Model for the regulation of size in the wing imaginal disc of Drosophila

For animal development it is necessary that organs stop growing after they reach a certain size. However, it is still largely unknown how this termination of growth is regulated. The wing imaginal disc of Drosophila serves as a commonly used model system to study the regulation of growth. Paradoxically, it has been observed that growth occurs uniformly throughout the disc, even though Decapentaplegic (Dpp), a key inducer of growth, forms a gradient. Here, we present a model for the control of growth in the wing imaginal disc, which can account for the uniform occurrence and termination of growth. A central feature of the model is that net growth is not only regulated by growth factors, but by mechanical forces as well. According to the model, growth factors like Dpp induce growth in the center of the disc, which subsequently causes a tangential stretching of surrounding peripheral regions. Above a certain threshold, this stretching stimulates growth in these peripheral regions. Since the stretching is not completely compensated for by the induced growth, the peripheral regions will compress the center of the disc, leading to an inhibition of growth in the center. The larger the disc, the stronger this compression becomes and hence the stronger the inhibiting effect. Growth ceases when the growth factors can no longer overcome this inhibition. With numerical simulations we show that the model indeed yields uniform growth. Furthermore, the model can also account for other experimental data on growth in the wing disc.     

The Drosophila gene zfh2 is required to establish proximal-distal domains in the wing disc

Three main events characterize the development of the proximal-distal axis of the Drosophila wing disc: first, generation of nested circular domains defined by different combinations of gene expression; second, activation of wingless (wg) gene expression in a ring of cells; and third, an increase of cell number in each domain in response to Wg. The mechanisms by which these domains of gene expression are established and maintained are unknown. We have analyzed the role of the gene zinc finger homeodomain 2 (zfh2). We report that in discs lacking zfh2 the limits of the expression domains of the genes tsh, nub, rn, dve and nab coincide, and expression of wg in the wing hinge, is lost. We show that zfh2 expression is delimited distally by Vg, Nub and Dpp signalling, and proximally by Tsh and Dpp. Distal repression of zfh2 permits activation of nab in the wing blade and wg in the wing hinge. We suggest that the proximal-most wing fate, the hinge, is specified first and that later repression of zfh2 permits specification of the distal-most fate, the wing blade. We propose that proximal-distal axis development is achieved by a combination of two strategies: on one hand a process involving proximal to distal specification, with the wing hinge specified first followed later by the distal wing blade; on the other hand, early specification of the proximal-distal domains by different combinations of gene expression. The results we present here indicate that Zfh2 plays a critical role in both processes.     

Dpp and Gbb exhibit different effective ranges in the establishment of the BMP activity gradient critical for Drosophila wing patterning

Morphogen gradients ensure the specification of different cell fates by dividing initially unpatterned cellular fields into distinct domains of gene expression. It is becoming clear that such gradients are not always simple concentration gradients of a single morphogen; however, the underlying mechanism of generating an activity gradient is poorly understood. Our data indicate that the relative contributions of two BMP ligands, Gbb and Dpp, to patterning the wing imaginal disc along its A/P axis, change as a function of distance from the ligand source. Gbb acts over a long distance to establish BMP target gene boundaries and a variety of cell fates throughout the wing disc, while Dpp functions at a shorter range. On its own, Dpp is not sufficient to mediate the low-threshold responses at the end points of the activity gradient, a function that Gbb fulfills. Given that both ligands signal through the Tkv type I receptor to activate the same downstream effector, Mad, the difference in their effective ranges must reflect an inherent difference in the ligands themselves, influencing how they interact with other molecules. The existence of related ligands with different functional ranges may represent a conserved mechanism used in different species to generate robust long range activity gradients.     

Spalt major controls the development of the notum and of wing hinge primordia of the Drosophila melanogaster wing imaginal disc

The Drosophila wing and the dorsal thorax develop from primordia within the wing imaginal disc. Here we show that spalt major (salm) is expressed within the presumptive dorsal body wall primordium early in wing disc development to specify notum and wing hinge tissue. Upon ectopic salm expression, dorsally located second leg disc cells develop notum and wing hinge tissue instead of sternopleural tissue. Similarly, by salm over-expression within the wing disc, wing blade formation is suppressed and a mirror-image duplication of the notum and wing hinge is formed. In large dorsal clones, which lack salm and its neighboring paralogue spalt related (salr), the cells of the notum primordium do not grow; these dorsal cells are not specified as notum, hence no notum outgrowth develops. These results suggest that the zinc finger factors encoded by the salm/salr complex play important roles in defining cells of the early wing disc as dorsal body wall cells, which develop into a large dorsal body wall territory and form mesonotum and some wing hinge tissue, and in delimiting the wing primordium. We also find that salm activity is down-regulated by its own product and by that of the Pax gene eyegone.     

