buttonhead

DEVELOPMENTAL BIOLOGY

Embryonic

The BTD transcript is first expressed in a stripe covering the head anlagen of the syncytial blastoderm embryo located between 65 and 77% egg-length (0% is the posterior pole). The BTD transcript overlaps the domain of Empty spiracles and Orthodenticle. The BTD strip persists until it decays in germ band extention. It is later expressed in a complex pattern (Wimmer, 1993).

During cellularization of the blastoderm a dorsal spot appears in the proneural region anterior to the head stripe. The head spot expression continues and splits up during germband retraction into several spots that become integrated into the developing brain, marking different brain areas. During the early phase of germ band extension btd starts to be expressed in a metameric pattern that decays at the fully extended germ band stage leaving single btd-expressing cells. The btd-expressing cells represent subgroups of neuroblasts, which finally end up in the ventral cord (Wimmer, 1996).

During germ band extension, a second metameric expression pattern of btd can be observed. It is restricted to the lateral region of the embryo, corresponding to the area of the proneural clusters from which the peripheral nervous system originates. During germ band extension btd is expressed in the leg anlagen located in the thoracic segments and in several restricted areas of the head. At this stage the pattern of btd expression resembles Distal-less, but btd expression is delayed compared to Dll expression. btd is expressed in the mandibular but not the labral segments, where Dll is expressed in the labral, but not in the mandibular segments (Wimmer, 1996).

Early tailless expression (blastoderm stage) covers the anlage of the entire brain. Beginning approximately with the onset of gastrulation, an anterior-dorsal region with a high expression level (called HL domain) can be distinguished from a posterior-ventral domain expressing tll at a somewhat lower level. The HL domain coincides with part of the central and anterior protocerebral neurectoderm. The low expression level LL domain covers the remaining part of the protocerebral neuroectoderm. orthodenticle is expressed in a circumferential domain of the cellular blastoderm but during gastrulation becomes restricted to a domain that encompasses most of the protocerebral neurectoderm and an adjacent part of the deuterocerebral neurectoderm. All neurobasts segregating from this domain transiently express otd during stages 10 and 11. buttonhead is initially expressed in a wide domain including the anlagen of the antennal, intercalary and mandibular segments, as well as the acron. With the beginning of gastrulation, expression disappears from most of the procephalon, except for small domains of the posterior part of the deuterocerebral and tritocerebral neurectoderm and a dorsoanterior patch that partially overlaps with the dorsoanterior protocerebrum. Both the late deutocerebral and tritocerebral btd domains contain few, if any neuroblasts. empty spiracles is in an asymmetric circumferential domain of the cellular blastoderm. During gastrulation, this pattern resolves into two stripes that occupy anterior portions of the deuterocerebral neuroectoderm and the mandibular metamere, respectively. In addition, a small circular domain corresponding to the tritocerebral neurectoderm appears ventral to the deuterocerebral stripe (Younossi-Hartenstein, 1997).

Loss of tll function results in the absence of all protocerebral neuroblasts and loss of all four coherent domains of Fas II expression in the protocerebrum. Also missing is the optic lobe. orthodenticle functions in a domain that includes a large part of the protocerebrum and a smaller part of the adjacent deuterocerebrum. Loss of otd results in loss of protocerebral P1, P2 and P4 coherent domains of Fas II expression. Also missing is a nerve that carries axons from the antennal organ. In buttonhead mutation the D/T cluster is missing; consequently a cervical connection is missing that normally sends nerves to the labral sensory organ, the hypopharyngeal sensory organ and the stomatogastric nervous system (Younossi-Hartenstein, 1997).

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).

Effects of Mutation or Deletion

BTD is required for development of the antennal, intercalary and mandibular segments of the head. In btd mutants these segments are lacking and head involution [Images] is incomplete (Wimmer, 1993).

btd mutants show reduced numbers of thoracic and abdominal chordotonal organs. Adult flies with chordotonal defects are disabled, showing uncoordinated or sedentary behavior. Such behavior is also seen in transgene-rescued btd mutants, i.e. they never fly nor mate, they rarely move and show very uncoordinated footwork when moving. Part, but not all of these defects may be explained by leg malformations, which vary in penetrance and expressivity (Wimmer, 1996).

