Dll expression pattern is built up in a series of stages, beginning during cellularization within the limb primordia of the gnathal segments or stripes (corresponding to future maxillary and labial segments). A second stripe corresponds to the antennal limb primordia. These two stripes flank the forming cephalic furrow [Image]. Once germ-band elongation is completed, maxillary and labial primordia have resolved into two distinct regions of expression. More anteriorly, primordium of the labrum also shows expression at onset of germ-band extension. During germ band extension, leg primordia show expression in the thoracic region (S.M. Cohen, 1990).

Arthropod appendages are thought to have evolved as outgrowths from the body wall of a limbless ancestor. Snodgrass, in his Principles of Insect Morphology (1935), proposed that, during evolution, expansion of the body wall would originate the base of the appendages, or coxopodite, upon which the most distal elements that represent the true outer limb, or telopodite, would develop. The homeobox gene Distal-less (Dll), which is required in the Drosophila appendages for development of distal regions is now thought to promote formation of telopodite structures above the evolutionary ground-state of non-limb or body wall. Another homeobox gene, extradenticle (exd), which is required for appropriate development of the trunk and the proximal parts of the appendages, represents a coxopodite gene. exd is transcribed in a pattern that surrounds and abuts Dll-expressing imaginal disc primordia in the ventrolateral epidermis of stage 14 Drosophila embryos. Early in embryogenesis, exd is broadly distributed throughout the embryo and colocalizes with Dll in the limb primordia. exd function is eliminated from the distal precursors in the developing limb and subsequently remains restricted to proximal precursors. This elimination is important because when ectopically expressed, exd prevents distal development and gives rise to truncated appendages lacking distal elements. This restriction of EXD protein to the peripheral parts of the disc is in contrast to its reported uniform mRNA distribution. EXD mRNA accumulates preferentially in the periphery of the leg disc, although lower levels are also detected in the central regions. This EXD mRNA in central regions may be responsible for the low levels of EXD protein detected in the cytoplasm, suggesting that the restriction of exd function to proximal leg parts may be controlled not only transcriptionally but also at the level of nuclear transport. The maintenance of exd expression during larval stages, contrary to Dll, does not require the hedgehog signaling pathway, suggesting that proximal regions of appendages develop independent of hh function. Targeting exd transcription to the distal limb, using a Dll promoter attached to the exd coding region, prevents distal leg development. Ectopic exd seems to interfere with cell proliferation in the central disc and results in cell death induction in the distal domain of the leg. Finally, in the crustacean Artemia, exd and Dll are expressed in comparable patterns as in Drosophila, suggesting a conserved genetic mechanism subdividing the arthropod limb (González-Crespo, 1996).

Wing and leg precursors of Drosophila are recruited from a common pool of ectodermal cells expressing the homeobox gene Dll. Induction by Dpp promotes this cell fate decision toward the wing and proximal leg. The receptor tyrosine kinase Egfr antagonizes the wing-promoting function of Dpp and allows recruitment of leg precursor cells from uncommitted ectodermal cells. By monitoring the spatial distribution of cells responding to Dpp and Egfr, it has been shown that nuclear transduction of the two signals peaks at different positions along the dorsoventral axis when the fates of wing and leg discs are specified and that the balance of the two signals assessed within the nucleus determines the number of cells recruited to the wing. Differential activation of the two signals and the cross talk between them critically affect this cell fate choice (Kubota, 2000).

In a screen for genes expressed in the embryonic limb primordia, rhomboid was found to be transiently expressed in the central part of Dll-expressing limb primordia in stage 11 embryos. rho transcription is the rate-limiting step of the activation of an EGFR ligand Spitz. As expected from the role of rho as a stimulator of Egfr, a transient expression of an activated, phosphorylated form of MAPK (dpMAPK) is detected in the nucleus of limb primordial cells surrounding the rho-expressing cells. The dpMAPK expression starts after the initiation of Dll transcription and diminishes before the separation of the wing and leg disc primordium. The dpMAPK expression is undetectable in null mutants of rho or Egfr. The peak of dpMAPK expression is located ventrally to the cells expressing dpp. The results suggest that rho-mediated stimulation of Egfr and MAPK occurs at the time of cell fate specification of wing and leg discs (Kubota, 2000).

The spatial distribution of cells responding to Dpp and its relationship to Egfr signals was studied. To this end, an antibody specific to phosphorylated C-terminal sequence of Mad was produced (the antibody is termed pSSVS. pSSVS is found mainly localized in the nucleus and distributed in regions a few cells wider in diameter than those of dpp-expressing cells. These properties are consistent with the previous findings that Mad transduces the Dpp signal to the nucleus. Double labeling of pSSVS and DLL mRNA shows that pSSVS expression is higher in the dorsal region of Dll-expressing cells. DLL mRNA is expressed in the entire limb primordium at stage 11 and becomes restricted to distal leg cells at stage 15. Combined with the double-labeling results of dpMAPK and Dll or dpp, it is concluded that cells responding to Dpp and Egfr overlap, but the peak of the responses are shifted. Such differential distribution of the two signals results in an arrangement of cells responding to a different strength of Dpp and Egfr along the dorsoventral axis (Kubota, 2000).

