Antenna and all gnathal appendages are similarly transformed by homothorax knock-down in the cricket Gryllus bimaculatus

Our understanding of the developmental mechanisms underlying the vast diversity of arthropod appendages largely rests on the peculiar case of the dipteran Drosophila melanogaster. In this insect, homothorax (hth) and extradenticle (exd) together play a pivotal role in appendage patterning and identity. We investigated the role of the hth homologue in the cricket Gryllus bimaculatus by parental RNA interference. This species has a more generalized morphology than Oncopeltus fasciatus, the one other insect besides Drosophila where homothorax function has been investigated. The Gryllus head appendages represent the morphologically primitive state including insect-typical mandibles, maxillae and labium, structures highly modified or missing in Oncopeltus and Drosophila. We depleted Gb’hth function through parental RNAi to investigate its requirement for proper regulation of other appendage genes (Gb’wingless, Gb’dachshund, Gb’aristaless and Gb’Distalless) and analyzed the terminal phenotype of Gryllus nymphs. Gb’hth RNAi nymphs display homeotic and segmentation defects similar to hth mutants or loss-of-function clones in Drosophila. Intriguingly, however, we find that in Gb’hth RNAi nymphs not only the antennae but also all gnathal appendages are homeotically transformed, such that all head appendages differentiate distally as legs and proximally as antennae. Hence, Gb’hth is not specifically required for antennal fate, but fulfills a similar role in the specification of all head appendages. This suggests that the role of hth in the insect antenna is not fundamentally different from its function as cofactor of segment-specific homeotic genes in more posterior segments.     

Bmdelta phenotype implies involvement of Notch signaling in body segmentation and appendage development of silkworm,Bombyx mori

The domesticated silkworm, Bombyx mori, belongs to the intermediate germband insects, in which the anterior segments are specified in the blastoderm, while the remaining posterior segments are sequentially generated from the cellularized growth zone. The pattern formation is distinct from Drosophila but somewhat resembles a vertebrate. Notch signaling is involved in the segmentation of vertebrates and spiders.Here, we studied the function of Notch signaling in silkworm embryogenesis via RNA interference (RNAi). Depletion of Bmdelta, the homolog of the Notch signaling ligand, led to severe defects in segment patterning, including a loss of posterior segments and irregular segment boundaries. The paired appendages on each segment were symmetrically fused along the ventral midline in Bmdelta RNAi embryos. An individual segment seemed to possess only one segmental appendage. Segmentation in prolegs could be observed.Our results show that Notch signaling is employed in not only appendage development but also body segmentation. Thus, conservation of Notch-mediated segmentation could also be extended to holometabolous insects. The involvement of Notch signaling seems to be the ancestral segmentation mechanism of arthropods.     

Appendage patterning in the primitively wingless hexapods Thermobia domestica (Zygentoma: Lepismatidae) and Folsomia candida (Collembola: Isotomidae)

Arthropod appendages are among the most diverse animal organs and have been adapted to a variety of functions. Due to this diversity, it can be difficult to recognize homologous parts in different appendage types and different species. Gene expression patterns of appendage development genes have been used to overcome this problem and to identify homologous limb portions across different species and their appendages. However, regarding the largest arthropod group, the hexapods, most of these studies focused on members of the winged insects (Pterygota), but primitively wingless groups like the springtails (Collembola) or silverfish and allies (Zygentoma) are underrepresented. We have studied the expression of a set of appendage patterning genes in the firebrat Thermobia domestica and the white springtail Folsomia candida. The expressions of Distal-less (Dll) and dachshund (dac) are generally similar to the patterns reported for pterygote insects. Modifications of gene regulation, for example, the lack of Dll expression in the palp of F. candida mouthparts, however, point to changes in gene function that can make the use of single genes and specific expression domains problematic for homology inference. Such hypotheses should therefore not rely on a small number of genes and should ideally also include information about gene function. The expression patterns of homothorax (hth) and extradenticle (exd) in both species are similar to the patterns of crustaceans and pterygote insects, but differ from those in chelicerates and myriapods. The proximal specificity of hth thus appears to trace from a common hexapod ancestor and also provides a link to the regulation of this gene in crustaceans.     

