs in vitro. Our results suggest that disc fragments and whole discs apparently respond differently in the culture system. With disc fragments, wound healing did not occur in vitro. When fragments were first cultured overnight in adult female hosts to allow initial wound healing prior to explantation in vitro, then some volume increase and regeneration of excised portions occurred during 2–3 weeks of culture in vitro. The extent of apparent growth was much less than that reported for whole discs, and the frequency of regeneration in vitro (19%), while highly significant relative to controls not cultured in vitro (0%), was much less than that observed for fragments cultured in vivo (84%). Furthermore the extent of regeneration which occurred in vitro was considerably smaller than that which occurs during regeneration in vivo.     

The wings ofBombyx mori develop from larval discs exhibiting an early differentiated state: a preliminary report

Lepidopteran insects present a complex organization of appendages which develop by various mechanisms. In the mulberry silkworm,Bombyx mori a pair of meso- and meta-thoracic discs located on either side in the larvae gives rise to the corresponding fore- and hind-wings of the adult. These discs do not experience massive cell rearrangements during metamorphosis and display the adult wing vein pattern. We have analysed wing development inB. mori by two approaches, viz., expression of patterning genes in larval wing discs, and regulatory capacities of larval discs following explantation or perturbation. Expression of Nubbin is seen all over the presumptive wing blade domains unlike inDrosophila, where it is confined to the hinge and the wing pouch. Excision of meso- and meta-thoracic discs during the larval stages resulted in emergence of adult moths lacking the corresponding wings without any loss of thoracic tissues suggesting independent origin of wing and thoracic primordia. The expression of wingless and distal-less along the dorsal/ventral margin in wing discs correlated well with their expression profile in adultDrosophila wings. Partially excised wing discs did not showin situ regeneration or duplication suggesting their early differentiation. The presence of adult wing vein patterns discernible in larval wing discs and the patterns of marker gene expression as well as the inability of these discs to regulate growth suggested that wing differentiation is achieved early inB. mori. The timings of morphogenetic events are different and the wing discs behave like presumptive wing buds opening out as wing blades inB. mori unlike evagination of only the pouch region as wing blades seen inDrosophila.     

Localized cell death in Drosophila imaginal wing disc epithelium caused by the mutation apterous-blot

Drosophila melanogaster carrying the mutation apterous-blot have blistered wings. Trypan blue stains a patch of dead cells localized to the wing pouch of imaginal discs and the same area shows acid phosphatase (AcPase) activity suggesting that the cell death is lysosomal. Autophagic vacuoles and other secondary lysosomes show AcPase activity within the disc epithelium and enzyme activity is found in fragments of dead cells which have been extruded basally. The cell death, although extensive and confined to the presumptive wing region, does not result in loss of adult structures.     

Mutations in tumour suppressor genes,l(2)gl andl(2)gd,alter the expression ofwingless inDrosophila

To understand the roles of two well known tumour suppressor genes.l(2)gl andl(2)gd in normal imaginal disc development inDrosophila, we have initiated a study to examine effect of mulations of these genes on the expression of genes involved in the patterning of the imaginal discs. In this study we show that the expression ofwingless, theDrosophila orthologue of the mammalian oncogeneWnt, is affected in the imaginal discs ofl(2)gl ⁴ andl(2)gd ¹ mutant individuals. In the tumourous wing imaginal discs froml(2)gl mutant larvae, the pattern ofwingless expression was progressively disrupted with an increase in the area of expression, Tumourous wing imaginal discs froml(2)gd homozygous individuals exhibited progressive broadening and extension of the wingless expressing domains. We suggest thatl(2)gl andl(2)gd might be involved in regulating post embryonic expression ofWingless.     

Role of Jun N-terminal Kinase (JNK) signaling in the wound healing and regeneration of a Drosophila melanogaster wing imaginal disc

When a fragment of a Drosophila imaginal disc is cultured in growth permissive conditions, it either regenerates the missing structures or duplicates the pattern present in the fragment. This kind of pattern regulation is known to be epimorphic, i.e. the new pattern is generated by proliferation in a specialized tissue called the blastema. Pattern regulation is accompanied by the healing of the cut surfaces restoring the continuous epithelia. Wound healing has been considered to be the inductive signal to commence regenerative cell divisions. Although the general outlines of the proliferation dynamics in a regenerating imaginal disc blastema have been well studied, little is known about the mechanisms driving cells into the regenerative cell cycles. In this study, we have investigated the role of Jun N-terminal Kinase (JNK) signaling in the wound healing and regeneration of a Drosophila wing imaginal disc. By utilizing in vivo and in vitro culturing of incised and fragmented discs, we have been able to visualize the dynamics in cellular architecture and gene expression involved in the healing and regeneration process. Our results directly show that homotypic wound healing is not a prerequisite for regenerative cell divisions. We also show that JNK signaling participates in imaginal disc wound healing and is regulated by the physical dynamics of the process, as well as in recruiting cells into the regenerative cell cycles. A model describing the determination of blastema size is discussed.     

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.     

Is regeneration inDrosophila the result of epimorphic regulation?

Summary It has been known for many years that when a wing disc ofDrosophila is bisected, and the fragments cultured in adult females, regulation occurs and either a complete disc is regenerated or the fragment is duplicated. We have investigated how this regeneration process occurs. To establish which cells contribute to the regenerate, and thus determine if regeneration is the result of epimorphic regulation, fragments of discs, after culture in an adult for one to five days, were exposed to³H-thymidine to label replicating cells. Imaginal discs, both whole and as regenerating fragments, undergo some DNA replication which is distributed throughout the disc, but cut discs frequently show clusters of labelled cells around the wound, indicating that regeneration is probably epimorphic.     