Association of tracheal placodes with leg primordia in Drosophila and implications for the origin of insect tracheal systems

Adaptation to diverse habitats has prompted the development of distinct organs in different animals to better exploit their living conditions. This is the case for the respiratory organs of arthropods, ranging from tracheae in terrestrial insects to gills in aquatic crustaceans. Although Drosophila tracheal development has been studied extensively, the origin of the tracheal system has been a long-standing mystery. Tracheal placodes and leg primordia arise from a common pool of cells in Drosophila, with differences in their fate controlled by the activation state of the wingless signalling pathway. Early events that trigger leg specification have been elucidated and it is shown that cryptic appendage primordia are associated with the tracheal placodes even in abdominal segments. The association between tracheal and appendage primordia in Drosophila is reminiscent of the association between gills and appendages in crustaceans. This similarity is strengthened by the finding that homologues of tracheal inducer genes are specifically expressed in the gills of crustaceans. It is concluded that crustacean gills and insect tracheae share a number of features that raise the possibility of an evolutionary relationship between these structures. An evolutionary scenario is proposed that accommodates the available data (Franch-Marro, 2006).

The Drosophila tracheal system has a clearly metameric origin, arising from clusters of cells, on either side of each thoracic and abdominal segment, that express the tracheal inducer genes trachealess (trh) and ventral veinless (vvl). Conversely, the leg precursors can be recognized as clusters of cells that express the Distal-less (Dll) gene, on either side of each thoracic segment; these will give rise both to the Keilin's Organs (KOs, the rudimentary legs of the larvae) and to the three pairs of imaginal discs that will give rise to the legs of the adult fly (Franch-Marro, 2006).

To investigate whether there is a direct physical association between the leg and tracheal primordia, Drosophila embryos co-stained for the expression of trh and early markers of leg primordia were examined. Although Dll is one of the most commonly used markers for the leg primordia, it is not the earliest gene required for their specification. Instead, a couple of related and apparently redundant genes, buttonhead (btd) and Sp1, act upstream of Dll in the specification of these primordia (Estella, 2003). Examining the specification of tracheal cells with respect to btd expression, tracheal cells were observed to appear in close apposition to btd-expressing cells, from the earliest stages of their appearance (by stage 9/early stage 10). Interestingly, unlike Dll, btd is initially expressed both in the thoracic and abdominal segments, and its expression is restricted to the thoracic segments later, under the influence of the BX-C. Thus, the cells of the respiratory system in Drosophila always arise in close proximity to the cells that are fated to give rise to the legs (Franch-Marro, 2006).

To fully endorse this conclusion it is necessary to show that the btd-expressing cells in the abdomen correspond to cryptic leg primordia. This may be a key point because, although many of the genes required for leg development are already known, it has not yet been possible to induce leg development in abdominal segments (except by transforming these segments into thoracic ones). In particular, although the Dll promoter contains BX-C binding sites that repress its expression in the abdominal segments, no ectopic appendage has been reported by misexpressing Dll in the abdomen. These observations have lead to some doubts as to whether a leg developmental program is at all compatible with abdominal segmental identity (Franch-Marro, 2006).

Since the initial expression of btd in the abdominal segments is downregulated by the BX-C genes, it was reasoned that sustained expression of btd might overcome the repressive effect of the BX-C genes and force the induction of leg structures in the abdomen. To test this, a btd-GAL4 driver was used to drive btd expression, expecting that the perdurance of the GAL4/UAS system would ensure a more persistent expression of btd in its endogenous expression domain. No sign was ever obtained of ectopic Dll expression or KOs in the abdominal segments, but the increased expression of btd had an effect on the KOs of the thoracic segments, which had more sensory hairs than the three normally found in wild-type KOs. Thus, on its own, btd seems unable to overcome BX-C repression of leg development (Franch-Marro, 2006).

One possibility would be that the BX-C genes could suppress appendage development in the abdomen by independently repressing both btd and Dll in this region. To assess this possibility, the same btd-GAL4 driver was used to simultaneously induce the expression of both btd and Dll. Under these circumstances, it was observed that KOs develop in otherwise normal abdominal segments; as in the previous experiment, the newly formed KOs have more than three sensory hairs. These results suggest that expression of btd and Dll in the btd-expressing abdominal primordia is sufficient to induce the development of leg structures in the abdomen, overcoming the repressive effect of the BX-C genes. Furthermore, these results demonstrate that these clusters of btd-expressing cells in the abdomen are indeed cryptic leg primordia. These results clearly show that tracheal cells are specified in close proximity to the leg primordia, in both thoracic and abdominal segments (Franch-Marro, 2006).

