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.     

Wnt/planar cell polarity signaling: an important mechanism to coordinate growth and patterning in the limb

The limb is one of the premier models for studying how a simple embryonic anlage develops into complex three-dimensional form. One of the key issues in the limb field has been to determine how the limb becomes patterned along its proximal (shoulder/hip) to distal (digits) axis. For decades it has been known that the apical ectodermal ridge (AER) plays a crucial role in distal outgrowth and patterning of the vertebrate embryonic limb. Most studies have explored the relationship between the AER and the progressive assignment of cell fates to mesenchyme along the proximal to distal (PD) axis. Comparatively few, however, have examined the additional role of the AER to regulate distal outgrowth of the limb and how this growth may also influence pattern along the PD axis. Here, I will review key studies that explore the role of growth in limb development. In particular, I will focus on a recent flurry of papers that examine the role of the Wnt/planar cell polarity (PCP) pathway in regulating directed growth of the limb mesenchyme. Finally, I will discuss a potential mechanism that relates the AER to the Wnt/PCP pathway and how directed growth can play a role in shaping the limb along the PD axis.     

Separable functions of wingless in distal and ventral patterning of the Tribolium leg

The gene wingless (wg) in Drosophila is an important factor in leg development. During embryonic development wg is involved in the allocation of the limb primordia. During imaginal disk development wg is involved in distal development and it has a separate role in ventral development. The expression pattern of wg is highly conserved in all arthropods (comprising data from insects, myriapods, crustaceans, and chelicerates), suggesting that its function in leg development is also conserved. However, recent work in other insects (e.g. the milkweed bug Oncopeltus fasciatus) argued against a role of wg in leg development. We have studied the role of wg in leg development of the flour beetle Tribolium castaneum. Using stage-specific staggered embryonic RNAi in wild-type and transgenic EGFP expressing enhancer trap lines we are able to demonstrate separable functions of Tribolium wg in distal and in ventral leg development. The distal role affects all podomeres distal to the coxa, whereas the ventral role is restricted to cells along the ventral midline of the legs. In addition, severe leg defects after injection into early embryonic stages are evidence that wg is also involved in proximal development and limb allocation in Tribolium. Our data suggest that the roles of wg in leg development are highly conserved in the holometabolous insects. Further studies will reveal the degree of conservation in other arthropod groups.     

A role for wingless in an early pupal cell death event that contributes to patterning the Drosophila eye

Programmed cell death (PCD) is utilized in a wide variety of tissues to refine structure in developing tissues and organs. However, little is understood about the mechanisms that, within a developing epithelium, combine signals to selectively remove some cells while sparing essential neighbors. One popular system for studying this question is the developing Drosophila pupal retina, where excess interommatidial support cells are removed to refine the patterned ommatidial array. In this paper, we present data indicating that PCD occurs earlier within the pupal retina than previously demonstrated. As with later PCD, this death is dependent on Notch activity. Surprisingly, altering Drosophila Epidermal Growth Factor Receptor or Ras pathway activity had no effect on this death. Instead, our evidence indicates a role for Wingless signaling to provoke this cell death. Together, these signals regulate an intermediate step in the selective removal of unneeded interommatidial cells that is necessary for a precise retinal pattern.     

Molecular patterning mechanism underlying metamorphosis of the thoracic leg in Manduca sexta

The tobacco hornworm Manduca sexta, like many holometabolous insects, makes two versions of its thoracic legs. The simple legs of the larva are formed during embryogenesis, but then are transformed into the more complex adult legs at metamorphosis. To elucidate the molecular patterning mechanism underlying this biphasic development, we examined the expression patterns of five genes known to be involved in patterning the proximal-distal axis in insect legs. In the developing larval leg of Manduca, the early patterning genes Distal-less and Extradenticle are already expressed in patterns comparable to the adult legs of other insects. In contrast, Bric-a-brac and dachshund are expressed in patterns similar to transient patterns observed during early stages of leg development in Drosophila. During metamorphosis of the leg, the two genes finally develop mature expression patterns. Our results are consistent with the hypothesis that the larval leg morphology is produced by a transient arrest in the conserved adult leg patterning process in insects. In addition, we find that, during the adult leg development, some cells in the leg express the patterning genes de novo suggesting that the remodeling of the leg involves changes in the patterning gene regulation.     

The role of Wg signaling in the patterning of embryonic leg primordium in Drosophila

Cellular interaction between the proximal and distal domains of the limb plays key roles in proximal-distal patterning. In Drosophila, these domains are established in the embryonic leg imaginal disc as a proximal domain expressing escargot, surrounding the Distal-less expressing distal domain in a circular pattern. The leg imaginal disc is derived from the limb primordium that also gives rise to the wing imaginal disc. We describe here essential roles of Wingless in patterning the leg imaginal disc. Firstly, Wingless signaling is essential for the recruitment of dorsal-proximal, distal, and ventral-proximal leg cells. Wingless requirement in the proximal leg domain appears to be unique to the embryo, since it was previously shown that Wingless signal transduction is not active in the proximal leg domain in larvae. Secondly, downregulation of Wingless signaling in wing disc is essential for its development, suggesting that Wg activity must be downregulated to separate wing and leg discs. In addition, we provide evidence that Dll restricts expression of a proximal leg-specific gene expression. We propose that those embryo-specific functions of Wingless signaling reflect its multiple roles in restricting competence of ectodermal cells to adopt the fate of thoracic appendages.     

