Spatial and temporal regulation of the homeotic selector gene Antennapedia is required for the establishment of leg identity in Drosophila

Antennapedia is one of the homeotic selector genes required for specification of segment identity in Drosophila. Dominant mutations that ectopically express Antennapedia cause transformation of antenna to leg. Loss-of-function mutations cause partial transformation of leg to antenna. Here we examine the role of Antennapedia in the establishment of leg identity in light of recent advances in our understanding of antennal development. In Antennapedia mutant clones in the leg disc, Homothorax and Distal-less are coexpressed and act via spineless to transform proximal femur to antenna. Antennapedia is negatively regulated during leg development by Distal-less, spineless, and dachshund and this reduced Antennapedia expression is needed for the proper development of distal leg elements. These findings suggest that the temporal and spatial regulation of the homeotic selector gene Antennapedia in the leg disc is necessary for normal leg development in Drosophila.     

Control of the spineless antennal enhancer: Direct repression of antennal target genes by Antennapedia

It is currently thought that antennal target genes are activated in Drosophila by the combined action of Distal-less, homothorax, and extradenticle, and that the Hox gene Antennapedia prevents activation of antennal genes in the leg by repressing homothorax. To test these ideas, we analyze a 62 bp enhancer from the antennal gene spineless that is specific for the third antennal segment. This enhancer is activated by a tripartite complex of Distal-less, Homothorax, and Extradenticle. Surprisingly, Antennapedia represses the enhancer directly, at least in part by competing with Distal-less for binding. We show that Antennapedia is required in the leg only within a proximal ring that coexpresses Distal-less, Homothorax and Extradenticle. We conclude that the function of Antennapedia in the leg is not to repress homothorax, as has been suggested, but to directly repress spineless and other antennal genes that would otherwise be activated within this ring.     

Early development of leg and wing primordia in the Drosophila embryo

The development of the leg and wing primordia in the Drosophila embryo has been traced using molecular markers. Distal-less and disconnected gene expression provide molecular labels for the leg primordia throughout embryonic development, disconnected expression in the developing leg primordia depends on Distal-less activity. The leg primordia arise as discrete clusters of cells that occupy well defined positions in the embryonic ectoderm. At later stages of embryogenesis the primordia become morphologically recognizable and are intimately associated with the development of the Keilin's organs. The presumptive leg disc and the Keilin's organ appear to derive from a common primordium. Similarly the Abnormal leg pattern gene provides a molecular label for the wing and haltere primordia. The dorsal thoracic primordia appear to be of independent origin from the legs.     

Coexpression of the homeobox genes Distal-less and homothorax determines Drosophila antennal identity

The Distal-less gene is known for its role in proximodistal patterning of Drosophila limbs. However, Distal-less has a second critical function during Drosophila limb development, that of distinguishing the antenna from the leg. The antenna-specifying activity of Distal-less is genetically separable from the proximodistal patterning function in that certain Distal-less allelic combinations exhibit antenna-to-leg transformations without proximodistal truncations. Here, we show that Distal-less acts in parallel with homothorax, a previously identified antennal selector gene, to induce antennal differentiation. While mutations in either Distal-less or homothorax cause antenna-to-leg transformations, neither gene is required for the others expression, and both genes are required for antennal expression of spalt. Coexpression of Distal-less and homothorax activates ectopic spalt expression and can induce the formation of ectopic antennae at novel locations in the body, including the head, the legs, the wings and the genital disc derivatives. Ectopic expression of homothorax alone is insufficient to induce antennal differentiation from most limb fields, including that of the wing. Distal-less therefore is required for more than induction of a proximodistal axis upon which homothorax superimposes antennal identity. Based on their genetic and biochemical properties, we propose that Homothorax and Extradenticle may serve as antenna-specific cofactors for Distal-less.     

