Imaginal Disc Cells Research Articles

Environmental control of the cell cycle in Drosophila: nutrition activates mitotic and endoreplicative cells by distinct mechanisms.

In newly hatched Drosophila larvae, quiescent cells reenter the cell cycle in response to dietary amino acids. To understand this process, we varied larval nutrition and monitored effects on cell cycle initiation and maintenance in the mitotic neuroblasts and imaginal disc cells, as well as the endoreplicating cells in other larval tissues. After cell cycle activation, mitotic and endoreplicating cells respond differently to the withdrawal of nutrition: mitotic cells continue to proliferate in a nutrition-independent manner, while most endoreplicating cells reenter a quiescent state. We also show that ectopic expression of Drosophila Cyclin E or the E2F transcription factor can drive quiescent endoreplicating cells, but not quiescent imaginal neuroblasts, into S-phase. Conversely, we demonstrate that quiescent imaginal neuroblasts, but not quiescent endoreplicating cells, can be induced to enter the cell cycle when co-cultured with larval fat body in vitro. These results demonstrate a fundamental difference in the control of cell cycle activation and maintenance in these two cell types, and imply the existence of a novel mitogen generated by the larval fat body in response to nutrition.

Antigenotoxicity studies in Drosophila melanogaster

The fruit fly Drosophila melanogaster with its well developed array of genotoxicity test systems has been used in a number of studies on antigenotoxicity of various compounds and mixtures. In recent years, the newly developed Somatic Mutation and Recombination Tests (SMART) have mainly been employed. These one-generation tests make use of the wing or eye imaginal disc cells in larvae and have proven to be very efficient and sensitive. They are based on the principle that the loss of heterozygosity of suitable recessive markers can lead to the formation of mutant clones of cells that are then expressed as spots on the wings or eyes of the adult flies. We have employed the wing spot test with the two markers multiple wing hairs ( mwh, 3-0.3) and flare ( flr, 3–38.8). Three-day-old larvae, trans-heterozygous for these markers, are treated chronically or acutely by oral administration with the test compound(s) or complex mixtures. For antigenotoxicity studies, chronic co-treatments can be used, as well as separate pre-treatments with an antigenotoxic agent followed by a chronic treatment with a genotoxin. After eclosion, the wings of the adult flies are scored for the presence of single and twin spots. These spots can be due to different genotoxic events: either mitotic recombination or mutation (deletion, point mutation, specific types of translocation, etc.). The analysis of two different genotypes (one with structurally normal chromosomes, one with a multiply inverted balancer chromosome) allows for a quantitative determination of the recombinagenic activity of genotoxins. Results of two separate studies are presented: (1) instant coffee has antirecombinagenic but not antimutagenic activity in the wing spot test; and (2) ascorbic acid and catechin are able to protect against in vivo nitrosation products of methyl urea in combination with sodium nitrite.

Somatic and germline clone analysis in mutants of the S-adenosylmethionine synthetase encoding gene in Drosophila melanogaster

We have analysed the phenotypic consequences of homozygous mutant clones in the S-adenosylmethionine synthetase encoding gene in Drosophila melanogaster. The results suggest that SamS function is required for cell proliferation/growth in embryonic/early larval cells and during development of imaginal disc cells. Homozygous SamS germline clones can, however, develop and give rise to viable heterozygous offspring. This offspring expresses a Minute-like phenotype. We suggest that this phenotype is caused by an obstruction of the polyamine biosynthesis.

Multiple developmental requirements of noisette, the Drosophila homolog of the U2 snRNP-associated polypeptide SP3a60.

