The Articulata hypothesis – or what is a segment?

The long held view that annelids and arthropods are closely related (Articulata) has been challenged recently by phylogenetic analyses using molecular data. The outcome of these studies is a clade of moulting animals (Ecdysozoa) comprising arthropods and some taxa of the nemathelminth worms. Monophyly of the Ecdysozoa has not yet been shown convincingly on morphological evidence, but is strongly supported by molecular data. The implication of the Ecdysozoa hypothesis is that the type of segmentation found in annelids and arthropods must be either convergent or an ancestral feature of protostomes or even bilaterians. The present review discusses aspects of segmentation in annelids and arthropods at the genetic, cellular, morphogenetic and morphological levels. Based on numerous similarities not shared with other bilaterian taxa it is suggested that segmentation of annelids and arthropods is homologous and apomorphic for a monophyletic Articulata. However, the challenge provided by the molecular analyses should stimulate research programmes gaining more data such as on additional genes, cleavage patterns, molecular developmental biology, and the comparison of nervous systems at the level of single neurons.     

Expression of engrailed proteins in arthropods, annelids, and chordates

engrailed is a homeobox gene that has an important role in Drosophila segmentation. Genes homologous to engrailed have been identified in several other organisms. Here we describe a monoclonal antibody that recognizes a conserved epitope in the homeodomain of engrailed proteins of a number of different arthropods, annelids, and chordates; we use this antibody to isolate the grasshopper engrailed gene. In Drosophila embryos, the antibody reveals engrailed protein in the posterior portion of each segment during segmentation, and in a segmentally reiterated subset of neuronal cells during neurogenesis. Other arthropods, including grasshopper and two crustaceans, have similar patterns of engrailed expression. However, these patterns of expression are not shared by the annelids or chordates we examined. Our results provide the most comprehensive view that has been obtained of how expression patterns of a regulatory gene vary during evolution. On the basis of these patterns, we suggest that engrailed is a gene whose ancestral function was in neurogenesis and whose function was co-opted during the evolution of segmentation in the arthropods, but not in the annelids and chordates.     

Expression of 'segmentation' genes during larval and juvenile development in the polychaetes Capitella sp. I and H. elegans

Polychaete annelids and arthropods are both segmented protostome invertebrates. To investigate whether the segmented body plan of these two phyla share a common molecular ground pattern, we report the developmental expression of orthologues of the arthropod segment polarity genes engrailed (en), hedgehog (hh), and wingless (wg/Wnt1) in larval and juvenile stages of the polychaete annelid Capitella sp. I and en in a second polychaete, Hydroides elegans. Temporally, neither Wnt1 nor hh are detected in the segmented region of the larval body until after morphological segmentation is apparent. Expression of CapI-Wnt1 is limited to a ring of ectoderm marking the future anus during larval segmentation. CapI-hh is expressed in a ring of the hindgut internal to that of CapI-Wnt1, as well as in a subset of ventral nerve cord neurons, anterior gut tissue, and mesoderm. In both H. elegans and Capitella sp. I, en is expressed in a spatially and temporally dynamic manner in segmentally iterated structures as well as a population of cells that migrate internally from ectoderm to mesoderm, possibly representing a population of ecto-mesodermal precursors. Significantly, the expression patterns we report for wg, en, and hh orthologues in Capitella sp. I and for en in larval development of H. elegans are not comparable to the highly conserved ectodermal segment polarity pattern observed in arthropods at any life history stage, consistent with distinct origins of segmentation between annelids and arthropods.     

Exploring myriapod segmentation: the expression patterns of even-skipped,engrailed, and wingless in a centipede

Segment formation is critical to arthropod development, yet there is still relatively little known about this process in most arthropods. Here, we present the expression patterns of the genes even-skipped (eve), engrailed, and wingless in a centipede, Lithobius atkinsoni. Despite some differences when compared with the patterns in insects and crustaceans, the expression of these genes in the centipede suggests that their basic roles are conserved across the mandibulate arthropods. For example, unlike the seven pair-rule stripes of eve expression in the Drosophila embryonic germband, the centipede eve gene is expressed strongly in the posterior of the embryo, and in only a few stripes between newly formed segments. Nonetheless, this pattern likely reflects a conserved role for eve in the process of segment formation, within the different context of a short-germband mode of embryonic development. In the centipede, the genes wingless and engrailed are expressed in stripes along the middle and posterior of each segment, respectively, similar to their expression in Drosophila. The adjacent expression of the engrailed and wingless stripes suggests that the regulatory relationship between the two genes may be conserved in the centipede, and thus this pathway may be a fundamental mechanism of segmental development in most arthropods.     

