The Drosophila segment polarity gene patched interacts with decapentaplegic in wing development.

The decapentaplegic (dpp) gene of Drosophila melanogaster encodes a polypeptide of the transforming growth factor-beta family of secreted factors. It is required for the proper development of both embryonic and adult structures, and may act as a morphogen in the embryo. In wing imaginal discs, dpp is expressed and required in a stripe of cells near the anterior-posterior compartment boundary. Here we show that viable mutations in the segment polarity genes patched (ptc) and costal-2 (cos2) cause specific alterations in dpp expression within the anterior compartment of the wing imaginal disc. The interaction between ptc and dpp is particularly interesting; both genes are expressed with similar patterns at the anterior-posterior compartment boundary of the disc, and mis-expressed in a similar way in segment polarity mutant backgrounds like ptc and cos2. This mis-expression of dpp could be correlated with some of the features of the adult mutant phenotypes. We propose that ptc controls dpp expression in the imaginal discs, and that the restricted expression of dpp near the anterior-posterior compartment boundary is essential to maintain the wild-type morphology of the wing disc.     

Targeted expression of the signaling molecule decapentaplegic induces pattern duplications and growth alterations in Drosophila wings.

In the wing imaginal disc, the decapentaplegic (dpp) gene is expressed in a stripe of anterior cells near the anterior-posterior compartment boundary, and it is required solely in these cells for the entire disc to develop. In some viable segment polarity mutants, alterations in dpp expression have been demonstrated that correlate with changes in wing morphology. To test the hypothesis that the abnormal patterns of dpp expression are responsible directly for the mutant phenotypes, we have expressed dpp in ectopic places in wing imaginal discs, and we have found that dpp is able to cause overgrowth and pattern duplications in both anterior and posterior compartments of the wing disc. The alterations of the anterior compartment are strikingly similar to those observed in some viable segment polarity mutants. Thus, ectopic dpp alone can account for the phenotype of these mutants. We also show that ectopic expression of the segment polarity gene hedgehog (hh) gives similar morphological changes and activates dpp expression in the anterior compartment. This strongly suggests that the organizating activity of hh is mediated by dpp. We propose that the expression of dpp near the anterior-posterior compartment boundary is directed by the interaction between patched and hh, and that dpp itself could act as a general organizer of the patterning in the wing imaginal disc.     

The relationship of decapentaplegic and engrailed expression in Drosophila imaginal disks: do these genes mark the anterior-posterior compartment boundary?

Imaginal disks, the primordia of the adult appendages in Drosophila, are divided into anterior and posterior compartments. However, the developmental role of such compartments remains unclear. The expression of decapentaplegic (dpp), a pattern formation gene required for imaginal disk development, has the intriguing property of being expressed in a line at or near the boundary between these compartments. Here, we compare the distribution of dpp-driven reporter gene expression to the pattern of expression of the engrailed (en) gene, known to be required for the maintenance of the compartment boundary. Using confocal microscopy to obtain single cell resolution, we have determined that the majority of the en+ imaginal disk cells expressing the dpp-driven reporter genes about those cells expressing en, while a small percentage of dpp reporter gene expressing cells also express en. In posterior regions of en mutant disks, where compartmentalization is abnormal, we observe ectopic expression of the dpp-driven reporter genes. We conclude that the pattern of dpp expression in imaginal disks is delimited in part through the direct or indirect repression by engrailed. Our results lead us to question the widely held assumption that the anterior edge of en expression demarcates the A/P compartment boundary.     

Engrailed expression in the anterior lineage compartment of the developing wing blade of Drosophila.

The developing wing of Drosophila melanogaster was examined at larval and pupal stages of development to determine whether the anterior-posterior lineage boundary, as identified by lineage restrictions, was congruent with the boundaries defined by the expression of posterior-specific (engrailed, invected), and anterior-specific (cubitus interruptus-D) genes. The lineage boundary was identified by marking mitotic recombinant clones, using an enhancer trap line with ubiquitous beta-gal expression in imaginal tissues; clones of +/+ cells were identified by their lack of beta-gal expression. Domains of gene expression were localized using antibodies and gene specific lacZ constructs. Surprisingly, it was found that engrailed expression extended a small distance into the anterior lineage compartment of the wing blade, as identified with anti-en/inv mAb, anti-en polyclonal antiserum, or an en-promoter-lacZ insert, ryxho25. This anterior expression was not present in early third instar discs, but appeared during subsequent larval and pupal development. In contrast, the expression of cubitus interruptus-D, as identified using the ci-Dplac insert, appeared to be limited to the anterior lineage compartment. Thus, en expression is not limited to cells from the posterior lineage compartment, and en and ci-D activities can overlap in a region just anterior to the lineage compartment boundary in the developing wing. The lineage boundary could also be identified by a line of aligned cells in the prospective wing blade region of wandering third instar discs. A decapentaplegic-lacZ construct was expressed in a stripe several cells anterior to the lineage boundary, and did not define or overlap into the posterior lineage compartment.     

