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.     

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.     

Fate map of the eye-antennal imaginal disc of the tumorous-head mutant of Drosophila melanogaster

In specific genetic backgrounds, a mutation in the tuh-3 gene results in the homeotic transformation of head structures to either leg disc derivatives or structures normally found in the extreme posterior end of wild-type animals. The origins of the homeotic structures were mapped to defined positions in the eye-antennal imaginal disc by transplanting abnormal regions of discs isolated from tuh-3 mutants into host mwh;e4 larvae. These metamorphosed implants were removed and differentiated structures were identified. Of 211 successfully recovered implants, 157 gave rise to homeotic tissue: abdominal tergite, male or female external genitalia and/or leg tissue. Transformations to abdominal tergite occurred primarily in cells taken from the eye region of the compound disc. Male and female genitalia arose most often in implants taken from the antennal portion of the disc, although some tissue taken from the lateral region of the eye disc also gave rise to external genitalia. Leg structures came exclusively from implants from the antennal region of the imaginal disc. These results suggest that cells from within specific regions of the eye-antennal compound disc are constrained in their developmental potential. An obvious constraint observed with this mutation is a dorsal/ventral one: Cells from the eye disc, a dorsal structure, primarily gave rise to other dorsal structures, abdominal tergite tissue. Cells from the antennal disc, a ventrally derived structure, primarily gave rise to other ventral structures including genital tissue and distal leg.     

The three leg imaginal discs of Drosophila: "Vive la différence"

The imaginal discs of Drosophila are the larval primordia for the adult cuticular structures of the adult fly. Fate maps of different discs have been generated that show the localization of prospective adult structures. Even though the three legs differ in their morphology, only the fate map for the T1 (prothoracic) leg disc has been generated. Here we present fate maps for the T2 (meso-) and T3 (metathoracic) leg discs. We show that there are many similarities to the map of the T1 leg disc. However, there are also significant differences in the contributions of each disc to the thorax, in the morphology of joints connecting the legs to the thorax, in bristle patterns, and in the positioning of some sensory organs. We also tested the developmental potential of disc fragments and observed that T2 and T3 leg discs have more limited plasticity and are unable to transdetermine. The differences in the cuticle patterns between legs are robust and conserved in many species of dipterans. While most previous analyses of imaginal disc development have not distinguished between the different leg discs, we believe that the underlying differences of the three leg discs demonstrated here cannot be ignored when studying leg disc development.     

New Components of Drosophila Leg Development Identified through Genome Wide Association Studies

The adult Drosophila melanogaster body develops from imaginal discs, groups of cells set-aside during embryogenesis and expanded in number during larval stages. Specification and development of Drosophila imaginal discs have been studied for many years as models of morphogenesis. These studies are often based on mutations with large developmental effects, mutations that are often lethal in embryos when homozygous. Such forward genetic screens can be limited by factors such as early lethality and genetic redundancy. To identify additional genes and genetic pathways involved in leg imaginal disc development, we employed a Genome Wide Association Study utilizing the natural genetic variation in leg proportionality found in the Drosophila Genetic Reference Panel fly lines. In addition to identifying genes already known to be involved in leg development, we identified several genes involved in pathways that had not previously been linked with leg development. Several of the genes appear to be involved in signaling activities, while others have no known roles at this time. Many of these uncharacterized genes are conserved in mammals, so we can now begin to place these genes into developmental contexts. Interestingly, we identified five genes which, when their function is reduced by RNAi, cause an antenna-to-leg transformation. Our results demonstrate the utility of this approach, integrating the tools of quantitative and molecular genetics to study developmental processes, and provide new insights into the pathways and networks involved in Drosophila leg development.     

Tissue-, age-, and stage-specific patterns of protein synthesis during the development of Drosophila melanogaster.

Patterns of polypeptide synthesis in the imaginal and larval tissues of wild-type larvae of different ages have been examined using a one-dimensional polyacrylamide gel electrophoresis and autofluorography. Tissue-, age, and stage-specific patterns of polypeptide bands have been observed and characterized. There are one or two bands unique to larval tissues and 13 bands unique to imaginal discs and brain. Each tissue has its own characteristic band pattern. The 160-hr wing disc contains all 13 disc and brain-specific bands plus another eight unique bands. The 160-hr leg disc contains 11 disc- and brain-specific bands plus another six unique bands. The 160-hr eye-antennal disc contains eight disc- and brain-specific bands. There are five bands common to all imaginal tissues at all times but not found in any of the larval tissues. There appears to be a much greater difference between the same tissue at different ages than there is between different tissues at the same age. There is also a marked change in the total body proteins extracted from different developmental stages. Eight (11%) of the bands detected appear to be common to all tissues at all times, while 13 bands (18%) appear to be specific to pairs of imaginal discs. In addition, there are major differences in the patterns observed between pulsed and pulse-chased animals. These results are discussed in relation to the concept of differential gene activity.     

