Invasive cell behavior during Drosophila imaginal disc eversion is mediated by the JNK signaling cascade

Drosophila imaginal discs are monolayered epithelial invaginations that grow during larval stages and evert at metamorphosis to assemble the adult exoskeleton. They consist of columnar cells, forming the imaginal epithelium, as well as squamous cells, which constitute the peripodial epithelium and stalk (PS). Here, we uncover a new morphogenetic/cellular mechanism for disc eversion. We show that imaginal discs evert by apposing their peripodial side to the larval epidermis and through the invasion of the larval epidermis by PS cells, which undergo a pseudo-epithelial-mesenchymal transition (PEMT). As a consequence, the PS/larval bilayer is perforated and the imaginal epithelia protrude, a process reminiscent of other developmental events, such as epithelial perforation in chordates. When eversion is completed, PS cells localize to the leading front, heading disc expansion. We found that the JNK pathway is necessary for PS/larval cells apposition, the PEMT, and the motile activity of leading front cells.     

Effect of the wingless (wg1) mutation on wing and haltere development in Drosophila melanogaster

Genetic and developmental studies of wingless (wg¹), a new second chromosome recessive mutation in Drosophila melanogaster, have shown that it affects not only wing and haltere development (giving rise to wingless and/or halterless flies), but also results in various abnormalities of the mesothorax. The larvae destined to develop into wingless and/or halterless flies possessed underdeveloped mesothoracic and/or metathoracic imaginal discs.     

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

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

Ultrabithorax gene expression in Drosophila imaginal discs and larval nervous system

Using a monoclonal antibody and image-processing procedures, the patterns of expression of the Ultrabithorax (Ubx) gene product have been characterized in Drosophila larvae. As reported previously, the metathoracic imaginal discs stain most intensely with anti-Ubx, with some mesothoracic and no prothoracic expression detectable. In the metathoracic discs, the greatest modulation in anti-Ubx staining is along the proximodistal axis. Ubx is generally expressed at higher levels in the posterior regions of metathoracic discs, although relatively high anterior expression is found in some areas. Expression in the mature wing disc is confined to the squamous peripodial membrane cells; in younger wings, Ubx expression fills the posterior half of the peripodial side of the disc. The mesothoracic leg stains with a pattern that is qualitatively similar (but not identical) to that of the metathoracic leg; Ubx is expressed in some anterior regions of the mesothoracic leg, in parasegment 4. Double staining with anti-Ubx and anti-engrailed reveals that discontinuities in Ubx expression that have been suggested to correspond to compartment borders do not coincide with the compartment boundaries in some cases. In the larval ventral ganglion, Ubx expression is greatest in parasegments 5 and 6, as in the embryonic nervous system.     

Segmental differences in the protein content of Drosophila imaginal discs

The protein content of various Drosophila imaginal discs was analysed by two-dimensional electrophoresis followed by silver-staining. Three proteins, identified as tropomyosins α and β and actin I, are more abundant in the metathoracic discs (haltere and third leg) than in the mesothoracic discs (wing and second leg). In the case of the wing disc, these proteins are probably contributed by the adepithelial (muscle precursor) cells, as indicated by their non-uniform localisation within the disc. Mutations in the bithorax complex have no effect on the difference between second and third leg discs. We conclude that there is a segmental difference in the protein content of homologous discs, that this difference is probably localized in the adepithelial cells, and that it is not under the direct control of known alleles of the bithorax complex.     

Wg and Egfr signalling antagonise the development of the peripodial epithelium in Drosophila wing discs

Imaginal discs contain a population of cells, known as peripodial epithelium, that differ morphologically and genetically from the rest of imaginal cells. The peripodial epithelium has a small contribution to the adult epidermis, though it is essential for the eversion of the discs during metamorphosis. The genetic mechanisms that control the identity and cellular morphology of the peripodial epithelia are poorly understood. In this report, we investigate the mechanisms that pattern the peripodial side of the wing imaginal disc during early larval development. At this time, the activities of the Wingless (Wg) and Epidermal growth factor receptor (Egfr) signalling pathways specify the prospective wing and notum fields, respectively. We show that peripodial epithelium specification occurs in the absence of Wingless and Egfr signalling. The ectopic activity in the peripodial epithelium of any of these signalling pathways transforms the shape of peripodial cells from squamous to columnar and resets their gene expression profile. Furthermore, peripodial cells where Wingless signalling is ectopically active acquire hinge identity, while ectopic Egfr activation results in notum specification. These findings suggest that suppression of Wg and Egfr activities is an early step in the development of the peripodial epithelium of the wing discs.     