Previous results have shown that the leg primordia are specified straddling the segmental stripes of wingless (wg) expression in the early embryonic ectoderm, whereas tracheal cells are specified in between these stripes. To investigate whether wg might play a role in determining the fate of these primordia, what happens when the normal pattern of wg expression is disrupted was studied. In wg mutant embryos, trh and vvl from the earliest stages of their expression are no longer restricted to separate clusters of cells; instead larger patches of expression add up to a continuous band of cells running along the anteroposterior axis of the embryo, while btd expression is suppressed in this part of the embryonic ectoderm. Conversely, ubiquitous expression of wg suppresses trh expression, while causing an expansion of btd expression along the embryo. Restricted activation or inactivation of the wg pathway by the expression of a constitutive form of armadillo or a dominant-negative form of dTCF, respectively, are also able to specifically induce or repress trh and btd expression. trh/vvl and btd seem to respond independently to wg signalling and there is no sign of cross-regulation among them, since btd expression is normal in trh vvl double mutants, and trh and vvl expression is normal in mutants for a deficiency uncovering btd and Sp1 (Franch-Marro, 2006).

The role of wg as a repressor of the tracheal fate is further illustrated by looking at the behaviour of transformed cells: the clusters of cells that have lost btd expression and gained trh and vvl expression in wg mutant embryos begin a process of invagination that is characteristic of tracheal cells. Furthermore, these cells also express the dof (stumps) gene, a target gene of both trh and vvl in the tracheal cells. Although further development of these cells is hard to ascertain because of gross abnormalities in wg- embryos, these results indicate that they have been specified as tracheal cells. Thus, wg appears to act as a genetic switch that decides between two mutually exclusive fates in this part of the embryonic ectoderm: the tracheal fate, which is followed in the absence of wg signalling; and the leg fate, which is followed upon activation of the wg pathway. Given that there are no cell lineage restrictions setting apart the cells of the tracheal and leg primordia, these two cell populations could be considered as a single equivalence group, with the differences in their fate controlled by the activation state of the wg signalling pathway (Franch-Marro, 2006).

A link between respiratory organs and appendages is also found in many primitively aquatic arthropods, like crustaceans, where gills typically develop as distinct dorsal branches (or lobes) of appendages called epipods. Following the current observations, which suggest a link between respiratory organs and appendages in Drosophila, whether further similarities could be found between insect tracheal cells and crustacean gills was examined. Specifically, whether homologues of the tracheal inducing genes might have a role in the development of appendage-associated gills in crustaceans was considered (Franch-Marro, 2006).

RT-PCR was used to clone fragments of the vvl and trh homologues from Artemia franciscana and from Parhyale hawaiensis, representing two major divergent groups of crustaceans (members of the branchiopod and malacostracan crustaceans, respectively). In the case of Artemia vvl, a fragment was cloned that corresponds to the APH-1 gene and an antibody was generated for immunochemical staining in developing Artemia larvae. It was observed that Artemia Vvl is initially absent from early limb buds; it becomes weakly and uniformly expressed while the limb is developing its characteristic branching morphology, and becomes strongly upregulated in one of the epipods as its cells begin to differentiate. Uniform weak expression persists in mature limbs, but expression levels in the epipod are always significantly higher. Expression of the trh homologue from Artemia appears to be restricted to the same epipod as Vvl. Similarly, homologues of vvl and trh were cloned from Parhyale hawaiensis and their expression was studied by in situ hybridization. Both genes are specifically expressed in the epipods of developing thoracic appendages. Besides epipods, the Artemia trh and vvl homologues are also expressed in the larval salt gland, an organ with osmoregulatory functions during early larval stages of Artemia development (Franch-Marro, 2006).

What is the significance of the two Drosophila tracheal inducer genes being specifically expressed in crustacean epipods/gills? One possibility is that the expression of these two genes was acquired independently in insect tracheae and in crustacean gills. Alternatively, tracheal systems and gills may have inherited these expression patterns from a common evolutionary precursor, perhaps a respiratory/osmoregulatory structure that was already present in the common ancestors of crustaceans and insects (Franch-Marro, 2006).

The latter possibility is considered unlikely by conventional views, because of the structural differences between gills and tracheae (external versus internal organs, discrete segmental organs versus fused network of tubes), and the difficulty to conceive a smooth transition between these structures. Yet, analogous transformations have occurred during arthropod evolution: tracheae can be organized as large interconnected networks or as isolated entities in each segment (as in some apterygote insects), invagination of external respiratory structures is well documented among groups that have made the transition from aquatic to terrestrial environments (terrestrial crustaceans, spiders and scorpions), and conversely evagination of respiratory surfaces is common in animals that have returned to an aquatic environment (tracheal gills or blood gills in aquatic insect larvae). A very similar (but independent) evolutionary transition is, in fact, thought to have occurred in arachnids, where gills have been internalised to give rise to book lungs, and these in turn have been modified to give rise to tracheae in some groups of spiders. Thus, a relationship between insect tracheae and crustacean gills is plausible (Franch-Marro, 2006).

A particular type of epipod/gill has also been proposed as the origin of insect wings, a hypothesis that has received support from the specific expression in a crustacean epipod of the pdm/nubbin (nub) and apterous (ap) genes - that have wing-specific functions in Drosophila. In fact, the Artemia nub and ap homologues are expressed in the same epipod as trh and vvl, raising questions as to the specific relationship of this epipod with either tracheae or wings. A resolution to this conundrum becomes apparent when one considers the different types of epipods/gills found in aquatic arthropods, and their relative positions with respect to other parts of the appendage (Franch-Marro, 2006).