Correlation of expression patterns of homothorax, dachshund, and Distal-less with the proximodistal segmentation of the cricket leg bud

We describe the expression pattern of Gryllus homothorax (Gbhth) and dachshund (Gbdac), a cricket homologue of Drosophila homothorax and dachshund, together with localization of Distal-less or Extradenticle protein during leg development. We correlated their expression patterns with the morphological segmentation of the leg bud. The boundary of Gbhth/GbDll subdivision is correlated with the segment boundary of the future trochanter/femur at early stages. Gbdac expression subdivides the leg bud into the presumptive femur and more distal region. During the leg proximodistal formation, although the early expression patterns of GbDll, Gbdac, and Gbhth significantly differ from those of Drosophila imaginal disc, their expression patterns in the fully segmented Gryllus leg were similar to those in the Drosophila late third instar disc.     

Fate map of the distal portion of Drosophila proboscis as inferred from the expression and mutations of basic patterning genes

The late-third-instar labial disc is comprised of two disc-proper cell layers, one representing mainly the ventral half of the anterior compartment (L-layer) and the other, the dorsal half of the anterior compartment and most, if not all, of the posterior compartment (M-layer). In the L-layer, Distal-less represses homothorax whereas no Distal-less-dependent homothorax repression occurs in the M-layer where Distal-less is coexpressed with homothorax. In wild-type labial discs, clawless, one of the two homeobox genes expressed in distal cells receiving maximum (Decapentaplegic+Wingless) signaling activity in leg and antennal discs, is specifically repressed by proboscipedia. A fate map, inferred from data on basic patterning gene expression in larval and pupal stages and mutant phenotypes, indicates the inner surface of the labial palpus, which includes the pseudotracheal region, to be a derivative of the distal portion of the M-layer expressing wingless, patched, Distal-less and homothorax. The outer surface of the labial palpus with more than 30 taste bristles derives from an L-layer area consisting of dorsal portions of the anterior and posterior compartments, each expressing Distal-less. Our analysis also indicates that, in adults and pupae, the anterior-posterior boundary, dividing roughly equally the outer surface of the distiproboscis, runs along the outer circumference of the inner surface of distiproboscis.     

Functional analyses of tiptop and antennapedia in the embryonic development of Oncopeltus fasciatus suggests an evolutionary pathway from ground state to insect legs

In insects, selector genes are thought to modify the development of a default, or 'ground state', appendage into a tagma-specific appendage such as a mouthpart, antenna or leg. In the best described example, Drosophila melanogaster, the primary determination of leg identity is thought to result from regulatory interactions between the Hox genes and the antennal-specifying gene homothorax. Based on RNA-interference, a functional analysis of the selector gene tiptop and the Hox gene Antennapedia in Oncopeltus fasciatus embryogenesis is presented. It is shown that, in O. fasciatus, tiptop is required for the segmentation of distal leg segments and is required to specify the identity of the leg. The distal portions of legs with reduced tiptop develop like antennae. Thus, tiptop can act as a regulatory switch that chooses between antennal and leg identity. By contrast, Antennapedia does not act as a switch between leg and antennal identity. This observation suggests a significant difference in the mechanism of leg specification between O. fasciatus and D. melanogaster. These observations also suggest a significant plasticity in the mechanism of leg specification during insect evolution that is greater than would have been expected based on strictly morphological or molecular comparisons. Finally, it is proposed that a tiptop-like activity is a likely component of an ancestral leg specification mechanism. Incorporating a tiptop-like activity into a model of the leg-specification mechanism explains several mutant phenotypes, previously described in D. melanogaster, and suggests a mechanism for the evolution of legs from a ground state.     

Evidence for canalization of Distal-less function in the leg of Drosophila melanogaster

A considerable body of theory pertaining to the evolution of canalization has emerged recently, yet there have been few empirical investigations of their predictions. To address this, patterns of canalization and trait correlation were investigated under the individual and joint effects of the introgression of a loss-of-function allele of the Distal-less gene and high-temperature stress on a panel of iso-female lines. Variation was examined for number of sex comb teeth and the length of the basi-tarsus on the pro-thoracic leg of male Drosophila melanogaster. I demonstrate that whereas there is evidence for trait canalization, there is no evidence to support the hypothesis of the evolution of genetic canalization as a response to microenvironmental canalization. Furthermore, I demonstrate that although there are genetic correlations between these traits, there is no association between their measures of canalization. I discuss the prospects of the evolutionary lability of the Distal-less gene within the context of changes in genetic variation and covariation.     