Tbx20-related genes, mid and H15, are required for tinman expression, proper patterning, and normal differentiation of cardioblasts in Drosophila

Tbx20-related T-box genes have been implicated in the regulation of heart development in several vertebrate species. In the present report, we demonstrate that a pair of genes representing Drosophila orthologs of Tbx20, midline (mid) and H15, have important functions during the development of the Drosophila equivalent of the heart, i.e. the dorsal vessel. We show that mid is among the earliest known genes that are specifically expressed in all cardioblasts during early embryogenesis, and H15 expression is subsequently activated in the same cells. Mutant embryos lacking the activity of mid, or both mid and H15, are able to form dorsal vessels with largely normal numbers of cardioblasts and pericardial cells. Furthermore, the mutant cardioblasts express several general cardioblast markers such as Mef2 and Toll at normal levels. However, the expression of tinman (tin), which normally occurs in four out of six cardioblasts in each hemisegment of the dorsal vessel, is almost abolished. Conversely, the expression of the Dorsocross (Doc) T-box genes, which is normally restricted to the two Tin-negative cardioblasts in each hemisegment, is strongly expanded into the majority of cardioblasts in mid mutant and mid+H15-deficient embryos. Altogether, the data from the loss-of-function phenotypes demonstrate that mid, and to a lesser degree H15, have important roles in establishing the metameric patterning of cardioblast identities, but not in specifying cardioblasts as such. Ectopic expression of mid causes ectopic tin expression and, less efficiently, produces extra cardioblasts. We propose that one of the major functions of mid and H15 during cardioblast development is the re-activation of tin expression at a stage when the induction of tin by Dpp in the dorsal mesoderm has ceased. Through this activity, mid and H15 are required for the normal functional diversification of cardioblasts and the expression of tin-dependent terminal differentiation genes within the dorsal vessel.     

Hedgehog and Dpp signaling induce cadherin Cad86C expression in the morphogenetic furrow during Drosophila eye development

During Drosophila eye development, cell differentiation is preceded by the formation of a morphogenetic furrow, which progresses across the epithelium from posterior to anterior. Cells within the morphogenetic furrow are apically constricted and shortened along their apical-basal axis. However, how these cell shape changes and, thus, the progression of the morphogenetic furrow are controlled is not well understood. Here we show that cells simultaneously lacking Hedgehog and Dpp signal transduction fail to shorten and do not enter the morphogenetic furrow. Moreover, we have identified a gene, cadherin Cad86C, which is highly expressed in cells of the leading flank of the morphogenetic furrow. Ectopic activation of either the Hedgehog or Dpp signal transduction pathway results in elevated Cad86C expression. Conversely, simultaneous loss of both Hedgehog and Dpp signal transduction leads to decreased Cad86C expression. Finally, ectopic expression of Cad86C in either eye-antennal imaginal discs or wing imaginal discs results in apical constriction and shortening of cells. We conclude that Hedgehog and Dpp signaling promote the shortening of cells within the morphogenetic furrow. Induction of Cad86C expression might be one mechanism through which Hedgehog and Dpp promote these cell shape changes.     

An endoderm-specific GATA factor gene, dGATAe, is required for the terminal differentiation of the Drosophila endoderm

GATA factors play an essential role in endodermal specification in both protostomes and deuterostomes. In Drosophila, the GATA factor gene serpent (srp) is critical for differentiation of the endoderm. However, the expression of srp disappears around stage 11, which is much earlier than overt differentiation occurs in the midgut, an entirely endodermal organ. We have identified another endoderm-specific Drosophila GATA factor gene, dGATAe. Expression of dGATAe is first detected at stage 8 in the endoderm, and its expression continues in the endodermal midgut throughout the life cycle. srp is required for expression of dGATAe, and misexpression of srp resulted in ectopic dGATAe expression. Embryos that either lacked dGATAe or were injected with double-stranded RNA (dsRNA) corresponding to dGATAe failed to express marker genes that are characteristic of differentiated midgut. Conversely, overexpression of dGATAe induced ectopic expression of endodermal markers even in the absence of srp activity. Transfection of the dGATAe cDNA also induced endodermal markers in Drosophila S2 cells. These studies provide an outline of the genetic pathway that establishes the endoderm in Drosophila. This pathway is triggered by sequential signaling through the maternal torso gene, a terminal gap gene, huckebein (hkb), and finally, two GATA factor genes, srp and dGATAe.     

The egghead gene is required for compartmentalization in Drosophila optic lobe development

The correct targeting of photoreceptor neurons (R-cells) in the developing Drosophila visual system requires multiple guidance systems in the eye-brain complex as well as the precise organization of the target area. Here, we report that the egghead (egh) gene, encoding a glycosyltransferase, is required for a compartment boundary between lamina glia and lobula cortex, which is necessary for appropriate R1-R6 innervation of the lamina. In the absence of egh, R1-R6 axons form a disorganized lamina plexus and some R1-R6 axons project abnormally to the medulla instead of the lamina. Mosaic analysis demonstrates that this is not due to a loss of egh function in the eye or in the neurons and glia of the lamina. Rather, as indicated by clonal analysis and cell-specific genetic rescue experiments, egh is required in cells of the lobula complex primordium which transiently abuts the lamina and medulla in the developing larval brain. In the absence of egh, perturbation of sheath-like glial processes occurs at the boundary region delimiting lamina glia and lobula cortex, and inappropriate invasion of lobula cortex cells across this boundary region disrupts the pattern of lamina glia resulting in inappropriate R1-R6 innervation. This finding underscores the importance of the lamina/lobula compartment boundary in R1-R6 axon targeting.     