The primary branches of arthropod appendages, the endopod/leg and exopod, develop straddling the anteroposterior (AP) compartment boundary, which corresponds to a widely conserved patterning landmark in all arthropods. Different types of epipods/gills, however, differ in their position with respect to this boundary. For example, in the thoracic appendages of the crayfish, some epipods develop spanning the AP boundary [visualized by engrailed (en) expression running across the epipod], whereas others develop exclusively from anterior cells (with no en expression). Given that wing primordia comprise cells from both the anterior and posterior compartments, wings probably derived from structures that were straddling the AP boundary. Conversely, given that tracheal primordia arise exclusively from cells of the anterior compartment (anterior to en and even wg-expressing cells), it seems probable that tracheal cells evolved from a population of cells that was located in the anterior compartment. In this respect, it is interesting to note that the former type of epipods express nub, whereas the latter do not (Franch-Marro, 2006).

In summary, it is suggested that the ancestors of arthropods had specific areas on the surface of their body that were specialized for osmoregulation and gas exchange. Homologues of trh and vvl were probably expressed in all of these cells and played a role in their specification, differentiation or function. Some of these structures were probably associated with appendages, in the form of epipods/gills or other types of respiratory surfaces. A particular type of gill, straddling the AP compartment boundary, is likely to have given rise to wings, whereas respiratory surfaces arising from anterior cells only may have given rise to the tracheal system of insects. Confirmation of this hypothetical scenario may ultimately come from the discovery of new fossils, capturing intermediate states in the transition of insects from an aquatic to a terrestrial lifestyle (Franch-Marro, 2006).


Thoracic imaginal primordia are allocated in response to signals from wingless and decapentaplegic. This activates Distal-less, which in turn marks the future position of legs and wings (B. Cohen, 1993).

The secreted protein Hedgehog has been identified as the signal transmitted along retinal axons which serves as the inductive signal triggering neurogenesis in the lamina (Huang, 1996). The target of HH in the developing visual system is wingless, which in turn targets decapentaplegic and Distal-less. The lamina neurons and the cortical neurons that contribute axons to the medulla neuropil derive from a neuroblast population (OPC or outer proliferation center) that divides throughout most of larval development. Although cells expressing wg constitute only a small fraction of the OPC, the inactivation of wg at early times results in the later absence of nearly the entire target structure.

Wingless regulates the onset and maintenance of dpp expression. Approximately 14 hr after the onset of wg expression, dpp expression begins in single cell domains immediately adjacent to the wg-expressing cells, and is maintained throughout larval development as these cell populations divide up to and including the period of retinal axon ingrowth. In dpp mutants many OPC progeny fail to down-regulate the expression of the cell adhesion molecule fasII, fail to express neuron markers, and fail to contribute axons to the medulla neuropil (Kaphingst, 1994).

Distal-less expression is found in wg-expressing cells adjacent to the dpp domains. dll expression is significantly greater in the dorsal domain. The involvement of dll in neurogenesis in Drosophila has yet to be documented (Kaphingst, 1994).

Homothorax and Extradenticle are expressed in the proximal domain of the leg (the Hth domain) : all the other factors studied (Wingless, Decapentaplegic and Distalless) are expressed in more distal regions (the Dll domain). Dachsund (Dac) is expressed in an intermediate domain, dorsal and lateral to the more distal Dll domain (the Dac domain). What follows is a more complete description of these domains. The expression of several targets of the signaling molecules Wg and Dpp were examined in relation to the hth expression domain. dpp expression in the leg disc at the early third larval instar stage consists of a sector that originates at the center of the disc, extends dorsally to the periphery and shows extensive overlap with Hth. omb, a target of the Dpp-signaling pathway, is expressed in a dorsal sector that, in contrast to dpp, extends dorsally only to abut, but not overlap with, the hth domain. wg expression consists of a ventral sector of cells that extends from the center to the periphery of the disc, whereas H15, an enhancer trap line that requires wg signaling for its activation, is largely not transcribed in the hth domain. The restriction of these Wg and Dpp target genes to non-hth-expressing cells suggests that hth restricts signaling by these two molecules. By the late third larval instar stage, there is a small degree of overlap between hth and omb expression as well as between hth and H15. This expression corresponds to the trochanter domain where gene activation can occur independent of the Wg- and Dpp-signaling pathways. Unlike omb and H15, the Dll and dac genes require input from both the Dpp and Wg signal transduction pathways to be activated in leg discs. Dll encodes a homeodomain protein present in the central portion of leg discs, and its activation requires the highest concentrations of Wg and Dpp. dac encodes a nuclear protein and a putative transcription factor whose expression is repressed by high concentrations, and activated by intermediate concentrations, of Wg and Dpp. By performing triple stains for the dacP-lacZ reporter gene, and Dll and Hth proteins at the early third larval instar stage, it was found that the leg disc is defined by three non-overlapping domains of gene expression. The distal-most domain of the leg disc contains Dll protein (the Dll domain). Dorsal and dorsolateral, but not ventral, to the Dll domain are cells that express dac (the Dac domain). The proximal-most cells of the disc, which surround the dac and Dll domains, express hth (the Hth domain). At the mid 3rd larval instar stage (~96 hours after egg lay, or AEL), the distal-most cells express only Dll and are surrounded by a ring of cells that express both Dll and dac. At this stage, there is also a dorsal patch of cells that express dac but not Dll. hth expression remains limited to the proximal-most cells of the disc and shows no overlap with dac or Dll. By the late 3rd larval instar stage (~120 hours AEL), hth is still not co-expressed with dac or Dll, with the exception of a thin band of cells corresponding to the trochanter domain, where all three genes are co-expressed. Gene expression in the trochanter domain is likely to represent secondary patterning events, because it is not dependent on Wg- or Dpp-signaling. At this stage dac expression also surrounds and partially overlaps the Dll expression domain. It is proposed that the Dll and Dac domains, where hth transcription is off and Exd is cytoplasmic, are Dpp- and/or Wg-responsive domains, as demonstrated by the ability of these cells to respond to these signals by activating the target genes Dll, dac, omb and H15. In contrast, the hth domain, where hth is active and Exd is nuclear, is a Wg- and/or Dpp-non- responsive domain, where these signals are present but cannot activate these targets (Abu-Shaar, 1998).

