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.     

The effects of X-rays on the proliferation dynamics of cells in the imaginal wing disc ofDrosophila melanogaster

Summary We report on the size distribution of clones marked by mitotic recombination induced by several different doses of X-rays applied to 72 h oldDrosophila larvae. The results indicate that the radiation significantly reduces the number of cells which undergo normal proliferation in the imaginal wing disc. We estimate that 1000 r reduces by 40–60% the number of cells capable of making a normal contribution to the development of the adult wing. Part of this reduction is due to severe curtailment in the proliferative ability of cells which nevertheless remain capable of adult differentiation; this effect is possibly due to radiation-induced aneuploidy. Cytological evidence suggests that immediate cell death also occurs as a result of radiation doses as low as 100 r. The surviving cells are stimulated to undergo additional proliferation in response to the X-ray damage so that the result is the differentiation of a normal wing.     

Differentiation capacities of the labial imaginal disc ofDrosophila melanogaster

Summary The mature labial disc, when implanted into a larva of the same age, undergoes metamorphosis along with the host and produces one lateral half of the medi- and distiproboscis. On the basis of results obtained from transplanted disc halves (including the separate peripodial membrane) a tentative fate map of the labial disc was constructed, which shows most of the presumptive mediproboscis to be located in the dorsal, and most of the presumptive distiproboscis in the ventral part of the disc. The distal protion of the peripodial membrane also contains imaginal anlagen, viz. part of the mediproboscis, prementum, and labellar cap anlagen. The involvement of this part of the peripodial membrane was checked by a careful histological analysis of labial disc development during the first ten hours after prepupation. The results were compared with the situation described forCalliphora imaginal discs.In addition, a detailed morphological analysis was made of the proboscis of the homoeotic mutantproboscipedia (pb). At 27°C,pb changes the distiproboscis into a telopodite (leg segments distal to the coxa); the (unchanged) prementum may therefore correspond to the coxa. At 15° C, the tarsus of this homoeotic telopodite is replaced to a greater or lesser extent by an arista. The present analysis thus confirms (a) the fundamental morphological correspondence of the medi- and distiproboscis with the labium of other insects, and (b) the fundamental developmental correspondence of the labial, antennal, and leg discs.K. K. was a member of the 8th International Research Group in Developmental Biology, and was the recipient of a UNESCO travel grant.)     

Experimental analysis of sensory nerve pathways inDrosophila

Summary The pathway of adult sensory nerves has been analysed in three experimental situations: (i) in flies with grossly abnormal thoracic morphology resulting from X-irradiation early during development, (ii) in flies which had been subjected to surgical operations late in the larval period, (iii) in homoeotic mutants. The results provide experimental support for a simple mechanism in which developing adult axons join the nearest larval nerve and are guided by it up to the central nervous system. In particular, experimental interference with normal development can result in nerves from different segments, or from dorsal and ventral appendages, joining each other and entering the central nervous system together.     

Transdetermination in leg imaginal discs ofDrosophila melanogaster undDrosophila nigromelanica

Summary The transdetermination capacities of leg discs ofDrosophila melanogaster were examined by mechanically disrupting and kneading whole discs from late third instar larvae and by culturing the resulting tissue mass for 10–14 days in adult female abdomens where the cells continued to divide. The grown implants were then dissected from the abdomens and injected into third instar larvae to undergo metamorphosis.After this treatment, prothoracic leg discs ofDrosophila melanogaster transdetermined with a high frequency (59% of all implants) to wing. Mesothoracic leg discs also transdetermined to wing, but at a very low frequency (4%). Metathoracic leg discs exhibited the same low frequency of transdetermination (4%), but in this case the direction of transdetermination was to haltere (Table 1,D. melanogaster).Very similar results were obtained with leg discs ofDrosophila nigromelanica (Table 1,D. nigromelanica), showing that the peculiar behavior of the three leg discs is not unique forDrosophila melanogaster.The homeotic mutation Polycomb (Pc ³) which partially transforms meso- and metathoracic legs into prothoracic legs did not significantly increase the frequencies of transdetermination in these leg dises and had clearly no effect on the direction of transdetermination (Table 1).We dedicate this publication to the memory of our teacher and advisor, the late Professor Ernst Hadorn, whose enthusiasm and interest stimulated our work     