The Drosophila JNK pathway controls the morphogenesis of imaginal discs during metamorphosis.

In Drosophila, the Jun-N-terminal Kinase-(JNK) signaling pathway is required for epithelial cell shape changes during dorsal closure of the embryo. In the absence of JNK pathway activity, as in the DJNKK/hemipterous (hep) mutant, the dorsolateral ectodermal cells fail both to elongate and move toward the dorsal midline, leading to dorsally open embryos. We show here that hep and the JNK pathway are required later in development, for correct morphogenesis of other epithelia, the imaginal discs. During metamorphosis, the imaginal discs undergo profound morphological changes, giving rise to the adult head and thoracic structures, including the cuticle and appendages. hep mutant pupae and pharate adults show severe defects in discs morphogenesis, especially in the fusion of the two lateral wing discs. We show that these defects are accompanied by a loss of expression of puckered (puc), a JNK phosphatase-encoding gene, in a subset of peripodial cells that ultimately delineates the margins of fusing discs. In further support of a role of puc in discs morphogenesis, pupal and adult hep phenotypes are suppressed by reducing puc function, indicative of a negative role of puc in disc morphogenesis. Furthermore, we show that the small GTPase Dcdc42, but not Drac1, is an activator of puc expression in a hep-dependent manner in imaginal discs. Altogether, these results demonstrate a new role for the JNK pathway in epithelial morphogenesis, and provide genetic evidence for a role of the peripodial membrane in disc morphogenesis. We discuss a general model whereby the JNK pathway regulates morphogenesis of epithelia with differentiated edges.     

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.     

The Homeotic Gene Sex Combs Reduced of Drosophila Melanogaster Is Differentially Regulated in the Embryonic and Imaginal Stages of Development

The Sex combs reduced (Scr) locus is unique among the genes contained within the Antennapedia complex (ANT-C) of Drosophila melanogaster in that it directs functions that are required for both cephalic and thoracic development in the embryo and the adult. Antibodies raised against protein encoded by Scr were used to follow the distribution of this gene product in embryos and imaginal discs of third instar larvae. Analysis of Scr protein accumulation in embryos hemizygous for breakpoint lesions mapping throughout the locus has allowed us to determine that sequences required for establishment of the Scr embryonic pattern are contained within a region of DNA that overlaps with the identified upstream regulatory region of the segmentation gene fushi tarazu (ftz). Gain-of-function mutations in Scr result in the presence of ectopic sex comb teeth on the first tarsal segment of mesothoracic and metathoracic legs of adult males. Heterozygous combinations of gain-of-function alleles with a wild-type Scr gene exhibit no evidence of ectopic protein localization in the second and third thoracic segments of embryos. However, mesothoracic and metathoracic leg imaginal discs can be shown to accumulate ectopically expressed Scr protein, implying a differential regulation of the Scr gene during these two periods of development. Additionally, we have found that the spatial pattern of Scr gene expression in imaginal tissues involved in the development of the adult thorax is governed in part by synapsis of homologous chromosomes in this region of the ANT-C. However, those imaginal discs that arise anteriorly to the prothorax do not appear to be sensitive to this form of gene regulation. Finally, we have demonstrated that the extent of Scr expression is influenced by mutations at the Polycomb (Pc) locus but not by mutant alleles of the zeste (z) gene. Taken together, our data suggests that Scr gene expression is differentially regulated both temporally and spatially in a manner that is sensitive to the structure of the locus.     

Formation of chimeric patterns and segregation of cells in varied combinations of thorax forming imaginai disks ofDrosophila melanogaster

Summary Genetically marked thoracic imaginal disks ofDrosophila melanogaster were partially dissociated and reaggregated. After metamorphosis, combinations of dorsal proand mesothoracic disks gave rise to mosaics, whereas no such mosaics were formed in combinations of dorsal meso- and metathoracic disks. Adventitious bristles are formed by the metathoracic disk, but these were never integrated in a common pattern with mesothoracic bristles.     

Developmental analysis of some mutants of the bithorax system ofDrosophila

Summary Mutants in the bithorax system ofDrosophila produce homeotic transformations that affect the mesothoracic, metathoracic and first abdominal segments. In the present report we describe a clonal analysis of the development of those mutants transforming the metathorax and first abdominal segments into mesothorax.The main results indicate that (1) The normal dorsal metathoracic (haltere) disk has similar developmental parameters to the dorsal mesothoracic disk. The main difference is that the initial and final numbers of cells are different in both disks. (2) In flies mutant forBithorax andpostbithorax (which transform the haltere into wing) the transformed haltere disk has the same initial and final number of cells as the normal wing disk. (3) In morphogenetic mosaics homozygousbithorax (andpostbithorax) clones express their genotype autonomously regardless of the genotype of surrounding haltere cells. This autonomy is expressed in a regulation of the number of adult cells per compartment, typical cell affinities and final cuticular differentiation.     