We report the cloning of the noisette gene (noi), which encodes the Drosophila melanogaster ortholog of a U2 snRNP-associated splicing factor, SF3a60 (SAP61) in humans and PRP9p in Saccharomyces cerevisiae. Antibodies raised against human SF3a60 recognized NOI in flies, showing a nuclear localization in all the stages examined, including the embryo, the dividing cells of imaginal discs, and the larval polyploid nuclei. NOI is expressed in somatic and germinal cells of both male and female gonads. By mobilization of P transposons, we have generated a large number of noi mutations. Complete loss of function resulted in lethality at the end of embryogenesis, without obvious morphological defects. Hypomorphic alleles revealed multiple roles of noi for the survival and differentiation of male germ cells, the differentiation of female germ cells, and the development of several adult structures.

4 Cell Determination and Transdetermination in Drosophila Imaginal Discs

Drosophila imaginal discs, the precursors of the adult fly appendages, are an important system for studying mechanisms of cell determination. How the different imaginal discs acquire and maintain their appendage-specific determined states are problems that have been addressed using experimental embryology as well as genetic and molecular approaches. Here we discuss the concept of cell determination and describe what is known about how determination is established and maintained in imaginal disc cells. The phenomenon of imaginal disc transdetermination, originally discovered in the 1960s, has remained an intriguing problem for understanding imaginal disc cell determination. We review the topic of imaginal disc transdetermination and describe how recent results from molecular genetic approaches have provided new insights into imaginal disc transdetermination and determination. We also discuss how an understanding of imaginal disc transdetermination can aid our understanding of parallel phenomena in other organisms, including human metaplasias.

A molecular basis for transdetermination in Drosophila imaginal discs: interactions between wingless and decapentaplegic signaling.

We are investigating how Drosophila imaginal disc cells establish and maintain their appendage-specific determined states. We have previously shown that ectopic expression of wingless (wg) induces leg disc cells to activate expression of the wing marker Vestigial (Vg) and transdetermine to wing cells. Here we show that ectopic wg expression non-cell-autonomously induces Vg expression in leg discs and that activated Armadillo, a cytosolic transducer of the Wg signal, cell-autonomously induces Vg expression in leg discs, indicating that this Vg expression is directly activated by Wg signaling. We find that ubiquitous expression of wg in leg discs can induce only dorsal leg disc cells to express Vg and transdetermine to wing. Dorsal leg disc cells normally express high levels of decapentaplegic (dpp) and its downstream target, optomotor-blind (omb). We find that high levels of dpp expression, which are both necessary and sufficient for dorsal leg development, are required for wg-induced transdetermination. We show that dorsalization of ventral leg disc cells, through targeted expression of either dpp or omb, is sufficient to allow wg to induce Vg expression and wing fate. Thus, dpp and omb promote both dorsal leg cell fate as well as transdetermination-competent leg disc cells. Taken together, our results show that the Wg and Dpp signaling pathways cooperate to induce Vg expression and leg-towing transdetermination. We also show that a specific vg regulatory element, the vg boundary enhancer, is required for transdetermination. We propose that an interaction between Wg and Dpp signaling can explain why leg disc cells transdetermine to wing and that our results have implications for normal leg and wing development.

Genetic characterization of the role of the two HOX proteins, Proboscipedia and Sex Combs Reduced, in determination of adult antennal, tarsal, maxillary palp and proboscis identities in Drosophila melanogaster.