A dual function of V0-ATPase a1 provides an endolysosomal degradation mechanism in Drosophila melanogaster photoreceptors

The vesicular adenosine triphosphatase (v-ATPase) is a proton pump that acidifies intracellular compartments. In addition, mutations in components of the membrane-bound v-ATPase V0 sector cause acidification-independent defects in yeast, worm, fly, zebrafish, and mouse. In this study, we present a dual function for the neuron-specific V0 subunit a1 orthologue v100 in Drosophila melanogaster. A v100 mutant that selectively disrupts proton translocation rescues a previously characterized synaptic vesicle fusion defect and vesicle fusion with early endosomes. Correspondingly, V100 selectively interacts with syntaxins on the respective target membranes, and neither synaptic vesicles nor early endosomes require v100 for their acidification. In contrast, V100 is required for acidification once endosomes mature into degradative compartments. As a consequence of the complete loss of this neuronal degradation mechanism, photoreceptors undergo slow neurodegeneration, whereas selective rescue of the acidification-independent function accelerates cell death by increasing accumulations in degradation-incompetent compartments. We propose that V100 exerts a temporally integrated dual function that increases neuronal degradative capacity.     

Role of programmed cell death in patterning the Drosophila antennal arista

Programmed cell death is a critical process for the patterning and sculpting of organs during development. The Drosophila arista, a feather-like structure at the tip of the antenna, is composed of a central core and several lateral branches. A homozygous viable mutation in the thread gene, which encodes an inhibitor of apoptosis protein, produces a branchless arista. We have found that mutations in the proapoptotic gene hid lead to numerous extra branches, suggesting that the level of cell death determines the number of branches in the arista. Consistent with this idea, we have found that thread mutants show excessive cell death restricted to the antennal imaginal disc during the middle third instar larval stage. These findings point to a narrow window of development in which regulation of programmed cell death is essential to the proper formation of the arista.     

The recombinant limb as a model for the study of limb patterning, and its application to muscle development

The recombinant limb is a model system that has proved fruitful for analyzing epithelial-mesenchymal interactions and understanding the functional properties of the components of the limb bud. Here we present an overview of some of the insights obtained through the use of this technique. Among these are the understanding that fore or hind limb identity is inherent to the limb bud mesoderm, that the apical ectodermal ridge (AER) is a permissive signaling center and that the limb bud ectoderm plays a central role in the control of dorsoventral polarity. Recombinant limb studies have also allowed the identification of the affected tissue component in several limb mutants. More recently this model has been applied to the study of regulation of gene expressions related to patterning. In this report we use recombinant limbs to analyze pattering of the Pax3 expressing limb muscle cell lineage in the early stages of limb development. In recombinant limbs made without the zone of polarizing activity (ZPA), myoblasts appear intermingled with other mesodermal cells at the beginning of the recombinant limb development. Rapidly thereafter, the muscle precursors segregate and organize around the central forming chondrogenic core of the recombinant. Although this segregation is reminiscent of that occurring during normal development, the myoblasts in the recombinant fail to proliferate appropriately and also fail to migrate distally. Consequently, the muscle pattern in the recombinant limb is defective indicating that normal patterning cues are absent. However, recombinant limbs polarized with a ZPA exhibited a larger mass of muscle cells and a more normal morphogenesis, supporting a role for this signaling center in limb muscle development. Finally, we have ruled out host somite contributions to recombinant limbs by grafting chick recombinant limbs to quail hosts. This initial report demonstrates the value of the recombinant limb model system for dissecting the environmental cues required for normal muscle limb patterning. Received: 31 August 1998 / Accepted: 29 September 1998     

Molecular organization and embryonic expression of the hedgehog gene involved in cell-cell communication in segmental patterning of Drosophila.

hedgehog is a segment polarity gene necessary to maintain the proper organization of each segment of the Drosophila embryo. We have identified the physical location of a number of rearrangement breakpoints associated with hedgehog mutations. The corresponding hh RNA is expressed in a series of segmental stripes starting at cellular blastoderm in the posterior portion of each segment. This RNA is localized predominantly within nuclei until stage 10, when the localization becomes primarily cytoplasmic. Expression of hh RNA in the posterior compartment is independent of most other segment polarity genes, including en, until the late extended germ-band stage (stage 11). Sequence analysis of the hedgehog locus suggests the protein product is a transmembrane protein, which may, therefore, be directly involved in cell-cell communication.     

Apical accumulation of the Drosophila PDGF/VEGF receptor ligands provides a mechanism for triggering localized actin polymerization

Epithelial tissue functions depend largely on a polarized organization of the individual cells. We examined the roles of the Drosophila PDGF/VEGF receptor (PVR) in polarized epithelial cells, with specific emphasis on the wing disc epithelium. Although the receptor is broadly distributed in this tissue, two of its ligands, PVF1 and PVF3 are specifically deposited within the apical extracellular space, implying that polarized apical activation of the receptor takes place. The apical localization of the ligands involves a specialized secretion pathway. Clones for null alleles of Pvr or expression of RNAi constructs showed no phenotypes in the wing disc or pupal wing, suggesting that Pvr plays a redundant role in this tissue. However, when uniform expression of a constitutively dimerizing receptor was induced, loss of epithelial polarity, formation of multiple adherens and septate junctions, and tumorous growth were observed in the wing disc. Elevation of the level of full-length PVR also gave rise to prominent phenotypes, characterized by higher levels of actin microfilaments at the basolateral areas of the cells and irregular folding of the tissue. Together, these results suggest that polarized PVR activation is necessary for the proper organization of the wing disc epithelium, by regulating the apical assembly of the actin cytoskeleton.