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.     

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.     

Effect of Abx, Bx and Pbx Mutations on Expression of Homeotic Genes in Drosophila Larvae

We have examined the patterns of expression of the homeotic gene Ubx in imaginal discs of Drosophila larvae carrying mutations in the abx, bx and pbx regulatory domains. In haltere discs, all five bx insertion mutations examined led to a general reduction in Ubx expression in the anterior compartment; for a given allele, the strength of the adult cuticle phenotype correlated with the degree of Ubx reduction. Deletions mapping near or overlapping the sites of bx insertions, including three abx alleles and the bx34e-prv(bx-prv) allele, showed greatly reduced Ubx expression in parts of the anterior compartment of the haltere disc; however, anterior patches of strong Ubx expression often remained, in highly variable patterns. As expected, the pbx1 mutation led to reduced Ubx expression in the posterior compartment of the haltere disc; surprisingly, pbx1 also led to altered expression of the en protein near the compartment border in the central region of the disc. In the metathoracic leg, all the bx alleles caused extreme reduction in Ubx expression in the anterior regions, with no allele-specific differences. In contrast, abx and bx-prv alleles resulted in patchy anterior reductions in third leg discs. In the larval central nervous system, abx but not bx alleles affected Ubx expression; the bx-prv deletion gave a wild-type phenotype, but it could not fully complement abx mutations. In the posterior wing disc, the bx-prv allele, and to a much lesser extent the bx34e chromosome from which it arose, led to ectopic expression of Ubx. Unlike other grain-of-function mutations in the BX-C, this phenotype appeared to be partially recessive to wild type. Finally, we asked whether the ppx transformation, which results from early lack of Ubx+ function in the mesothorax and is seen in abx animals, is due to ectopic Scr expression. Some mesothoracic leg and wing discs from abx2 larvae displayed ectopic expression of Scr, which was variable in extent but always confined to the posterior compartment.     

The lateral thoracico-abdominal region in adults and fifth instar nymphs of an aquatic bug,Notonecta undulata say (Insecta,Heteroptera)

Skeletal differences in the lateral thoracico-abdominal regions of fifth instar and adult Notonecta appear to reflect respiratory differences in the two stages. Changes in the epidermis of this region during the last instar are described, and the possible relationships between the epidermis, nymphal cuticle, and imaginal cuticle are discussed.Explanation of Figures A Abdominal segment - AC Antecosta (anterior boundary of abdominal segment) - AP Abdominal projection of second abdominal segment - AT Anterior tracheolar branch - B Posteromedial boundary of posterior projection - C Metacoxa - CM Metacoxal membrane - CP Metacoxal process - CS Coxal-subalar muscle - DL Dorsal longitudinal muscle of abdomen - EL External lateral muscle of abdomen - EM Metathoracic epimeron - ES Metathoracic episternum - EV Posterior evagination of metanotum - F Fold of metacoxal membrane - FM Functional thoracico-abdominal membrane - FW Metathoracic wing or wingpad - H Horizontal ridge or sulcus on metathoracic postnotum - HW Metathoracic wing or wingpad - HWM Costal margin of hindwing - ILL Internal lateral muscle of abdomen, lateral portion - ILM Internal lateral muscle of abdomen, medial portion - IN Insertions of wingbase muscles on subalar epidermis - LE Lobe of metathoracic episternum - MM Posterior moulting muscle - MP Process for attachment of Muscle EL 1 - MSP Pleuron of mesothorax - MST Tergum of mesothorax - N Metathoracic notum - P Metathoracic pleural ridge or sulcus - PA Postalar portion of metathorax - PAB Metathoracic postalar bridge - PH Third phragma - PN Metathoracic postnotum - PP Posterior projection of metathoracic epimeron - PT Posterior tracheolar branch - PW Posterior end of metathoracic wingbase - S Abdominal spiracle - SA Subalar portion of metathorax - SAM Subalar membrane - SAS Subalar sclerite - SAV Widened ventrolateral portion of subalar air space - SL Scutellum of metathoracic notum - SM Spiracular membrane - ST Spiracular tracheole - SU Scutum of metathoracic notum - T Tendon of metacoxal remotor muscle - TA Thoracico-abdominal sclerite - TAA Anterior portion of thoracicoabdominal sclerite - TAC Posterior portion of thoraeicoabdominal sclerite - TAM Membranous portion of lateral thoracico-abdominal region - TS Metathoracic spiracle - VA Anterior end of ventral abdominal air channel - WB Junction between subalar wall and metathoracic wing or wingpad - WH Hairs on margin of mesothoracic wingpad     

