Distal antenna and distal antenna related encode nuclear proteins containing pipsqueak motifs involved in antenna development in Drosophila

Legs and antennae are considered to be homologous appendages. The fundamental patterning mechanisms that organize spatial pattern are conserved, yet appendages with very different morphology develop. A genetic hierarchy for specification of antennal identity has been partly elucidated. We report identification of a novel family of genes with roles in antennal development. The distal antenna (dan) and distal antenna-related (danr) genes encode novel nuclear proteins that are expressed in the presumptive distal antenna, but not in the leg imaginal disc. Ectopic expression of dan or danr causes partial transformation of distal leg structure toward antennal identity. Mutants that remove dan and danr activity cause partial transformation of antenna toward leg identity. Therefore we suggest that dan and danr contribute to differentiation of antenna-specific characteristics. Antenna-specific expression of dan and danr depends on a regulatory hierarchy involving homothorax and Distal-less, as well as cut and spineless. We propose that dan and danr are effector genes that act downstream of these genes to control differentiation of distal antennal structures.     

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.     

The pleiohomeotic gene is required for maintaining expression of genes functioning in ventral appendage formation in Drosophila melanogaster

Polycomb group (PcG) proteins are negative regulators that maintain the expression of homeotic genes and affect cell proliferation. Pleiohomeotic (Pho) is a unique PcG member with a DNA-binding zinc finger motif and was proposed to recruit other PcG proteins to form a complex. The pho null mutants exhibited several mutant phenotypes such as the transformation of antennae to mesothoracic legs. We examined the effects of pho on the identification of ventral appendages and proximo-distal axis formation during postembryogenesis. In the antennal disc of the pho mutant, Antennapedia (Antp), which is a selector gene in determining leg identity, was ectopically expressed. The homothorax (hth), dachshund (dac) and Distal-less (Dll) genes involved in proximo-distal axis formation were also abnormally expressed in both the antennal and leg discs of the pho mutant. The engrailed (en) gene, which affects the formation of the anterior-posterior axis, was also misexpressed in the anterior compartment of antennal and leg discs. These mutant phenotypes were enhanced in the mutant background of Posterior sex combs (Psc) and pleiohomeotic-like (phol), which are another PcG genes. These results suggest that pho functions in maintaining expression of genes involved in the formation of ventral appendages and the proximo-distal axis.     

Leg development in flies versus grasshoppers: differences in dpp expression do not lead to differences in the expression of downstream components of the leg patterning pathway

All insect legs are structurally similar, characterized by five primary segments. However, this final form is achieved in different ways. Primitively, the legs developed as direct outgrowths of the body wall, a condition retained in most insect species. In some groups, including the lineage containing the genus Drosophila, legs develop indirectly from imaginal discs. Our understanding of the molecular mechanisms regulating leg development is based largely on analysis of this derived mode of leg development in the species D. melanogaster. The current model for Drosophila leg development is divided into two phases, embryonic allocation and imaginal disc patterning, which are distinguished by interactions among the genes wingless (wg), decapentaplegic (dpp) and distalless (dll). In the allocation phase, dll is activated by wg but repressed by dpp. During imaginal disc patterning, dpp and wg cooperatively activate dll and also indirectly inhibit the nuclear localization of Extradenticle (Exd), which divide the leg into distal and proximal domains. In the grasshopper Schistocerca americana, the early expression pattern of dpp differs radically from the Drosophila pattern, suggesting that the genetic interactions that allocate the leg differ between the two species. Despite early differences in dpp expression, wg, Dll and Exd are expressed in similar patterns throughout the development of grasshopper and fly legs, suggesting that some aspects of proximodistal (P/D) patterning are evolutionarily conserved. We also detect differences in later dpp expression, which suggests that dpp likely plays a role in limb segmentation in Schistocerca, but not in Drosophila. The divergence in dpp expression is surprising given that all other comparative data on gene expression during insect leg development indicate that the molecular pathways regulating this process are conserved. However, it is consistent with the early divergence in developmental mode between fly and grasshopper limbs.     

