And Lophotrochozoa makes three: Notch/Hes signaling in annelid segmentation

Segmentation is unquestionably a major factor in the evolution of complex body plans, but how this trait itself evolved is unknown. Approaching this problem requires comparing the molecular mechanisms of segmentation in diverse segmented and unsegmented taxa. Notch/Hes signaling is involved in segmentation in sequentially segmenting vertebrates and arthropods, as judged by patterns of expression of one or more genes in this network and by the disruption of segmental patterning when Notch/Hes signaling is disrupted. We have previously shown that Notch and Hes homologs are expressed in the posterior progress zone (PPZ), from which segments arise, in the leech Helobdella robusta, a sequentially segmenting lophotrochozoan (phylum Annelida). Here, we show that disrupting Notch/Hes signaling disrupts segmentation in this species as well. Thus, Notch/Hes functions in either the maintenance of the PPZ and/or the patterning processes of segmentation in representatives of all three superphyla of bilaterally symmetric animals. These results are consistent with two evolutionary scenarios. In one, segmentation was already present in the ancestor of all three superphyla. In the other, Notch/Hes signaling functioned in axial growth by terminal addition in an unsegmented bilaterian ancestor, and was subsequently exapted to function in segmentation as that process evolved independently in two or more taxa.     

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.     

Trilobite body patterning and the evolution of arthropod tagmosis

Preservation permitting patterns of developmental evolution can be reconstructed within long extinct clades, and the rich fossil record of trilobite ontogeny and phylogeny provides an unparalleled opportunity for doing so. Furthermore, knowledge of Hox gene expression patterns among living arthropods permit inferences about possible Hox gene deployment in trilobites. The trilobite anteroposterior body plan is consistent with recent suggestions that basal euarthropods had a relatively low degree of tagmosis among cephalic limbs, possibly related to overlapping expression domains of cephalic Hox genes. Trilobite trunk segments appeared sequentially at a subterminal generative zone, and were exchanged between regions of fused and freely articulating segments during growth. Homonomous trunk segment shape and gradual size transition were apparently phylogenetically basal conditions and suggest a single trunk tagma. Several derived clades independently evolved functionally distinct tagmata within the trunk, apparently exchanging flexible segment numbers for greater regionally autonomy. The trilobite trunk chronicles how different aspects of arthropod segmentation coevolved as the degree of tagmosis increased.     

The segmented urbilateria: a testable scenario

The idea that the last common ancestor of bilaterian animals(Urbilateria) was segmented has been raised recently on evidencecoming from comparative molecular embryology. Leaving asidethe complex debate on the value of genetic evidence, the morphologicaland developmental evidence in favor of a segmented Urbilateriaare discussed in the light of the emerging molecular phylogenyof metazoans. Applying a cladistic character optimization procedureto the question of segmentation is vastly complicated by theproblem of defining without ambiguity what segmentation is andto what taxa this definition applies. An ancestral segmentationmight have undergone many complex derivations in each differentphylum, thus rendering the cladistics approaches problematic.Taking the most general definitions of coelom and segmentationhowever, some remarkably similar patterns are found across thebilaterian tree in the way segments are formed by the posterioraddition of mesodermal segments or somites. Postulating thatthese striking similarities in mesodermal patterns are ancestral,a scenario for the diversification of bilaterians from a metamericancestor is presented. Several types of evolutionary mechanisms(specialization, tagmosis, progenesis) operating on a segmentedancestral body plan would explain the rapid emergence of bodyplans during the Cambrian. We finally propose to test this hypothesisby comparing genes involved in mesodermal segmentation.     

Segmentation: mono- or polyphyletic?