The role of buttonhead and Sp1 in the development of the ventral imaginal discs

The related genes buttonhead (btd) and Drosophila Sp1 (the Drosophila homolog of the human SP1 gene) encode zinc-finger transcription factors known to play a developmental role in the formation of the Drosophila head segments and the mechanosensory larval organs. A novel function of btd and Sp1 is reported: they induce the formation and are required for the growth of the ventral imaginal discs. They act as activators of the headcase (hdc) and Distal-less (Dll) genes, which allocate the cells of the disc primordia. The requirement for btd and Sp1 persists during the development of ventral discs: inactivation by RNA interference results in a strong reduction of the size of legs and antennae. Ectopic expression of btd in the dorsal imaginal discs (eyes, wings and halteres) results in the formation of the corresponding ventral structures (antennae and legs). However, these structures are not patterned by the morphogenetic signals present in the dorsal discs; the cells expressing btd generate their own signalling system, including the establishment of a sharp boundary of engrailed expression, and the local activation of the wingless and decapentaplegic genes. Thus, the Btd product has the capacity to induce the activity of the entire genetic network necessary for ventral imaginal discs development. It is proposed that this property is a reflection of the initial function of the btd/Sp1 genes that consists of establishing the fate of the ventral disc primordia and determining their pattern and growth (Estella, 2003).

In a search for genes with restricted expression in the adult cuticle, the MD808 Gal4 line was found to direct expression in the ventral derivatives of the adult body; proboscis, antennae, legs and genitalia. In the abdomen and analia no clear expression was discerned. It was also noticed that the insertion was located in the first chromosome and associated with a lethal mutation. The mutant larvae showed a head phenotype resembling that described for mutants at the btd gene: loss of antennal organ and the ventral arms of the cephalopharyngeal skeleton, and complementation analysis indicated that the chromosome carrying the insert contained a mutation at btd. The expression pattern found in MD808/UAS-lacZ embryos was also similar to that reported for btd, suggesting that the Gal4 insertion was located at this gene. In addition, the imaginal expression of MD808 and of btd was largely coincident (Estella, 2003).

Further to the genetic analysis and the expression data, DNA fragments at the insertion site were cloned to map the position of the P-element on the genome. It is located 753 bp 5' of the btd gene. The related gene Sp1 is immediately adjacent. It is likely that btd and Sp1 have originated by a tandem duplication of a primordial btd-like gene (Estella, 2003).

In early embryos btd is expressed in the head region, but by the extended germ band stage the expression domain has expanded to the ventral region of cephalic, thoracic and abdominal segments. During germ band retraction most of the abdominal and thoracic expression is lost, except in derivatives of the peripheral nervous system and the primordia of the imaginal discs. Sp1 is not expressed in early embryos, but from stage 11 onwards it shows the same pattern as btd (Estella, 2003).

Special attention was paid to the btd/Sp1 expression domain in the thoracic imaginal discs primordia, as it may suggest a novel function related to imaginal development. Double labelling with Dll and btd probes indicates that btd precedes Dll expression, but by stage 12 the two genes are co-expressed in a group of thoracic cells. However, the Dll domain is smaller and is included within the btd/Sp1 domain: there are cells expressing btd that do not show Dll activity, although all the cells expressing Dll express btd (Estella, 2003).

The ventral disc primordia include not only cells expressing Dll but also other cells containing expression of escargot (esg) and hdc, markers of the diploid cells that form the imaginal primordia. In late embryonic stages, esg is expressed in a ring domain surrounding the Dll-expressing cells and hdc is expressed in a similar pattern. Double label experiments were carried out with btd, hdc and esg probes; the expression of the two latter genes overlaps with that of btd (and with Sp1) in the thoracic disc primordia (Estella, 2003).

The overlap of the btd and of esg domains indicates that btd is also expressed in the hth domain, which is coincident with that of esg. As the hth/esg domain marks the precursor cells of the proximal region of the adult leg the embryonic expression data indicate that btd and Sp1 are active in the entire primordia of the ventral adult structures, including the distal and the proximal parts (Estella, 2003).

In the mature antennal disc, btd expression is restricted mostly to the region corresponding to the second antennal segment, where it co-localizes with both Dll and hth. In the leg disc btd also overlaps in part with Dll and with hth. The latter result is significant, for the expression of Dll and hth define two major genetic domains, which are kept apart by antagonistic interactions. The fact that btd is expressed in the two domains suggests that its regulation and function is independent from the interactions between the two domains. This observation is consistent with the results obtained in embryos and suggests that the btd domain includes the precursors of the whole ventral thoracic region from the beginning of development (Estella, 2003).

