Appendage patterning in the South American bird spider <Emphasis Type="Italic">Acanthoscurria geniculata</Emphasis> (Araneae: Mygalomorphae)

Pattern formation by the genes dachshund (dac), Distal-less (Dll), extradenticle (exd) and homothorax (hth) in spider appendages has been studied previously only in members of the higher spiders (Araneomorphae). In order to study the diversity and conservation of pattern formation in spiders as a whole, we studied homologs of these genes in embryos of the bird spider Acanthoscurria geniculata, which belongs to the Mygalomorphae, a more primitive spider group. We show that the patterns of dac and Dll are largely conserved in all spiders studied so far. We find a duplication of hth and exd genes as previously identified in the higher spider Cupiennius salei. These data suggest that pattern formation shows little diversity in all spiders, including the duplication of hth and exd that likely occurred before the split of Mygalomorphae and Araneomorphae. We also find that the legs and pedipalps bear endites of which only the pedipalpal endite expresses Dll and is retained in the adult. Similarly, the limb buds of the posterior spinnerets express Dll and become segmented appendages in the adult, whereas the anterior spinnerets lack Dll expression and are absent in postembryonic stages. In both cases, the expression of Dll or the lack of it indicates structures which will be retained as adult traits or rudimentary structures that degenerate, respectively. The presence of embryonic rudiments of leg endites in Acanthoscurria and the leg-like pattern formation in the posterior spinnerets are interpreted as primitive traits that have been lost in the Araneomorphae.     

Expression of arthropod distal limb-patterning genes in the onychophoran Euperipatoides kanangrensis

A current hypothesis states that the ancestral limb of arthropods is composed of only two segments. The proximal segment represents the main part of the modern leg, and the distal segment represents the tarsus and claw of the modern leg. If the distal part of the limb is an ancestral feature, one would expect conserved regulatory gene networks acting in distal limb development in all arthropods and possibly even their sister group, the onychophorans. We investigated the expression patterns of six genes known to function during insect distal limb development in the onychophoran Euperipatoides kanangrensis, i.e., clawless (cll), aristaless (al), spineless (ss), zinc finger homeodomain 2 (zfh2), rotund (rn), and Lim1. We find that all investigated genes are expressed in at least some of the onychophoran limbs. The expression patterns of most of these genes, however, display crucial differences to the known insect patterns. The results of this study question the hypothesis of conserved distal limb evolution in arthropods and highlight the need for further studies on arthropod limb development.     

Patterning defects in the primary axonal scaffolds caused by the mutations of the extradenticle and homothorax genes in the embryonic Drosophila brain

During early brain development in Drosophila a highly stereotyped pattern of axonal scaffolds evolves by precise pioneering and selective fasciculation of neural fibers in the newly formed brain neuromeres. Using an axonal marker, Fasciclin II, we show that the activities of the extradenticle (exd) and homothorax (hth) genes are essential to this axonal patterning in the embryonic brain. Both genes are expressed in the developing brain neurons, including many of the tract founder cluster cells. Consistent with their expression profiles, mutations of exd and hth strongly perturb the primary axonal scaffolds. Furthermore, we show that mutations of exd and hth result in profound patterning defects of the developing brain at the molecular level including stimulation of the orthodenticle gene and suppression of the empty spiracles and cervical homeotic genes. In addition, expression of a Drosophila Pax6 gene, eyeless, is significantly suppressed in the mutants except for the most anterior region. These results reveal that, in addition to their homeotic regulatory functions in trunk development, exd and hth have important roles in patterning the developing brain through coordinately regulating various nuclear regulatory genes, and imply molecular commonalities between the developmental mechanisms of the brain and trunk segments, which were conventionally considered to be largely independent. Received: 4 October 1999 / Accepted: 10 January 2000     

Deciphering the onychophoran ‘segmentation gene cascade’: Gene expression reveals limited involvement of pair rule gene orthologs in segmentation,but a highly conserved segment polarity gene network

