Expansion-repression mechanism for scaling the Dpp activation gradient in Drosophila wing imaginal discs

Maintaining a proportionate body plan requires the adjustment or scaling of organ pattern with organ size. Scaling is a general property of developmental systems, yet little is known about its underlying molecular mechanisms. Using theoretical modeling, we examine how the Dpp activation gradient in the Drosophila wing imaginal disc scales with disc size. We predict that scaling is achieved through an expansion-repression mechanism [1] whose mediator is the widely diffusible protein Pentagone (Pent). Central to this mechanism is the repression of pent expression by Dpp signaling, which provides an effective size measurement, and the Pent-dependent expansion of the Dpp gradient, which adjusts the gradient with tissue size. We validate this mechanism experimentally by demonstrating that scaling requires Pent and further, that scaling is abolished when pent is ubiquitously expressed. The expansion-repression circuit can be readily implemented by a variety of molecular interactions, suggesting its general utilization for scaling morphogen gradients during development.     

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.     

Peripodial cells regulate proliferation and patterning of Drosophila imaginal discs

Cells employ a diverse array of signaling mechanisms to establish spatial patterns during development. Nowhere is this better understood than in Drosophila, where the limbs and eyes arise from discrete epithelial sacs called imaginal discs. Molecular-genetic analyses of pattern formation have generally treated discs as single epithelial sheets. Anatomically, however, discs comprise a columnar cell monolayer covered by a squamous epithelium known as the peripodial membrane. Here we demonstrate that during development, peripodial cells signal to disc columnar cells via microtubule-based apical extensions. Ablation and targeted gene misexpression experiments demonstrate that peripodial cell signaling contributes to growth control and pattern formation in the eye and wing primordia. These findings challenge the traditional view of discs as monolayers and provide foundational evidence for peripodial cell function in Drosophila appendage development.     

Control of growth and patterning of the Drosophila wing imaginal disc by EGFR-mediated signaling

The subdivision of the Drosophila wing imaginal disc into dorsoventral (DV) compartments and limb-body wall (wing-notum) primordia depends on Epidermal Growth Factor Receptor (EGFR) signaling, which heritably activates apterous (ap) in D compartment cells and maintains Iroquois Complex (Iro-C) gene expression in prospective notum cells. We examine the source, identity and mode of action of the EGFR ligand(s) that specify these subdivisions. Of the three known ligands for the Drosophila EGFR, only Vein (Vn), but not Spitz or Gurken, is required for wing disc development. We show that Vn activity is required specifically in the dorsoproximal region of the wing disc for ap and Iro-C gene expression. However, ectopic expression of Vn in other locations does not reorganize ap or Iro-C gene expression. Hence, Vn appears to play a permissive rather than an instructive role in organizing the DV and wing-notum segregations, implying the existance of other localized factors that control where Vn-EGFR signaling is effective. After ap is heritably activated, the level of EGFR activity declines in D compartment cells as they proliferate and move ventrally, away from the source of the instructive ligand. We present evidence that this reduction is necessary for D and V compartment cells to interact along the compartment boundary to induce signals, like Wingless (Wg), which organize the subsequent growth and differentiation of the wing primordium.     

Analysis of cell proliferation in Drosophila wing imaginal discs using mosaic clones

Experimental data on spatial and temporal distributions of mosaic clones in Drosophila wing imaginal disc were analyzed. Long-lived proliferation centers (PR1, PR2, and PR3) and areas with decreased proliferation activity were found in the notum region of the disc. Simulation of the growth kinetics of mosaic patches demonstrated that the cell cycle in proliferation centers PR2 and PR3 was shorter than the average cycle in the disc and in the center PR1. A nonrandom clustering of rapidly dividing cells was observed in the PR2, but not in the other cases. The reason why the cell-cycle duration and the clustering of dividing cells may not coincide is discussed in terms of the recruitment of nondividing cells into the cell cycle. The simulation of the time course of the first and second moments of the size distribution of mosaic clones allowed the variance of cell-cycle progression rates to be determined and demonstrated that a model with a continuous cell-cycle rates gave a better fit to the data than the transition probability model of Smith and Martin.     