Drawing a Stripe in Drosophila Imaginal Disks: Negative Regulation of Decapentaplegic and Patched Expression by Engrailed

During development of the Drosophila adult appendage precursors, the larval imaginal disks, the decapentaplegic (dpp) gene is expressed in a stripe just anterior to the anterior/posterior (A/P) compartment boundary. Here, we investigate the genetic controls that lead to production of this stripe. We extend previous observations on leaky engrailed (en) mutations by showing that mutant clones completely lacking both en and invected (inv) activity ectopically express dpp-lacZ reporter genes in the posterior compartment, where dpp activity ordinarily is repressed. Similarly, patched (ptc) is also ectopically expressed in such posterior compartment en(-)inv(-) null clones. In contrast, these en(-)inv(-) clones exhibit loss of hedgehog (hh) expression. We suggest that the absence of dpp expression in the posterior compartment is due to direct repression by en. Ubiquitious expression of en in imaginal disks, produced by a hs-en construct, eliminates the expression of dpp-lacZ in its normal A/P boundary stripe. We identify three in vitro Engrailed binding sites in one of our dpp-lacZ reporter gene. Mutagenesis of these Engrailed binding sites results in ectopic expression of this reporter gene, but does not alter the normal stripe of expression at the A/P boundary. We propose that the en-hh-ptc regulatory loop that is responsible for segmental expression of wingless in the embryo is reutilized in imaginal disks to create a stripe of dpp expression along the A/P compartment boundary.     

The range of spalt-activating Dpp signalling is reduced in endocytosis-defective Drosophila wing discs.

Pattern formation along the anterior-posterior (A/P) axis of the developing Drosophila wing depends on Decapentaplegic (Dpp), a member of the conserved transforming growth factor beta (TGFbeta) family of secreted proteins. Dpp is expressed in a stripe along the A/P compartment boundary of the wing imaginal disc and forms a long-range concentration gradient with morphogen-like properties which generates distinct cell fates along the A/P axis. We have monitored Dpp expression and Dpp signalling in endocytosis-mutant wing imaginal discs which develop severe pattern defects specifically along the A/P wing axis. The results show that the size of the Dpp expression domain is expanded in endocytosis-mutant wing discs. However, this expansion did not result in a concomitant expansion of the functional range of Dpp activity but rather its reduction as indicated by the reduced expression domain of the Dpp target gene spalt. The data suggest that clathrin-mediated endocytosis, a cellular process necessary for membrane recycling and vesicular trafficking, participates in Dpp action during wing development. Genetic interaction studies suggest a link between the Dpp receptors and clathrin. Impaired endocytosis does not interfere with the reception of the Dpp signal or the intracellular processing of the mediation of the signal in the responder cells, but rather affects the secretion and/or the distribution of Dpp in the developing wing cells.     

Context-dependent relationships between the BMPs gbb and dpp during development of the Drosophila wing imaginal disk

The Drosophila BMP5/6/7/8 homolog, glass bottom boat (gbb), has been shown to be involved in proliferation and vein patterning in the wing disk. To better understand the roles for gbb in wing development, as well as its relationship with the Drosophila BMP2/4 homolog decapentaplegic (dpp), we have used clonal analysis to define the functional foci of gbb during wing development. Our results show that gbb has both local and long-range functions in the disk that coincide both spatially and functionally with the established functions of dpp, suggesting that both BMPs contribute to the same processes during wing development. Indeed, comparison of the mutant phenotypes of dpp and gbb hypomorphs and null clones shows that both BMPs act locally along the longitudinal and cross veins to affect the process of vein promotion during pupal development, and long-range from a single focus along the A/P compartment boundary to affect the processes of disk proliferation and vein specification during larval development. Moreover, we show that duplications of dpp are able to rescue many of the phenotypes associated with gbb mutants and clones, indicating that the functions of gbb are at least partially redundant with those of dpp. While this relationship is similar to that described for dpp and the BMP screw (scw) in the embryo, we show that the mechanisms underlying both local and long-range functions of gbb and dpp in the wing are different. For the local foci, gbb function is confined to the regions of the veins that require the highest levels of dpp signaling, suggesting that gbb acts to augment dpp signaling in the same way as scw is proposed to do in the embryo. However, unlike scw-dependent signals in the embryo, these gbb signals are not transduced by the Type I receptor saxophone (sax), thus, the cooperativity between gbb and dpp is not achieved by signaling through distinct receptor complexes. For the long-range focus along the A/P compartment boundary, gbb function does not appear to affect the high point of the dpp gradient, but, rather, appears to be required for low points, which is the reciprocal of the relationship between dpp and scw in the embryo. Moreover, these functions of gbb also do not require the Type I receptor sax. Given these results, we conclude that the relationships between gbb and dpp in the wing disk represent novel paradigms for how multiple BMP ligands signal during development, and that signaling by multiple BMPs involves a variety of different inter-ligand relationships that depend on the developmental context in which they act.     