Functional conservation of the wingless-engrailed interaction as shown by a widely applicable baculovirus misexpression system

BACKGROUND: The expression patterns of the segment polarity genes wingless and engrailed are conserved during segmentation in a variety of arthropods, suggesting that the regulatory interactions between these two genes are also evolutionarily conserved. Hypotheses derived from such comparisons of gene expression patterns are difficult to test experimentally as genetic manipulation is currently possible for only a few model organisms. RESULTS: We have developed a system, using recombinant baculoviruses, that can be applied to a wide variety of organisms to study the effects of ectopic expression of genes. As a first step, we studied the range and type of infection of several reporter viruses in the embryos of two arthropod and one vertebrate species. Using this system to express wingless, we were able to induce expression of engrailed in the anterior half of each parasegment in embryos of the fruit fly Drosophila melanogaster. Virus-mediated wingless expression also caused ectopic naked ventral cuticle formation in wild-type Drosophila larvae. In the flour beetle, Tribolium castaneum, ectopic wingless also induced engrailed expression. As in Drosophila, this expression was only detectable in the anterior half of the parasegment. CONCLUSIONS: The functional interaction between wingless and engrailed, and the establishment of cells competent to express engrailed, appears to be conserved between Drosophila and Tribolium. The data on the establishment of an engrailed-competent domain also support the idea that prepatterning by pair-rule genes is conserved between these two insects. The recombinant baculovirus technology reported here may help answer other long-standing comparative evolutionary questions.     

Parasegmental organization of the spider embryo implies that the parasegment is an evolutionary conserved entity in arthropod embryogenesis

Spiders belong to the chelicerates, which is a basal arthropod group. To shed more light on the evolution of the segmentation process, orthologs of the Drosophila segment polarity genes engrailed, wingless/Wnt and cubitus interruptus have been recovered from the spider Cupiennius salei. The spider has two engrailed genes. The expression of Cs-engrailed-1 is reminiscent of engrailed expression in insects and crustaceans, suggesting that this gene is regulated in a similar way. This is different for the second spider engrailed gene, Cs-engrailed-2, which is expressed at the posterior cap of the embryo from which stripes split off, suggesting a different mode of regulation. Nevertheless, the Cs-engrailed-2 stripes eventually define the same border as the Cs-engrailed-1 stripes. The spider wingless/Wnt genes are expressed in different patterns from their orthologs in insects and crustaceans. The Cs-wingless gene is expressed in iterated stripes just anterior to the engrailed stripes, but is not expressed in the most ventral region of the germ band. However, Cs-Wnt5-1 appears to act in this ventral region. Cs-wingless and Cs-Wnt5-1 together seem to perform the role of insect wingless. Although there are differences, the wingless/Wnt-expressing cells and en-expressing cells seem to define an important boundary that is conserved among arthropods. This boundary may match the parasegmental compartment boundary and is even visible morphologically in the spider embryo. An additional piece of evidence for a parasegmental organization comes from the expression domains of the Hox genes that are confined to the boundaries, as molecularly defined by the engrailed and wingless/Wnt genes. Parasegments, therefore, are presumably important functional units and conserved entities in arthropod development and form an ancestral character of arthropods. The lack of by engrailed and wingless/Wnt-defined boundaries in other segmented phyla does not support a common origin of segmentation.     

Parasegmental appendage allocation in annelids and arthropods and the homology of parapodia and arthropodia

The new animal phylogeny disrupts the traditional taxon Articulata (uniting arthropods and annelids) and thus calls into question the homology of the body segments and appendages in the two groups. Recent work in the annelid Platynereis dumerilii has shown that although the set of genes involved in body segmentation is similar in the two groups, the body units of annelids correspond to arthropod parasegments not segments. This challenges traditional ideas about the homology of "segmental" organs in annelids and arthropods, including their appendages. Here I use the expression of engrailed, wingless and Distal-less in the arthropod Artemia franciscana to identify the parasegment boundary and the appendage primordia. I show that the early body organization including the appendage primordia is parasegmental and thus identical to the annelid organization and by deriving the different adult appendages from a common ground plan I suggest that annelid and arthropod appendages are homologous structures despite their different positions in the adult animals. This also has implications for the new animal phylogeny, because it suggests that Urprotostomia was not only parasegmented but also had parasegmental appendages similar to extant annelids, and that limb-less forms in the Protostomia are derived from limb-bearing forms.     