Both Proboscipedia (PB) and Sex Combs Reduced (SCR) activities are required for determination of proboscis identity. Here we show that simultaneous removal of PB and SCR activity results in a proboscis-to-antenna transformation. Dominant negative PB molecules inhibit the activity of SCR indicating that PB and SCR interact in a multimeric protein complex in determination of proboscis identity. These data suggest that the expression pattern of PB and SCR and the ability of PB and SCR to interact in a multimeric complex control the determination of four adult structures. The absence of PB and SCR expression leads to antennal identity; expression of only PB leads to maxillary palp identity; expression of only SCR leads to tarsus identity; and expression of both PB and SCR, which results in the formation of a PB-SCR-containing complex, leads to proboscis identity. However, the PB-SCR interaction is not detectable in vitro and is not detectable genetically in the head region during embryogenesis, indicating the PB-SCR interaction may be regulated and indirect. This regulation may also explain why ectopic expression of SCR(Q50K) and SCR do not result in the expected transformation of the maxillary palp to an antennae and proboscis, respectively. Previous analysis of the requirements of SCR activity for adult pattern formation has shown that ectopic expression of SCR results in an antenna-to-tarsus transformation, but removal of SCR activity in a clone of cells does not result in a tarsus-to-arista transformation. Here we show in five independent assays the reason for this apparent contradictory requirement of SCR activity in tarsus determination. SCR activity is required cell nonautonomously for tarsus determination. Specifically, we propose that SCR activity is required in the mesodermal adepithelial cells of all leg imaginal discs at late second/early third instar larval stage for the synthesis of a mesoderm-specific, tarsus-inducing, signaling factor, which after secretion from the adepithelial cells acts on the overlaying ectodermal cells determining tarsus identity. This study characterizes a combinatorial interaction between two HOX proteins; a mechanism that may have a major role in patterning the anterior-posterior axis of other animals.

Effect of age on the growth and response of a Drosophila cell line to moulting hormone

Insect cell lines in culture are used for a variety of studies. In this laboratory imaginal disc cell lines have been established from primary cultures from third instar larvae, and used for a number of experiments. The effect of ageing on the morphology and physiology of Drosophila cell lines has received very little attention, although problems of genotypic or phenotypic changes in cell lines with age are recognized in other areas of animal cell culture. We tested our cell line CI8+ for any difference in growth, morphology and response to 20-hydroxyecdysone (20HE) at different ages (passage numbers). The cells were found to multiply faster, adhere less firmly to the substrate and to lose the tendency to aggregate at higher passages. The response to 20HE in terms of cell numbers and induction of β-galactosidase was similar at all passage numbers but morphological changes in hormone-treated cells were less obvious in the higher passages. Cell lines are likely to vary in the extent of ageing effects but workers are advised to be aware of the possibilities. We suggest the effects of age on cell lines should be established, and passage numbers noted in experimental reports.

Role of knot (kn) in wing patterning in Drosophila.

We have undertaken a genetic analysis of new strong alleles of knot (kn). The original kn1 mutation causes an alteration of wing patterning similar to that associated with mutations of fused (fu), an apparent fusion of veins 3 and 4 in the wing. However, unlike fu, strong kn mutations do not affect embryonic segmentation and indicate that kn is not a component of a general Hh (Hedgehog)-signaling pathway. Instead we find that kn has a specific role in those cells of the wing imaginal disc that are subject to ptc-mediated Hh-signaling. Our results suggest a model for patterning the medial portion of the Drosophila wing, whereby the separation of veins 3 and 4 is maintained by kn activation in the intervening region in response to Hh-signaling across the adjacent anterior-posterior compartment boundary.

ArrowheadEncodes a LIM Homeodomain Protein That Distinguishes Subsets ofDrosophilaImaginal Cells

TheArrowheadgene encodes a LIM-homeodomain transcription factor required for establishment of a subset of imaginal tissues: the abdominal histoblasts and the salivary gland imaginal rings. Consistent with its role in development, during embryogenesisArrowheadis expressed in each abdominal segment and in the labial segment. Late in embryonic development, expression is refined to the abdominal histoblasts and salivary gland imaginal ring cells themselves. When ectopically expressed in imaginal disc cells,Arrowheadcauses programmed cell death and loss of corresponding adult structures. Therefore,Arrowheadexpression is required for development of one set of imaginal cells and is incompatible with development of another, emphasizing the specificity ofArrowheadand the sensitivity of different target cells to its expression. Loss-of-function mutations inArrowheadaffect conserved or invariant amino acids in the LIM- and homeo-domains demonstrating the importance of these residues in LIM homeodomain protein activity.

Ectopic mitotic recombination in Drosophila probed with bacterial beta-galactosidase gene-based reporter transgenes.