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. A novel lineage-tagging method was used to address the reversibility of cell fate specification by morphogen gradients. Responses to Hedgehog and Dpp in the wing disc are readily reversible. In the leg, 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 so long as the imaginal discs are growing. In other systems, where growth and patterning are uncoupled, nonreversible specification events or ‘ratchet’ effects may be of functional significance (Weigmann, 1999).

In the developing leg disc, some responses to Wg and Dpp are readily reversible, while others are not. Lineage tracing of cells born in the TshGAL4 domain (proximal) suggests that cells readily lose Tsh (and Hth) expression and instead express Dll and Dac (distal markers). In young discs, a small proportion of cells expressing low levels of both Dll and Tsh are found at the edge between these domains. It is possible that these are cells in transition between the domains. These results suggest that cells born in the presumptive body wall readily contribute to formation of more distal leg regions. Under normal circumstances cells born in the Dll-expressing distal domain of the leg do not contribute to the body wall. However, they are not prohibited from doing so when given a strong growth advantage. The progeny of Dll-expressing cells in second instar are mostly fated to give rise to the tarsus and do not contribute to femur. In contrast, femur, tibia and tarsal segments (the distal segments) derive from cells that have expressed Dll in early third instar. The difference between these stages suggests that new cells must be induced to turn on Dll in order to provide the population of cells that contribute to the femur (the most proximal of the distal segments). These cells must derive from the Tsh domain in second instar and acquire Dll and Dac expression. At later stages, Dll is expressed at very low levels in the femur, where it may be repressed by Dac. Downregulation of Dll expression in the femur is unlikely to be a direct response to a lowering of Wg and Dpp signaling, because clones of cells unable to respond to these signals do not show abnormal Dll or Dac expression. This contrasts with the situation in the wing where removal of Dpp signaling leads to loss of Spalt expression. The low level of Dll expression in the femur is in part due to Dac activity, since dac mutant clones show elevated levels of Dll. The transient induction of Dll in the precursors of the femur is consistent with genetic analyses showing that formation of all leg segments except coxa depends on Dll activity in early development, whereas the low level of Dll expressed later is apparently not required for normal femur development (Weigmann, 1999).

When the distal part of a leg imaginal disc, or of an amphibian or a cockroach leg is removed, distal structures will regenerate from the cut edge. If the distal part of the leg disc is cultured in isolation, distal structures will regenerate from the cut edge, leading to a duplication of the fragment. The fact that distal structures regenerate but proximal structures do not has been termed distal transformation. These classical experiments have shown that cells have a general tendency to distalize, whereas their capability to proximalize is restricted. The current experiments show that distal transformation happens during normal development. Some proximal cells switch their pattern of gene expression as the disc grows and they acquire distal fate. Distal cells do not normally switch to proximal fate, but can do so if forced during early development. The classical regeneration studies suggest that the ability to shift from distal to proximal fate may be lost as development proceeds (Weigmann, 1999).