Gene expression suggests decoupled dorsal and ventral segmentation in the millipede Glomeris marginata (Myriapoda: Diplopoda)

Diplopods (millipedes) are known for their irregular body segmentation. Most importantly, the number of dorsal segmental cuticular plates (tergites) does not match the number of ventral structures (e.g., sternites). Controversial theories exist to explain the origin of this so-called diplosegmentation. We have studied the embryology of a representative diplopod, Glomeris marginata, and have analyzed the segmentation genes engrailed (en), hedgehog (hh), cubitus-interruptus (ci), and wingless (wg). We show that dorsal segments can be distinguished from ventral segments. They differ not only in number and developmental history, but also in gene expression patterns. engrailed, hedgehog, and cubitus-interruptus are expressed in both ventral and dorsal segments, but at different intrasegmental locations, whereas wingless is expressed only in the ventral segments, but not in the dorsal segments. Ventrally, the patterns are similar to what has been described from Drosophila and other arthropods, consistent with a conserved role of these genes in establishing parasegment boundaries. On the dorsal side, however, the gene expression patterns are different and inconsistent with a role in boundary formation between segments, but they suggest that these genes might function to establish the tergite borders. Our data suggest a profound and rather complete decoupling of dorsal and ventral segmentation leading to the dorsoventral discrepancies in the number of segmental elements. Based on gene expression, we propose a model that may resolve the hitherto controversial issue of the correlation between dorsal tergites and ventral leg pairs in basal diplopods (e.g., Glomeris) and is suggestive also for derived, ring-forming diplopods (e.g., Juliformia).     

The segmental pattern of otx, gbx, and Hox genes in the annelid Platynereis dumerilii

SUMMARY Annelids and arthropods, despite their distinct classification as Lophotrochozoa and Ecdysozoa, present a morphologically similar, segmented body plan. To elucidate the evolution of segmentation and, ultimately, to align segments across remote phyla, we undertook a refined expression analysis to precisely register the expression of conserved regionalization genes with morphological boundaries and segmental units in the marine annelid Platynereis dumerilii. We find that Pdu-otx defines a brain region anterior to the first discernable segmental entity that is delineated by a stripe of engrailed-expressing cells. The first segment is a "cryptic" segment that lacks chaetae and parapodia. This and the subsequent three chaetigerous larval segments harbor the anterior expression boundary of gbx, hox1, hox4, and lox5 genes, respectively. This molecular segmental topography matches the segmental pattern of otx, gbx, and Hox gene expression in arthropods. Our data thus support the view that an ancestral ground pattern of segmental identities existed in the trunk of the last common protostome ancestor that was lost or modified in protostomes lacking overt segmentation.     

The polychaete Platynereis dumerilii (Annelida): a laboratory animal with spiralian cleavage, lifelong segment proliferation and a mixed benthic/pelagic life cycle

Platynereis dumerilii, a marine polychaetous annelid with indirect development, can be continuously bred in the laboratory. Here, we describe its spectacular reproduction and development and address a number of open research problems. Oogenesis is easily studied because the oocytes grow while floating in the coelom. Unlike the embryos of other model spiralians, the Platynereis embryo is transparent giving insight into the dynamic structures and processes inside the cells that accompany the prevailing anisotropic cleavages. Functional studies on cell specification and differential gene expression in embryos, larvae, and later stages are underway. Lifelong proliferation of uniform trunk segments qualifies Platynereis as a model for the study of gene expression and of the functional circuitry of this process. Platynereis can also become a stepping stone in the comparison of segmentation between annelids and arthropods because it comes closer to the putative ancestral morphology and style of development than other model annelids.     