Understanding the evolutionary origins of segmented body plans in the metazoa has been a long-standing fascination for scientists. Competing hypotheses explaining the presence of distinct segmented taxa range from the suggestion that all segmentation in the metazoa is homologous to the proposal that segmentation arose independently many times, even within an individual clade or species. A major new source of information regarding the extent of homology vs. homoplasy of segmentation in recent years has been an examination of the extent to which molecular mechanisms underlying the segmentation process are conserved, the rationale being that a shared history will be apparent by the presence of common molecular components of a developmental program that give rise to a segmented body plan. There has been substantial progress recently in understanding the molecular mechanisms underlying the segmentation process in many groups, specifically within the three overtly segmented phyla: Annelida, Arthropoda and Chordata. This review will discuss what we currently know about the segmentation process in each group and how our understanding of the development of segmented structures in distinct taxa have influenced the hypotheses explaining the presence of a segmented body plan in the metazoa.     

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.     

Why are arthropods segmented?

SUMMARY Segmentation as an attribute of organisms is being increasingly discussed in the recent literature because (1) new phylogenies suggest that organisms classically considered to be segmented may lie in separate clades; (2) the molecular basis of segmental development has been much studied; (3) various theories of bilaterian origins place weight on segmentation as a primitive character; (4) there has been recent stress on the importance of modularity as an evolutionary topic. However, the definition and extent of segmentation are highly ambiguous and usually typological. Here, segmentation is regarded as an attribute of organs, not organisms. The evolution of just one system, the arthropod epidermis, is examined on the basis of the fossil record and the extant euarthropods, tardigrades, and onychophorans. It may be seen to have become segmented in a complex pathway that necessitated shifts in function, redundancy, and changes in associated organs. This complexity must inevitably reflect on, and to an extent have primacy over, the genetic basis for the changes involved. Evolutionary functional morphology has been relatively little considered in the context of the evolution of development, but may play an important role in defining the framework within which this evolution occurs.     

The evolution of the Ecdysozoa

Ecdysozoa is a clade composed of eight phyla: the arthropods, tardigrades and onychophorans that share segmentation and appendages and the nematodes, nematomorphs, priapulids, kinorhynchs and loriciferans, which are worms with an anterior proboscis or introvert. Ecdysozoa contains the vast majority of animal species and there is a great diversity of body plans among both living and fossil members. The monophyly of the clade has been called into question by some workers based on analyses of whole genome datasets. We review the evidence that now conclusively supports the unique origin of these phyla. Relationships within Ecdysozoa are also controversial and we discuss the molecular and morphological evidence for a number of monophyletic groups within this superphylum.     

Unlocking the early fossil record of the arthropod central nervous system

Extant panarthropods (euarthropods, onychophorans and tardigrades) are hallmarked by stunning morphological and taxonomic diversity, but their central nervous systems (CNS) are relatively conserved. The timing of divergences of the ground pattern CNS organization of the major panarthropod clades has been poorly constrained because of a scarcity of data from their early fossil record. Although the CNS has been documented in three-dimensional detail in insects from Cenozoic ambers, it is widely assumed that these tissues are too prone to decay to withstand other styles of fossilization or geologically older preservation. However, Cambrian Burgess Shale-type compressions have emerged as sources of fossilized brains and nerve cords. CNS in these Cambrian fossils are preserved as carbon films or as iron oxides/hydroxides after pyrite in association with carbon. Experiments with carcasses compacted in fine-grained sediment depict preservation of neural tissue for a more prolonged temporal window than anticipated by decay experiments in other media. CNS and compound eye characters in exceptionally preserved Cambrian fossils predict divergences of the mandibulate and chelicerate ground patterns by Cambrian Stage 3 (ca 518 Ma), a dating that is compatible with molecular estimates for these splits.     

Ether-induced segmentation disturbances in Drosophila melanogaster

Summary Drosophila embryos, exposed to ether between 1 and 4 h after oviposition, develop defects ranging from the complete lack of segmentation to isolated gaps in single segments. Between these extremes are varying extents of incomplete and abnormal segmentation. On the basis of both their temporal and spatial characteristics, five major phenotype classes may be distinguished: headless — unsegmented or incompletely segmented anteriorly; gap — interruptions of segmentation not obviously periodic; alternating segment gaps — interruptions with double segment periodicities; fused segments; and short segments — truncations with single segment periodicities. Many defects resemble known mutant phenotypes. The disturbances in segmentation are predominantly global and frequently accompanied by alterations in segment specification, such that the segments obtained show no resemblance to the normal homologues. These features, together with the distinctive spatiotemporal characteristics of the defects, all point to segmentation as a dynamic process. The regular spacing of the segments and the fact that the entire range of defects is inducible by ether are further consistent with the hypothesis that at least part of the segmentation process may consist of physicochemical reactions coordinated over the whole body. The relationship between our data and data from genetic and other analyses are briefly discussed.     