This work demonstrates a novel and also redundant function of btd and Sp1: they are responsible for the formation of the ventral imaginal discs by activating the genetic network necessary for their development. Furthermore, Btd protein retains the capacity of inducing the entire ventral genetic network during the larval period. It is proposed that the activation of btd/Sp1 is the crucial event in the determination of the ventral structures of the adult fly (Estella, 2003).

This argument is based on the finding that btd and Sp1 appear to mediate all events connected with the formation of the ventral discs. The discussion deals with the leg disc, but there is evidence that antennal primordium also requires btd. Moreover, the genital primordium is lacking in Df(1)C52 embryos, suggesting that this disc is also under the same control. Most of the experiments concern the function of btd but given the expression and functional similarities between the two genes, it is assumed that Sp1 fulfils the same or a very similar role. Therefore, btd/Sp1 will be considered to carry out a single function (Estella, 2003).

One crucial feature is that btd is an upstream activator of Dll and hdc, which are considered developmental markers of disc primordia: (1) btd expression precedes that of Dll and of hdc; (2) the btd expression domain includes those of Dll and hdc; (3) in btd mutants, Dll and hdc activity is much reduced, and completely absent in Df(1)C52 embryos; (4) ectopic btd function induces ectopic activation of Dll and hdc (Estella, 2003).

The role of btd in embryogenesis can be illustrated in the light of the models of Dll regulation. Dll is activated by wg and its expression modulated by the EGF spitz and by dpp, whereas it is repressed in the abdominal segments by the BX-C genes. The current experiments suggest that Dll regulation is mediated by btd: in wg mutants there is no btd expression and hence neither Dll nor hdc activity. In dpp mutant embryos, btd expands to the dorsal region resembling the effect on Dll. In Ubx^- embryos there is an additional group of cells in the first abdominal segment expressing btd; the same cells that also express Dll in those embryos. The interpretation of the role of btd mediating Dll regulation by Ubx is complicated by previous observations showing direct repression of Dll by the Ubx protein. It is possible that Ubx regulates Dll both directly and by controlling btd activity (Estella, 2003).

It is proposed that the localization of btd/Sp1 activity to a group of ventral cells is a major event in the specification of adult structures. btd and Sp1 are activated in response to spatial cues from Wg, Dpp, EGF and BX-C, and in turn their function induces the activity of the genes necessary for ventral imaginal development (Estella, 2003).

This hypothesis is strongly supported by the results obtained inducing ectopic btd activity in the dorsal discs; just the presence of the Btd product alone is sufficient to bring about ventral disc development. In the wing and the haltere discs, Btd induces a transformation into leg, whereas in the eye it induces antennal development. This indicates that it specifies ventral disc identity jointly with other factors, e.g., the Hox genes, possibly through the activation of subsidiary genes such as Dll, known to contribute to ventral appendage identity in combination with Hox genes (Estella, 2003).

The requirement for btd and Sp1 activity appears to be restricted only to the ventral discs, even during the early phases of the thoracic disc primordia. In this context it is worth considering the observation that in Df(1)C52 embryos there is esg expression in the wing and haltere disc primordia, even though it is absent in the leg discs. Thus, the wing and haltere discs are formed in the absence of btd and Sp1. Because in these embryos there is an almost complete lack of Dll expression, this observation raises the question of the origin of the dorsal thoracic discs, which are currently considered to derive from the original ventral primordium, formed by cells expressing Dll. Although some of the original group of ventral cells may contribute to the dorsal disc primordia, the data suggest that there may be cells recruited to form the dorsal discs that do not originate in the initial ventral primordium. Accordingly, it is worth considering that in the absence of Dll activity the leg and wing discs are formed, although the leg only differentiates proximal disc derivatives. Thus, the activity of Dll cannot be considered a reliable marker for imaginal discs (Estella, 2003).

