Compartments and the control of growth in the Drosophila wing imaginal disc

The mechanisms that control organ growth are among the least known in development. This is particularly the case for the process in which growth is arrested once final size is reached. We have studied this problem in the wing disc of Drosophila, the developmental and growth parameters of which are well known. We have devised a method to generate entire fast-growing Minute(+) (M(+)) discs or compartments in slow developing Minute/+ (M/+) larvae. Under these conditions, a M(+) wing disc gains at least 20 hours of additional development time. Yet it grows to the same size of Minute/+ discs developing in M/+ larvae. We have also generated wing discs in which all the cells in either the anterior (A) or the posterior (P) compartment are transformed from M/+ to M(+). We find that the difference in the cell division rate of their cells is reflected in autonomous differences in the developmental progression of these compartments: each grows at its own rate and manifests autonomous regulation in the expression of the developmental genes wingless and vestigial. In spite of these differences, ;mosaic' discs comprising fast and slow compartments differentiate into adult wings of the correct size and shape. Our results demonstrate that imaginal discs possess an autonomous mechanism with which to arrest growth in anterior and posterior compartments, which behave as independent developmental units. We propose that this mechanism does not act by preventing cell divisions, but by lengthening the division cycle.     

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.     

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.     

Regulative Properties of Wing Discs from the Vestigial Mutant of Drosophila melanogaster

The vestigial (vg) mutant of Drosophila melanogaster shows reduced wing size and lacks margin structures from the wing blade. The expressivity is temperature-sensitive, more structures being formed at 29°C than at 25°C. There is cell death in the third instar wing disc which to some extent parallels the fate map locations of the structures absent in the adult.
Vestigial wing discs are unable to regenerate margin structures even when given extra time for growth by culturing them in an adult abdomen before metamorphosis. If the region of cell death is excised from the disc before culture, there is still no regeneration of margin structures, indicating that the dead cells do not physically prevent regulation. Furthermore, by metamorphosing young vg wing discs, it was discovered that cells never acquire competence to make margin during wing disc development. Experiments mixing fragments of vg wing disc with non- vg wing disc fragments of ebony multiple wing hairs (e mwh) genotype showed that the vg cells interacted with the e mwh cells and wing blade was intercalated of both genotypes. However, structures such as wing margin, and alar lobe, usually affected in vg wings, were always made from e mwh cells and not from vg cells. Analysis of mutants which are unable to differentiate particular cell types may help us to understand the mechanism of pattern establishment in developing imaginal discs.     

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.     

Intrinsic growth control in the imaginal primordia of Drosophila, and the autonomous action of a lethal mutation causing overgrowth

Cell proliferation in Drosophila imaginal discs appears to be regulated by a disc-intrinsic mechanism involving local cell interactions that also control the formation of patterns of differentiation. This growth-control mechanism breaks down in animals homozygous for the mutation lethal (2) giant discs (l(2)gd) which remain as larvae for up to 9 days longer than normal. During this time cell proliferation continues in the imaginal discs as well as in the imaginal rings for the salivary glands, foregut, and hindgut, so that these tissues become greatly overgrown. When wild-type wing discs from mid-third instar larvae were removed and cultured for up to 28 days in wild-type female adult hosts, they grew and terminated growth at a cell number close to that which would be attained in situ by the time of pupariation. On the other hand, wing discs from l(2)gd homozygotes grew rapidly and continuously when cultivated in wild-type hosts, reached an enormous size, and acquired abnormal folding patterns. Overgrowth of mutant imaginal rings also continued during culture of these tissues in wild-type hosts. We conclude that overgrowth in this mutant is due to an autonomous defect in the imaginal primordia, which requires an extended larval period for its expression in situ.     

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.     