Localized transcription factors specify the identity of developmental domains. The function of the Teashirt zinc finger protein, which is expressed in the proximal domain of the Drosophila leg, has been analyzed. At stage 10 of embryogenesis, Distal-less is detected in the putative distal part of the primordia of the leg in each of the thoracic hemisegments of the embryo. Tsh is coexpressed with Dll at this stage. By stage 15 the cells of the presumptive leg imaginal discs have invaginated inside the embryo and Tsh is not detected in the most distal part of the leg primordium, where Dll is expressed alone. However, Tsh is still coexpressed with Dll in a ring of cells at the periphery of the Dll domain. At the beginning of the third instar, Dll occupies a distinct distal ring of cells in the disc; Tsh is expressed in a proximal ring. These territories are separated by 2 or 3 cells in ventral and lateral regions and up to 10 cells in dorsal parts. The Dachshund transcription factor is expressed in the intermediate ring of cells overlapping the Dll expression domain by, at the most, 1 or 2 cells. By mid-third instar Dll is expressed in a new 4-cell-wide proximal ring that is destined to make the proximal femur and possibly the distal edge of the trochanter. Tsh overlaps with this new Dll domain at the proximal edge, which persists until the late third instar stage. By ectopic expression of a teashirt transgene it has been shown that Teashirt contributes to the differences in cell-cell adhesion between proximal and distal leg cells. Whereas clones of cells expressing the teashirt transgene survive in the endogenous Teashirt domain, most cells expressing Teashirt in an ectopic distal position are lost from the epithelium. In clones which were recovered in the distal domain, different effects were seen, dependent on position with respect to the dorsal-ventral axis. In the ventral region, where Wingless is signaling, surviving clones express Teashirt and cause abnormalities in the adult leg. Contrarily, lateral and dorsal clones generally do not accumulate Teashirt and have no effect on patterning. One exception to the differential dorsal-ventral effects occurs at the boundary between Teashirt-expressing and -nonexpressing cells. Both ectopic and hypomorphic loss of teashirt affects patterning at all positions at the boundary, suggesting that Teashirt plays a crucial role in boundary formation. The results are discussed with respect to the roles of transcriptional and posttranscriptional mechanisms in proximal-distal axis patterning of the Drosophila legs. It is suggested that because Arm binds to Tsh, this binding stablizes Tsh in cells within the Wg signaling domain (Erkner, 1999).

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. Essential roles of Wingless in patterning the leg imaginal disc are described. (1) 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 has previously been shown that Wingless signal transduction is not active in the proximal leg domain in larvae. (2) 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, evidence is provided that Dll restricts expression of a proximal leg-specific gene expression. It is proposed 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 (Kubota, 2003).

At embryonic stage 11, the early expression of D11 expression marks the entire limb primordium that gives rise to both wing and leg discs. After separation of wing and leg discs at stage 12, Dll expression becomes restricted to the center of the leg disc. Double labeling of stage 15 leg discs reveals that there is still a significant number of cells that coexpress Dll and the proximal leg marker Esg, suggesting that expression of Dll and Esg is not a strictly exclusive event. Rather, the result suggests that those marker genes respond differentially to inductive signals in the leg primordium (Kubota, 2003).

In the leg disc, Hth defines the trunk and proximal cell identities, and its expression is excluded in the distal leg domain in the larval stage. Double labeling with antibodies against Esg and Hth reveal that the Esg expression overlaps with Hth expression. Esg is used as a marker uniquely labeling the distinct cell identity of the proximal leg domain in the trunk region (Kubota, 2003).

The expression domain of wg and the position of wing and leg primordia were compared. Wg expression in the trunk ectoderm starts as stripes along the anterior side of the compartment boundaries. At early stage 11, most of the limb primordia marked with Dll protein expression overlap with wg stripes, as revealed by the wg-lacZ reporter. At late stage 11, wg-lacZ stripes break up into dorsal patches and ventromedial stripes. By late stage 12, expression of Dll protein becomes limited to a group of cells partially overlapping the dorsal edge of ventromedial wg stripes. The ventromedial wg stripe also overlaps with proximal leg cells that are labeled with anti-Esg at stage 15. The ventral half of proximal leg cells is nearly completely included within the ventral wg stripes. The dorsal half of leg cells is also located adjacent to, but not included in, the dorsal edge of the wg stripes. On the other hand, a reciprocal relationship between wg expression and wing primordia was observed. When wing primordia are first recognizable at stage 12 as cells expressing Vestigial (Vg), they do not overlap with the stripe of wg. Dorsal cell migration further separates wing primordia from the source of Wg at stage 15. The absence of Wg expression near wing primordia suggests that Wg does not play a positive role in wing disc development (Kubota, 2003).

To confirm whether Wg signaling is required cell autonomously for leg disc development, the dominant-negative forms of Drosophila TCF (DTCFdeltaN) or Drosophila axin (Daxin) were expressed in the limb primordia. The Dll-Gal4 driver, which is turned on in the limb primordium at stage 11 and continues to be active in leg and wing discs, was used. In Dll-GAL4 embryos carrying UAS-DTCFdeltaN or UAS-Daxin, the overall size of leg discs was reduced. Expression of Esg was preferentially reduced in the dorsal side. The drastic reduction of Dll mRNA and protein in distal leg cells in the armH8.6 mutants as well as in DTCFdeltaN- and Daxin-expressing embryos demonstrate that Wg signaling is required for both proximal and distal leg cells. In arm mutants, Hth-expressing cells expand to the distal domain. This observation suggests that, upon loss of Wg signaling, prospective leg disc cells lose their identity and adopt the fate of trunk ectoderm. However, disc-specific reduction of Wg signaling does not affect wing disc formation, although the Dll-Gal4 driver is active in the wing primordium. It is concluded that late function of Wg signaling promotes formation of the leg disc with a higher requirement in the proximal domain, but is dispensable for wing disc formation (Kubota, 2003).