The development of the sensory neuron pattern in the antennal disc of wild-type and mutant (lz3, ssa) Drosophila melanogaster

The development of the sensory neuron pattern in the antennal disc of Drosophila melanogaster was studied with a neuron-specific monoclonal antibody (22C10). In the wild type, the earliest neurons become visible 3 h after pupariation, much later than in other imaginal discs. They lie in the center of the disc and correspond to the neurons of the adult aristal sensillum. Their axons join the larval antennal nerve and seem to establish the first connection towards the brain. Later on, three clusters of neurons appear in the periphery of the disc. Two of them most likely give rise to the Johnston's organ in the second antennal segment. Neurons of the olfactory third antennal segment are formed only after eversion of the antennal disc (clusters t1-t3). The adult pattern of antennal neurons is established at about 27% of metamorphosis. In the mutant lozenge3 (lz3), which lacks basiconic antennal sensilla, cluster t3 fails to develop. This indicates that, in the wild type, a homogeneous group of basiconic sensilla is formed by cluster t3. The possible role of the lozenge gene in sensillar determination is discussed. The homeotic mutant spineless-aristapedia (ssa) transforms the arista into a leg-like tarsus. Unlike leg discs, neurons are missing in the larval antennal disc of ssa. However, the first neurons differentiate earlier than in normal antennal discs. Despite these changes, the pattern of afferents in the ectopic tarsus appears leg specific, whereas in the non-transformed antennal segments a normal antennal pattern is formed. This suggests that neither larval leg neurons nor early aristal neurons are essential for the outgrowth of subsequent afferents.     

Homologies of positional information in thoracic imaginal discs ofDrosophila melanogaster

Summary The regulative behavior of fragments of the imaginal discs of the wing and first leg was studied when these fragments were combined with fragments of other thoracic imaginal discs. A fragment of the wing disc which does not normally regenerate when cultured could be stimulated to regenerate by combination with certain fragments of the haltere disc. When combined with a haltere disc fragment thought to be homologous by the criteria of morphology and the pattern of homoeotic transformation, such stimulated intercalary regeneration was not observed. Combinations of first and second leg disc fragments showed that a lateral first leg fragment could be stimulated to regenerate medial structures when combined with a medial second leg disc fragment but not when combined with a lateral second leg disc fragment. Combinations of wing and second leg disc fragments showed that one fragment of the second leg disc is capable of stimulating regeneration from a wing disc fragment while another second leg disc fragment fails to stimulate such regeneration. It is suggested that absence of intercalary regeneration in combinations of fragments of different thoracic imaginal discs is a result of homology or identity of the positional information residing in the cells of the fragments. The pattern of correspondence of positional information revealed by this analysis is consistant with the pattern of homology determined by morphological observation and by analysis of the positional specificity of homoeotic transformation among serially homologous appendages. The implications of the existence of homologous positional information in wing and second leg discs which share a common cell lineage early in development are discussed.     

Lineage-tracing cells born in different domains along the PD axis of the developing Drosophila leg.

Patterning of the developing limbs by the secreted signaling proteins Wingless, Hedgehog and Dpp takes place while the imaginal discs are growing rapidly. Cells born in regions of high ligand concentration may be displaced through growth to regions of lower ligand concentration. We have used a novel lineage-tagging method to address the reversibility of cell fate specification by morphogen gradients. We find that responses to Hedgehog and Dpp in the wing disc are readily reversible. In the leg, we find that cells readily adopt more distal fates, but do not normally shift from distal to proximal fate. However, they can do so if given a growth advantage. These results indicate that cell fate specification by morphogen gradients remains largely reversible while the imaginal discs grow. In other systems, where growth and patterning are uncoupled, nonreversible specification events or 'ratchet' effects may be of functional significance.     

Transdetermination in Drosophila imaginal discs: a model for understanding pluripotency and selector gene maintenance

Drosophila imaginal disc cells have the ability to undergo transdetermination, a process whereby determined disc cells change fate to that of another disc identity. For example, leg disc cells can transdetermine to develop as wing cells. Such events can occur after mechanical disc fragmentation and subsequent regeneration. A subset of transdetermination events can be induced in situ by misexpression of the signaling gene wingless. Both fragmentation and wingless induce transdetermination by altering the expression of selector genes, which drive disc-specific developmental programs. An important future goal is to address how signaling pathways interact with chromatin structure to regulate and maintain the proper expression of selector genes.     

Transdetermination: Drosophila imaginal disc cells exhibit stem cell-like potency

Drosophila imaginal discs, the primordia of the adult fly appendages, are an excellent system for studying developmental plasticity. Cells in the imaginal discs are determined for their disc-specific fate (wingness, legness) during embryogenesis. Disc cells maintain their determination during larval development, a time of extensive growth and proliferation. Only when prompted to regenerate do disc cells exhibit lability in their determined identity. Regeneration in the disc is mediated by a localized region of cell division, known as the regeneration blastema. Most regenerating disc cells strictly adhere to their disc-specific identity; some cells however, switch fate in a phenomenon known as transdetermination. Similar regeneration and transdetermination events can be induced in situ by misexpression of the signaling molecule wingless. Recent studies indicate that the plasticity of disc cells during regeneration is associated with high morphogen activity and the reorganization of chromatin structure. Here we provide both a historical perspective of imaginal disc transdetermination, as well as discuss recent findings on how imaginal disc cells acquire developmental plasticity and multipotency. We also highlight how an understanding of imaginal disc transdetermination can enhance an understanding of developmental potency exhibited by stem cells.     