The roles of wingless and decapentaplegic in axis and appendage development in the red flour beetle, Tribolium castaneum

Axis patterning and appendage development have been well studied in Drosophila melanogaster, a species in which both limb and segment morphogenesis are derived. In Drosophila, positional information from genes important in anteroposterior and dorsoventral axis formation, including wingless (wg) and decapentaplegic (dpp), is required for allocating and patterning the appendage primordia. We used RNA interference to characterize the functions of wg and dpp in the red flour beetle, Tribolium castaneum, which retains more ancestral modes of limb and segment morphogenesis. We also characterized the expression of potential targets of the WG and DPP signaling pathways in these embryos. Tribolium embryos in which dpp had been downregulated had defects in the dorsalmost body wall, but did not appear to have been globally repatterned and had normal appendages. Downregulation of wg led to the loss of segment boundaries, gnathal and thoracic appendages, and lateral head lobes, and to changes in the expression of dpp, Distal-less, and Engrailed. The functions of wg varied along both the anteroposterior and dorsoventral axes of the embryo. Phylogenetic comparisons indicate that the role of WNT signaling in segment boundary formation is evolutionarily old, but that its role in appendage allocation originated in the common ancestor of holometabolous insects.     

Head patterning and Hox gene expression in an onychophoran and its implications for the arthropod head problem

The arthropod head problem has puzzled zoologists for more than a century. The head of adult arthropods is a complex structure resulting from the modification, fusion and migration of an uncertain number of segments. In contrast, onychophorans, which are the probable sister group to the arthropods, have a rather simple head comprising three segments that are well defined during development, and give rise to the adult head with three pairs of appendages specialised for sensory and food capture/manipulative purposes. Based on the expression pattern of the anterior Hox genes labial, proboscipedia, Hox3 and Deformed, we show that the third of these onychophoran segments, bearing the slime papillae, can be correlated to the tritocerebrum, the most anterior Hox-expressing arthropod segment. This implies that both the onychophoran antennae and jaws are derived from a more anterior, Hox-free region corresponding to the proto and deutocerebrum of arthropods. Our data provide molecular support for the proposal that the onychophoran head possesses a well-developed appendage that corresponds to the anterior, apparently appendage-less region of the arthropod head.     

Patterning mechanisms in the body trunk and the appendages of Drosophila.

During evolution, many animal groups have developed specialised outgrowths of the body wall, limbs or appendages. The type of appendage depends on the identity of the segment where they appear, indicating that the Hox genes contribute to appendage specification. Moreover, work carried out principally in Drosophila has identified the gene products and the mechanisms involved in pattern formation in the appendages. In this essay, we compare the morphogenetic processes in the appendages and the body wall; the function of the Hox genes and the response to the signalling molecules involved in local patterning. We speculate that, although the basic mechanisms are similar, there are significant differences in the manner the body trunk and appendages respond to them.     

Trilobite tagmosis and body patterning from morphological and developmental perspectives

The Trilobita were characterized by a cephalic region in whichthe biomineralized exoskeleton showed relatively high morphologicaldifferentiation among a taxonomically stable set of well definedsegments, and an ontogenetically and taxonomically dynamic trunkregion in which both exoskeletal segments and ventral appendageswere similar in overall form. Ventral appendages were homonomousbiramous limbs throughout both the cephalon and trunk, exceptfor the most anterior appendage pair that was antenniform, preoral,and uniramous, and a posteriormost pair of antenniform cerci,known only in one species. In some clades trunk exoskeletalsegments were divided into two batches. In some, but not all,of these clades the boundary between batches coincided withthe boundary between the thorax and the adult pygidium. Therepeated differentiation of the trunk into two batches of segmentsfrom the homonomous trunk condition indicates an evolutionarytrend in aspects of body patterning regulation that was achievedindependently in several trilobite clades. The phylogeneticplacement of trilobites and congruence of broad patterns oftagmosis with those seen among extant arthropods suggest thatthe expression domains of trilobite cephalic Hox genes may haveoverlapped in a manner similar to that seen among extant arachnates.This, coupled with the fact that trilobites likely possessedten Hox genes, presents one alternative to a recent model inwhich Hox gene distribution in trilobites was equated to eightputative divisions of the trilobite body plan.     

From development to biodiversity—Tribolium castaneum, an insect model organism for short germband development

Insect embryogenesis is best understood in the fruit fly Drosophila. However, Drosophila embryogenesis shows evolutionary-derived features: anterior patterning is controlled by a highly derived Hox gene bicoid, the body segments form almost simultaneously and appendages develop from imaginal discs. In contrast, embryogenesis of the red flour beetle Tribolium castaneum displays typical features in anterior patterning, axis and limb formation shared with most insects, other arthropods as well as with vertebrates. Anterior patterning depends on the conserved homeobox gene orthodenticle, the main body axis elongates sequentially and limbs grow continuously starting from an appendage bud. Thus, by analysing developmental processes in the beetle at the molecular and cellular level, inferences can be made for similar processes in other arthropods. With the completion of sequencing the Tribolium genome, the door is now open for post-genomic studies such as RNA expression profiling, proteomics and functional genomics to identify beetle-specific gene circuits.     