Cell fate maintenance of proximal leg requires continuous signaling by Wg. The requirement for arm and wg is higher in the proximal leg domain. arm mutations nearly eliminate all Esg expression, but leave some Dll-positive cells. wgts is a hypomorph at the restrictive temperature and leaves distal leg cells nearly intact while significantly affecting proximal leg cells, especially those at the dorsal side of the disc. Dorsal cells are far from the source of Wg and are first to lose identity upon reduction of Wg activity. Since Esg expression in ventral proximal cells overlaps with the wg stripe, it is proposed that the localized expression of Wg and its range of diffusion are major determinants of the site of proximal cell formation. It is likely that dorsal-proximal cells require a higher level of Wg to be produced to reach their position (Kubota, 2003).

Dll expression is initially found in the entire limb primordia and becomes restricted to the edge of the Wg stripe that becomes the center of the leg disc. One candidate for an additional factor that places Dll in this position is Dpp, is expressed in stripes abutting the Wg stripe; Dpp is known to be required for distal leg development (Kubota, 2003).

Finally, the center of the embryonic leg disc is devoid of the expression of proximal cell markers Esg and Hth, marking the distal leg domain. Separation of the proximal and distal leg domains is a slow process, taking several hours to complete. One model for the mechanism regulating this separation process is that proximal gene expression is downregulated by a distal gene, as shown by ectopic Dll expression repressing Esg expression. Since expression of Esg is also regulated by positive input from Wg signaling, Esg expression does not necessary mirror the absence of Dll. In support of this idea, Dll is known to repress proximal genes in larval leg discs. The second possibility is a restriction of proximal cell movement into the distal domain. Cells in the Hth-expressing proximal domain in the larval leg disc have distinct cell-adhesive properties from those in the Dll-expressing distal domain, and by extension, cells with high levels of Dll or Hth may not mix well in the embryo as well. Since Hth is widely expressed in the embryonic ectoderm, Dll-expressing cells may be forced to localize at the center of the leg disc (Kubota, 2003).

Notch-dependent epithelial fold determines boundary formation between developmental fields in the Drosophila antenna

Compartment boundary formation plays an important role in development by separating adjacent developmental fields. Drosophila imaginal discs have proven valuable for studying the mechanisms of boundary formation. This study examined the boundary separating the proximal A1 segment and the distal segments, defined respectively by Lim1 and Dll expression in the eye-antenna disc. Sharp segregation of the Lim1 and Dll expression domains precedes activation of Notch at the Dll/Lim1 interface. By repressing bantam miRNA and elevating the actin regulator Enabled, Notch signaling then induces actomyosin-dependent apical constriction and epithelial fold. Disruption of Notch signaling or the actomyosin network reduces apical constriction and epithelial fold, so that Dll and Lim1 cells become intermingled. These results demonstrate a new mechanism of boundary formation by actomyosin-dependent tissue folding, which provides a physical barrier to prevent mixing of cells from adjacent developmental fields (Ku, 2017).

This study attempted to unravel the molecular and cellular mechanisms of boundary formation in the Drosophila head. Focus was placed on the antennal A1 fold that separates the A1 and A2-Ar segments. The results showed that the expression of the selector genes Lim1 and Dll, which are expressed in A1 and A2-Ar, respectively, was sharply segregated. This step was followed by differential expression of Dl, Ser and Fng, as well as activation of N signaling at the interface between A1 and A2. N signaling then induced apical constriction and epithelial fold, possibly through repression of bantam to allow levels of the bantam target Ena to become elevated, with this latter inducing the actomyosin network. The actomyosin-dependent epithelial fold then provided a mechanical force to prevent cell mixing. When N signaling or actomyosin was disrupted, or when bantam was overexpressed, the epithelial fold was disrupted and Dll and Lim1 cells become mixed. Thus this study describes a clear temporal and causal sequence of events leading from selector gene expression to the establishment of a lineage-restricting boundary (Ku, 2017).

Sharp segregation of Dll/Lim1 expressions began before formation of the A1 fold, suggesting that fold formation is not the driving force for segregation of Dll/Lim1 expression. Instead, the fold functions to safeguard the segregated lineages from mixing. Whether Dll/Lim1 segregated expression is due to direct or indirect antagonism between the two proteins is not known (Ku, 2017).

Actomyosin-dependent apical constriction is an important mechanism for tissue morphogenesis in diverse developmental processes, e.g., gastrulation in vertebrates, neural closure and Drosophila gastrulation, as well as dorsal closure and formation of the ventral furrow and segmental groove in embryos. This study describes a new function of actomyosin, i.e., the formation of lineage-restricting boundaries via apical constriction during development (Ku, 2017).