A dual role for homothorax in inhibiting wing blade development and specifying proximal wing identities in Drosophila

The Drosophila wing imaginal disc gives rise to three body parts along the proximo-distal (P-D) axis: the wing blade, the wing hinge and the mesonotum. Development of the wing blade initiates along part of the dorsal/ventral (D/V) compartment boundary and requires input from both the Notch and wingless (wg) signal transduction pathways. In the wing blade, wg activates the gene vestigial (vg), which is required for the wing blade to grow. wg is also required for hinge development, but wg does not activate vg in the hinge, raising the question of what target genes are activated by wg to generate hinge structures. Here we show that wg activates the gene homothorax (hth) in the hinge and that hth is necessary for hinge development. Further, we demonstrate that hth also limits where along the D/V compartment boundary wing blade development can initiate, thus helping to define the size and position of the wing blade within the disc epithelium. We also show that the gene teashirt (tsh), which is coexpressed with hth throughout most of wing disc development, collaborates with hth to repress vg and block wing blade development. Our results suggest that tsh and hth block wing blade development by repressing some of the activities of the Notch pathway at the D/V compartment boundary.     

Eyeless collaborates with Hedgehog and Decapentaplegic signaling in Drosophila eye induction

eyeless (ey) is a key regulator of the eye development pathway in Drosophila. Ectopic expression of ey can induce the expression of several eye-specification genes (eya, so, and dac) and induce eye formation in multiple locations on the body. However, ey does not induce eye formation everywhere where it is ectopically expressed, suggesting that EY needs to collaborate with additional factors for eye induction. We examined ectopic eye induction by EY in the wing disc and found that eye induction was spatially restricted to the posterior compartment and the anterior-posterior (A/P) compartmental border, suggesting a requirement for both HH and DPP signaling. Although EY in the anterior compartment induced dpp and dac, these were not sufficient for eye induction. Coexpression experiments show that EY needs to collaborate with high level of HH and DPP to induce ectopic eye formation. Ectopic eye formation also requires the activation of an eye-specific enhancer of the endogenous hh gene.     

Notch and Wingless modulate the response of cells to Hedgehog signalling in the Drosophila wing

During Drosophila wing development, Hedgehog (Hh) signalling is required to pattern the imaginal disc epithelium along the anterior-posterior (AP) axis. The Notch (N) and Wingless (Wg) signalling pathways organise the dorsal-ventral (DV) axis, including patterning along the presumptive wing margin. Here, we describe a functional hierarchy of these signalling pathways that highlights the importance of competing influences of Hh, N, and Wg in establishing gene expression domains. Investigation of the modulation of Hh target gene expression along the DV axis of the wing disc revealed that collier/knot (col/kn), patched (ptc), and decapentaplegic (dpp) are repressed at the DV boundary by N signalling. Attenuation of Hh signalling activity caused by loss of fused function results in a striking down-regulation of col, ptc, and engrailed (en) symmetrically about the DV boundary. We show that this down-regulation depends on activity of the canonical Wg signalling pathway. We propose that modulation of the response of cells to Hh along the future proximodistal (PD) axis is necessary for generation of the correctly patterned three-dimensional adult wing. Our findings suggest a paradigm of repression of the Hh response by N and/or Wnt signalling that may be applicable to signal integration in vertebrate appendages.     

