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Sequence and expression of IMP-L1, an ecdysone-inducible gene expressed during Drosophila imaginal disc morphogenesis.

Drosophila imaginal discs are induced by the steroid hormone 20-hydroxyecdysone to initiate morphogenesis leading to formation of the adult appendages and thoracic epidermis at the end of the third larval instar. Ecdysone-dependent transcriptional activation of a set of genes that encode imaginal disc transcripts found on membrane-bound polysomes precedes and may be responsible for some aspects of the cellular changes that mediate epithelial morphogenesis in this system. A 1.35 kb transcript from one of these genes, IMP-L1, is first observed in vivo at or just prior to pupariation, as ecdysone titers are peaking and beginning to decline. Expression is initiated in proximal areas of the antennal disc, later spreading to a more widespread but nonuniform distribution throughout other thoracic imaginal discs. IMP-L1 is not, however, expressed in other ecdysone target tissues such as salivary glands or fat body. The IMP-L1 gene encodes a novel protein product containing a signal peptide, a possible transmembrane domain, two highly charged domains and a proline rich C-terminal domain. We suggest that the delayed timing of expression of this secondary response gene is necessary for proper ordering of cellular events associated with disc morphogenesis.

Amino Acid Sequence↗

Localization of the Antennapedia protein in Drosophila embryos and imaginal discs.

Antibodies have been raised against a fusion protein containing the 3' region of the coding sequence of the Antennapedia (Antp) gene fused to beta-galactosidase. The distribution of the protein on whole mount embryos and imaginal discs of third instar larvae was examined by immunofluorescence. In young embryos, expression of the Antp protein was limited to the thoracic segments in the epidermis, whereas it was found in all neuromeres of head, thorax and abdomen. At the end of embryogenesis, the Antp protein mainly accumulated in the ventral nervous system in certain parts of the thoracic neuromeres, from posterior T1 to anterior T3, with a gap in posterior T2. Comparison of Antp protein distribution in nervous systems from wild-type and Df P9 embryos, lacking the genes of the Bithorax-complex (BX-C), revealed a pattern of expression which indicated that the BX-C represses Antp in the posterior segments with the exception of the last abdominal neuromeres (A8-9) which are regulated independently. The protein pattern in nervous systems from Sex combs reduced(Scr) mutant embryos was indistinguishable from that found in wild-type embryos; thus, neurogenic expression of Antp in T1 and the more anterior segments does not appear to be under the control of Scr. All imaginal discs derived from the three thoracic segments express Antp protein. The distribution was distinct in each disc; strongest expression was observed in the proximal parts of the discs. In the leg discs the protein distribution seemed to be compartmentally restricted, whereas in the wing disc this was not the case. Antp protein was not detected in the eye-antennal disc. In embryos, as well as in imaginal discs, the protein is localized in the nucleus.

Journal Article↗

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.

Animals↗

Development of the eye-antenna imaginal disc and morphogenesis of the adult head in Drosophila melanogaster.

We have studied the organization and development of the eye-antenna imaginal disc of Drosophila melanogaster. We examined the pattern of gynandromorph mosaicism and determined the "sturt distances" between 42 different structures of the head, antenna, and maxillary palpus. A morphogenetic map based on these sturt distances resembles more closely in size and shape that of a single thoracic segment than that of two or more adjacent segments, suggesting that the eye-antenna disc is derived from a single embryonic body segment. We examined the morphology of the eye-antenna discs in situ in late third-instar larvae in serial cross sections. The two discs are connected medially by a thin cellular membrane that probably serves to join the two discs during evagination and morphogenesis of the adult head. A fate map of the imaginal disc was established by cutting the mature disc into fragments and transplanting the fragments into host larvae for metamorphosis. The peripodial layer of the eye-antenna disc is thickened in several regions, and our data suggest that these thickened areas represent primordia of adult head structures. A comparison of the location of precursors in the imaginal disc with those of the differentiated structures of the adult head revealed the nature of the morphogenetic movements that must occur during evagination and differentiation.

Animals↗

The effect of imaginal exposure length on outcome of treatment for PTSD.

The effects of prolonged imaginal exposure sessions (60 minutes; n=60) were compared with those of shorter exposure sessions (30 minutes, n=32) for patients with chronic posttraumatic stress disorder (PTSD). Consistent with the authors' hypothesis, patients who received 30-minute imaginal exposure sessions showed less within-session habituation than patients who received 60-minute exposure sessions. However, no differences between patients who received 60-minute and 30-minute exposure sessions emerged on improvement in PTSD-symptoms, state anxiety, depression, and end-state functioning, both at posttreatment and at 1-month follow-up. No group differences were found with regard to between-sessions habituation, number of sessions, and dropout rate. Results suggest that 30-minute imaginal exposure sessions are as effective as 60-minute exposure sessions and that within-session habituation may not be a necessary condition for successful treatment of PTSD. Future research is needed to replicate these findings and extend them to other clinical populations.