Growth is required for cell competition in the imaginal discs ofDrosophila melanogaster

Summary Imaginal wing discs from late third-instar larvae were gammairradiated to induce clones of rapidly growingMinute ^– cells in a background of slowly growingMinute cells and culturedin vivo for periods up to 18 days. Clones in discs cultured for 16 to 18 days did not grow significantly larger than clones in uncultured controls, indicating that competition between populations of cells having potentially different mitotic rates does not occur in imaginal discs after their growth is completed.     

Enzyme distribution patterns in the imaginal wing disc ofDrosophila melanogaster and other diptera

Summary Analysis of the development of the aldehyde oxidase (AO) pattern in the wing pouch ofD. melanogaster showed that the extension of areas with AO activity occurs in steps. This indicates that the activation of this enzyme is regulated in groups of cells. It is proposed to use the term territory for such a cell group. In the wing pouches ofD. melanogaster, D. simulans andMusca, corresponding parts of the disc become AO positive at comparable developmental stages. This indicates that AO becomes active in individual territories in a specific sequence.Borderlines of the distribution pattern of different enzymes in the wing pouch ofDrosophila and other dipteran species are in agreement with those found for the development of the AO pattern or are complementary to them. This indicates the existence of a common set of territories in the wing pouches of all higher diptera. Borderlines of patterns, as caused by different genetic constitution, are also in accord with this set of territories. The borderlines of some territories coincide with the compartmental A/P or D/V boundary. The results support the idea that both the location of compartmental boundaries and that of borderlines of enzyme territories are determined by a single mechanism.The distribution and the shape of the territories in the wing pouch is best explained by the reaction-diffusion model proposed by Meinhardt (1980), which involves three different gradients.     

The expression of PS integrins in Drosophila melanogaster imaginal disc cell lines

Summary Drosophila imaginal disc cell lines show a characteristic pattern of aggregation in culture, which appears to be due to cell-cell rather than cell-substrate interactions. We have examined the distribution of PS integrins in wing and leg cell lines, and find that these integrin homologues are expressed preferentially in aggregates. Cell sheets, small cell clumps and chains of cells express antigen at points of cell-cell contact only.     

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.     

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.     

Correlation between adult transformation and aldehyde oxidase staining pattern in wing discs ofApterous genotypes inDrosophila melanogaster

Summary The aldehyde oxidase staining pattern in wing discs ofDrosophila melanogaster bearing the genotypesap ^blt /ap ^blt andap ^blt andap ^blt /ap ⁷³ⁿ showns changes from the wild-type pattern. Extensive areas of the presumptive dorsal posterior wing blade, which are normally unstained, have enzyme activity in these mutants. In wings of these genotypes, dorsal posterior structures are replaced by dorsal anterior wing structures. A strong correlation has been found between the frequencies of various staining patterns in the discs and the extent of transformation in the cuticular structures of the wing, which is consistent with the idea that aldehyde oxidase activity can be used as an indicator in the wing disc of this transformation. Unlike the homoeotic mutationengrailed, apterous has not been interpreted as a selector gene yet the work reported here shows thatapterous alleles can cause changes resembling those of theengrailed phenotype both in aldehyde oxidase staining behaviour and in the cuticular transformation.     

Cell degeneration and elimination in the imaginal wing disc,caused by the mutationsvestigial andUltravestigial ofDrosophila melanogaster

Summary The mutationsvestigial (vg; recessive) andUltravestigial (vg ^U; dominant) ofDrosophila melanogaster give rise to identical mutant adult phenotypes in which much of the cases this results from cell death in the presumptive wing margin of the wing disc in the third larval instar, but the process of cell degeneration is quite different in the two mutants. Invg cell death occurs continuously throughout the third larval instar, while invg ^U it occurs only in the early third instar. Cells fragment and some of the fragments condense, becoming electron dense (apoptosis). Both condensed and ultrastructurally normal cell fragments are extruded to the basal side of thevg disc epithelium. They accumulate under the basal lamina in the wing pouch area until they are phagocytosed by blood cells entering the wing pouch during the six hours following pupariation. Fragments are not extruded from thevg ^U epithelium but are apparently phagocytosed by neighboring epithelial cells. The basal lamina undergoes mophological changes following pupariation and is phagocytosed by blood cells in both wild-type andvestigial, but investigial the degenerated cell fragments are also engulfed by the same blood cells.     