Partial co-option of the appendage patterning pathway in the development of abdominal appendages in the sepsid fly Themira biloba

The abdominal appendages on male Themira biloba (Diptera: Sepsidae) are complex novel structures used during mating. These abdominal appendages superficially resemble the serially homologous insect appendages in that they have a joint and a short segment that can be rotated. Non-genital appendages do not occur in adult pterygote insects, so these abdominal appendages are novel structures with no obvious ancestry. We investigated whether the genes that pattern the serially homologous insect appendages have been co-opted to pattern these novel abdominal appendages. Immunohistochemistry was used to determine the expression patterns of the genes extradenticle (exd), Distal-less (Dll), engrailed (en), Notch, and the Bithorax Complex in the appendages of T. biloba during pupation. The expression patterns of Exd, En, and Notch were consistent with the hypothesis that a portion of the patterning pathway that establishes the coxopodite has been co-opted to pattern the developing abdominal appendages. However, Dll was only expressed in the bristles of the developing appendages and not the proximal–distal axis of the appendage itself. The lack of Dll expression indicates the absence of a distal domain of the appendage suggesting that sepsid abdominal appendages only use genes that normally pattern the base of segmental appendages.     

Divergent and conserved roles of extradenticle in body segmentation and appendage formation, respectively, in the cricket Gryllus bimaculatus

The cricket Gryllus bimaculatus is a typical hemimetabolous intermediate germ insect, in which the processes of segmentation and appendage formation differ from those in Drosophila, a holometabolous long germ insect. In order to compare their developmental mechanisms, we have focused on Gryllus orthologs of the Drosophila developmental regulatory genes and studied their functions. Here, we report a functional analysis of the Gryllus ortholog of extradenticle (Gb′exd) using embryonic and parental RNA interference (RNAi) techniques. We found the following: (1) RNAi suppression of Gb′exd results in the deletion or fusion of body segments. Especially the head was often very severely affected. This gap-like phenotype may be related to reduced expression of the gap genes hunchback and Krüppel in early RNAi germbands. (2) In the appendages, several segments (podomeres) were fused. (3) Head appendages including the antenna were transformed to a leg-like structure consisting of at least one proximal podomere as well as several tarsomeres. The defects in appendages are reminiscent of the phenotype caused by large exd clones in Drosophila antennal discs. These findings led us to the conclusion that (1) Gb′exd is required for segment patterning in the gnathal to abdominal region, acting in a gap gene-like manner in the anterior region. (2) Gb′exd plays important roles in formation of the appendages and the determination of their identities, acting as a regulatory switch that chooses between the fates of head appendages versus the appendage ground state. Although functions of Gb′exd in appendage patterning appear fundamentally conserved between Gryllus and Drosophila, its role in body segmentation may differ from that of Drosophila exd.     

Gene expression patterns in an onychophoran reveal that regionalization predates limb segmentation in pan‐arthropods

SUMMARY In arthropods, such as Drosophila melanogaster, the leg gap genes homothorax (hth), extradenticle (exd), dachshund (dac), and Distal‐less (Dll) regionalize the legs in order to facilitate the subsequent segmentation of the legs. We have isolated homologs of all four leg gap genes from the onychophoran Euperipatoides kanangrensis and have studied their expression. We show that leg regionalization takes place in the legs of onychophorans even though they represent simple and nonsegmented appendages. This implies that leg regionalization evolved for a different function and was only later co‐opted for a role in leg segmentation. We also show that the leg gap gene patterns in onychophorans (especially of hth and exd) are similar to the patterns in crustaceans and insects, suggesting that this is the plesiomorphic state in arthropods. The reversed hth and exd patterns in chelicerates and myriapods are therefore an apomorphy for this group, the Myriochelata, lending support to the Myriochelata and Tetraconata clades in arthropod phylogeny.     