This actomyosin-dependent epithelial fold provides a mechanism distinctly different from other known types of boundary formation. The cells at the A1 fold still undergo mitosis, suggesting that mitotic quiescence is not involved. Perhaps epithelial fold as a lineage barrier is needed in situations in which mitotic quiescence does not happen. Mechanically and physically, epithelial folds could serve as stronger barriers than intercellular cables when mitotic activity is not suppressed. The drastic and sustained morphological changes, including reduced apical area and cell volume, may be accompanied by increased cortical tension of cells along the A1 fold, with such high interfacial tension then preventing cell intermingling and ensuring Dll and Lim1 cell segregation. Although similar to actomyosin boundaries, the epithelial fold in the A1 boundary is distinctly different from the supracellular actomyosin cable structure in fly parasegmental borders, the wing D/V border, and the interrhombomeric boundaries of vertebrates. The adherens junction protein Echinoid, which is known to promote the formation of supracellular actomyosin cables, is not involved in A1 fold formation. Although actomyosin is enriched in a ring of cells in the A1 fold, it does not exert a centripetal force to close the ring, unlike the circumferential cable described in dorsal closure and wound healing (see review. In the A1 fold, the constricting cells become smaller in both their apical and basolateral domains, thus differing from ventral furrow cells where cell volume remains constant (Ku, 2017).

A tissue fold probably provides a strong physical or mechanical barrier to prevent cell mixing. In addition, whereas in a flat tissue where the boundary involves only one to two rows of cells, the tissue fold involves more cells engaging in cell-cell communication. The close apposition of cells within the fold may allow efficient signaling within a small volume. This may be an evolutionarily conserved mechanism for boundary formation that corresponds to stable morphological constrictions such as the joints in the antennae and leg segments (Ku, 2017).

Although N signaling has been reported to be involved in many developmental processes, a role in inducing actomyosin-dependent apical constriction and epithelial fold is a novel described function for N. For the A1 boundary, N activity is possibly mediated through repression of bantam and consequent upregulation of Ena. In the wing D/V boundary, N signaling is also mediated through bantam and Ena, but the outcome is formation of actomyosin cables, i.e., without apical constriction and epithelial fold [19]. Thus, the N/bantam/Ena pathway for tissue morphological changes is apparently context-dependent (Ku, 2017).

Tissue constriction also occurs later in joint formation of the legs and antennae. N activation also occurs in the joints of the leg disc and is required for joint formation. This role is conserved from holometabolous insects like the fruitfly Drosophila melanogaster and the red flour beetle Tribolium castaneum to the hemimetabolous cricket Gryllus bimaculatus. It is possible that for segmented structures that telescope out in the P/D axis, like the antennae, legs, proboscis and genitalia, N signaling is used to demarcate the boundaries between segments, which are characterized by tissue constriction. N-dependent epithelial fold morphogenesis has also been reported in mice cilia body development without affecting cell fate, suggesting that such N-dependent regulation in morphogenesis is evolutionarily-conserved (Ku, 2017).

It is proposed that N signaling is important in all boundaries that involve stable tissue morphogenesis. For those boundaries corresponding to stable morphological constrictions, e.g., the joints in insect appendages, N acts via actomyosin-mediated epithelial fold. The wing D/V boundary represents a different type of stable tissue morphogenesis. It becomes bent into the wing margin and involves N signaling via actomyosin cables, rather than apical constriction. In contrast, actomyosin-dependent apical constrictions do not involved N signaling and are involved in transient tissue morphogenesis, such as gastrulation in vertebrates, neural closure, Drosophila gastrulation, dorsal closure, as well as formation of the ventral furrow, eye disc morphogenetic furrow, and segmental groove in embryos (Ku, 2017).

N signaling is also involved in the boundary between new bud and the parent body of Hydra, where it is required for sharpening of the gene expression boundary and tissue constriction at the base of the bud [78]. Whether the role of N in these tissue constrictions is due to actomyosin-dependent apical constriction and epithelial fold is not known (Ku, 2017).

Boundaries may be established early in development. As the tissue grows in size through cell divisions and growth, boundary maintenance become essential. This study found that N activity is maintained by actomyosin, suggesting feedback regulation to stably maintain the boundary. Mechanical tension generated by actomyosin networks has been suggested to enhance actomyosin assembly in a feedback manner. Interestingly, the N-mediated wing A/P and D/V boundaries, which form actomyosin cables rather than tissue folds, did not exhibit such positive feedback regulation. Instead, the stability of the Drosophila wing D/V boundary is maintained by a complex gene regulatory network involving N, Wg, N ligands and Cut. Perhaps this is necessary for a boundary not involving tissue morphogenesis (Ku, 2017).

The segmented appendages of arthropods (antennae, legs, mouth parts) are homologous structures of common evolutionary origin. It has been proposed that the generalized arthropod appendage is composed of a proximal segment called the coxopodite and a distal segment called the telopodite, either of which can further develop into more segments. The coxopodite is believed to be an extension of the body wall, whereas the telopodite represents the true limb, and thus represents an evolutionary addition. Dll mutants lack all distal segments except for the coxa in legs and the A1 segment in antennae. Lineage tracing studies have shown that Dll-expressing cells contributed to all parts of the legs except the coxa. These results indicate that the leg coxa and antenna A1 segment correspond to the Dll-independent coxopodite, and that Dll is the selector gene for the telopodite. Therefore, the antennal A1 fold is the boundary between the coxopodite and telopodite. It is postulated that the same N-mediated epithelial fold mechanism also operates in the coxopodite/telopodite boundary of legs and other appendages (Ku, 2017).

Distal-less: Biological Overview | Evolutionary Homologs | Regulation | Effects of Mutation | References

Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.