Homologs of wingless and decapentaplegic display a complex and dynamic expression profile during appendage development in the millipede Glomeris marginata (Myriapoda: Diplopoda)

BACKGROUND: The Drosophila genes wingless (wg) and decapentaplegic (dpp) comprise the top level of a hierarchical gene cascade involved in proximal-distal (PD) patterning of the legs. It remains unclear, whether this cascade is common to the appendages of all arthropods. Here, wg and dpp are studied in the millipede Glomeris marginata, a representative of the Myriapoda. RESULTS: Glomeris wg (Gm-wg) is expressed along the ventral side of the appendages compatible with functioning during the patterning of both the PD and dorsal-ventral (DV) axes. Gm-wg may also be involved in sensory organ formation in the gnathal appendages by inducing the expression of Distal-less (Dll) and H15 in the organ primordia. Expression of Glomeris dpp (Gm-dpp) is found at the tip of the trunk legs as well as weakly along the dorsal side of the legs in early stages. Taking data from other arthropods into account, these results may be interpreted in favor of a conserved mode of WG/DPP signaling. Apart from the main PD axis, many arthropod appendages have additional branches (e.g. endites). It is debated whether these extra branches develop their PD axis via the same mechanism as the main PD axis, or whether branch-specific mechanisms exist. Gene expression in possible endite homologs in Glomeris argues for the latter alternative. CONCLUSION: All available data argue in favor of a conserved role of WG/DPP morphogen gradients in guiding the development of the main PD axis. Additional branches in multibranched (multiramous) appendage types apparently do not utilize the WG/DPP signaling system for their PD development. This further supports recent work on crustaceans and insects, that lead to similar conclusions.     

Timing of Wingless signalling distinguishes maxillary and antennal identities in Drosophila melanogaster

The Drosophila adult head mostly derives from the composite eye-antenna imaginal disc. The antennal disc gives rise to two adult olfactory organs: the antennae and maxillary palps. Here, we have analysed the regional specification of the maxillary palp within the antennal disc. We found that a maxillary field, defined by expression of the Hox gene Deformed, is established at about the same time as the eye and antennal fields during the L2 larval stage. The genetic program leading to maxillary regionalisation and identity is very similar to the antennal one, but is distinguished primarily by delayed prepupal expression of the ventral morphogen Wingless (Wg). We find that precociously expressing Wg in the larval maxillary field suffices to transform it towards antennal identity, whereas overexpressing Wg later in prepupae does not. These results thus indicate that temporal regulation of Wg is decisive to distinguishing maxillary and antennal organs. Wg normally acts upstream of the antennal selector spineless (ss) in maxillary development. However, mis-expression of Ss can prematurely activate wg via a positive-feedback loop leading to a maxillary-to-antenna transformation. We characterised: (1) the action of Wg through ss selector function in distinguishing maxillary from antenna; and (2) its direct contribution to identity choice.     

Functional stability of the aristaless gene in appendage tip formation during evolution

The appendages of an insect are subdivided into distinct segments or podomeres. Many genes responsible for the regionalization of the growing limb into subdomains have been isolated from Drosophila. So far, only one gene is known in the leg that is solely required for specifying the distal-most pattern element—the pretarsal claw. In Drosophila, the gene aristaless is expressed in the centre of the antennal and leg imaginal disc that represents the most distal position of appendages, and in a proximal region. When Drosophila aristaless function is impaired, antennae and legs develop without their distal-most structures—the arista and the claw. We describe here the analysis of aristaless in the beetle Tribolium—an insect that shows a different, more ancestral mode of appendage formation than Drosophila. In Tribolium, appendages grow out continuously during embryogenesis, and no imaginal discs are formed. Tribolium aristaless (Tc-al) expression starts midway during appendage elongation, and is seen in a distal and a proximal position of head and trunk appendages. At the end of embryogenesis, Tc-al is seen in four expression domains in the leg, in the dorsal epidermis, and ventrally in every segment in lateral groups of cells, presumably the histoblasts. Like in the Drosophila adult, Tc-al is required in the larva for the formation of the most distal structures of the leg and the antenna as revealed by RNAi experiments. We conclude that aristaless is evolutionarily robust, meaning that it has retained its expressional and functional characteristics, although a heterochronic change of the process of appendage elongation took place towards the evolution of the highly derived diptera.Edited by D. Tautz     