Adult↗

Proteomic analysis of the wing imaginal discs of Drosophila melanogaster.

We have combined high-resolution two-dimensional (2-D) gel electrophoresis and mass spectrometry with the aim of identifying proteins represented in the 2-D gel database of the wing imaginal discs of Drosophila melanogaster. First, we obtained a high-resolution 2-D gel pattern of [35S]methionine + [35S]cysteine-labeled polypeptides of Schneider cells, a permanent cell line of Drosophila embryonic origin, and compared it with the standard pattern of polypeptides of the wing imaginal disc. These studies reveal qualitative and quantitative differences between the two samples, but have more than 600 polypeptides in common. Second, we carried out preparative 2-D polyacrylamide gel electrophoresis using Schneider cells mixed with radioactively labeled wing imaginal discs in order to isolate some of the shared polypeptides and characterize them by matrix-assisted laser desorption/ionization-time of flight MALDI-TOF analysis. Using this strategy we identified 100 shared proteins represented in the database, and in each case confirmed their identity by MALDI-TOF/TOF analysis.

Animals↗

Temporal regulation of Drosophila imaginal disc morphogenesis: a hierarchy of primary and secondary 20-hydroxyecdysone-responsive loci.

The release of 20-hydroxyecdysone at the end of the third larval instar provides a temporal signal that triggers specific developmental programs in hormone target tissues in Drosophila at metamorphosis. Imaginal discs respond to the steroid hormone by initiating morphogenesis leading to the formation of the adult head structures, appendages, and thoracic epidermis. The cellular events of morphogenesis are preceded and accompanied by 20-hydroxyecdysone-dependent activation of a set of genes encoding Inducible Membrane-bound Polysomal transcripts, the IMP-genes. Analysis of expression characteristics in imaginal discs cultured in vitro reveals that the IMP-E1 gene is expressed within 15-30 min after exposure to 20-hydroxyecdysone while the expression of the IMP-L1 gene is delayed 6-8 hr. Induction studies in the presence of cycloheximide establish that IMP-E1 is a primary response locus while IMP-L1 transcription is a secondary response. These genes are regulated at the level of transcription initiation. Differences between the induction characteristics of IMP-E1 and the early 20-hydroxyecdysone-responsive gene E74 lead us to propose an addition to the Ashburner model for the 20-hydroxyecdysone regulatory hierarchy. We suggest that the sequential temporal expression of steroid hormone-responsive genes in imaginal discs is important in organizing cellular mechanisms involved in morphogenesis of the epithelium.

Animals↗

Epithelial hyperplasia of imaginal discs induced by mutations in Drosophila tumor suppressor genes: growth and pattern formation in genetic mosaics.

Lethal mutations in the giant discs (lgd) and fat (ft) tumor suppressor genes of Drosophila cause epithelial hyperplasia in all imaginal discs. By contrast, mutations in the vestigial (vg) gene adversely affect cell viability in the wing imaginal discs and consequently cause loss of pattern in the adult wings. However, combining homozygous lgd or ft mutations with homozygous vg1 increases the size of the wing imaginal discs and partially restores the bristle pattern in the wings of pharate adults. Comparable pattern restoration in vg1 wings is also induced by a newly isolated weak hypomorphic lgd3 allele. Further, mosaic analysis revealed that whereas lgd clones generated by the Minute technique display abnormal differentiation, those induced in a homozygous vg1 background exhibit autonomous restoration of wing pattern. These results suggest that pattern restoration in vg1 wings can serve as an assay for hyperplasia induced by mutations in Drosophila tumor suppressor genes.

Animals↗

Regeneration of Sarcophaga imaginal discs in vitro: implication of 20-hydroxyecdysone.

When the 3/4 sectors of leg imaginal discs of Sarcophaga were cultured in vitro in the presence of 2.5 x 10(-8) M 20-hydroxyecdysone, wound healing and restoration of their morphology occurred. This concentration of ecdysone was critical for wound healing and was 40 times lower than that necessary for inducing differentiation of imaginal discs in vitro. Lost positional values revealed by expression of the wingless gene were found to show partial recovery under these conditions. These results suggest that a low titer of ecdysone is essential for the regeneration of imaginal discs.