Plasmids were constructed to investigate homologous mitotic recombination in Drosophila cells. Heteroalleles containing truncated but overlapping segments of the bacterial beta-galactosidase gene (lacZ) were positioned either on separate plasmids or as direct repeats on the same chromosome. Recombination reconstituted a functional lacZgene leading to expression of LacZ+activity detectable by histochemical staining. High extrachromosomal recombination (ECR) frequencies between unlinked heteroalleles were observed upon transient co-transfection into Drosophila melanogaster Schneider line 2 (S2) cells. Stably transfected cells containing the lacZ heteroalleles linked on a chromosome exhibited intrachromosomal recombination (ICR) frequencies two orders of magnitude lower than ECR frequencies. Recombination was inducible by exposing the cells to ethyl methanesulphonate or mitomycin C. Recombination products were characterized by multiplex PCR analysis and unequal sister chromatid recombination was found as the predominant mechanism reconstituting the lacZ gene. To investigate recombination in vivo imaginal disc cells from transgenic larvae carrying the reporter gene on the X chromosome were isolated and stained for LacZ+ activity. The presence of a few LacZ+ clones indicated that mitotic recombination events occurred at frequencies two orders of magnitude lower than the corresponding event in cultured cells and late during larval development.

Dual pathways for induction of wingless expression by protein kinase A and Hedgehog in Drosophila embryos.

The secreted Drosophila Hedgehog (Hh) protein induces transcription of specific genes by an unknown mechanism that requires the serpentine transmembrane protein Smoothened (Smo) and the transcription factor Cubitus interruptus (Ci). Protein kinase A (PKA) has been implicated in the mechanism of Hh signal transduction because it acts to repress Hh target genes in imaginal disc cells that express Ci. Changes in Ci protein levels, detected by an antibody that recognizes an epitope in the carboxy-terminal half of Ci, have been suggested to mediate the positive effects of Hh and the negative effects of PKA on Hh target gene expression in imaginal discs. Here we show that PKA inhibition, like Hh, leads to increased "carboxy-terminal" Ci staining and Hh target gene expression in embryos. In addition, we find that Hh and Smo can stimulate target gene expression at constant Ci levels and that increased PKA activity can induce ectopic Hh target gene expression in a manner that requires Smo and Ci activities but does not involve changes in Ci protein concentration. This suggests a branching pathway of Hh signal transduction downstream of Smo and that PKA exerts opposite effects on the two branches. Finally we show that Hh signaling in embryos does not depend on cAMP-dependent regulation of PKA activity.

The effect of dominant vestigial alleles upon vestigial-mediated wing patterning during development of Drosophila melanogaster

The vestigial gene product is required for the completion of wing development in Drosophila melanogaster. In the absence of vestigial gene expression, cells within the larval wing and haltere imaginal discs fail to proliferate normally thus producing adults with severely reduced wings. Of a large number of vestigial mutations that have been characterized, only tow are currently known to exist, vestigial U and vestigial W , which manifest a significant dominant phenotype. Both are associated with chromosomal inversions that fuse the majority of the vestigial coding regions to other genes; mastermind in vestigial U and invected in vestigial W . Examination of vestigial expression in the presence of these dominant alleles shows alterations in the disc-specific expression of vestigial during later stages of larval development. These patterning disruptions are specific to cells of the wing imaginal disc, as significant suppression of total levels of vestigial expression within entire larvae could not be detected. This dominant interference of vestigial patterning appears to be mediated in part by the vestigial coding sequences that are within the gene fusions. Further evidence that the dominant phenotype is the result of disrupted vestigial patterning comes from observations that the dominant alleles can be partially suppressed by mutations within the Drosophila-epidermal growth factor receptor gene. Mutagenesis of vestigial U and vestigial W produced a series of alleles with partially dominant phenotypes that restored various amounts of the adult wing. These phenotypes can be correlated with alterations in specific portions of the vestigial sequences associated with the dominant alleles. In the presence of these partially dominant alleles, wing imaginal discs have significantly more cells which express vestigial compared with the number associated with the original dominant phenotype. Additionally, eliminating some of the dominant effect causes alterations in the patterns of early stage apoptotic cell death associated with dominant vestigial alleles. Utilizing these new vestigial alleles, it is possible to correlate the consequence of altered vestigial expression to subsequent changes in patterning of the wing disc.