Limb type-specific regulation of bric a brac contributes to morphological diversity

The insect antenna and leg are considered homologous structures, likely to have arisen via duplication and divergence from an ancestral limb. Consistent with this, the antenna and leg are derived from primordia with similar developmental potentials. Nonetheless, the adult structures differ in both form and function. In Drosophila, one conspicuous morphological difference is that the antenna has fewer distal segments than the leg. We propose that this is due in part to the variations in the regulation of bric a brac. bric a brac is required for joint formation, and loss of bric a brac function leads to fusion of distal antennal and leg segments, resulting in fewer total segments. Here, we address how bric a brac is regulated to generate the mature expression patterns of two concentric rings in the antenna versus four concentric rings in the leg. We find that bric a brac expression is activated early throughout most of the Distal-less domain in both antenna and leg and subsequently is restricted to the distal portion and into rings. Although bric a brac expression in the antenna and in all four tarsal rings of the leg requires Distal-less, only the proximal three tarsal rings are Spineless-dependent. Thus bric a brac is regulated differentially even within a single appendage type. The restriction of bric a brac expression to the distal portion of the Distal-less domain is a consequence of negative regulation by distinct sets of genes in different limb types. In the leg, the proximal boundary of bric a brac is established by the medial-patterning gene dachshund, but dachshund alone is insufficient to repress bric a brac, and the expression of the two genes overlaps. In the antenna, the proximal boundary of bric a brac is established by an antenna-specifying gene, homothorax, in conjunction with dachshund and spalt, and there is much less overlap between the bric a brac and the dachshund domains. Thus tissue-specific expression of other patterning genes that differentially repress bric a brac accounts for antenna-leg differences in bric a brac pattern. We propose that the limb type-specific variations in expression of bric a brac repressors contribute to morphological variations by controlling distal limb segment number.     

A complex role for distal-less in crustacean appendage development.

The developing leg of Drosophila is initially patterned by subdivision of the leg into proximal and distal domains by the activity of the homeodomain proteins Extradenticle (Exd) and Distal-less (Dll). These early domains of gene expression are postulated to reflect a scenario of limb evolution in which an undifferentiated appendage outgrowth was subdivided into two functional parts, the coxapodite and telopodite. The legs of most arthropods have a more complex morphology than the simple rod-shaped leg of Drosophila. We document the expression of Dll and Exd in two crustacean species with complex branched limbs. We show that in these highly modified limbs there is a Dll domain exclusive of Exd but there is also extensive overlap in Exd and Dll expression. While arthropod limbs all appear to have distinct proximal and distal domains, those domains do not define homologous structures throughout arthropods. In addition, we find a striking correlation throughout the proximal/distal extent of the leg between setal-forming cells and Dll expression. We postulate that this may reflect a pleisiomorphic function of Dll in development of the peripheral nervous system. In addition, our results confirm previous observations that branch formation in multiramous arthropod limbs is not regulated by a simple iteration of the proximal/distal patterning module employed in Drosophila limb development.     

The Hox gene Abdominal-B antagonizes appendage development in the genital disc of Drosophila

In Drosophila, the Hox gene Abdominal-B is required to specify the posterior abdomen and the genitalia. Homologues of Abdominal-B in other species are also needed to determine the posterior part of the body. We have studied the function of Abdominal-B in the formation of Drosophila genitalia, and show here that absence of Abdominal-B in the genital disc of Drosophila transforms male and female genitalia into leg or, less frequently, into antenna. These transformations are accompanied by the ectopic expression of genes such as Distal-less or dachshund, which are normally required in these appendages. The extent of wild-type and ectopic Distal-less expression depends on the antagonistic activities of the Abdominal-B gene, as a repressor, and of the decapentaplegic and wingless genes as activators. Absence of Abdominal-B also changes the expression of Homothorax, a Hox gene co-factor. Our results suggest that Abdominal-B forms genitalia by modifying an underlying positional information and repressing appendage development. We propose that the genital primordia should be subdivided into two regions, one of them competent to be transformed into an appendage in the absence of Abdominal-B.     

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

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

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

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