Evolution of networks for body plan patterning; interplay of modularity, robustness and evolvability

A major goal of evolutionary developmental biology (evo-devo) is to understand how multicellular body plans of increasing complexity have evolved, and how the corresponding developmental programs are genetically encoded. It has been repeatedly argued that key to the evolution of increased body plan complexity is the modularity of the underlying developmental gene regulatory networks (GRNs). This modularity is considered essential for network robustness and evolvability. In our opinion, these ideas, appealing as they may sound, have not been sufficiently tested. Here we use computer simulations to study the evolution of GRNs' underlying body plan patterning. We select for body plan segmentation and differentiation, as these are considered to be major innovations in metazoan evolution. To allow modular networks to evolve, we independently select for segmentation and differentiation. We study both the occurrence and relation of robustness, evolvability and modularity of evolved networks. Interestingly, we observed two distinct evolutionary strategies to evolve a segmented, differentiated body plan. In the first strategy, first segments and then differentiation domains evolve (SF strategy). In the second scenario segments and domains evolve simultaneously (SS strategy). We demonstrate that under indirect selection for robustness the SF strategy becomes dominant. In addition, as a byproduct of this larger robustness, the SF strategy is also more evolvable. Finally, using a combined functional and architectural approach, we determine network modularity. We find that while SS networks generate segments and domains in an integrated manner, SF networks use largely independent modules to produce segments and domains. Surprisingly, we find that widely used, purely architectural methods for determining network modularity completely fail to establish this higher modularity of SF networks. Finally, we observe that, as a free side effect of evolving segmentation and differentiation in combination, we obtained in-silico developmental mechanisms resembling mechanisms used in vertebrate development.     

Developmental architecture of the nervous system in Themiste lageniformis (Sipuncula): New evidence from confocal laser scanning microscopy and gene expression

Sipuncula is a clade of unsegmented marine worms that are currently placed among the basal radiation of conspicuously segmented Annelida. Their new location provides a unique opportunity to reinvestigate the evolution and development of segmented body plans. Neural segmentation is clearly evident during ganglionic ventral nerve cord (VNC) formation across Sedentaria and Errantia, which includes the majority of annelids. However, recent studies show that some annelid taxa outside of Sedentaria and Errantia have a medullary cord, without ganglia, as adults. Importantly, neural development in these taxa is understudied and interpretation can vary widely. For example, reports in sipunculans range from no evidence of segmentation to vestigial segmentation as inferred from a few pairs of serially repeated neuronal cell bodies along the VNC. We investigated patterns of pan-neuronal, neuronal subtype, and axonal markers using immunohistochemistry and whole mount in situ hybridization (WMISH) during neural development in an indirect-developing sipunculan, Themiste lageniformis. Confocal imaging revealed two clusters of 5HT⁺ neurons, two pairs of FMRF⁺ neurons, and Tubulin⁺ peripheral neurites that appear to be serially positioned along the VNC, similar to other sipunculans, to other annelids, and to spiralian taxa outside of Annelida. WMISH of a synaptotagmin1 ortholog in T. lageniformis (Tl-syt1) showed expression throughout the centralized nervous system (CNS), including the VNC where it appears to correlate with mature 5HT⁺ and FMRF⁺ neurons. An ortholog of elav1 (Tl-elav1) showed expression in differentiated neurons of the CNS with continuous expression in the VNC, supporting evidence of a medullary cord, and refuting evidence of ontogenetic segmentation during formation of the nervous system. Thus, we conclude that sipunculans do not exhibit any signs of morphological segmentation during development.     

Cell lineage analysis of pattern formation in the Tubifex embryo. I. Segmentation in the mesoderm.