G2 arrest of cell cycle ensures a determination process of sensory mother cell formation in Drosophila.

In Drosophila, the sensory mother cells of macrochaetes are chosen from among the mitotically quiescent clusters of cells in wing imaginal discs, where other cells are proliferating. The pattern of cyclin A, one of the G2 cyclins, reveals that mitotically quiescent clusters of cells are arrested in G2. When precocious mitoses are induced during sensory mother cell determination by the ectopic expression of string, a known G2/M transition regulator, the formation of sensory mother cells is disturbed, resulting in the loss of macrochaetes in the adult notum. This suggests that G2 arrest of the cell cycle ensures the proper determination of sensory mother cells.

Overexpression of the mouse dishevelled-1 protein inhibits GSK-3β-mediated phosphorylation of tau in transfected mammalian cells

Tau is a neuronal microtubule-associated protein whose function is modulated by phosphorylation. GSK-3β is a tau kinase. GSK-3β is part of the wingless signalling pathway and stimulation by wingless is predicted to down-regulate GSK-3β activity. In Drosophila imaginal disc cells, overexpression of dishevelled, a component of the wingless pathway, mimics the wingless signal. We have therefore studied the effect that overexpression of the murine dishevelled-1 protein has on GSK-3β-mediated phosphorylation of tau in transfected CHO cells. We find that co-transfection with dishevelled-1 is inhibitory to GSK-3β-mediated tau phosphorylation. Tau is hyperphosphorylated in Alzheimer's disease and the possible relevance of these findings to Alzheimer's disease pathogenesis are discussed.

Autonomous and non-autonomous phenotypic expression of Notch mutant cells in Drosophila embryogenesis.

Transplantation of single homozygous Notch - cells into the ventral neuroectoderm of Drosophila wild-type embryos reveals a non-autonomous behaviour of these cells. However, mitotic recombination events induced in heterozygous cells in imaginal discs lead to generation of homozygous Notch - cells which differentiate cell autonomously. We have tested various possible explanations for the non-autonomous behaviour of Notch mutant cells following transplantation. We confirm the previous results using different Notch alleles. Moreover, we find that increasing the number of wild-type Notch copies in a cell increases probability that it will take on an epidermal fate. However, single Notch mutant cells behave differently depending on whether they are placed in the ventral neuroectoderm, the procephalic neuroectoderm or the proctodeal anlage. Following transplantation into host embryos devoid of mesoderm, almost all single Notch mutant cells behave autonomously. These results suggest an influence of the mesoderm on ectodermal development. Finally, we provide further evidence that DELTA acts as the signal source in lateral inhibition.

The effects of several ecdysteroids and ecdysteroid agonists on two Drosophila imaginal disc cell lines.

Two Drosophila imaginal disc cell lines, C18+ (sensitive to 20-hydroxyecdysone, 20HE) and C18R (resistant to 20HE) were exposed to the ecdysteroid agonists RH5849 and RH5992 and the ecdysteroids inokosterone, makisterone A and muristerone A. All compounds tested were found to have similar effects on the cells, comparable to the effects of 20HE, although at different concentrations. C18R showed resistance to all compounds, again at varying concentrations. We conclude that it is likely that all the compounds tested use the same receptors as 20HE, but show maximum effectiveness at different concentrations.