The specification of a highly derived arthropod appendage, the Drosophila labial palps, requires the joint action of selectors and signaling pathways

The remarkable diversity of form in arthropods reflects flexible genetic programs deploying many conserved genes. In the insect model Drosophila melanogaster, diversity of form can be observed between serially homologous appendages or when a single appendage is transformed by homeotic mutations, such as the adult labial mouthparts that can present alternative antennal, prothoracic, or maxillary identities. We have examined the roles of the Hox selector genes proboscipedia (pb) and Sex combs reduced (Scr), and the antennal selectors homothorax (hth) and spineless (ss) in labial specification, by tissue-directed mitotic recombination. Whereas loss of pb function transforms labium to prothoracic leg, loss of Scr, hth, or ss functions results in little or no change in labial specification. Results of analysis of single and multiple mutant combinations support a genetic hierarchy in which the homeotic pb gene possesses a primary role. It is surprising to note that while loss of ss activity alone had no detected effect, all mutant combinations lacking both pb and ss yielded the most severe phenotype observed: stunted, apparently tripartite legs that may correspond to a default state. The roles of the four selector genes are functionally linked to a cell nonautonomous mechanism involving the coupled activities of the decapentaplegic (dpp)/TGF-β and wingless (wg)/Wnt signaling pathways. Accordingly, several mutant combinations impaired in dpp signaling were seen to reorient labial-to-leg transformations toward antennal aristae. A crucial aspect of selector function in development and evolution may be in regulating diffusible signals, including those emitted by dpp and wg.     

Cephalic and Limb Anatomy of a New Isoxyid from the Burgess Shale and the Role of “Stem Bivalved Arthropods” in the Disparity of the Frontalmost Appendage

We herein describe Surusicaris elegans gen. et sp. nov. (in Isoxyidae, amended), a middle (Series 3, Stage 5) Cambrian bivalved arthropod from the new Burgess Shale deposit of Marble Canyon (Kootenay National Park, British Columbia). Surusicaris exhibits 12 simple, partly undivided biramous trunk limbs with long tripartite caeca, which may illustrate a plesiomorphic “fused” condition of exopod and endopod. We construe also that the head is made of five somites (= four segments), including two eyes, one pair of anomalocaridid-like frontalmost appendages, and three pairs of poorly sclerotized uniramous limbs. This fossil may therefore be a candidate for illustrating the origin of the plesiomorphic head condition in euarthropods, and questions the significance of the “two-segmented head” in, e.g., fuxianhuiids. The frontalmost appendage in isoxyids is intriguingly disparate, bearing similarities with both dinocaridids and euarthropods. In order to evaluate the relative importance of bivalved arthropods, such as Surusicaris, in the hypothetical structuro-functional transition between the dinocaridid frontal appendage and the pre-oral—arguably deutocerebral—appendage of euarthropods, we chose a phenetic approach and computed morphospace occupancy for the frontalmost appendages of 36 stem and crown taxa. Results show different levels of evolutionary decoupling between frontalmost appendage disparity and body plans. Variance is greatest in dinocaridids and “stem bivalved” arthropods, but these groups do not occupy the morphospace homogeneously. Rather, the diversity of frontalmost appendages in “stem bivalved” arthropods, distinct in its absence of clear clustering, is found to link the morphologies of “short great appendages,” chelicerae and antennules. This find fits the hypothesis of an increase in disparity of the deutocerebral appendage prior to its diversification in euarthropods, and possibly corresponds to its original time of development. The analysis of this pattern, however, is sensitive to the—still unclear—extent of polyphyly of the “stem bivalved” taxa.     

Evolution of the chelicera: a dachshund domain is retained in the deutocerebral appendage of Opiliones (Arthropoda,Chelicerata)

The proximo‐distal axis of the arthropod leg is patterned by mutually antagonistic developmental expression domains of the genes extradenticle, homothorax, dachshund, and Distal‐less. In the deutocerebral appendages (the antennae) of insects and crustaceans, the expression domain of dachshund is frequently either absent or, if present, is not required to pattern medial segments. By contrast, the dachshund domain is entirely absent in the deutocerebral appendages of spiders, the chelicerae. It is unknown whether absence of dachshund expression in the spider chelicera is associated with the two‐segmented morphology of this appendage, or whether all chelicerates lack the dachshund domain in their chelicerae. We investigated gene expression in the harvestman Phalangium opilio, which bears the plesiomorphic three‐segmented chelicera observed in “primitive” chelicerate orders. Consistent with patterns reported in spiders, in the harvestman chelicera homothorax, extradenticle, and Distal‐less have broadly overlapping developmental domains, in contrast with mutually exclusive domains in the legs and pedipalps. However, unlike in spiders, the harvestman chelicera bears a distinct expression domain of dachshund in the proximal segment, the podomere that is putatively lost in derived arachnids. These data suggest that a tripartite proximo‐distal domain structure is ancestral to all arthropod appendages, including deutocerebral appendages. As a corollary, these data also provide an intriguing putative genetic mechanism for the diversity of arachnid chelicerae: loss of developmental domains along the proximo‐distal axis.     