Transdetermination: Drosophila imaginal disc cells exhibit stem cell-like potency

Drosophila imaginal discs, the primordia of the adult fly appendages, are an excellent system for studying developmental plasticity. Cells in the imaginal discs are determined for their disc-specific fate (wingness, legness) during embryogenesis. Disc cells maintain their determination during larval development, a time of extensive growth and proliferation. Only when prompted to regenerate do disc cells exhibit lability in their determined identity. Regeneration in the disc is mediated by a localized region of cell division, known as the regeneration blastema. Most regenerating disc cells strictly adhere to their disc-specific identity; some cells however, switch fate in a phenomenon known as transdetermination. Similar regeneration and transdetermination events can be induced in situ by misexpression of the signaling molecule wingless. Recent studies indicate that the plasticity of disc cells during regeneration is associated with high morphogen activity and the reorganization of chromatin structure. Here we provide both a historical perspective of imaginal disc transdetermination, as well as discuss recent findings on how imaginal disc cells acquire developmental plasticity and multipotency. We also highlight how an understanding of imaginal disc transdetermination can enhance an understanding of developmental potency exhibited by stem cells.     

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.     

Regenerative interactions between Drosophila imaginal discs of different types.

We have tested the ability of fragments of one type of imaginal disc to stimulate regeneration of another type. It has been shown by others that, when extreme proximal and distal fragments of the wing disc are combined, intercalary regeneration of the missing tissue ensues. Each fragment, if cultured alone, will merely duplicate its structures. We now find that distal fragments of other thoracic discs, haltere and leg, while retaining their autonomy for differentiation, also interact with proximal wing tissue to promote regeneration of more distal wing structures. The proximal wing tissue used in these experiments was the wingless abnormal wing disc which, in the absence of interaction, yields only proximal wing structures. These results suggest that spatial organization is controlled by similar systems in the various thoracic discs. In contrast, head and genital disc material provided no regenerative stimulus to the mutant wing disc tissue.     

Transdetermination in the homeotic eye--antenna imaginal disc of Drosophila melanogaster

The effects of homeotic mutations on transdetermination in eye-antenna imaginal discs of Drosophila melanogaster were studied. After 12 days of culture in vivo, antenna discs transformed to ventral mesothorax by AntpNs or AntpZ, transdetermined to notum and wing structures four to five times more frequently than the corresponding wild-type antenna discs. Likewise, eye discs transformed to dorsal mesothorax by eyopt transdetermined to leg structures, also extremely frequently (90%). It seems that, during culture, homeotic antenna as well as homeotic eye discs tend to complete the structural inventory of the mesothoracic segment. Transdetermination in the homeotic disc parts is interpreted as a regeneration process which reestablishes an entire segment, i.e., the ventral mesothoracic portion (leg) in the antenna disc regenerates dorsal mesothoracic parts, and the dorsal mesothoracic portion in the eye disc (wing) regenerates ventral mesothoracic parts, respectively. This implies that antenna and leg discs (ventral qualities) as well as eye and wing discs (dorsal qualities) are serially homologous. The transdetermination frequency of the untransformed eye disc to notum and wing structures is enhanced by Antp to the same extent as is the transdetermination frequency of the antenna disc. The first allotypic wing disc structure formed by the eye disc is notum, followed by structures of the anterior wing compartment and finally by posterior wing structures. No evidence for such a sequence was found in the transdetermination pattern of the antenna disc.     

Components of positional information in the developing wing margin of the Lyra mutant of Drosophila

Summary A number of parameters characteristic of the wing margin precursor in imaginal discs of Drosophila are known: the zone of non-proliferating cells or ZNC (O'Brochta and Bryant 1985), aldehyde oxidase (AO) and other enzyme staining patterns (Sprey et al. 1982), E1C antigen localization in a narrow band along the margin (Piovant and Lena 1988). To test our hypothesis that such parameters, and others, act in concert to determine margin identity and the positional information that specifies the bristles and hairs appropriate to the anterior, posterior and distal margins, we have examined these parameters in the dominant mutant Lyra, in which much of the anterior and posterior margins is missing. After establishing that Lyra phenotype is already evident in the early pupal wing, we tested the known imaginal disc parameters and found that only Mab E1C (Piovant and Lena 1988) distribution differs from wild type, suggesting that E1C antigen may be a component of positional information. Sibatani's (1983) model for specification of positional information (PI) applied to wing discs predicts the Lyra adult wing shape as well as the reduced distribution of E1C antigen in Lyra wing discs. The model is based on the assumption that specification of positional information depends on interactions of multiple, independent factors. Clonal analysis with shaggy (Simpson et al. 1988 and Ripoll et al. 1988) indicates that factors in addition to E1C antigen contribute to margin PI in Lyra wings and should allow us to test the multi-component hypothesis further.     

Egfr/Ras pathway mediates interactions between peripodial and disc proper cells in Drosophila wing discs

All imaginal discs in Drosophila are made up of a layer of columnar epithelium or the disc proper and a layer of squamous epithelium called the peripodial membrane. Although the developmental and molecular events in columnar epithelium or the disc proper are well understood, the peripodial membrane has gained attention only recently. Using the technique of lineage tracing, we show that peripodial and disc proper cells arise from a common set of precursors cells in the embryo, and that these cells diverge in the early larval stages. However, peripodial and disc proper cells maintain a spatial relationship even after the separation of their lineages. The peripodial membrane plays a significant role during the regional subdivision of the wing disc into presumptive wing, notum and hinge. The Egfr/Ras pathway mediates this function of the peripodial membrane. These results on signaling between squamous and columnar epithelia are particularly significant in the context of in vitro studies using human cell lines that suggest a role for the Egfr/Ras pathway in metastasis and tumour progression.