Growth dynamics in the regeneration of a fragment of the wing imaginal disc of Drosophila melanogaster

Regen rating fragments of wing imaginal discs were cultured in vivo for various periods up to 1 week. At specified times the fragments were removed, macerated, and the resulting cell counts were compared to similar counts made on the contralateral intact disc. Significant growth was seen beginning on the second day if the hosts were transferred to fresh media daily, while seen only on Day 4 and not thereafter if hosts were maintained on the same media throughout the culture period.     

Cell proliferation and DNA replication in the imaginal wing disc of Drosophila melanogaster

Cell proliferation in the imaginal wing disc of Drosophila has been analyzed by both pulse and chronic labeling with [3H]thymidine. We find neither spatial nor temporal variation in the fraction of S phase cells during the third instar. At or near the time of white prepupae formation the fraction of S phase cells falls sharply. Our chronic labeling experiments have demonstrated that almost all (and perhaps all) of the cells in a mid third instar wing disc are cycling. By examining sectioned material from such experiments we have found that the collumnar epithelial cell and the adepithetial cell populations become labeled with similar kinetics. The peripodial membrane cell population becomes labeled more slowly. We have also obtained estimates of cell cycle parameters for the imaginal wing disc cells.     

Dpp gradient formation in the Drosophila wing imaginal disc

The secreted signaling protein Dpp acts as a morphogen to pattern the anterior-posterior axis of the Drosophila wing. Dpp activity is required in all cells of the developing wing imaginal disc, but the ligand gradient that supports this activity has not been characterized. Here we make use of a biologically active form of Dpp tagged with GFP to examine the ligand gradient. Dpp-GFP forms an unstable extracellular gradient that spreads rapidly in the wing disc. The activity gradient visualized by MAD phosphorylation differs in shape from the ligand gradient. The pMAD gradient adjusted to compartment size when this was experimentally altered. These observations suggest that the Dpp activity gradient may be shaped at the level of receptor activation.     

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.     

Subdivision of the Drosophila wing imaginal disc by EGFR-mediated signaling

Growth and patterning of the Drosophila wing imaginal disc depends on its subdivision into dorsoventral (DV) compartments and limb (wing) and body wall (notum) primordia. We present evidence that both the DV and wing-notum subdivisions are specified by activation of the Drosophila Epidermal Growth Factor Receptor (EGFR). We show that EGFR signaling is necessary and sufficient to activate apterous (ap) expression, thereby segregating the wing disc into D (ap-ON) and V (ap-OFF) compartments. Similarly, we demonstrate that EGFR signaling directs the expression of Iroquois Complex (Iro-C) genes in prospective notum cells, rendering them distinct from, and immiscible with, neighboring wing cells. However, EGFR signaling acts only early in development to heritably activate ap, whereas it is required persistently during subsequent development to maintain Iro-C gene expression. Hence, as the disc grows, the DV compartment boundary can shift ventrally, beyond the range of the instructive EGFR signal(s), in contrast to the notum-wing boundary, which continues to be defined by EGFR input.     

Absence of distal to proximal intercalary regeneration in imaginal wing discs of Drosophila melanogaster.

The fate of an imaginal disc cell of Drosophila can be affected by the associations and interactions that it has with other cells in the disc. A fragment of an imaginal disc, not regenerating under conditions allowing a complementary fragment to do so, can be stimulated to regenerate by interactions with cells of the complementary fragment [Haynie, J. L., and Bryant, P. J. (1976) Nature (London)259, 659–662]. We report here that one nonregenerating fragment of an imaginal wing disc cannot be stimulated to regenerate by interactions with cells from other parts of the disc. This fragment, containing the anlagen of the distal wing, fails to regenerate proximally when combined with a proximal fragment even though this association stimulates some proximal fragments to regenerate distally. We suggest that this may be a phenomenon similar to that observed in cockroach legs by H. Bohn (1970, Wilhelm Roux Arch. Entwicklungsmech. Organismen165, 303–341), in which proximal regeneration from grafted distal leg segments proceeds only to a limited extent. We consider the possibility that there exist reiterated sets of positional information arranged concentrically in the wing disc.     