Sodium azide induces mitotic recombination in Drosophila melanogaster larvae

Sodium azide (NaN 3), a potent mutagen for bacteria and barley, was tested for somatic mutation and mitotic recombination induction in wing imaginal disc cells of Drosophila melanogaster. Comparisons were made among inversion-free flr 3/mwh , inversion-heterozygous TM3, Ser/mwh, and inversion-free, high bioactivation OR(R), flr 3/mwh flies. Third instar larvae were exposed chronically for 48 h to sodium azide at 0.5, 0.63. 0.75, 0.88 and 1.0 mM. The frequencies of spots per wing obtained in the three kinds of progeny scored were compared. In inversion-free flies, sodium azide induced large single and total spots at all concentrations tested, and small single and twin spots at 0.75 mM and higher concentrations. In contrast, it failed to increase the frequency of small and large single spots in inversion-heterozygous flies. In high bioactivation flies (which are inversion-free), sodium azide increased the frequency of large single spots at 0.63, 0.88 and 1.0 mM and the frequency of total spots at 0.63 mM. From the absence of genotoxic activity observed in inversion-heterozygous flies it is concluded that sodium azide induces exclusively mitotic recombination in wing somatic cells of Drosophila melanogaster larvae after chronic exposure. This recombinogenic activity is reduced in the presence of high bioactivation capacity.

Homeotic transformation of legs to mouthparts by proboscipedia expression in Drosophila imaginal discs

The Drosophila homeotic gene proboscipedia ( pb) specifies labial identity and directs formation of the adult distiproboscis from the labial imaginal discs. pb null alleles result in the homeotic transformation of the distiproboscis into prothoracic (T1) legs [ Kaufman (1978) Genetics 90, 579–596; Pultz et al. (1988) Genes Dev. 2, 901–920]. Homology with other transcription factors, localization to the nucleus, and restricted embryonic and imaginal expression implicate the pb protein (PB) as a transcription factor. In order to examine the possible roles that PB may play in the specification of adult mouthparts, we have expressed PB in cells of wing, leg and eye-antennal imaginal discs and assayed for effects on the development of adult structures. We report here that the ectopic expression of PB in the imaginal discs under the control of the inducible GAL4 system [ Brand and Perrimon (1993) Development 118, 401–415] alters the developmental program of adult legs into maxillary or labial palps. These homeotic transformations have an equal effect on all three sets of legs, indicating an activity that is not solely dependent upon the unique combinations of other homeotic genes present in each of the leg discs. Segment polarity genes required for establishing the AP compartment boundary were found to be undisturbed by ectopic PB. Furthermore, normal patterns of apoptosis are observed in animals expressing ectopic PB, indicating that PB does not alter or affect cell death. These results suggest that molecular events occurring downstream of the establishment of the compartment boundary are affected by ectopic PB expression in imaginal discs and point to a general role in ‘palp’ formation in addition to the specification of labial identity.

ELAV, a Drosophila neuron-specific protein, mediates the generation of an alternatively spliced neural protein isoform

Background Tissue-specific alternative pre-mRNA splicing is a widely used mechanism for gene regulation and the generation of different protein isoforms, but relatively little is known about the factors and mechanisms that mediate this process. Tissue-specific RNA-binding proteins could mediate alternative pre-mRNA splicing. In Drosophila melanogaster, the RNA-binding protein encoded by the elav (embryonic lethal abnormal visual system) gene is a candidate for such a role. The ELAV protein is expressed exclusively in neurons, and is important for the formation and maintenance of the nervous system.Results In this study, photoreceptor neurons genetically depleted of ELAV, and elav-null central nervous system neurons, were analyzed immunocytochemically for the expression of neural proteins. In both situations, the lack of ELAV corresponded with a decrease in the immunohistochemical signal of the neural-specific isoform of Neuroglian, which is generated by alternative splicing. Furthermore, when ELAV was expressed ectopically in cells that normally express only the non-neural isoform of Neuroglian, we observed the generation of the neural isoform of Neuroglian.ConclusionsDrosophila ELAV promotes the generation of the neuron-specific isoform of Neuroglian by the regulation of pre-mRNA splicing. The findings reported in this paper demonstrate that ELAV is necessary, and the ectopic expression of ELAV in imaginal disc cells is sufficient, to mediate neuron-specific alternative splicing.