Double-stranded RNA interference in the spider Cupiennius salei: the role of Distal-less is evolutionarily conserved in arthropod appendage formation

Chelicerates represent a basal arthropod group, which makes them an excellent system for the study of evolutionary processes in arthropods. To enable functional studies in chelicerates, we developed a double-stranded RNA-interference (RNAi) protocol for spiders while studying the function of the Distal-less gene. We isolated the Distal-less gene from the spider Cupiennius salei. Cs-Dll gene expression is first seen in cells of the prosomal segments before the outgrowth of the appendages. After the appendages have formed, Cs-Dll is expressed in the distal portion of the prosomal appendages, and in addition, in the labrum, in two pairs of opisthosmal (abdominal) limb buds, in the head region, and at the posterior-most end of the spider embryo. In embryos, in which Dll was silenced by RNAi, the distal part of the prosomal appendages was missing and the labrum was completely absent. Thus, Dll also plays a crucial role in labrum formation. However, the complete lack of labrum in RNAi embryos may point to a different nature of the labrum from the segmental appendages. Our data show that the expression of Dll in the appendages is conserved among arthropods, and furthermore that the role of Dll is evolutionarily conserved in the formation of segmental appendages in arthropods.     

A revision of brain composition in Onychophora (velvet worms) suggests that the tritocerebrum evolved in arthropods

Background  

The composition of the arthropod head is one of the most contentious issues in animal evolution. In particular, controversy surrounds the homology and innervation of segmental cephalic appendages by the brain. Onychophora (velvet worms) play a crucial role in understanding the evolution of the arthropod brain, because they are close relatives of arthropods and have apparently changed little since the Early Cambrian. However, the segmental origins of their brain neuropils and the number of cephalic appendages innervated by the brain - key issues in clarifying brain composition in the last common ancestor of Onychophora and Arthropoda - remain unclear.     

Hox genes and the phylogeny of the arthropods

The arthropods are the most speciose, and among the most morphologically diverse, of the animal phyla. Their evolution has been the subject of intense research for well over a century, yet the relationships among the four extant arthropod subphyla - chelicerates, crustaceans, hexapods, and myriapods - are still not fully resolved. Morphological taxonomies have often placed hexapods and myriapods together (the Atelocerata) [1, 2], but recent molecular studies have generally supported a hexapod/crustacean clade [2-9]. A cluster of regulatory genes, the Hox genes, control segment identity in arthropods, and comparisons of the sequences and functions of Hox genes can reveal evolutionary relationships [10]. We used Hox gene sequences from a range of arthropod taxa, including new data from a basal hexapod and a myriapod, to estimate a phylogeny of the arthropods. Our data support the hypothesis that insects and crustaceans form a single clade within the arthropods to the exclusion of myriapods. They also suggest that myriapods are more closely allied to the chelicerates than to this insect/crustacean clade.     

Hox gene duplications correlate with posterior heteronomy in scorpions

The evolutionary success of the largest animal phylum, Arthropoda, has been attributed to tagmatization, the coordinated evolution of adjacent metameres to form morphologically and functionally distinct segmental regions called tagmata. Specification of regional identity is regulated by the Hox genes, of which 10 are inferred to be present in the ancestor of arthropods. With six different posterior segmental identities divided into two tagmata, the bauplan of scorpions is the most heteronomous within Chelicerata. Expression domains of the anterior eight Hox genes are conserved in previously surveyed chelicerates, but it is unknown how Hox genes regionalize the three tagmata of scorpions. Here, we show that the scorpion Centruroides sculpturatus has two paralogues of all Hox genes except Hox3, suggesting cluster and/or whole genome duplication in this arachnid order. Embryonic anterior expression domain boundaries of each of the last four pairs of Hox genes (two paralogues each of Antp, Ubx, abd-A and Abd-B) are unique and distinguish segmental groups, such as pectines, book lungs and the characteristic tail, while maintaining spatial collinearity. These distinct expression domains suggest neofunctionalization of Hox gene paralogues subsequent to duplication. Our data reconcile previous understanding of Hox gene function across arthropods with the extreme heteronomy of scorpions.