Hedgehog creates a gradient of DPP activity in Drosophila wing imaginal discs

Hedgehog (HH) and Decapentaplegic (DPP) direct anteroposterior patterning in the developing Drosophila wing by functioning as short- and long-range morphogens, respectively. Here, we show that the activity of DPP is graded and is directly regulated by a novel HH-dependent mechanism. DPP activity was monitored by visualizing the activated form of Mothers against dpp (MAD), a cytoplasmic transducer of DPP signaling. We found that activated MAD levels are highest near the source of DPP but are unexpectedly low in the cells that express dpp. HH induces dpp in these cells; it also attenuates their response to DPP by downregulating expression of the DPP receptor thick veins (tkv). We suggest that regulation of tkv by HH is a key part of the mechanism that controls the level and distribution of DPP.     

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.     

Ultrastructure of cell death in gamma- or X-irradiated imaginal wing discs of Drosophila

When Drosophila larvae were irradiated with 1300-1500 R of gamma rays both apoptotic and necrotic cell death were observed in imaginal wing discs. The ultrastructure of cell death by apoptosis was characterized by fragmentation of dead cells into highly condensed, membrane-bound particles. The ultrastructure of cell death by necrosis was characterized by cell lysis and organelle degeneration. Marked contrast was also seen in the distribution of the two types of cell death: apoptosis was universal in irradiated discs and affected widely distributed single cells, or small groups of cells, whereas necrosis formed lesions by afflicting large numbers of contiguous cells. It was noted that even where there were large lesions in the epithelial cell layer, which is the primary component of imaginal discs, the basement membrane associated with this epithelium always remained intact. Lesions could be identified in freshly extirpated discs by staining with trypan blue and were found in 50-70% of irradiated discs (depending on the larval age at the time of irradiation). Lesions were seen in all regions of the wing disc and varied greatly in size. In spite of extensive necrotic cell death wing discs developed into normal adult wings. Regenerative growth in this case would appear to require significant reorganization of cells. Implications of this for the appropriate interpretation of clonal analysis are discussed.     

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.     

Control of Drosophila wing growth by the vestigial quadrant enhancer

Following segregation of the Drosophila wing imaginal disc into dorsal (D) and ventral (V) compartments, the wing primordium is specified by activity of the selector gene vestigial (vg). In the accompanying paper, we present evidence that vg expression is itself driven by three distinct inputs: (1) short-range DSL (Delta/Serrate/LAG-2)-Notch signaling across the D-V compartment boundary; (2) long-range Wg signaling from cells abutting the D-V compartment boundary; and (3) a short-range signal sent by vg-expressing cells that entrains neighboring cells to upregulate vg in response to Wg. Furthermore, we showed that these inputs define a feed-forward mechanism of vg autoregulation that initiates in D-V border cells and propagates from cell to cell by reiterative cycles of vg upregulation. Here, we provide evidence that this feed-forward mechanism is required for normal wing growth and is mediated by two distinct enhancers in the vg gene. The first is a newly defined ;priming' enhancer (PE), that provides cryptic, low levels of Vg in most or all cells of the wing disc. The second is the previously defined quadrant enhancer (QE), which we show is activated by the combined action of Wg and the short-range vg-dependent entraining signal, but only if the responding cells are already primed by low-level Vg activity. Thus, entrainment and priming constitute distinct signaling and responding events in the Wg-dependent feed-forward circuit of vg autoregulation mediated by the QE. We posit that Wg controls the expansion of the wing primordium following D-V segregation by fueling this autoregulatory mechanism.     

Control of cuticle formation by wing imaginal discs in vitro.

Wing discs from late final-instar Ephestia larvae form only pupal cuticle when immediately implanted into pupae which subsequently undergo metamorphosis. However, either pupal or adult structures are made in vitro depending on (1) the ecdysterone dose and/or (2) disc cell proliferation. Continuous culture in ecdysterone (0.5–5.0 μg/ml) results in the appearance of transparent cuticle. On the basis of several criteria, this untanned cuticle is postulated to be scaleless adult cuticle. Discs pulsed with 0.5 μg/ml ecdysterone for 48–120 hr, or with 5.0 μg/ml for 24 hr, formed tanned cuticle. Lower doses of ecdysterone (i.e., 0.5 μg/ml for 24 hr or continuous exposure to 0.05 μg/ml) trigger adult scale formation. Enhancement of [³H]thymidine incorporation by these latter doses suggests the occurrence of disc cell divisions and polyploidization. The choice between pupal and adult pathways by wing discs of this age can be controlled exclusively by ecdysterone; juvenile hormone need not be involved in vitro.     

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.     

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.