Wound healing and regeneration in the imaginal wing disc ofDrosophila

Summary When complementary fragments of an imaginal disc ofDrosophila are cultured for several days prior to metamorphosis, usually one fragment will regenerate while the other will duplicate. It has been proposed that wound healing plays an important part in disc regulation (French et al. 1976; Reinhardt et al. 1977) by initiating cell proliferation and determining the mode of regulation. We tried to delay the wound healing process by leaving a region of dead cells between the wound edges. In 06 fragments (Bryant 1975a) wound healing has occurred after 1–2 days of culture and the regeneration of missing structures after 2–4 days of culture. We observed that leaving a region of dead cells between the wound edges delays both wound healing and the regeneration of missing structures by 2 days.When disc fragments are cultured in female abdomens and then exposed to³H-thymidine to label replicating cells, then the label is found to be localised around the wound. We observed that delaying wound healing does not delay this localisation of labelled nuclei indicating that wound healing may not be required to initiate DNA replication.     

Changes in the distribution of gap junctions inDrosophila melanogaster wing discs during the third larval and early pupal stages of development

Summary Developmental changes in the distribution of gap junctions in early, mid and late third larval stage wing discs and in pupariation+6 h and pupariation+24 h stage wing discs fromDrosophila melanogaster were analyzed by quantitative electron microscopy. Gap junctions occur in all 12 intradisc regions examined in each of the five developmental stages. Their distribution is non-random and changes during development which suggests that they are developmentally regulated. The gap junctions are not static structures, rather they grow and regress during development. The changes tend to be gradual ones without sudden increases or decreases. Gap junctions continuously form and grow in size throughout the third larval stage and during the first 6 h following pupariation. Their surface density, number, percent of the lateral plasma membrane area, and absolute area as well as the lateral plasma membrane surface density all increase during this time. Between pupariation+ 6 h and pupariation+24 h all but one of these parameters decrease indicative of gap junctional breakdown. Gap junctions are most numerous and change least during development in the apical cell regions where intercellular contacts are close and stable. They change most in the basal cell regions where intercellular contacts tend to be looser and change during development. The most dramatic change is in the absolute area which increases by a factor of 23 between the early third larval stage and pupariation+24 h. At pupariation the rate of gap junction growth undergoes a transient increase before the phase of disassembly begins. Developmental changes in gap junction surface density are closely coupled with changes in the lateral plasma membrane surface density which suggests that these may be coregulated. Evidence from mutants suggests that when the number and density of gap junctions fail to increase in proportion to lateral plasma membrane growth, wing disc development will be abnormal. Our results support the idea that some minimum gap junction density is required for normal development and that this must increase as development proceeds. The results are consistent with the notion that gap junctions are involved in pattern formation and growth control and are discussed with respect to the acquisition of competence for metamorphosis, disc growth, disc morphogenesis and changes in the hormonal environment.     

Evagination of imaginal discs in the fleshflySarcophaga crassipalpis: Hormonal control in vivo

Summary The hormonal control of evagination of the imaginal leg discs of the fleshflySarcophaga crassipalpis was studied by means of ectopic transplantation of discs from mature larvae or prepupae onto prepupal hosts. Evagination was influenced by the age of the donor and the developmental commitment of the host. Discs from non-diapuse as well as diapause-committed donors did not differ in their capacity to evaginate and differentiate. Mature larval discs from either type of donor could evaginate only when transplanted onto non-diapause hosts; they failed to evaginate on diapause-committed hosts even though host discs evaginated in situ about 40 h after transplantation. Discs from white prepupae evaginated whether transplanted onto non-diapause or onto diapause-committed hosts.     