The hallmark of the arthropods is their segmented body, although origin of segmentation, however, is unresolved. In order to shed light on the origin of segmentation we investigated orthologs of pair rule genes (PRGs) and segment polarity genes (SPGs) in a member of the closest related sister-group to the arthropods, the onychophorans. Our gene expression data analysis suggests that most of the onychophoran PRGs do not play a role in segmentation. One possible exception is the even-skipped (eve) gene that is expressed in the posterior end of the onychophoran where new segments are likely patterned, and is also expressed in segmentation-gene typical transverse stripes in at least a number of newly formed segments. Other onychophoran PRGs such as runt (run), hairy/Hes (h/Hes) and odd-skipped (odd) do not appear to have a function in segmentation at all. Onychophoran PRGs that act low in the segmentation gene cascade in insects, however, are potentially involved in segment-patterning. Most obvious is that from the expression of the pairberry (pby) gene ortholog that is expressed in a typical SPG-pattern. Since this result suggested possible conservation of the SPG-network we further investigated SPGs (and associated factors) such as Notum in the onychophoran. We find that the expression patterns of SPGs in arthropods and the onychophoran are highly conserved, suggesting a conserved SPG-network in these two clades, and indeed also in an annelid. This may suggest that the common ancestor of lophotrochozoans and ecdysozoans was already segmented utilising the same SPG-network, or that the SPG-network was recruited independently in annelids and onychophorans/arthropods.     

Conservation, innovation, and the evolution of horned beetle diversity

Beetle horns represent an evolutionary novelty exhibiting remarkable diversity above and below the species level. Here, we show that four typical appendage patterning genes, extradenticle (exd), homothorax (hth), dachshund (dac), and Distal-less (Dll) are expressed in the context of the development of sexually dimorphic thoracic horns in three Onthophagus species. At least two of these genes, Dll and hth, exhibited expression patterns consistent with a conservation of patterning function during horn development relative to their known roles in the development of insect legs. exd, hth, and dac expression patterns during horn development were largely invariable across species or sexes within species. In contrast, Dll expression was far more discrete and exhibited consistent differences between sexes and species. Most importantly, differences in location and domain size of Dll expression tightly correlated with the degree to which prepupal horn primordia were retained or resorbed before the final adult molt. Our results lend further support to the hypothesis that the origin of beetle horns relied, at least in part, on the redeployment of already existing developmental mechanisms, such as appendage patterning processes and that changes in the exact location and domain size of Dll expression may represent important modifier mechanisms that modulate horn expression in different species or sexes. If correct, this would imply that certain components of genetic basis of horn development may be able to diversify rapidly within lineages and largely independent of phylogenetic distance. We present a first model that integrates presently available data on the genetic regulation of horn development and diversity.     

Gene expression suggests conserved mechanisms patterning the heads of insects and myriapods

Segmentation, i.e. the subdivision of the body into serially homologous units, is one of the hallmarks of the arthropods. Arthropod segmentation is best understood in the fly Drosophila melanogaster. But different from the situation in most arthropods in this species all segments are formed from the early blastoderm (so called long-germ developmental mode). In most other arthropods only the anterior segments are formed in a similar way (so called short-germ developmental mode). Posterior segments are added one at a time or in pairs of two from a posterior segment addition zone. The segmentation mechanisms are not universally conserved among arthropods and only little is known about the genetic patterning of the anterior segments. Here we present the expression patterns of the insect head patterning gene orthologs hunchback (hb), orthodenticle (otd), buttonhead-like (btdl), collier (col), cap-n-collar (cnc) and crocodile (croc), and the trunk gap gene Krüppel (Kr) in the myriapod Glomeris marginata. Conserved expression of these genes in insects and a myriapod suggests that the anterior segmentation system may be conserved in at least these two classes of arthropods. This finding implies that the anterior patterning mechanism already existed in the last common ancestor of insects and myriapods.     