Pattern formation in imaginal discs of Drosophila melanogaster after irradiation of embryos and young larvae

Embryos and first instar larvae of Drosophila melanogaster were X-irradiated in order to study pattern formation in discs damaged at early stages. After treatment, appendages were found in which some pattern elements were duplicated and others were absent. In some strains legs were preferentially duplicated, and in others, antennae were preferentially duplicated. Duplicated appendages were mirror images and resulted most frequently when animals were irradiated during late embryonic or early larval stages. Appendages varied from those which showed complete duplication of only the distal parts (claws or aristae), to those which showed duplication of almost the entire appendage. Examination of the cuticular patterns in duplicated mesothoracic legs showed that in legs with complete duplication solely in distal regions, only extreme lateral leg parts were duplicated, and medial leg parts were absent. In legs with duplication extending into proximal regions, much of the lateral side was duplicated, and only extreme medial parts were missing. The situation for partially duplicated antennae was similar. Prothoracic legs were found fused in some X-rayed flies. The cuticular patterns were almost perfect mirror images, although the amount of fusion varied widely between different individuals. Apparently the pattern forming processes in the right and left first leg discs are coordinated in X-rayed animals. The results were consistent with a model embracing a gradient of developmental capacity in the early disc similar to that postulated to exist in the late third instar leg disc. This model is also consistent with results of various surgical experiments reported in the literature. Several predictions of the model are explained including the possible mode of action of a specific class of mutants which may affect pattern formation by altering a morphogenetic gradient.     

Projection of sensory neurons from a homeotic mutant appendage,Antennapedia, in Drosophila melanogaster

Central projections of sensory neurons from homeotic mutant appendages (Antennapedia) of Drosophila melanogaster were compared with those of wild-type antennae and wild-type legs by means of degeneration and cobalt backfilling methods. Sensory axons originating from wild-type thoracic legs terminate within the ventral ipsilateral half of the corresponding neuropile segment and do not project to the brain. Sensory fibers from the third antennal segment (AIII) of wild-type animals project into the ipsilateral antennal glomerulus (AG) and to a lesser extent into the contralateral AG, whereas those from the second antennal segment terminate principally within the ipsilateral posterior antennal center. The sensory terminals of femur, tibia, and tarsi of the homeotic leg show a distribution very similar to that of the homologous wild-type antennal segment AIII, differing to a minor degree only in the size and precise localization of terminals within the antennal glomeruli. No degenerating axons were evident in ultrastructural examination of neck connectives after removal of homeotic legs. It is thus very improbable that any sensory fibers of the homeotic leg project to normal leg projection areas in the thoracico-abdominal ganglion. Several alternative explanations are offered for the apparent retention of antennal specificity by axons from the transformed appendage.     

Correlation of diversity of leg morphology in Gryllus bimaculatus (cricket) with divergence in dpp expression pattern during leg development

Insects can be grouped into mainly two categories, holometabolous and hemimetabolous, according to the extent of their morphological change during metamorphosis. The three thoracic legs, for example, are known to develop through two overtly different pathways: holometabolous insects make legs through their imaginal discs, while hemimetabolous legs develop from their leg buds. Thus, how the molecular mechanisms of leg development differ from each other is an intriguing question. In the holometabolous long-germ insect, these mechanisms have been extensively studied using Drosophila melanogaster. However, little is known about the mechanism in the hemimetabolous insect. Thus, we studied leg development of the hemimetabolous short-germ insect, Gryllus bimaculatus (cricket), focusing on expression patterns of the three key signaling molecules, hedgehog (hh), wingless (wg) and decapentaplegic (dpp), which are essential during leg development in Drosophila. In Gryllus embryos, expression of hh is restricted in the posterior half of each leg bud, while dpp and wg are expressed in the dorsal and ventral sides of its anteroposterior (A/P) boundary, respectively. Their expression patterns are essentially comparable with those of the three genes in Drosophila leg imaginal discs, suggesting the existence of the common mechanism for leg pattern formation. However, we found that expression pattern of dpp was significantly divergent among Gryllus, Schistocerca (grasshopper) and Drosophila embryos, while expression patterns of hh and wg are conserved. Furthermore, the divergence was found between the pro/mesothoracic and metathoracic Gryllus leg buds. These observations imply that the divergence in the dpp expression pattern may correlate with diversity of leg morphology.     