Roles for scalloped and vestigial in regulating cell affinity and interactions between the wing blade and the wing hinge

The scalloped and vestigial genes are both required for the formation of the Drosophila wing, and recent studies have indicated that they can function as a heterodimeric complex to regulate the expression of downstream target genes. We have analyzed the consequences of complete loss of scalloped function, ectopic expression of scalloped, and ectopic expression of vestigial on the development of the Drosophila wing imaginal disc. Clones of cells mutant for a strong allele of scalloped fail to proliferate within the wing pouch, but grow normally in the wing hinge and notum. Cells overexpressing scalloped fail to proliferate in both notal and wing-blade regions of the disc, and this overexpression induces apoptotic cell death. Clones of cells overexpressing vestigial grow smaller or larger than control clones, depending upon their distance from the dorsal-ventral compartment boundary. These studies highlight the importance of correct scalloped and vestigial expression levels to normal wing development. Our studies of vestigial-overexpressing clones also reveal two further aspects of wing development. First, in the hinge region vestigial exerts both a local inhibition and a long-range induction of wingless expression. These and other observations imply that vestigial-expressing cells in the wing blade organize the development of surrounding wing-hinge cells. Second, clones of cells overexpressing vestigial exhibit altered cell affinities. Our analysis of these clones, together with studies of scalloped mutant clones, implies that scalloped- and vestigial-dependent cell adhesion contributes to separation of the wing blade from the wing hinge and to a gradient of cell affinities along the dorsal-ventral axis of the wing.     

Expression of Patella vulgata orthologs of engrailed and dpp-BMP2/4 in adjacent domains during molluscan shell development suggests a conserved compartment boundary mechanism

The engrailed gene is well known from its role in segmentation and central nervous system development in a variety of species. In molluscs, however, engrailed is involved in shell formation. So far, it seemed that engrailed had been co-opted uniquely for this particular process in molluscs. Here, we show that, in the gastropod mollusc Patella vulgata, an engrailed ortholog is expressed in the edge of the embryonic shell and in the anlage of the apical sensory organ. Surprisingly, a dpp-BMP2/4 ortholog is expressed in cells of the ectoderm surrounding, but not overlapping, the engrailed-expressing shell-forming cells. It is also expressed in the anlage of the eyes. Earlier it was shown that a compartment boundary exists between the cells of the embryonic shell and the adjacent ectoderm. We conclude that engrailed and dpp are most likely involved in setting up a compartment boundary between these cells, very similar to the situation in, for example, the developing wing imaginal disc in Drosophila. We suggest that engrailed became involved in shell formation because of its ancestral role, which is to set up compartment boundaries between embryonic domains.     

An anterior/posterior communication compartment border in engrailed wing discs: possible implications for Drosophila pattern formation

Our previous studies have suggested that all the known lineage compartment borders in the wing imaginal disc of Drosophila are coincident with boundaries of reduced gap junctional communication (communication compartment borders). Since engrailed discs have a disrupted anterior/posterior (A/P) lineage border (G. Morata and P. A. Lawrence, 1975, Nature (London) 255, 614-617), it was of great interest to determine if their A/P communication restriction boundary is similarly disrupted. Examination of gap-junction-mediated exchange of small fluorescent molecules between cells in the engrailed wing disc revealed a boundary of restricted communication that appeared to be identical to the wild-type A/P communication restriction boundary. This result suggests that lineage compartments are not required for the formation of A/P communication restrictions. Furthermore, we suggest that perhaps communication compartments are the domains within which information is provided for specifying the formation of lineage compartments.     

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.     

msh specifies dorsal cell fate in the Drosophila wing

Drosophila limbs develop from imaginal discs that are subdivided into compartments. Dorsal-ventral subdivision of the wing imaginal disc depends on apterous activity in dorsal cells. Apterous protein is expressed in dorsal cells and is responsible for (1) induction of a signaling center along the dorsal-ventral compartment boundary (2) establishment of a lineage restriction boundary between compartments and (3) specification of dorsal cell fate. Here, we report that the homeobox gene msh (muscle segment homeobox) acts downstream of apterous to confer dorsal identity in wing development.     

Genetic mosaics of the rudimentary locus of Drosophila melanogaster: a genetical investigation into the physiology of pyrimidine synthesis.

Genetic mosaics have been utilized to investigate each of the three pleiotropic effects which result from mutation of the rudimentary locus. The wing abnormality results from a requirement for endogenous pyrimidine synthesis within the wing imaginal disk itself. Mosaic analysis indicates the defect in the wing can be rescued by pyrimidine synthesis any place within the imaginal disk and that no more than 10% of the cells of the disk need be genetically capable of producing pyrimidines. This requirement for endogenous synthesis is probably the result of a reduction or complete elimination of pyrimidine uptake at a particular stage during development. Ovary transplantations indicate that the sterility of homozygous rudimentary females results from a requirement for pyrimidine synthesis within the ovary itself, and mosaic analysis of the auxotrophic phenotype indicates that survival of larvae on pyrimidine-free medium depends on pyrimidine synthesis within a variety of different tissues.