A comparative study of embryonic gene expression inDrosophila andEphestia

Summary The ontogeny of allozyme patterns has been studied in embryos ofDrosophilamelanogaster, which are doubly heterozygous for alleles specifying the slow and fast forms of alcohol dehydrogenase (ADH) and -glycerophosphate dehydrogenase (GPDH). The ontogeny of esterase-2 was studied in embryos and young larvae of the flour mothEphestia kühniella, which are heterozygous for two of the three existing esterase-2 alleles. In freshly laidDrosophila eggs only the maternal enzyme forms are present and during the first 15 hours of development the staining of these forms becomes progressively fainter. After 16 and 17 h, the paternal and hybrid bands of ADH and GPDH respectively become obvious. Before hatching, the intensity distribution in the three-banded pattern of reciprocal hybrids is asymmetric in favour of the persisting maternal enzyme form. InEphestia embryos, however, there is no persistence of the maternal esterases. In all reciprocal heterozygotes a three-banded pattern suddenly appears 96 h after egg deposition, indicating synchronous activation of both parental alleles. The relative intensity distribution in the hybrid patterns approaches that of the mature larvae stepwise and in an allele-specific manner. This result and the fact that the various heterozygous types exhibit unequal total activities suggest that the Esterase-2 alleles have different activities, which are fixed late in embryogenesis.     

Cell interactions during the fusionin vitro ofDrosophila eye-antennal imaginal discs

Summary The fusion of the eye-antennal discs during culturein vitro has been investigated, and the complex morphogenetic movements which occur during the formation of the head capsule of the insect are described. The initial contact between the eye anlagen is by means of cell processes spanning the gap between the two discs. Subsequently the two epithelia become firmly apposed, and then the integrity of the epithelium in the region of fusion breaks down, cells appearing to move to new positions in order to form an epithelium which unites the two discs. The epithelium eventually secretes a pattern of cuticular structures which is continuous between the derivatives of the two discs. Bristles on either side of the line of fusion are perfectly aligned, and structures such as the median ocellus, which are formed jointly by the cells of the two discs, differentiate normally. This is also found when left and right eye-antennal discs of different genotypes are placed side-by-side, indicating that processes of pattern regulation can occur in culture.     

Positional information in imaginal discs transformed by homoeotic mutations

Summary Mutations of the bithorax complex result in segmental transformations in the thorax and abdomen ofDrosophila. The haltere discs from larvae homozygous forbx ³ orpbx are transformed so that the discs contain cells that will produce wing cuticle as well as cells that produce haltere cuticle. The pattern regulation behavior of these discs has been examined. The fate maps of the two discs were established, and then the regulative behavior of a number of fragments from both types of mutant discs was established by culturing the fragments in vivo prior to metamorphosis. The most important conclusion from this work is that the cells producing, haltere cuticle and wing cuticle within the same disc share the same positional information and that they communicate during pattern regulation.     

JNK signaling pathway required for wound healing in regenerating Drosophila wing imaginal discs

We have examined wound healing during regeneration of Drosophila wing imaginal discs fragments by confocal microscopy and assessed the role of components of the JNK pathway in this process. After cutting, columnar and peripodial epithelia cells at the wound edge start to close the wound through formation and contraction of an actin cable. This is followed by a zipping process through filopodial protrusions from both epithelia knitting the wound edges from proximal to distal areas of the disc. Activation of the JNK pathway is involved in such process. puckered (puc) expression is induced in several rows of cells at the edge of the wound, whereas absence of JNK pathway activity brought about by hemipterous, basket, and Dfos mutants impair wound healing. These defects are accompanied by lowered or loss of expression of puc. In support of a role of puc in wound healing, hep mutant phenotypes are rescued by reducing puc function, whereas overexpression of puc inhibits wound healing. Altogether, these results demonstrate a role for the JNK pathway in imaginal disc wound healing, similar to that reported for other healing processes such as embryonic dorsal closure, thoracic closure, and adult epithelial wound healing in Drosophila. Differences with such processes are also highlighted.