Role of Jun N-terminal Kinase (JNK) signaling in the wound healing and regeneration of a Drosophila melanogaster wing imaginal disc

When a fragment of a Drosophila imaginal disc is cultured in growth permissive conditions, it either regenerates the missing structures or duplicates the pattern present in the fragment. This kind of pattern regulation is known to be epimorphic, i.e. the new pattern is generated by proliferation in a specialized tissue called the blastema. Pattern regulation is accompanied by the healing of the cut surfaces restoring the continuous epithelia. Wound healing has been considered to be the inductive signal to commence regenerative cell divisions. Although the general outlines of the proliferation dynamics in a regenerating imaginal disc blastema have been well studied, little is known about the mechanisms driving cells into the regenerative cell cycles. In this study, we have investigated the role of Jun N-terminal Kinase (JNK) signaling in the wound healing and regeneration of a Drosophila wing imaginal disc. By utilizing in vivo and in vitro culturing of incised and fragmented discs, we have been able to visualize the dynamics in cellular architecture and gene expression involved in the healing and regeneration process. Our results directly show that homotypic wound healing is not a prerequisite for regenerative cell divisions. We also show that JNK signaling participates in imaginal disc wound healing and is regulated by the physical dynamics of the process, as well as in recruiting cells into the regenerative cell cycles. A model describing the determination of blastema size is discussed.     

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

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

Developmental compartments in the Drosophila melanogaster wing disc

Distribution of the enzyme aldehyde oxidase (AO) within the pouch of the mature wing disc is precise and differential. General locations of compartmental boundaries have been identified by fate mapping and studies of AO distribution. The suspected locations of the boundaries were verified by analyzing the distribution of AO-negative cells within an AO-stained background in gynandromorphs and in X-ray-induced clones of AO-negative cells. The anterior/posterior border appeared slightly anterior to the junction of the AO⁺ anterior presumptive wing surfaces and AO^? posterior wing surfaces. A narrow band of AO⁺ cells extending proximodistally on both presumptive wing surfaces belongs to the posterior compartment. Two dorsal/ventral (dor./vent.) restrictions were found. The dor./vent. restriction equivalent to the dor./vent. border found in the adult wing was located at the ventral most edge of the AO-stained presumptive wing margin. A second restriction which was less strictly obeyed was found on the dorsal edge of the wing margin. We conclude that the whole presumptive wing margin is part of the dorsal compartment. Within the anterior wing margin an intensively stained oval was also found to be clonally restrictive. Therefore, territories were found within the prospective wing margin for which no such features have been identified in the adult Drosophila melanogaster wing.     

Origin and proliferation of blastema cells during regeneration of Drosophila wing imaginal discs

Following a period of neglect, there has been a resurgence of interest in Drosophila imaginal discs as a model with which to analyze the relationships between growth and pattern formation during regeneration. To broaden our understanding of this process, we used cell lineage techniques to trace the origin of blastema cells and the early and late boundaries of the blastema in regenerating 3/4 wing disc fragments, examined the distribution of S-phase, mitotic and dead cells, and undertook clonal analysis to determine the topology of cell proliferation and its relationship to pattern formation. Using lineage tagging with the JNK phosphatase puckered (puc), we demonstrate that a substantial number of blastema cells arise from cells in which JNK is activated. Furthermore, we show that DNA synthesis and mitosis are activated well before wound healing is completed, in a region where the JNK pathway is activated; later, DNA synthesis and mitosis are observed in scattered cells throughout the blastema. Finally, clonal analysis shows a close relationship between the size and shape of clones and disparities in the positional values of the apposed surfaces.     

