Fast cycling culture of the annelid model Platynereis dumerilii.

Platynereis dumerilii, a marine annelid, is a model animal that has gained popularity in various fields such as developmental biology, biological rhythms, nervous system organization and physiology, behaviour, reproductive biology, and epigenetic regulation. The transparency of P. dumerilii tissues at all developmental stages makes it easy to perform live microscopic imaging of all cell types. In addition, the slow-evolving genome of P. dumerilii and its phylogenetic position as a representative of the vast branch of Lophotrochozoans add to its evolutionary significance. Although P. dumerilii is amenable to transgenesis and CRISPR-Cas9 knockouts, its relatively long and indefinite life cycle, as well as its semelparous reproduction have been hindrances to its adoption as a reverse genetics model. To overcome this limitation, an adapted culturing method has been developed allowing much faster life cycling, with median reproductive age at 13-14 weeks instead of 25-35 weeks using the traditional protocol. A low worm density in boxes and a strictly controlled feeding regime are important factors for the rapid growth and health of the worms. This culture method has several advantages, such as being much more compact, not requiring air bubbling or an artificial moonlight regime for synchronized sexual maturation and necessitating only limited water change. A full protocol for worm care and handling is provided.

The Nereid on the rise: Platynereis as a model system.

The Nereid Platynereis dumerilii (Audouin and Milne Edwards (Annales des Sciences Naturelles 1:195-269, 1833) is a marine annelid that belongs to the Nereididae, a family of errant polychaete worms. The Nereid shows a pelago-benthic life cycle: as a general characteristic for the superphylum of Lophotrochozoa/Spiralia, it has spirally cleaving embryos developing into swimming trochophore larvae. The larvae then metamorphose into benthic worms living in self-spun tubes on macroalgae. Platynereis is used as a model for genetics, regeneration, reproduction biology, development, evolution, chronobiology, neurobiology, ecology, ecotoxicology, and most recently also for connectomics and single-cell genomics. Research on the Nereid started with studies on eye development and spiralian embryogenesis in the nineteenth and early twentieth centuries. Transitioning into the molecular era, Platynereis research focused on posterior growth and regeneration, neuroendocrinology, circadian and lunar cycles, fertilization, and oocyte maturation. Other work covered segmentation, photoreceptors and other sensory cells, nephridia, and population dynamics. Most recently, the unique advantages of the Nereid young worm for whole-body volume electron microscopy and single-cell sequencing became apparent, enabling the tracing of all neurons in its rope-ladder-like central nervous system, and the construction of multimodal cellular atlases. Here, we provide an overview of current topics and methodologies for P. dumerilii, with the aim of stimulating further interest into our unique model and expanding the active and vibrant Platynereis community.

Globins in the marine annelid Platynereis dumerilii shed new light on hemoglobin evolution in bilaterians.

BACKGROUND: How vascular systems and their respiratory pigments evolved is still debated. While many animals present a vascular system, hemoglobin exists as a blood pigment only in a few groups (vertebrates, annelids, a few arthropod and mollusk species). Hemoglobins are formed of globin sub-units, belonging to multigene families, in various multimeric assemblages. It was so far unclear whether hemoglobin families from different bilaterian groups had a common origin. RESULTS: To unravel globin evolution in bilaterians, we studied the marine annelid Platynereis dumerilii, a species with a slow evolving genome. Platynereis exhibits a closed vascular system filled with extracellular hemoglobin. Platynereis genome and transcriptomes reveal a family of 19 globins, nine of which are predicted to be extracellular. Extracellular globins are produced by specialized cells lining the vessels of the segmental appendages of the worm, serving as gills, and thus likely participate in the assembly of a previously characterized annelid-specific giant hemoglobin. Extracellular globin mRNAs are absent in smaller juveniles, accumulate considerably in growing and more active worms and peak in swarming adults, as the need for O2 culminates. Next, we conducted a metazoan-wide phylogenetic analysis of globins using data from complete genomes. We establish that five globin genes (stem globins) were present in the last common ancestor of bilaterians. Based on these results, we propose a new nomenclature of globins, with five clades. All five ancestral stem-globin clades are retained in some spiralians, while some clades disappeared early in deuterostome and ecdysozoan evolution. All known bilaterian blood globin families are grouped in a single clade (clade I) together with intracellular globins of bilaterians devoid of red blood. CONCLUSIONS: We uncover a complex "pre-blood" evolution of globins, with an early gene radiation in ancestral bilaterians. Circulating hemoglobins in various bilaterian groups evolved convergently, presumably in correlation with animal size and activity. However, all hemoglobins derive from a clade I globin, or cytoglobin, probably involved in intracellular O2 transit and regulation. The annelid Platynereis is remarkable in having a large family of extracellular blood globins, while retaining all clades of ancestral bilaterian globins.

Cell lineage and cell cycling analyses of the 4d micromere using live imaging in the marine annelid Platynereis dumerilii.

Cell lineage, cell cycle, and cell fate are tightly associated in developmental processes, but in vivo studies at single-cell resolution showing the intricacies of these associations are rare due to technical limitations. In this study on the marine annelid Platynereis dumerilii, we investigated the lineage of the 4d micromere, using high-resolution long-term live imaging complemented with a live-cell cycle reporter. 4d is the origin of mesodermal lineages and the germline in many spiralians. We traced lineages at single-cell resolution within 4d and demonstrate that embryonic segmental mesoderm forms via teloblastic divisions, as in clitellate annelids. We also identified the precise cellular origins of the larval mesodermal posterior growth zone. We found that differentially-fated progeny of 4d (germline, segmental mesoderm, growth zone) display significantly different cell cycling. This work has evolutionary implications, sets up the foundation for functional studies in annelid stem cells, and presents newly established techniques for live imaging marine embryos.