Segmenting the fly embryo: logical analysis of the role of the segment polarity cross-regulatory module

Initially activated by the pair-rule genes, the expression patterns of the segment polarity genes engrailed and wingless become consolidated through inter-cellular interactions between juxtaposed cells. We delineate a logical model focusing on a dozen molecular components at the core of the regulatory network controlling this process. Our model leads to the following conclusions: (1) the pair-rule signals, which activate engrailed and wingless genes independently of each other, need to be operative until the inter-cellular circuit involving these two genes is functional. This implies that the pair-rule pattern is instrumental both in determining the activation of the genes engrailed and wingless in rows of adjacent cells, and in consolidating these expression patterns; (2) the consolidation of engrailed and wingless expression patterns requires the simultaneous activation of both autocrine and paracrine Wingless-pathways, and the Hedgehog pathway; (3) protein kinase A plays at least two roles through the phosphorylation of Cubitus interruptus, the effector molecule of the Hedgehog signalling pathway and (4) the roles of Sloppy-paired and Naked in the delineation of the engrailed and wingless expression domains are emphasized as being important for segmental boundary formation. Moreover, the application of an original computational method leads to the delineation of a subset of crucial regulatory circuits enabling the coexistence of specific expression states at the cellular level, as well as specific combination of cellular states inter-connected through Wingless and Hedgehog signalling. Finally, the simulation of altered expressions of segment polarity genes leads to results consistent with the published data.     

Orthologs of key vertebrate neural genes are expressed during neurogenesis in the annelid Platynereis dumerilii

SUMMARY The molecular mechanisms underlying the formation and patterning of the nervous system are relatively poorly understood for lophotrochozoans (like annelids) as compared with ecdysozoans (especially Drosophila ) and deuterostomes (especially vertebrates). Therefore, we have undertaken a candidate gene approach to study aspects of neurogenesis in a polychaete annelid Platynereis dumerilii . We determined the spatiotemporal expression for Platynereis orthologs of four genes ( SoxB, Churchill, prospero / Prox , and SoxC) known to play key roles in vertebrate neurogenesis. During Platynereis development, SoxB is expressed in the neuroectoderm and its expression switches off when committed neural precursors are formed. Subsequently, Prox is expressed in all differentiating neural precursors in the central and peripheral nervous systems. Finally, SoxC and Churchill are transcribed in patterns consistent with their involvement in neural differentiation. The expression patterns of Platynereis SoxB and Prox closely resemble those in Drosophila and vertebrates—this suggests that orthologs of these genes play similar neurogenic roles in all bilaterians. Whereas Platynereis SoxC , like its vertebrate orthologs, plays a role in neural cell differentiation, related genes in Drosophila do not appear to be involved in neurogenesis. Finally, conversely to Churchill in Platynereis , vertebrate orthologs of this gene are expressed during neuroectoderm formation, but not later during nerve cell differentiation; in the insect lineage, homologs of these genes have been secondarily lost. In spite of such instances of functional divergence or loss, the present study shows conspicuous similarities in the genetic control of neurogenesis among bilaterians. These commonalities suggest that key features of the genetic program for neurogenesis are ancestral to bilaterians.     

Secretion and localized transcription suggest a role in positional signaling for products of the segmentation gene hedgehog.

The segment polarity genes engrailed and wingless are expressed in neighboring stripes of cells on opposite sides of the Drosophila parasegment boundary. Each gene is mutually required for maintenance of the other's expression; continued expression of both also requires several other segment polarity genes. We show here that one such gene, hedgehog, encodes a protein targeted to the secretory pathway and is expressed coincidently with engrailed in embryos and in imaginal discs; maintenance of the hedgehog expression pattern is itself dependent upon other segment polarity genes including engrailed and wingless. Expression of hedgehog thus functions in, and is sensitive to, positional signaling. These properties are consistent with the non-cell autonomous requirement for hedgehog in cuticular patterning and in maintenance of wingless expression.     

The consequences of ubiquitous expression of the wingless gene in the Drosophila embryo.

The segment polarity gene wingless has an essential function in cell-to-cell communication during various stages of Drosophila development. The wingless gene encodes a secreted protein that affects gene expression in surrounding cells but does not spread far from the cells where it is made. In larvae, wingless is necessary to generate naked cuticle in a restricted part of each segment. To test whether the local accumulation of wingless is essential for its function, we made transgenic flies that express wingless under the control of a hsp70 promoter (HS-wg flies). Uniform wingless expression results in a complete naked cuticle, uniform armadillo accumulation and broadening of the engrailed domain. The expression patterns of patched, cubitus interruptus Dominant and Ultrabithorax follow the change in engrailed. The phenotype of heatshocked HS-wg embryos resembles the segment polarity mutant naked, suggesting that embryos that overexpress wingless or lack the naked gene enter similar developmental pathways. The ubiquitous effects of ectopic wingless expression may indicate that most cells in the embryo can receive and interpret the wingless signal. For the development of the wild-type pattern, it is required that wingless is expressed in a subset of these cells.     