Patterning of the branched head appendages in Schistocerca americana and Tribolium castaneum

Much of our understanding of arthropod limb development comes from studies on the leg imaginal disc of Drosophila melanogaster. The fly limb is a relatively simple unbranched (uniramous) structure extending out from the body wall. The molecular basis for this outgrowth involves the overlap of two signaling molecules, Decapentaplegic (Dpp) and Wingless (Wg), to create a single domain of distal outgrowth, clearly depicted by the expression of the Distal-less gene (Dll). The expression of wg and dpp during the development of other arthropod thoracic limbs indicates that these pathways might be conserved across arthropods for uniramous limb development. The appendages of crustaceans and the gnathal appendages of insects, however, exhibit a diverse array of morphologies, ranging from those with no distal elements, such as the mandible, to appendages with multiple distal elements. Examples of the latter group include branched appendages or those that possess multiple lobes; such complex morphologies are seen for many crustacean limbs as well as the maxillary and labial appendages of many insects. It is unclear how, if at all, the known patterning genes for making a uniramous limb might be deployed to generate these diverse appendage forms. Experiments in Drosophila have shown that by forcing ectopic overlaps of Wg and Dpp signaling it is possible to generate artificially branched legs. To test whether naturally branched appendages form in a similar manner, we detailed the expression patterns of wg, dpp, and Dll in the development of the branched gnathal appendages of the grasshopper, Schistocerca americana, and the flour beetle, Tribolium castaneum. We find that the branches of the gnathal appendages are not specified through the redeployment of the Wg-Dpp system for distal outgrowth, but our comparative studies do suggest a role for Dpp in forming furrows between tissues.     

The control of antennal movements by leg proprioceptors in the rock lobster,Palinurus vulgaris

Summary The antennae of the rock lobster,Palinurus vulgaris, show systematic responses to movements of the legs on a tilting footboard. Myographic recordings in muscles of the first antennal segment have been used in an analysis of the sensory basis of these reactions. Antennal posture is modified in the experimental apparatus, although its relation to the change in loading conditions of the legs is uncertain. The motor control of the antennal equilibrium responses involves a complete reciprocation between both excitatory and inhibitory motoneurones to the antagonist muscle groups in the two antennae. Sensory inputs from single legs produce movements of both antennae, but a stronger drive ipsilaterally. Leg receptor inputs also modulate antennal resistance reflexes in a systematic manner, providing a sensitive test for the involvement of particular receptor organs in the leg. Movement at the coxo-basal leg joint is a major source of sensory input, and ablation/ stimulation experiments have established that stimulation of the CB chordotonal organ is a necessary but not sufficient condition to produce the antennal equilibrium reactions. The possibility is discussed that other receptors at the coxo-basal joint are also involved.D.M.N. was supported by a grant from The Max-Planck Institut to Professor H. Schöne.     

RNAi analysis of nubbin embryonic functions in a hemimetabolous insect,Oncopeltus fasciatus

SUMMARY Although the expression of the POU homeodomain gene nubbin (nub) has been examined in several arthropod species, its function has been studied only in Drosophila. Here, we provide the first insight into functional roles of this gene in a hemimetabolous insect species, Oncopeltus fasciatus. The analysis of its function using RNAi resulted in the altered morphology of antennae and labial tubes in the head, legs in the thorax, and, most notably, the growth of ectopic appendages originating from abdominal segments A2–A6. This change in the morphology of the abdomen can largely be attributed to the altered expression patterns of two hox genes, Ubx and abd‐A, in RNAinub embryos. First, abd‐A expression is completely abolished in A3–A6. Second, weak Ubx expression expands posteriorly to encompass novel domains in A2 and A3. Concomitant with these changes, limbs on A2 and A3 are small and less developed, whereas limbs on A4–A6 are large thoracic‐like legs. These results show that nub function is necessary for normal abd‐A expression and thus plays a critical role in suppressing leg formation on the abdomen. The loss of this regulation leads to upregulation of Distal‐less, and subsequent development of appendages. In Drosophila, however, abd‐A expression is unaffected in a nub‐depleted background, indicating that no such regulatory relationship exists between these two genes in the fruit fly. These differences reveal that variation exists in the genetic mechanisms that maintain an ancient insect feature, the limbless abdomen.     