Interaction of the Stubble-Stubbloid Locus and the Broad-Complex of Drosophila Melanogaster

The 2B5 region on the X chromosome of Drosophila melanogaster forms an early ecdysone puff at the end of the third instar. The region is coextensive with a complex genetic locus, the Broad-Complex (BR-C). The BR-C is a regulatory gene that contains two major functional domains, the br domain and the l(1)2Bc domain. BR-C mutants prevent metamorphosis, including morphogenesis of imaginal discs; br mutants prevent elongation and eversion of appendages and l(1)2Bc mutants prevent fusion of the discs. The Stubble-stubbloid (Sb-sbd) locus at 89B9-10 is best known for the effects of its mutants on bristle structure. Mutants of the BR-C and the Sb-sbd locus interact to produce severe malformation of appendages. Viable heteroallelic and homoallelic combinations of Sb-sbd mutants, including loss-of-function mutants, affect the elongation of imaginal disc appendages. Thus, the Sb-sbd(+) product is essential for normal appendage elongation. Sb-sbd mutants, however, do not affect eversion or fusion of discs. Correspondingly, only BR-C mutants deficient in br function interact with Sb-sbd mutants. The interaction occurs in deficiency heterozygotes using single, wild-type doses of the BR-C, of the Sb-sbd locus, or of both loci. These last results are formally consistent with the possibility that the BR-C acts as a positive regulator of the Sb-sbd locus. The data do not exclude other possible nonregulatory interactions between the two loci, e.g., interactions between the products of both genes.     

The effects of 20-hydroxyecdysone on the metabolic labeling of membrane proteins in Drosophila imaginal discs

20-Hydroxyecdysone induces evagination of imaginal discs of Drosophila melanogaster cultured in vitro. The possible involvement of cell-surface proteins in this process has prompted us to study the synthesis of membrane proteins in imaginal discs. A procedure is reported for the isolation of membrane vesicle fractions from discs that are enriched for the plasma membrane enzyme, Na+/K+-ATPase, and that label with the surface-labeling reagent [125I]iodosulfanilic acid. 20-Hydroxyecdysone alters the pattern of [35S]methionine incorporation into polypeptides in these membrane vesicle fractions. Increased and decreased incorporation as well as changes in migration on two-dimensional gels of specific polypeptides are caused by the hormone. These changes parallel in time the onset and the continuation of evagination.     

Interactions and developmental effects of mutations in the Broad-Complex of Drosophila melanogaster

The 2B5 region on the X chromosome of Drosophila melanogaster forms an early ecdysone puff at the end of the third larval instar. The region contains a complex genetic locus, the Broad-Complex (BR-C) composed of four groups of fully complementing (br, rbp, l(1)2Bc, and l(1)2Bd) alleles, and classes of noncomplementing (npr 1) and partially noncomplementing l(1)2Bab alleles. BR-C mutants prevent metamorphosis, including the morphogenesis of imaginal discs. Results are presented that indicate that the BR-C contains two major functional domains. One, the br domain is primarily, if not exclusively, involved in the elongation and eversion of appendages by imaginal discs. The second, the l(1)2Bc domain, is primarily involved in the fusion of discs to form a continuous adult epidermis. Nonetheless, the two domains may encode products with related functions because in some situations mutants in both domains appear to affect similar developmental processes.     

Drosophila peripodial cells,more than meets the eye?

Drosophila imaginal discs (appendage primordia) have proved invaluable for deciphering cellular and molecular mechanisms of animal development. By combining the accessibility of the discs with the genetic tractability of the fruit fly, researchers have discovered key mechanisms of growth control, pattern formation and long-range signaling. One of the principal experimental attractions of discs is their anatomical simplicity - they have long been considered to be cellular monolayers. During larval stages, however, the growing discs are 2-sided sacs composed of a columnar epithelium on one side and a squamous 'peripodial' epithelium on the other. Recent studies suggest important roles for peripodial epithelia in processes previously assumed to be confined to columnar cell monolayers.