Computer simulation of compartment maintenance in the Drosophila wing imaginal disc

A new method for modelling cell division is reported which uses a cellular representation based on graph theory. This allows us to model the adjacencies of non-regular dividing cells accurately, avoiding the rigid geometrical constraints present in earlier simulations. We use this system to simulate compartment boundary maintenance in the Drosophila wing imaginal disc. We show that a boundary of minimum length between two growing polyclones of cells could depend on sorting between cells in the different polyclones. We also investigate the response of the model to differential cell division rates within polyclones. This is the first demonstration that cell sorting can generate a smooth boundary in a dividing cell mass. We suggest that biological analogs of our computer sorting rules are responsible for the similar straight polyclone borders seen in the real wing disc. A possible strategy for showing the existence of these analogs is also given.     

2D gene expression parameters of wing imaginal disc of Drosophila for developmental analysis

 By using high resolution two-dimensional (2D) gel electrophoresis coupled with computer-analysis we have established a quantitative Drosophila wing imaginal disc protein database of third instar larvae as a reference to be used for comparative purposes in genetic studies. A general catalogue integrated by 1,184 ³⁵S-methionine-labelled polypeptides from wing imaginal disc has been obtained. The level of expression for all the proteins has been quantitatively determined. The quantitative reproducibility of the analysis system has been estimated and all the controls studied as database reference to interpret the results of experiments with mutant discs. One example, corresponding to iro ¹ mutation, has been used to show how some of the changes observed with mutant discs clearly extend out of the limits defined by the controls. This enables us to generate comparative parameters for the study of proliferation, morphogenesis and differentiation of Drosophila and opens the possibility of rapidly defining the nature and quantity of changes in patterns of gene expression in developmental genetic studies. Received: 21 June 1996 / Accepted: 27 September 1996     

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.     

Function and regulation of homothorax in the wing imaginal disc of Drosophila

The gene homothorax (hth) is originally expressed uniformly in the wing imaginal disc but, during development, its activity is restricted to the cells that form the thorax and the hinge, where the wing blade attaches to the thorax, and eliminated in the wing pouch, which forms the wing blade. We show that hth repression in the wing pouch is a prerequisite for wing development; forcing hth expression prevents growth of the wing blade. Both the Dpp and the Wg pathways are involved in hth repression. Cells unable to process the Dpp (lacking thick veins or Mothers against Dpp activity) or the Wg (lacking dishevelled function) signal express hth in the wing pouch. We have identified vestigial (vg) as a Wg and Dpp response factor that is involved in hth control. In contrast to its repressing role in the wing pouch, wg upregulates hth expression in the hinge. We have also identified the gene teashirt (tsh) as a positive regulator of hth in the hinge. tsh plays a role specifying hinge structures, possibly in co-operation with hth.     

Overexpression of transglutaminase in the Drosophila wing imaginal disc induced an extra wing crossvein phenotype

To determine the roles of Drosophila transglutaminase-A (dTG-A), we examined a phenotype induced through ectopic expression of dTG-A. Overexpression of dTG-A in the wing imaginal disc induced an extra wing crossvein phenotype. This phenotype was suppressed by crossing with epidermal growth factor receptor (Egfr) signaling pathway mutant flies. These results indicate that this phenotype, induced by dTG-A, is related to enhancement of the Egfr signaling pathway.     

The roles of miRNAs in wing imaginal disc development in Drosophila

During development, it is essential for gene expression to occur in a very precise spatial and temporal manner. There are many levels at which regulation of gene expression can occur, and recent evidence demonstrates the importance of mRNA stability in governing the amount of mRNA that can be translated into functional protein. One of the most important discoveries in this field has been miRNAs (microRNAs) and their function in targeting specific mRNAs for repression. The wing imaginal discs of Drosophila are an excellent model system to study the roles of miRNAs during development and illustrate their importance in gene regulation. This review aims at discussing the developmental processes where control of gene expression by miRNAs is required, together with the known mechanisms of this regulation. These developmental processes include Hox gene regulation, developmental timing, growth control, specification of SOPs (sensory organ precursors) and the regulation of signalling pathways.