Taste sensitivity in homeotic mutants of the drosophila leg-arista-wing complex

It is well established that taste chemoreceptors in Drosophila are located on the tarsi of the first leg pair. In order to investigate the influence of the novel homeotic arista-tarsus transformation on behavior, an analysis of taste perception in the lawc-mutants, characterized by the transformation of the arista into tarsus elements, was carried out. The data were subjected to thorough statistical treatment. It was shown that elements of an additional leg that appeared as a result of homeotic transformation of the arista were sensitive to gustatory stimuli. Analysis of the innervation of the homeotic organs by means of cobalt staining of afferent projections showed that the afferents starting from the homeotic leg reached the thoracic ganglion.     

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.     

Drosophila twin spot clones reveal cell division dynamics in regenerating imaginal discs

Cell proliferation is required for tissue regeneration, yet the dynamics of proliferation during regeneration are not well understood. Here we investigated the proliferation of eye and leg regeneration in fragments of Drosophila imaginal discs. Using twin spot clones, we followed the proliferation and fates of sister cells arising from the same mother cell in the regeneration blastema. We show that the mother cell gives rise to two sisters that participate equally in regeneration. However, when cells switch disc identity and transdetermine to another fate, they fail to turn off the cell cycle and continue dividing long after regeneration is complete. We further demonstrate that the regeneration blastema moves as a sweep of proliferation, in which cells are displaced. Our results suggest that regenerating cells stop dividing once the missing parts are formed, but if they undergo a switch in cell fate, the proliferation clock is reset.     

Regulation of cellular plasticity in Drosophila imaginal disc cells by the Polycomb group, trithorax group and lama genes

Drosophila imaginal disc cells can switch fates by transdetermining from one determined state to another. We analyzed the expression profiles of cells induced by ectopic Wingless expression to transdetermine from leg to wing by dissecting transdetermined cells and hybridizing probes generated by linear RNA amplification to DNA microarrays. Changes in expression levels implicated a number of genes: lamina ancestor, CG12534 (a gene orthologous to mouse augmenter of liver regeneration), Notch pathway members, and the Polycomb and trithorax groups of chromatin regulators. Functional tests revealed that transdetermination was significantly affected in mutants for lama and seven different PcG and trxG genes. These results validate our methods for expression profiling as a way to analyze developmental programs, and show that modifications to chromatin structure are key to changes in cell fate. Our findings are likely to be relevant to the mechanisms that lead to disease when homologs of Wingless are expressed at abnormal levels and to the manifestation of pluripotency of stem cells.     

Dissociation and sorting out of Drosophila imaginal disc cells

Previous attempts to study sorting out of Drosophila imaginal disc cells have been hampered by an inability to thoroughly dissociate these cells and the need to use cuticular markers which require several days of in vivo culture. This study overcomes these limitations by using a new dissociation procedure and a genetic marker for undifferentiated cells, the succinate dehydrogenase8 (sdh8) mutation. Dissociated and reaggregated cells from wing and leg imaginal discs segregated or "sorted out" from one another after only 24 hr of in vivo culture. It was also found that leg cells from different body segments may sort out, but to a lesser degree than wing and leg cells. Mixtures of wing and haltere cells did not sort out, in contrast to previous reports. These results constitute the first unambiguous study of sorting out with Drosophila imaginal disc cells and indicate that dorsally situated imaginal cells share a recognition specificity which is different from that of ventral imaginal cells.     

The morphostatic actions of juvenile hormone

The maintenance of "status quo" in larvae by juvenile hormone (JH) involves both the programming of ecdysteroid-dependent synthesis during the molt and the suppression of morphogenetic growth during the intermolt. The latter morphostatic action does not require ecdysteroids, and has been studied in the formation of imaginal discs in Manduca sexta. Preultimate larval instars have both invaginated discs and imaginal primordia, both of which grow isomorphically with the larva. In the last instar, the young discs/primordia initiate the morphogenesis and patterning that results in a mature disc. JH suppresses both the initiation and progression of the signaling that transforms immature discs or primordia into a fully patterned imaginal disc. This transformation normally occurs in the context of the rapid growth of the last larval stage, and nutrient-dependent factors appear to be able to override the JH suppression. The morphostatic action of JH may have been important for the evolution of the larval stage. Studies on embryos of basal, hemimetabolous insects show that their premature exposure to JH can truncate patterning programs and cause precocious tissue maturation, factors essential for organizing a novel larval form. This suppression of embryonic patterning then results in embryonic fields that remain dormant as long as JH is present. These are the primordia that can transform into imaginal discs once JH disappears in preparation for metamorphosis.