RNA interference experiments also indicate that both btd and Sp1 are required for the growth of the antennal and leg discs. When the two gene functions are reduced simultaneously, leg segments fuse and there is an overall reduction in the size of antennae and legs. The reduction of growth affects the proximal and distal regions of the appendage, and assigns a role to the expression observed in the imaginal discs. The two genes are able to perform this function on their own, for the inactivation of only one is not sufficient to impair growth. This conclusion is also supported by the observation that mutant btd clones do not have any effect; they still possess Sp1 activity, which is sufficient for normal development. At this point the mechanism by which btd/Sp1 may affect growth is not known (Estella, 2003).

One particularly significant result about the mode of action of btd comes from the analysis of the ectopic leg patterns observed with ectopic btb expression in the wing and halteres. The clones of cells ectopically expressing btd tend to recapitulate the complete development of leg and antennal discs. For example, the whole genetic network necessary to make a leg appears to be activated. btd induces the activity of hth, dac and Dll, the domains of which account for the entire disc. Furthermore, hth, dac and Dll are activated in a spatially discriminated manner. The formation of the dac and Dll domains is dependent on signalling from Wg and Dpp, although they require different signal thresholds. In one clone, for example hth is expressed only in the peripheral region, resembling the normal expression in the leg disc; in another clone the discriminate expressions of dac and Dll define three distinct regions. The formation of the dac and Dll domains is dependent on signalling from Wg and Dpp, although they require different signal thresholds, but the hth domain is independent from Wg and Dpp (Estella, 2003).

The generation of distinct hth, dac and Dll domains within the clones suggested that btd-expressing cells in the wing and haltere generate their own signalling process. Indeed, within these clones there is local activation of en, the transcription factor that initiates Hh/Wg/Dpp signalling in imaginal discs. btd-expressing clones also acquire wg and dpp activity in subsets of cells. It is probably in the boundary of en-expressing with non expressing cells where the Wg and Dpp signals are generated de novo; subsequently, their diffusion initiates the same patterning mechanism which operates during normal leg development. The result of this process is that the hth, dac and Dll genes are expressed in different domains contributing to form leg patterns containing DV and PD axes. One question for which there is no clear answer is how the initial asymmetry is generated, so that a few cells within the group gain (or lose) en activity. The cells expressing en within the clones are those closer to the posterior compartment cells. It is conceivable that there might be an external signal, perhaps mediated by Hh, which triggers the initial asymmetry (Estella, 2003).

The ability of cells expressing btd to build their own patterning mechanism is also indicated by the observation that inducing btd activity in different parts of the wing disc results in the production of similar sets of leg pattern elements. For example, in MD743/UAS-btd and omb-Gal4/UAS-btd flies, btd is induced in different, non-overlapping wing regions, and yet all leg pattern elements are produced in both genotypes. Thus, the effect of btd is by and large independent of the position where it is induced, e.g., it does not depend on local positional signals (Estella, 2003).

A relevant issue is whether the ability of the Btd product to induce the formation of the full array of ventral structures has a functional significance in normal development. This property may be a faithful reflection of the original btd/Sp1 function: the activation of the developmental program to build the ventral adult patterns. btd/Sp1 function can be envisaged as follows. During the embryonic period, the conjunction of several regulatory factors (Wg, Dpp, EGF, Hox genes) allows activation of btd/Sp1 in a group of cells in each thoracic segment (it is assumed that a similar process takes place in the head). These cells become the precursors of the ventral imaginal discs and will eventually form the ventral thorax of the adult -- these include the trunk (the hth domain) and appendage (the Dll domain) regions. The activity of btd/Sp1 is instrumental in segregating these ventral discs precursors from those of the larval epidermis and determining their imaginal fate. It is involved in specifying their segment identity (in collaboration with the Hox genes) and in establishing their pattern and growth. To achieve the latter role btd/Sp1 induces the production of the growth signals Wg and Dpp, probably in response to localized activation of en and subsequent signalling by hedgehog (hh) (Estella, 2003).