Amino Acid Sequence↗

DWnt4 and wingless elicit similar cellular responses during imaginal development.

Wnt genes encode evolutionarily conserved secreted proteins that provide critical functions during development. Although Wnt proteins share highly conserved features, they also show sequence divergence, which almost certainly contributes to the variety of their signaling activities. We previously reported that DWnt4 and wingless (wg), two divergent clustered Wnt genes, can have either antagonist or distinct functions during Drosophila embryogenesis. Here we provide evidence that both genes can elicit similar cellular responses during imaginal development. Ectopic expression of DWnt4 along the anterior/posterior (A/P) boundary of imaginal discs alters morphogenesis of adult appendages. In the wing disc, DWnt4 phenocopies ectopic Wg activity by inducing notum to wing transformation, suggesting similar signaling capabilities of both molecules. In support of this, we demonstrate that DWnt4 can rescue wg loss-of-function phenotypes in the antenna and haltere and is able to substitute for Wg in wing field specification. We also show that both genes are transcribed in overlapping domains in imaginal discs, suggesting that DWnt4 may cooperate with wg during limb patterning.

Animals↗

Cell lines from imaginal discs of Drosophila melanogaster.

New cell lines, designated as ML-DmD1-10, were established from dissociated imaginal discs of Drosophila melanogaster. The culture medium was prepared by mixing in a 1:1 ratio Cross and Sang's M3(BF) medium, supplemented with 10% heat inactivated fetal bovine serum (FBS), with the supernatant of a primary embryonic cell culture made in the M3(BF) medium and supplementing this mixture with insulin. One cell line was established in the medium containing larval hemolymph instead of the primary culture supernatant, and another was established in fresh M3(BF) medium supplemented with insulin and FBS. In these mediums, imaginal disc cells first formed aggregates and cellular vesicles within a few weeks followed by the proliferation of thin-layered cells around them after about 1 mo. Ten cell lines have so far been established from two kinds of imaginal discs and disc mixtures. The ploidy of these cell lines was predominantly diploid. Population doubling time was about 50 to 70 h at 3 to 10 mo. after initiation of the culture. When the cell aggregates formed in vitro were implanted in metamorphosing larvae, they differentiated at high frequency into adult cuticular structures in the early phase of the primary culture. This differentiation of aggregates was also observed, though at low frequency, in a culture maintained by dilution-transfer for 6 to 15 mo. in vitro.

Animals↗

DNA replication and pattern regulation in the imaginal wing disc of Drosophila.

Imaginal disc cells of Drosophila undergo pattern regulation when part of a disc is removed by microsurgery. I have analyzed DNA replication in imaginal disc cells during pattern regulation using both pulse and chronic labeling with tritiated thymidine. Both types of experiments have shown a local increase in the fraction of labeled cells associated with pattern regulation. In addition I have found that when pattern regulation takes place during chronic thymidine labeling approximately 15% of the cells in a "regenerated" region are unlabeled. Thus DNA replication may not be required for respecification of "positional values" in imaginal disc cells. These results are consistent with patterning information being important in controlling cell growth during normal development.

Animals↗

In vitro culture of Drosophila imaginal disc cells: aggregation, sorting out, and differentiative abilities.

This paper describes the aggregation in vitro of cells dissociated from imaginal discs and demonstrates the sorting out of undifferentiated cells from different imaginal discs and from differently determined regions of the same imaginal disc, as well as the abilities of such cells to undergo pattern reconstruction when injected into larvae. Dissociated cells begin to aggregate by 1.5 hr of rotation. By 5 hr of rotation, large aggregates of loosely associated cells appear. By 18 hr the aggregates have condensed and taken on a characteristic epithelial structure. To study sorting out in undifferentiated cells, we combined a histochemical stain for acid phosphatase with the use of the acid phosphatase null mutant acphn-11. We performed cell mixing experiments with 0-2 (prospective notum) and 2-8 (prospective wing) fragments, with the A and P (prospective anterior and posterior) fragments of the dorsal mesothoracic disc and with mixtures of cells from ventral prothoracic and dorsal mesothoracic discs. We found that prospective anterior and posterior dorsal mesothoracic cells do not sort out, but that prospective notum and wing and leg and wing cells do. The results from differentiated implants are consistent with those from undifferentiated mixes.

Acid Phosphatase↗

Extracellular protease production by Drosophila imaginal discs.