Onychophoran‐like myoanatomy of the Cambrian gilled lobopodian Pambdelurion whittingtoni

Arthropods are characterized by a rigid, articulating, exoskeleton operated by a lever‐like system of segmentally arranged, antagonistic muscles. This skeletomuscular system evolved from an unsegmented body wall musculature acting on a hydrostatic skeleton, similar to that of the arthropods’ close relatives, the soft‐bodied onychophorans. Unfortunately, fossil evidence documenting this transition is scarce. Exceptionally‐preserved panarthropods from the Cambrian Lagerstätte of Sirius Passet, Greenland, including the soft‐bodied stem‐arthropod Pambdelurion whittingtoni and the hard‐bodied arthropods Kiisortoqia soperi and Campanamuta mantonae, are unique in preserving extensive musculature. Here we show that Pambdelurion's myoanatomy conforms closely to that of extant onychophorans, with unsegmented dorsal, ventral and longitudinal muscle groups in the trunk, and extrinsic and intrinsic muscles controlling the legs. Pambdelurion also possesses oblique musculature, which has previously been interpreted as an arthropodan characteristic. However, this oblique musculature appears to be confined to the cephalic region and first few body segments, and does not represent a shift towards arthropodan myoanatomy. The Sirius Passet arthropods, Kiisortoqia and Campanamuta, also possess large longitudinal muscles in the trunk, although, unlike Pambdelurion, they are segmentally divided at the tergal boundaries. Thus, the transition towards an arthropodan myoanatomy from a lobopodian ancestor probably involved the division of the peripheral longitudinal muscle into segmented units.     

A congruent solution to arthropod phylogeny: phylogenomics,microRNAs and morphology support monophyletic Mandibulata

While a unique origin of the euarthropods is well established, relationships between the four euarthropod classes—chelicerates, myriapods, crustaceans and hexapods—are less clear. Unsolved questions include the position of myriapods, the monophyletic origin of chelicerates, and the validity of the close relationship of euarthropods to tardigrades and onychophorans. Morphology predicts that myriapods, insects and crustaceans form a monophyletic group, the Mandibulata, which has been contradicted by many molecular studies that support an alternative Myriochelata hypothesis (Myriapoda plus Chelicerata). Because of the conflicting insights from published molecular datasets, evidence from nuclear-coding genes needs corroboration from independent data to define the relationships among major nodes in the euarthropod tree. Here, we address this issue by analysing two independent molecular datasets: a phylogenomic dataset of 198 protein-coding genes including new sequences for myriapods, and novel microRNA complements sampled from all major arthropod lineages. Our phylogenomic analyses strongly support Mandibulata, and show that Myriochelata is a tree-reconstruction artefact caused by saturation and long-branch attraction. The analysis of the microRNA dataset corroborates the Mandibulata, showing that the microRNAs miR-965 and miR-282 are present and expressed in all mandibulate species sampled, but not in the chelicerates. Mandibulata is further supported by the phylogenetic analysis of a comprehensive morphological dataset covering living and fossil arthropods, and including recently proposed, putative apomorphies of Myriochelata. Our phylogenomic analyses also provide strong support for the inclusion of pycnogonids in a monophyletic Chelicerata, a paraphyletic Cycloneuralia, and a common origin of Arthropoda (tardigrades, onychophorans and arthropods), suggesting that previous phylogenies grouping tardigrades and nematodes may also have been subject to tree-reconstruction artefacts.     

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.     

The delineation of the fourth walking leg segment is temporally linked to posterior segmentation in the mite Archegozetes longisetosus (Acari: Oribatida,Trhypochthoniidae)

Acari (mites and ticks) lack external segmentation, with the only indication of segmentation being the appendages of the prosoma (chelicerae, pedipalps, and four pairs of walking legs). Acari also have a mode of development in which the formation of the fourth walking leg is suppressed until the nymphal stages, following a hexapodal larva. To determine the number of segments in the posterior body region (opisthosoma) of mites, and to also determine when the fourth walking leg segment is delineated during embryogenesis, we followed the development of segmentation in the oribatid mite Archegozetes longisetosus using time‐lapse and scanning electron microscopy, as well as in situ hybridizations of the A. longisetosus orthologues of the segmentation genes engrailed and hedgehog. Our data show that A. longisetosus patterns only two opisthosomal segments, indicating a large degree of segmental fusion or loss. Also, we show that the formation of the fourth walking leg segment is temporally tied to opisthosomal segmentation, the first such observation in any arachnid.     