The BEAF insulator regulates genes involved in cell polarity and neoplastic growth

Boundary Element Associated Factor-32 (BEAF-32) is an insulator protein predominantly found near gene promoters and thought to play a role in gene expression. We find that mutations in BEAF-32 are lethal, show loss of epithelial morphology in imaginal discs and cause neoplastic growth defects. To investigate the molecular mechanisms underlying this phenotype, we carried out a genome-wide analysis of BEAF-32 localization in wing imaginal disc cells. Mutation of BEAF-32 results in miss-regulation of 3850 genes by at least 1.5-fold, 794 of which are bound by this protein in wing imaginal cells. Up-regulated genes encode proteins involved in cell polarity, cell proliferation and cell differentiation. Among the down-regulated genes are those encoding components of the wingless pathway, which is required for cell differentiation. Miss-regulation of these genes explains the unregulated cell growth and neoplastic phenotypes observed in imaginal tissues of BEAF-32 mutants.     

patufet, the gene encoding the Drosophila melanogaster homologue of selenophosphate synthetase, is involved in imaginal disc morphogenesis

Proliferation in imaginal discs requires cell growth and is linked to patterning processes controlled by secreted cell-signalling molecules. To identify new genes involved in the control of cell proliferation we have screened a collection of P-lacW insertion mutants that result in lethality in the larval/pupal stages, and characterized a novel gene, patufet (ptuf). Inactivation of ptuf by a P element insertion in the 5′ untranslated region leads to aberrant imaginal disc morphology characterized by a reduction in mass of discs and disorganisation of disc cells where no folding or patterning can be detected. Moreover, apoptotic cells can be observed in these small and abnormal mutant discs. To examine the role of ptuf we have studied its clonal behaviour in genetic mosaics generated by mitotic recombination. The mutation causes reduced cell viability, smaller cell size and stops vein differentiation. Non-autonomous effects, such as abnormal differentiation of wild-type cells surrounding the clones, are also observed. We have cloned the ptuf gene of Drosophila melanogaster and found that it encodes a selenophosphate synthetase, which is the first identified in insects. Mutant flies transformed with the full-length cDNA show complete reversion of lethality and disc phenotype. Northern blot analysis and in situ hybridization indicate that the ptuf gene is expressed in imaginal discs as well as at different stages of development. The synthesis of selenoproteins by the selenophosphate synthetase, the role of selenoproteins in the maintenance of the oxidant/antioxidant balance of the cell and its possible implications in imaginal disc morphogenesis are discussed.     

Localized cell death caused by mutations in a Drosophila gene coding for a transforming growth factor-beta homolog

Recessive mutations (dppdisk) in one region of the decapentaplegic (dpp) gene of Drosophila, which codes for a transforming growth factor-beta homolog, cause loss of distal parts from adult appendages. Different dppdisk alleles cause effects of different severity, the milder alleles removing distal parts and the more severe alleles removing progressively more proximal structures. In the wing disc derivatives, the most extreme dppdisk genotype removes the entire wing and leaves only a thorax fragment. We show that structures are lost in these mutants as a result of massive apoptotic cell death in the corresponding regions of the imaginal discs during the mid-third larval instar. The remaining disc fragments do not regenerate when cultured alone in the growth-permissive environment of the adult abdomen, but they can be made to regenerate by coculturing them with appropriate fragments of wild-type wing discs. This nonautonomous development is interpreted as showing that a product of dpp+, presumably the TGF-beta homolog, is secreted by the normal cells and can rescue the mutant cells in the mixed tissue.     

Targeted expression of the signaling molecule decapentaplegic induces pattern duplications and growth alterations in Drosophila wings.

In the wing imaginal disc, the decapentaplegic (dpp) gene is expressed in a stripe of anterior cells near the anterior-posterior compartment boundary, and it is required solely in these cells for the entire disc to develop. In some viable segment polarity mutants, alterations in dpp expression have been demonstrated that correlate with changes in wing morphology. To test the hypothesis that the abnormal patterns of dpp expression are responsible directly for the mutant phenotypes, we have expressed dpp in ectopic places in wing imaginal discs, and we have found that dpp is able to cause overgrowth and pattern duplications in both anterior and posterior compartments of the wing disc. The alterations of the anterior compartment are strikingly similar to those observed in some viable segment polarity mutants. Thus, ectopic dpp alone can account for the phenotype of these mutants. We also show that ectopic expression of the segment polarity gene hedgehog (hh) gives similar morphological changes and activates dpp expression in the anterior compartment. This strongly suggests that the organizating activity of hh is mediated by dpp. We propose that the expression of dpp near the anterior-posterior compartment boundary is directed by the interaction between patched and hh, and that dpp itself could act as a general organizer of the patterning in the wing imaginal disc.     