We are investigating the role of extracellular proteases in imaginal disc eversion to understand the mechanism that controls cell rearrangements within epithelia. We have identified three cation-dependent neutral proteases released by Drosophila leg discs everting in culture. Serine protease inhibitors block disc eversion and inhibit activity of disc proteases. The pattern of extracellular proteases changes when eversion is blocked with added protease inhibitors. Changes in protease activity occur when released disc proteases are treated with trypsin. Trypsin treatment of intact imaginal discs releases protease and inhibitor activities to the medium, indicating their presence on the cell surface before release. Our results suggest that extracellular proteases are required for imaginal disc morphogenesis and are regulated by more than one mechanism.

Animals↗

Possible migration of imaginal myoblasts from adjacent nerve sheath into the developing flight muscle of Chironomus.

The emplacement of the first imaginal myoblasts along the larval muscles which are precursors of the dorsal longitudinal flight muscles, has been studied in Chironomus (Diptera, Nematocera), by light and electron microscopy. At the beginning of larval life there are no imaginal myoblasts stored along these muscles. These cells are discerned only at the beginning of the last larval instar. They first appear in the median region of the muscles near the neuromuscular junction. Prior to this, however, there are cells possessing the same cytological characteristics as the imaginal myoblasts inside the sheath of the motor nerves that supply the muscles. These observations suggest that myoblasts could arrive by the nerve sheath. The presence of a thick, continuous basal lamina around the larval muscles seems to exclude all other possibility of access to these muscles. The extension of this hypothesis to the Cyclorrhaphan Diptera is discussed.

Animals↗

Hedgehog is a signaling protein with a key role in patterning Drosophila imaginal discs.

The segment polarity genes hedgehog and engrailed are expressed in identical posterior-compartment-specific patterns in both Drosophila embryos and imaginal discs. We show here that the hedgehog protein is secreted, and it can cross embryo parasegment borders and the anterior-posterior compartment border of imaginal discs to neighboring cells that express neither engrailed nor hedgehog. In these cells, it is localized in discrete punctate structures that are sequestered within the polarized epithelium. Analysis of animals that have expressed hedgehog ectopically, or of a mutant that expresses hedgehog abnormally in the anterior compartment of the wing disc, indicates that hedgehog is involved in regulating patched. In the embryo, hedgehog regulation of patched apparently facilitates patched and wingless expression. In the discs, hedgehog regulation of patched and other genes in the anterior compartment helps to establish the proximodistal axis. We propose that the cell-cell communication mediated by hedgehog links the special properties of compartment borders with specification of the proximodistal axis in imaginal development.

Animals↗

Function of protein kinase A in hedgehog signal transduction and Drosophila imaginal disc development.

Reduced protein kinase A (PKA) activity in anterior imaginal disc cells leads to cell-autonomous induction of decapentaplegic (dpp), wingless (wg), and patched (ptc) transcription that is independent of hedgehog (hh) gene activity. The resulting nonautonomous adult wing and leg pattern duplications are largely due to induced dpp and wg expression and resemble phenotypes elicited by ectopic hh expression. Inhibition of PKA in anterior cells close to the posterior compartment can substitute for hh activity to promote growth of imaginal discs, whereas overexpression of PKA can counteract transcriptional induction of ptc by hh in these cells. PKA therefore appears to be an integral component of the mechanism by which hh regulates the expression of key patterning molecules in imaginal discs.

Animals↗

Distribution of cuticular protein mRNAs in silk moth integument and imaginal discs.

The distributions of mRNAs for two cuticular proteins of Hyalophora cecropia were examined with RT-PCR and in situ hybridization. For major regions of larval and pupal cuticle, there was a strong correspondence between the type of cuticle and the predominant cuticular protein message found. Epidermal cells underlying soft cuticle had mRNA for HCCP12, with a RR-1 consensus attributed to soft cuticle, while the epidermal cells associated with hard cuticle had predominantly mRNA for HCCP66, a protein with the RR-2 consensus attributed to hard cuticle. Both messages were found in all areas of the pupal fore- and hind-wings, with modest area-specific difference in concentration being much less than differences in the relative abundance of these cuticular proteins.mRNA for HCCP12 was present in imaginal discs of feeding larvae of H cecropia. Data from Bombyx mori available at SilkBase (http://www.ab.a.u-tokyo.ac.jp/silkbase/) revealed that imaginal discs from feeding larvae had abundant mRNA for RR-1 cuticular proteins, representing six distinct gene products. Only discs from spinning larvae had mRNAs that coded for RR-2 proteins arising from 10 distinct genes. Thus, lepidopteran wing imaginal discs can no longer be regarded as inactive in larval cuticle production.

Amino Acid Sequence↗