A problem with this model is that in normal development all the genes involved, hth, en, hh, wg and dpp, are expressed in embryos prior to btd/Sp1. Why should a new round of activation be necessary? Although a totally satisfactory answer can not be provided, it is noted that clones of btd-expressing cells in wing or haltere lose their memory of en expression. Those that originated in the A compartment had no previous en expression, but gained it in some cells. Conversely, all cells in P compartment clones contained en activity but some lose it. The best explanation for this unexpected behavior is that btd provokes a 'naïve' cell state in which the previous commitment for en activity is lost. Later, en activity is re-established. This phenomenon may reflect the process that occurs in normal development. The initial btd/Sp1 domain may not inherit the previous developmental commitments and has to build a new developmental program. It is worth considering that the btd/Sp1function appears to determine ventral imaginal fate as different from larval fate. This is a major developmental decision, which may require de novo establishment of the genetic system responsible for pattern and growth. A key aspect of this would be the localized activation of en in part of the btd/Sp1 domain. It is speculated that there might be a short-range signal, perhaps Hh, emanating from nearby en-expressing embryonic cells, that acts as an inducer in the btd/Sp1 primordium. There is evidence that Hh can induce en activity (Estella, 2003).

The origins of the Drosophila leg revealed by the cis-regulatory architecture of the Distalless gene

Limb development requires the elaboration of a proximodistal (PD) axis, which forms orthogonally to previously defined dorsoventral (DV) and anteroposterior (AP) axes. In arthropods, the PD axis of the adult leg is subdivided into two broad domains, a proximal coxopodite and a distal telopodite. This study shows that the progressive subdivision of the PD axis into these two domains occurs during embryogenesis and is reflected in the cis-regulatory architecture of the Distalless (Dll) gene. Dll protein in the thorax was first detected during embryonic stage 11, and continues to be visualized in this region until the end of embryogenesis. Early Dll expression, governed by the Dll304 enhancer, is in cells that can give rise to both domains of the leg as well as to the entire dorsal (wing) appendage. A few hours after Dll304 is activated, the activity of this enhancer fades, and two later-acting enhancers assume control over Dll expression. The LT enhancer is expressed in cells that will give rise to the entire telopodite, and only the telopodite. By contrast, cells that activate the DKO ("Distalless Keilin Organ") enhancer will give rise to a leg-associated larval sensory structure known as the Keilin's organ (KO). Cells that activate neither LT nor DKO, but had activated Dll304, will give rise to the coxopodite. In addition, the trans-acting signals controlling the LT and DKO enhancers are described; surprisingly, the coxopodite progenitors begin to proliferate ~24 hours earlier than the telopodite progenitors. Together, these findings provide a complete and high-resolution fate map of the Drosophila appendage primordia, linking the primary domains to specific cis-regulatory elements in Dll (McKay, 2009).

To determine how each of the cell fates in the limb primordia is specified, genetic experiments were carried out to identify the regulators of the LT and DKO enhancers. Consistent with LT's dependency on wg and dpp for leg disc expression, LT is activated in the embryo in cells that receive both inputs, as monitored by anti-Wg and anti-PMad staining. To determine whether wg is required for LT activity, a temperature-sensitive allele of wg was used to allow earlier Dll activation. Switching the embryos to the restrictive temperature at stage 11 resulted in the absence of LT activity, despite the presence of Dll protein (probably derived from Dll304 activity. In addition, ectopic activation of the wg pathway [using an activated form of armadillo (arm^*)] resulted in more LT-lacZ-expressing cells (McKay, 2009).

Like wg, the dpp pathway is necessary for LT-lacZ expression in leg discs. Paradoxically, dpp signaling represses Dll in the embryo because dpp mutants show an expansion in Dll304-lacZ expression. By contrast, LT-lacZ is not expressed in dpp null embryos. LT-lacZ, but not Dll protein, was also repressed by two dpp pathway repressors, Dad and brk. Conversely, stimulation of the dpp pathway [using an activated form of the Dpp receptor (Tkv^QD)] resulted in ectopic activation of LT ventrally (McKay, 2009).

Taken together, these data demonstrate that LT is activated by Wg and Dpp in the embryonic limb primordia, just as it (and Dll) is in the leg disc. Similarly, DKO activity also requires Wg and Dpp input (McKay, 2009).

Although LT is activated by wg and dpp in the leg primordia, these signals are also present in each abdominal segment. Consequently, there must be additional factors that restrict LT activity to the thorax. One possibility is that LT is repressed by the abdominal Hox factors, such as Dll304. Alternatively, LT might be regulated by Dll, itself. In Dll null embryos LT-lacZ was initially expressed in a stripe of cells instead of a ring, but then expression decayed. Ectopic expression of Dll resulted in weak ectopic expression of LT-lacZ in the thorax and abdomen. These data suggest that LT activity is restricted to the thorax in part because of the earlier restriction of Dll304 activity to the thorax (McKay, 2009).