Towards Long Term Cultivation of Drosophila Wing Imaginal Discs In Vitro

The wing imaginal disc of Drosophila melanogaster is a prominent experimental system for research on control of cell growth, proliferation and death, as well as on pattern formation and morphogenesis during organogenesis. The precise genetic methodology applicable in this system has facilitated conceptual advances of fundamental importance for developmental biology. Experimental accessibility and versatility would gain further if long term development of wing imaginal discs could be studied also in vitro. For example, culture systems would allow live imaging with maximal temporal and spatial resolution. However, as clearly demonstrated here, standard culture methods result in a rapid cell proliferation arrest within hours of cultivation of dissected wing imaginal discs. Analysis with established markers for cells in S- and M phase, as well as with RGB cell cycle tracker, a novel reporter transgene, revealed that in vitro cultivation interferes with cell cycle progression throughout interphase and not just exclusively during G1. Moreover, quantification of EGFP expression from an inducible transgene revealed rapid adverse effects of disc culture on basic cellular functions beyond cell cycle progression. Disc transplantation experiments confirmed that these detrimental consequences do not reflect fatal damage of imaginal discs during isolation, arguing clearly for a medium insufficiency. Alternative culture media were evaluated, including hemolymph, which surrounds imaginal discs during growth in situ. But isolated larval hemolymph was found to be even less adequate than current culture media, presumably as a result of conversion processes during hemolymph isolation or disc culture. The significance of prominent growth-regulating pathways during disc culture was analyzed, as well as effects of insulin and disc co-culture with larval tissues as potential sources of endocrine factors. Based on our analyses, we developed a culture protocol that prolongs cell proliferation in cultured discs.     

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.     

Regulation of imaginal disc cell size, cell number and organ size by Drosophila class I(A) phosphoinositide 3-kinase and its adaptor.

BACKGROUND: Class I(A) phosphoinositide 3-kinases (PI 3-kinases) have been implicated in the regulation of several cellular processes including cell division, cell survival and protein synthesis. The size of Drosophila imaginal discs (epithelial structures that give rise to adult organs) is maintained by factors that can compensate for experimentally induced changes in these PI 3-kinase-regulated processes. Overexpression of the gene encoding the Drosophila class I(A) PI 3-kinase, Dp110, in imaginal discs, however, results in enlarged adult organs. These observations have led us to investigate the role of Dp100 and its adaptor, p60, in the control of imaginal disc cell size, cell number and organ size. RESULTS: Null mutations in Dp110 and p60 were generated and used to demonstrate that they are essential genes that are autonomously required for imaginal disc cells to achieve their normal adult size. In addition, modulating Dp110 activity increases or reduces cell size in the developing imaginal disc, and does so throughout the cell cycle. The inhibition of Dp110 activity reduces the rate of increase in cell number in the imaginal discs, suggesting that Dp110 normally promotes cell division and/or cell survival. Unlike direct manipulation of cell-cycle progression, manipulation of Dp110 activity in one compartment of the disc influences the size of that compartment and the size of the disc as a whole. CONCLUSIONS: We conclude that during imaginal disc development, Dp110 and p60 regulate cell size, cell number and organ size. Our results indicate that Dp110 and p60 signalling can affect growth in multiple ways, which has important implications for the function of signalling through class I(A) PI 3-kinases.     