A conserved genetic mechanism specifies deutocerebral appendage identity in insects and arachnids

The segmental architecture of the arthropod head is one of the most controversial topics in the evolutionary developmental biology of arthropods. The deutocerebral (second) segment of the head is putatively homologous across Arthropoda, as inferred from the segmental distribution of the tripartite brain and the absence of Hox gene expression of this anterior-most, appendage-bearing segment. While this homology statement implies a putative common mechanism for differentiation of deutocerebral appendages across arthropods, experimental data for deutocerebral appendage fate specification are limited to winged insects. Mandibulates (hexapods, crustaceans and myriapods) bear a characteristic pair of antennae on the deutocerebral segment, whereas chelicerates (e.g. spiders, scorpions, harvestmen) bear the eponymous chelicerae. In such hexapods as the fruit fly, Drosophila melanogaster, and the cricket, Gryllus bimaculatus, cephalic appendages are differentiated from the thoracic appendages (legs) by the activity of the appendage patterning gene homothorax (hth). Here we show that embryonic RNA interference against hth in the harvestman Phalangium opilio results in homeonotic chelicera-to-leg transformations, and also in some cases pedipalp-to-leg transformations. In more strongly affected embryos, adjacent appendages undergo fusion and/or truncation, and legs display proximal defects, suggesting conservation of additional functions of hth in patterning the antero-posterior and proximo-distal appendage axes. Expression signal of anterior Hox genes labial, proboscipedia and Deformed is diminished, but not absent, in hth RNAi embryos, consistent with results previously obtained with the insect G. bimaculatus. Our results substantiate a deep homology across arthropods of the mechanism whereby cephalic appendages are differentiated from locomotory appendages.     

A molecular view of onychophoran segmentation

This paper summarizes our current knowledge on the expression and assumed function of Drosophila and (other) arthropod segmentation gene orthologs in Onychophora, a closely related outgroup to Arthropoda. This includes orthologs of the so-called Drosophila segmentation gene cascade including the Hox genes, as well as other genetic factors and pathways involved in non-drosophilid arthropods.Open questions about and around the topic are addressed, such as the definition of segments in onychophorans, the unclear regulation of conserved expression patterns downstream of non-conserved factors, and the potential role of mesodermal patterning in onychophoran segmentation.     

Notch-mediated segmentation of the appendages is a molecular phylotypic trait of the arthropods

Arthropod limbs are arguably the most diverse organs in the animal kingdom. Morphological diversity of the limbs is largely based on their segmentation, because this divides the limbs into modules that can evolve separately for new morphologies and functions. Limb segmentation also distinguishes the arthropods from related phyla (e.g. onychophorans) and thus forms an important evolutionary innovation in arthropods. Understanding the genetic basis of limb segmentation in arthropods can thus shed light onto the mechanisms of macroevolution and the origin of a character (articulated limbs) that defines a new phylum (arthropods). In the fly Drosophila limb segmentation and limb growth are controlled by the Notch signaling pathway. Here we show that the Notch pathway also controls limb segmentation and growth in the spider Cupiennius salei, a representative of the most basally branching arthropod group Chelicerata, and thus this function must trace from the last common ancestor of all arthropods. The similarities of Notch and Serrate function between Drosophila and Cupiennius are extensive and also extend to target genes like odd-skipped, nubbin, AP-2 and hairy related genes. Our data confirm that the jointed appendages, which are a morphological phylotypic trait of the arthropods and the basis for naming the phylum, have a common developmental genetic basis. Notch-mediated limb segmentation is thus a molecular phylotypic trait of the arthropods.     

The morphological and molecular processes of onychophoran brain development show unique features that are neither comparable to insects nor to chelicerates

The phylogenetic position of onychophorans is still being debated; however, most phylogenies suggest that onychophorans are a sister group to the arthropods. Here we have analysed neurogenesis in the brain of the onychophoran Euperipatoides kanangrensis. We show that the development of the onychophoran brain is considerably different from arthropods. Neural precursors seem to be generated at random positions rather than in distinct spatio-temporal domains as has been shown in insects and chelicerates. The different mode of neural precursor formation is reflected in the homogenous expression of the proneural and neurogenic genes. Furthermore, the morphogenetic events that generate the three-dimensional structure of the onychophoran brain are significantly different from arthropods. Despite the different mode of neural precursor formation in insects and chelicerates (neuroblasts versus neural precursor groups), brain neurogenesis shares more similarities in these arthropods as compared to the onychophoran. Our data show that the developmental processes that generate the brain have considerably diverged in onychophorans and arthropods.     

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.     

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.     

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.