The related zinc-finger transcription factors encoded by buttonhead (btd) and Sp1 are also expressed in the limb primordia and are also required for ventral appendage specification. In strong btd hypomorphs, the activity of LT was still detected but the number of cells expressing LT-lacZ was decreased and its pattern was disrupted. LT-lacZ expression was completely eliminated in animals bearing a large deficiency that removes both btd and Sp1. By contrast, Dll304 was activated normally in these animals (data not shown). Importantly, LT-lacZ expression was rescued by expressing btd in these deficiency embryos. By contrast, expressing Dll, tkv^QD, or arm^* did not rescue LT expression in these deficiency embryos. Ectopic expression of btd resulted in weak ectopic activation of LT-lacZ in cells of the thorax and abdomen. Strikingly, the simultaneous expression of Dll and btd resulted in robust ectopic expression of LT-lacZ in abdominal segments in the equivalent ventrolateral position as the thoracic limb primordia. btd and Dll were not sufficient to activate LT in wg null embryos (data not shown). These data indicate that the thoracic-specific expression of the LT enhancer is controlled by the combined activities of btd and/or Sp1, Dll and the wg and dpp pathways (McKay, 2009).

Although the data suggest that LT is activated by a combination of Wg, Dpp, Btd and Dll, these activators are also present in the precursors of the KO, which activate DKO instead of LT. Because the KO is a sensory structure, the role of members of the achaete-scute complex (ASC) that are expressed in these cells was tested. In embryos hemizygous for a deficiency that removes the achaete-scute complex, LT-lacZ expression was expanded at the expense of the Ct-expressing cells. Consistently, ectopic expression of the ASC gene asense (ase) repressed LT and increased the number of Ct-expressing cells. These data suggest that there is a mutual antagonism between the progenitors of the telopodite and those of the KO. It was also found that DKO-lacZ expression in the leg primordia was lost in Dll or btd null embryos, consistent with the loss of KOs in these mutants. DKO activity was also lost from the limb primordia in embryos deficient for the ASC. These results indicate that DKO is activated by the same genes that promote LT expression but, in addition, requires proneural input from the ASC (McKay, 2009).

One of the most interesting findings from this work is that the temporal control of Dll expression in the limb primordia by three cis-regulatory elements is linked to cell-type specification. The earliest acting element, Dll304, is active throughout the appendage primordia. At the time Dll304 is active, the cells are multipotent and can give rise to any part of the dorsal or ventral appendages, or KO. A few hours later, Dll304 activity fades, and two alternative cis-regulatory elements become active. Together, these two elements allow for the uninterrupted and uniform expression of Dll within the appendage primordia. However, their activation correlates with a higher degree of refinement in cell fate potential: LT, active in only the outer ring of the appendage primordia, is only expressed in the progenitors of the telopodite. By contrast, DKO, active in the cells within the LT ring, is only expressed in the progenitors of the KO. Thus, although the pattern of Dll protein appears unchanged, the control over Dll expression has shifted from singular control by Dll304 to dual control by LT and DKO. Moreover, not only is there a molecular handoff from Dll304 to LT and DKO, the two later enhancers both require the earlier expression of Dll. Thus, the logic of ventral primordia refinement depends on a cascade of Dll regulatory elements in which the later ones depend on the activity of an earlier one (McKay, 2009).

The high-resolution view of the embryonic limb primordia provided in this study allows clarification of some contradictions that currently exist in the literature. Initial expression of Dll in the thorax overlaps entirely with Hth-nExd (referring to nuclear Extradenticle). Subsequently, hth expression is lost from most, but not all, of the Dll-expressing cells of the leg primordia. The first reports describing these changes failed to recognize the persistent overlap between Dll and Hth-nExd in some cells. As a result, and partly because of the analogy with the third instar leg disc, the predominant view of this fate map became that the Dll-positive, Hth-nExd-negative cells of the embryonic primordia gave rise to the telopodite, while the surrounding Hth-positive cells gave rise to the coxopodite. The expression pattern of esg, a gene required for the maintenance of diploidy, was also misinterpreted as being a marker exclusively of proximal leg fates. Counter to these earlier studies, the current experiments unambiguously show that the Dll-positive, Hth-nExd-negative cells in the center of the primordia give rise to the KO, the ring of Dll-positive, Esg-positive, Hth-nExd-positive cells gives rise to the telopodite, and the remaining Esg-positive, Dll-negative cells give rise to the coxopodite (McKay, 2009).