The tumour suppressor gene l(2)giant discs is required to restrict the activity of Notch to the dorsoventral boundary during Drosophila wing development

During the development of the Drosophila wing, the activity of the Notch signalling pathway is required to establish and maintain the organizing activity at the dorsoventral boundary (D/V boundary). At early stages, the activity of the pathway is restricted to a small stripe straddling the D/V boundary, and the establishment of this activity domain requires the secreted molecule fringe (fng). The activity domain will be established symmetrically at each side of the boundary of Fng-expressing and non-expressing cells. Here, I present evidence that the Drosophila tumour-suppressor gene lethal (2) gaint discs (lgd) is required to restrict the activity of Notch to the D/V boundary. In the absence of lgd function, the activity of Notch expands from its initial domain at the D/V boundary. This expansion requires the presence of at least one of the Notch ligands, which can activate Notch more efficiently in the mutants. The results further suggest that Lgd appears to act as a general repressor of Notch activity, because it also affects vein, eye, and bristle development.     

wing blister, a new Drosophila laminin alpha chain required for cell adhesion and migration during embryonic and imaginal development

We report the molecular and functional characterization of a new alpha chain of laminin in Drosophila. The new laminin chain appears to be the Drosophila counterpart of both vertebrate alpha2 (also called merosin) and alpha1 chains, with a slightly higher degree of homology to alpha2, suggesting that this chain is an ancestral version of both alpha1 and alpha2 chains. During embryogenesis, the protein is associated with basement membranes of the digestive system and muscle attachment sites, and during larval stage it is found in a specific pattern in wing and eye discs. The gene is assigned to a locus called wing blister (wb), which is essential for embryonic viability. Embryonic phenotypes include twisted germbands and fewer pericardial cells, resulting in gaps in the presumptive heart and tracheal trunks, and myotubes detached from their target muscle attachment sites. Most phenotypes are in common with those observed in Drosophila laminin alpha3, 5 mutant embryos and many are in common with those observed in integrin mutations. Adult phenotypes show blisters in the wings in viable allelic combinations, similar to phenotypes observed in integrin genes. Mutation analysis in the eye demonstrates a function in rhabdomere organization. In summary, this new laminin alpha chain is essential for embryonic viability and is involved in processes requiring cell migration and cell adhesion.     

The detached locus encodes Drosophila Dystrophin, which acts with other components of the Dystrophin Associated Protein Complex to influence intercellular signalling in developing wing veins

Dystrophin and Dystroglycan are the two central components of the multimeric Dystrophin Associated Protein Complex, or DAPC, that is thought to provide a mechanical link between the extracellular matrix and the actin cytoskeleton, disruption of which leads to muscular dystrophy in humans. We present the characterization of the Drosophila ‘crossveinless’ mutation detached (det), and show that the gene encodes the fly ortholog of Dystrophin. Our genetic analysis shows that, in flies, Dystrophin is a non-essential gene, and the sole overt morphological defect associated with null mutations in the locus is the variable loss of the posterior crossvein that has been described for alleles of det. Null mutations in Drosophila Dystroglycan (Dg) are similarly viable and exhibit this crossvein defect, indicating that both of the central DAPC components have been co-opted for this atypical function of the complex. In the developing wing, the Drosophila DAPC affects the intercellular signalling pathways involved in vein specification. In det and Dg mutant wings, the early BMP signalling that initiates crossvein specification is not maintained, particularly in the pro-vein territories adjacent to the longitudinal veins, and this results in the production of a crossvein fragment in the intervein between the two longitudinal veins. Genetic interaction studies suggest that the DAPC may exert this effect indirectly by down-regulating Notch signalling in pro-vein territories, leading to enhanced BMP signalling in the intervein by diffusion of BMP ligands from the longitudinal veins.     

Tissue remodeling during maturation of the Drosophila wing

The final step in morphogenesis of the adult fly is wing maturation, a process not well understood at the cellular level due to the impermeable and refractive nature of cuticle synthesized some 30 h prior to eclosion from the pupal case. Advances in GFP technology now make it possible to visualize cells using fluorescence after cuticle synthesis is complete. We find that, between eclosion and wing expansion, the epithelia within the folded wing begin to delaminate from the cuticle and that delamination is complete when the wing has fully expanded. After expansion, epithelial cells lose contact with each other, adherens junctions are disrupted, and nuclei become pycnotic. The cells then change shape, elongate, and migrate from the wing into the thorax. During wing maturation, the Timp gene product, tissue inhibitor of metalloproteinases, and probably other components of an extracellular matrix are expressed that bond the dorsal and ventral cuticular surfaces of the wing following migration of the cells. These steps are dissected using the batone and Timp genes and ectopic expression of alphaPS integrin, inhibitors of Armadillo/beta-catenin nuclear activity and baculovirus caspase inhibitor p35. We conclude that an epithelial-mesenchymal transition is responsible for epithelial delamination and dissolution.