Interactions between fused, a segment-polarity gene in Drosophila, and other segmentation genes.

Fused (fu) is a segment polarity gene whose product is maternally required in the posterior part of each segment. To define further the role of fused and determine how it interacts with other segmentation genes, we examined the phenotypes obtained by combining fused with mutations of pair rule, homeotic and other segment polarity loci. When it was possible, we also looked at the distribution of corresponding proteins in fused mutant embryos. We observed that fused-naked (fu;nkd) double mutant embryos display a phenotypic suppression of simple mutant phenotypes: both naked cuticle and denticle belts, which would normally have been deleted by one of the two mutants alone, were restored. In fused mutant embryos, engrailed (en) and wingless (wg) expression was normal until germ band extension, but partially and completely disappeared respectively during germ band retraction. In the fu;nkd double mutant embryo, en was expressed as in nkd mutant at germ band extension, but later this expression was restricted and became normal at germ band retraction. On the contrary, wg expression disappeared as in fu simple mutant embryos. We conclude that the requirements for fused, naked and wingless activities for normal segmental patterning are not absolute, and propose mechanisms by which these genes interact to specify anterior and posterior cell fates.     

Cell patterning in the Drosophila segment: spatial regulation of the segment polarity gene patched

Intrasegmental patterning in the Drosophila embryo requires the activity of the segment polarity genes. The acquisition of positional information by cells during embryogenesis is reflected in the dynamic patterns of expression of several of these genes. In the case of patched, early ubiquitous expression is followed by its repression in the anterior portion of each parasegment; subsequently each broad band of expression splits into two narrow stripes. In this study we analyse the contribution of other segment polarity gene functions to the evolution of this pattern; we find that the first step in patched regulation is under the control of engrailed whereas the second requires the activity of both cubitus interruptusD and patched itself. Furthermore, the products of engrailed, wingless and hedgehog are essential for maintaining the normal pattern of expression of patched.     

Segmental expression of an engrailed-class gene during early development and neurogenesis in an annelid.

ht-en protein, an annelid homolog of the Drosophila engrailed protein, is expressed during both early development and neurogenesis in embryos of the leech, Helobdella triserialis. In Helobdella as in Drosophila, early expression is in segmentally iterated stripes of cells within the posterior portion of the segment and later expression is in cells of the segmental ganglia. These findings suggest that dual expression of an en-class gene was present in a common ancestor of annelids and arthropods.     

Suppressor of Hairless and Presenilin phenotypes imply involvement of canonical Notch-signalling in segmentation of the spider Cupiennius salei

Arthropods, vertebrates, and annelids all have a segmented body. Our recent discovery of involvement of Notch-signalling in spider segmentation revived the discussion on the origin of segmented body plans and suggests the sharing of a common genetic program in a common ancestor. Here, we analysed the spider homologues of the Suppressor of Hairless and Presenilin genes, which encode components of the canonical Notch-pathway, to further explore the role of Notch-signalling in spider segmentation. RNAi silencing of two spider Suppressor of Hairless homologues and the spider Presenilin homologue causes severe segmentation phenotypes. The most prominent defect is the consistent breakdown of segmentation after the formation of three (Suppressor of Hairless) or five (Presenilin) opisthosomal segments. These phenotypes indicate that Notch-signalling during spider segmentation likely involves the canonical pathway via Presenilin and Suppressor of Hairless. Furthermore, it implies that Notch-signalling influences both the formation and patterning of the spider segments: it is required for the specification of the posterior segments and for proper specification of the segment boundaries. We argue that alternative, partly redundant, pathways might act in the formation of the anterior segments that are not active in the posterior segments. This suggests that at least some differences exist in the specification of anterior and posterior segments of the spider, a finding that may be valid for most short germ arthropods. Our data provide additional evidence for the similarities of Notch-signalling in spider segmentation and vertebrate somitogenesis and strengthen our previous notion that the formation of the segments in arthropods and vertebrates might have shared a genetic program in a common ancestor.