The spurious expression of DKO-lacZ in Dll-non-expressing cells outside the leg primorida complicates the interpretation of several experiments. Attempts to refine DKO activity by changing the size of the cloned fragment proved unsuccessful. Nevertheless, the evidence supports the idea that DKO-positive, Dll-positive cells of the leg primordia give rise to the Keilin's organ, and not the adult appendage (McKay, 2009).

The progenitors of the coxopodite begin to proliferate at approximately 48 hours of development, consistent with previous measurements of leg imaginal disc growth, whereas the progenitors of the telopodite do not resume proliferating for an additional 12 to 24 hours. According to estimates of the cell cycle time in leg discs, this difference in the onset of proliferation results in one to two additional cell divisions in the coxopodite, consistent with images of late second instar leg discs presented in this study. Why might the telopodite and coxopodite begin proliferation at different times? One possibility is that the cells of the coxopodite give rise to the peripodial epithelium that covers the leg imaginal disc, and therefore require additional cell divisions relative to the telopodite. It is also possible that the telopodite is delayed because the neurons of the Keilin's organ serve a pathfinding role for larval-born neurons that innervate the adult limb. Perhaps this pathfinding function requires that the KO and telopodite remain associated with each other through the second instar. Consistently, the leg is the only imaginal disc that has not invaginated as a sac-like structure in newly hatched first instar larvae (McKay, 2009).

A possible explanation for the delay in the onset of telopodite proliferation is the persistent co-expression of hth and Dll in these cells; hth (and tsh) expression is turned off in these cells at about the same time they begin to proliferate. Consistent with this idea, maintaining the expression of hth throughout the primordia blocks the proliferation of the telopodite. Also noteworthy is the finding that the genes no ocelli and elbow have been shown to mediate the ability of Wg and Dpp to repress coxopodite fates. Together with the current findings, it is possible that the activation of these two genes in the LT-expressing progenitors is the trigger that turns off hth and tsh in these cells (McKay, 2009).

The experiments suggest that once LT is activated, and under normal growth conditions, there is a lineage restriction between the telopodite and coxopodite. By contrast, previous lineage-tracing experiments using tsh-Gal4 concluded that the progeny of proximal cells could adopt more distal leg fates. However, tsh is still expressed in the telopodite progenitors far into the second instar, providing an explanation for these results. In contrast to this early restriction, there is no evidence for a later lineage restriction within the telopodite. For example, the progeny of a Dll-positive cell can lose Dll expression and contribute to the dac-only domain (McKay, 2009).

Interestingly, the lineage restriction between coxopodite and telopodite is not defined by the presence or absence of Hth-nExd or Tsh because both progenitor populations express hth and tsh after their fates have been specified. By contrast, when these two domains are specified, the telopodite expresses Dll, while the coxopodite does not, suggesting that Dll may be important for the lineage restriction. However, later in development, some cells in the telopodite lose Dll expression and express dac, but continue to respect the coxopodite-telopodite boundary. Thus, either Dll expression in the telopodite is somehow remembered or the telopodite-coxopodite boundary can be maintained by dac, which is expressed in place of Dll immediately adjacent to the telopodite-coxopodite boundary. Also noteworthy is the finding that clones originating in the coxopodite can contribute to the trochanter, the segment inbetween the proximal and distal components of the adult leg that expresses both Dll and hth in third instar imaginal discs. However, the progeny of such clones do not contribute to fates more distal than the trochanter. Likewise, a clone originating in the telopodite can also contribute to the trochanter, but will not grow more proximally into the coxa. Thus, the lineage restriction uncovered here seems to be determined by distinct combinations of transcription factors expressed in the coxopodite and telopodite progenitors at stage 14. The progeny of cells that express Dll, tsh and hth can populate the telopodite or trochanter, whereas the progeny of cells that express tsh and hth, but not Dll, can populate the coxopodite or trochanter. In light of Minute-positive results, however, the lineage restriction between coxopodite and telopodite does not satisfy the classical definition of a compartment boundary. A similar non-compartment lineage restriction has also been documented along the PD axis of the developing Drosophila wing (McKay, 2009).

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date revised: 20 February 2010

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

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