Regulation of Organ Growth by Morphogen Gradients

Morphogen gradients play a fundamental role in organ patterning and organ growth. Unlike their role in patterning, their function in regulating the growth and the size of organs is poorly understood. How and why do morphogen gradients exert their mitogenic effects to generate uniform proliferation in developing organs, and by what means can morphogens impinge on the final size of organs? The decapentaplegic (Dpp) gradient in the Drosophila wing imaginal disc has emerged as a suitable and established system to study organ growth. Here, we review models and recent findings that attempt to address how the Dpp morphogen contributes to uniform proliferation of cells, and how it may regulate the final size of wing discs.     

The Drosophila segment polarity gene patched interacts with decapentaplegic in wing development.

The decapentaplegic (dpp) gene of Drosophila melanogaster encodes a polypeptide of the transforming growth factor-beta family of secreted factors. It is required for the proper development of both embryonic and adult structures, and may act as a morphogen in the embryo. In wing imaginal discs, dpp is expressed and required in a stripe of cells near the anterior-posterior compartment boundary. Here we show that viable mutations in the segment polarity genes patched (ptc) and costal-2 (cos2) cause specific alterations in dpp expression within the anterior compartment of the wing imaginal disc. The interaction between ptc and dpp is particularly interesting; both genes are expressed with similar patterns at the anterior-posterior compartment boundary of the disc, and mis-expressed in a similar way in segment polarity mutant backgrounds like ptc and cos2. This mis-expression of dpp could be correlated with some of the features of the adult mutant phenotypes. We propose that ptc controls dpp expression in the imaginal discs, and that the restricted expression of dpp near the anterior-posterior compartment boundary is essential to maintain the wild-type morphology of the wing disc.     

Duration of the cell cycle phases in mutants for the tumor suppressor Merlin in Drosophila melanogaster

The cell cycle duration was estimated in Drosophila melanogaster mutants for the tumor suppressor Merlin with the use of different approaches. Experiments on induction of mosaic clones in tissues of the larval wing imaginal disc showed that the cell cycle in mutant discs is shorter than that in control. Flow fluorescence cytometry revealed no differences between mutant and normal animals in the relative duration of the cell cycle phases, which suggests proportional shortening of the cell cycle phases. The study with pulse-labeled mitoses confirmed these results and showed that the length of the cell cycle is 7 h (S phase duration 3 h) in control individuals and 5 h (S phase duration 2 h) in Merlin gene mutants.     

patufet , the gene encoding the Drosophila melanogaster homologue of selenophosphate synthetase, is involved in imaginal disc morphogenesis

Proliferation in imaginal discs requires cell growth and is linked to patterning processes controlled by secreted cell-signalling molecules. To identify new genes involved in the control of cell proliferation we have screened a collection of P-lacW insertion mutants that result in lethality in the larval/pupal stages, and characterized a novel gene, patufet (ptuf). Inactivation of ptuf by a P element insertion in the 5′ untranslated region leads to aberrant imaginal disc morphology characterized by a reduction in mass of discs and disorganisation of disc cells where no folding or patterning can be detected. Moreover, apoptotic cells can be observed in these small and abnormal mutant discs. To examine the role of ptuf we have studied its clonal behaviour in genetic mosaics generated by mitotic recombination. The mutation causes reduced cell viability, smaller cell size and stops vein differentiation. Non-autonomous effects, such as abnormal differentiation of wild-type cells surrounding the clones, are also observed. We have cloned the ptuf gene of Drosophila melanogaster and found that it encodes a selenophosphate synthetase, which is the first identified in insects. Mutant flies transformed with the full-length cDNA show complete reversion of lethality and disc phenotype. Northern blot analysis and in situ hybridization indicate that the ptuf gene is expressed in imaginal discs as well as at different stages of development. The synthesis of selenoproteins by the selenophosphate synthetase, the role of selenoproteins in the maintenance of the oxidant/antioxidant balance of the cell and its possible implications in imaginal disc morphogenesis are discussed. Received: 22 August 1997 / Accepted: 9 September 1997     

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.     

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.