Annelids are strongly segmented animals that display a high degree of metamerism in their body plan. The embryonic origin of metameric segmentation was examined in an oligochaete annelid Tubifex using lineage tracers. Segmental organization arises sequentially in the anterior-to-posterior direction along the longitudinal axis of the mesodermal germ band, a coherent column of primary blast cells that are produced from the mesodermal teloblast. Shortly after its birth, each primary blast cell undergoes a spatiotemporally stereotyped sequence of cell divisions to generate three classes of cells (in terms of cell size), which together give rise to a distinct cell cluster. Each cluster is composed of descendants of a single primary blast cell; there is no intermingling of cells between adjacent clusters. Relatively small-sized cells in each cluster become localized at its periphery, and they form coelomic walls including an intersegmental septum to establish individuality of segments. A set of cell ablation experiments showed that these features of mesodermal segmentation are not affected by the absence of the overlying ectodermal germ band. These results suggest that each primary blast cell serves as a founder cell of each mesodermal segment and that the boundary between segments is determined autonomously. It is concluded that the metameric body plan of Tubifex arises from an initially simple organization (i.e., a linear series) of segmental founder cells.     

The functional relationship between ectodermal and mesodermal segmentation in the crustacean, Parhyale hawaiensis

In arthropods, annelids and chordates, segmentation of the body axis encompasses both ectodermal and mesodermal derivatives. In vertebrates, trunk mesoderm segments autonomously and induces segmental arrangement of the ectoderm-derived nervous system. In contrast, in the arthropod Drosophila melanogaster, the ectoderm segments autonomously and mesoderm segmentation is at least partially dependent on the ectoderm. While segmentation has been proposed to be a feature of the common ancestor of vertebrates and arthropods, considering vertebrates and Drosophila alone, it is impossible to conclude whether the ancestral primary segmented tissue was the ectoderm or the mesoderm. Furthermore, much of Drosophila segmentation occurs before gastrulation and thus may not accurately represent the mechanisms of segmentation in all arthropods. To better understand the relationship between segmented germ layers in arthropods, we asked whether segmentation is an intrinsic property of the ectoderm and/or the mesoderm in the crustacean Parhyale hawaiensis by ablating either the ectoderm or the mesoderm and then assaying for segmentation in the remaining tissue layer. We found that the ectoderm segments autonomously. However, mesoderm segmentation requires at least a permissive signal from the ectoderm. Although mesodermal stem cells undergo normal rounds of division in the absence of ectoderm, they do not migrate properly in respect to migration direction and distance. In addition, their progeny neither divide nor express the mesoderm segmentation markers Ph-twist and Ph-Even-skipped. As segmentation is ectoderm-dependent in both Parhyale and holometabola insects, we hypothesize that segmentation is primarily a property of the ectoderm in pancrustacea.     

A three-phase model of arthropod segmentation

Molecular and morphological evidence (expression patterns of pair-rule genes and segmental position of the genital openings and other segmental markers) suggest that the segmental units of the arthropod body are specified, in early ontogeny, by three spatially and/or temporally distinct mechanisms and do not appear in a strict antero-posterior sequence. A first anterior set of indivisible segments (naupliar segments, possibly three in all arthropods) is followed by a set of more caudal (post-naupliar) primary units (eosegments, possibly ten in all arthropods) which then undergo a process of secondary segmentation, thus giving rise to a higher number of definitive segments (merosegments). The number of merosegments deriving from each eosegment is characteristic of the different arthropod clades and is mostly stable at the level of the traditional arthropodan classes or subclasses. All their segmentation patterns, however, including those found in the segmental organisation of highly segmented forms (such as centipedes and millipedes, notostracan, lipostracan and anostracan crustaceans, and trilobites) are reducible to the basic groundplan with three naupliar and ten postnaupliar segments. These basic units of arthropod segmentation may also have an equivalent in other Ecdysozoa, despite the lack of any segmentation (nematodes) or, at least, of an overt segmentation (kinorhynchs).     

The origin of crustaceans: new evidence from the Early Cambrian of China.