Functional analyses in the hemipteran Oncopeltus fasciatus reveal conserved and derived aspects of appendage patterning in insects

The conservation of expression of appendage patterning genes, particularly Distal-less, has been shown in a wide taxonomic sampling of animals. However, the functional significance of this expression has been tested in only a few organisms. Here we report functional analyses of orthologues of the genes Distal-less, dachshund, and homothorax in the appendages of the milkweed bug Oncopeltus fasciatus (Hemiptera). This hemimetabolous insect has typical legs but highly derived mouthparts. Distal-less, dachshund, and homothorax are conserved in their individual expression patterns and functions in the legs of Oncopeltus, but their functions in other appendages are in some cases divergent. We find that specification of antennal identity does not require wild-type Distal-less activity in Oncopeltus as it does in Drosophila. Additionally, the mouthparts of Oncopeltus show novel patterns of gene expression and function, relative to other insects. Expression of Distal-less in the maxillary stylets of Oncopeltus does not seem necessary for proper development of this appendage, while dachshund and homothorax are crucial for formation of the mandibular and maxillary stylets. These data are used to evaluate hypotheses for the evolution of hemipteran mouthparts and the evolution of developmental mechanisms in insect appendages in general.     

Spatial regulation of DELTA expression mediates NOTCH signalling for segmentation of Drosophila legs.

The Notch (N) signalling pathway is recruited for segregation of cell fates in a number of Drosophila tissue types. We show here that N dependent segmentation of Drosophila legs is regulated by a dynamic pattern of expression of its ligand, DELTA (DL). During third larval instar and early stages of pupation, high levels of DL expression is seen in stripes of cells in the leg imaginal discs which later form the proximal borders of leg joints. These domains also displayed heightened Dl enhancer activity. During subsequent stages of pupation, following segmentation of the leg primordium, DL expression becomes uniform throughout these segments barring the joints. We further show that regulatory Dl mutations or mis-expression of DL abolish leg segmentation. Domains of N signalling for segmentation of legs of flies are thus set up by a stringent spatial regulation of expression of its ligand at the segment border. Further, a comparable role of DL in antennal development reveals a common paradigm of DL-N signalling for segmentation of appendages in flies.     

Pattern formation and determination in the antenna of the homoeotic mutant Antennapedia of Drosophila melanogaster

The development of the antenna in the antennal-leg homoeotic mutant Antennapedia (Antp^R) was investigated using somatic crossing-over to mark clones of cells in Antp^R antennal appendages. Antp^R antennae ranged from a nearly normal antenna to a nearly normal leg. The arrangement of clones of marked bristles and cuticle in the more antennalike antennae was similar to the wild type antenna, and that of the leglike antennae was similar to the wild-type leg. The contiguity of clones argued against extensive individual cell migration. The regions occupied by homoeotic leg varied considerably between different Antp^R antennae. Observation of Antp^R antennae in these phenotypic mosaics showed that specific leg parts replaced specific antennal parts. Even small groups of leg sensilla appeared only at precise locations in the antenna. These results suggest that homoeotic leg cells and antennal cells can both respond to the same positional information or prepattern. An analysis of clone size provided estimates for cell number in the Antp^R antenna. It was found that cell numbers in the wild-type and Antp^R antennae are about the same until the third instar, when the Antp^R cells start dividing more rapidly than wild type. Previous work had shown that clonal inheritance of a commitment for homoeotic leg also did not occur prior to the early third instar. It is suggested that determination for homoeotic leg occurs in the early third instar, and that thereafter this commitment is inherited by the progeny of the determined cells. The increase in growth rate is probably due to a faster growth rate in cells with a leg commitment than in cells with an antennal commitment. The results suggest that, once initiated, determination may be of two types—a clonally inherited determination (for example, to be homoeotic leg) and an environmental determination (for example, to be a specific part of a homoeotic leg). Clonal inheritance of determination in normal embryonic development and in sex determination in intersexes is discussed.