One of the smallest arthropods recently discovered in the Early Cambrian Maotianshan Shale Lagerstätte is described. Ercaia gen. nov. has an untagmatized trunk bearing serially repeated biramous appendages (long and segmented endopods and flap-like exopods), a head with an acron bearing stalked lateral eyes and a sclerite and two pairs of antennae. The position of this 520 million-year-old tiny arthropod within the Crustacea is supported by several anatomical features: (i) a head with five pairs of appendages including two pairs of antennae, (ii) highly specialized antennae (large setose fans with a possible function in feeding), and (iii) specialized last trunk appendages (segmented pediform structures fringed with setae). The segmentation pattern of Ercaia (5 head and 13 trunk) is close to that of Maxillopoda but lacks the trunk tagmosis of modern representatives of the group. Ercaia is interpreted as a possible derivative of the stem group Crustacea. Ercaia is likely to have occupied an ecological niche similar to those of some Recent meiobenthic organisms (e.g. copepods living in association with sediment). This new fossil evidence supports the remote ancestry of crustaceans well before the Late Cambrian and shows, along with other fossil data (mainly Early Cambrian in China), that a variety of body plans already coexisted among the primitive crustacean stock.     

The evolutionary history of crustacean segmentation: a fossil-based perspective

The evolution of segmentation in Crustacea, that is, the formation of sclerotized and jointed body somites and arrangement of somites into tagmata, is viewed in light of historical traits and functional constraints. The set of Early to Late Cambrian 'Orsten' arthropods have informed our current views of crustacean evolution considerably. These three-dimensionally preserved fossils document ancient morphologies, as opposed to purely hypothetical models and, because of the unusual preservation of larval stages, provide us with unparalleled insight into the morphogenesis of body somites and their structural equipment. The variety of evolutionary levels represented in the 'Orsten' including lobopodians, tardigrades, and pentastomids also allows phylogenetic interpretations far beyond the Crustacea. The 'Orsten' evidence and data from representatives of the Lower Cambrian Chengjiang biota in southwestern China, including phylogenetically earlier forms, form the major source of our morphology-based review of structural and functional developments that led toward the Crustacea. The principal strategy of arthropods is the simultaneous development of head somites, as expressed in a basal "head larva," and a successive addition of postcephalic somites from a preterminal budding zone with progressive maturation of metameric structures. This can be recognized in the developmental patterns of extant and fossil representatives of several euarthropod taxa, particularly crustaceans, trilobites, and chelicerates (at least basally). The development of these taxa points to an early somite-poor and free-living hatching stage. Embryonic development to a late stage within an egg, as occurring in recent onychophorans and certain in-group euarthropods, is regarded as achieved several times convergently.     

Decoupling elongation and segmentation: Notch involvement in anostracan crustacean segmentation

Repeated body segments are a key feature of arthropods. The formation of body segments occurs via distinct developmental pathways within different arthropod clades. Although some species form their segments simultaneously without any accompanying measurable growth, most arthropods add segments sequentially from the posterior of the growing embryo or larva. The use of Notch signaling is increasingly emerging as a common feature of sequential segmentation throughout the Bilateria, as inferred from both the expression of proteins required for Notch signaling and the genetic or pharmacological disruption of Notch signaling. In this study, we demonstrate that blocking Notch signaling by blocking γ‐secretase activity causes a specific, repeatable effect on segmentation in two different anostracan crustaceans, Artemia franciscana and Thamnocephalus platyurus. We observe that segmentation posterior to the third or fourth trunk segment is arrested. Despite this marked effect on segment addition, other aspects of segmentation are unaffected. In the segments that develop, segment size and boundaries between segments appear normal, engrailed stripes are normal in size and alignment, and overall growth is unaffected. By demonstrating Notch involvement in crustacean segmentation, our findings expand the evidence that Notch plays a crucial role in sequential segmentation in arthropods. At the same time, our observations contribute to an emerging picture that loss‐of‐function Notch phenotypes differ significantly between arthropods suggesting variability in the role of Notch in the regulation of sequential segmentation. This variability in the function of Notch in arthropod segmentation confounds inferences of homology with vertebrates and lophotrochozoans.