Centrin diversity and basal body patterning across evolution: new insights from Paramecium.

First discovered in unicellular eukaryotes, centrins play crucial roles in basal body duplication and anchoring mechanisms. While the evolutionary status of the founding members of the family, Centrin2/Vfl2 and Centrin3/cdc31 has long been investigated, the evolutionary origin of other members of the family has received less attention. Using a phylogeny of ciliate centrins, we identify two other centrin families, the ciliary centrins and the centrins present in the contractile filaments (ICL centrins). In this paper, we carry on the functional analysis of still not well-known centrins, the ICL1e subfamily identified in Paramecium, and show their requirement for correct basal body anchoring through interactions with Centrin2 and Centrin3. Using Paramecium as well as a eukaryote-wide sampling of centrins from completely sequenced genomes, we revisited the evolutionary story of centrins. Their phylogeny shows that the centrins associated with the ciliate contractile filaments are widespread in eukaryotic lineages and could be as ancient as Centrin2 and Centrin3.

The Notch pathway in the annelid Platynereis: insights into chaetogenesis and neurogenesis processes.

Notch is a key signalling pathway playing multiple and varied functions during development. Notch regulates the selection of cells with a neurogenic fate and maintains a pool of yet uncommitted precursors through lateral inhibition, both in insects and in vertebrates. Here, we explore the functions of Notch in the annelid Platynereis dumerilii (Lophotrochozoa). Conserved components of the pathway are identified and a scenario for their evolution in metazoans is proposed. Unexpectedly, neither Notch nor its ligands are expressed in the neurogenic epithelia of the larva at the time when massive neurogenesis begins. Using chemical inhibitors and neural markers, we demonstrate that Notch plays no major role in the general neurogenesis of larvae. Instead, we find Notch components expressed in nascent chaetal sacs, the organs that produce the annelid bristles. Impairing Notch signalling induces defects in chaetal sac formation, abnormalities in chaetae producing cells and a change of identity of chaeta growth accessory cells. This is the first bilaterian species in which the early neurogenesis processes appear to occur without a major involvement of the Notch pathway. Instead, Notch is co-opted to pattern annelid-specific organs, likely through a lateral inhibition process. These features reinforce the view that Notch signalling has been recruited multiple times in evolution due to its remarkable 'toolkit' nature.

A metameric origin for the annelid pygidium?

BACKGROUND: Segmented body organizations are widely represented in the animal kingdom. Whether the last common bilaterian ancestor was already segmented is intensely debated. Annelids display broad morphological diversity but many species are among the most homonomous metameric animals. The front end (prostomium) and tail piece (pygidium) of annelids are classically described as non-segmental. However, the pygidium structure and development remain poorly studied. RESULTS: Using different methods of microscopy, immunolabelling and a number of molecular markers, we describe the neural and mesodermal structures of the pygidium of Platynereis dumerilii. We establish that the pygidium possesses a complicated nervous system with a nerve ring and a pair of sensory ganglia, a complex intrinsic musculature, a large terminal circular blood sinus and an unusual unpaired torus-shaped coelomic cavity. We also describe some earlier steps of pygidial development and pygidial structure of mature animals after epitokous transformation. CONCLUSIONS: We describe a much more complex organization of the pygidium of P. dumerilii than previously suggested. Many of the characteristics are strikingly similar to those found in the trunk segments, opening the debate on whether the pygidium and trunk segments derive from the same ancestral metameric unit. We analyze these scenarios in the context of two classical theories on the origin of segmentation: the cyclomeric/archicoelomate concept and the colonial theory. Both theories provide possible explanations for the partial or complete homology of trunk segments and pygidium.

History of a prolific family: the Hes/Hey-related genes of the annelid Platynereis.

BACKGROUND: The Hes superfamily or Hes/Hey-related genes encompass a variety of metazoan-specific bHLH genes, with somewhat fuzzy phylogenetic relationships. Hes superfamily members are involved in a variety of major developmental mechanisms in metazoans, notably in neurogenesis and segmentation processes, in which they often act as direct effector genes of the Notch signaling pathway. RESULTS: We have investigated the molecular and functional evolution of the Hes superfamily in metazoans using the lophotrochozoan Platynereis dumerilii as model. Our phylogenetic analyses of more than 200 Metazoan Hes/Hey-related genes revealed the presence of five families, three of them (Hes, Hey and Helt) being pan-metazoan. Those families were likely composed of a unique representative in the last common metazoan ancestor. The evolution of the Hes family was shaped by many independent lineage specific tandem duplication events. The expression patterns of 13 of the 15 Hes/Hey-related genes in Platynereis indicate a broad functional diversification. Nevertheless, a majority of these genes are involved in two crucial developmental processes in annelids: neurogenesis and segmentation, resembling functions highlighted in other animal models. CONCLUSIONS: Combining phylogenetic and expression data, our study suggests an unusual evolutionary history for the Hes superfamily. An ancestral multifunctional annelid Hes gene may have undergone multiples rounds of duplication-degeneration-complementation processes in the lineage leading to Platynereis, each gene copies ensuring their maintenance in the genome by subfunctionalisation. Similar but independent waves of duplications are at the origin of the multiplicity of Hes genes in other metazoan lineages.

Segment formation in Annelids: patterns, processes and evolution.

The debate on the origin of segmentation is a central question in the study of body plan evolution in metazoans. Annelids are the most conspicuously metameric animals as most of the trunk is formed of identical anatomical units. In this paper, I summarize the various patterns of evolution of the metameric body plan in annelids, showing the remarkable evolvability of this trait, similar to what is also found in arthropods. I then review the different modes of segment formation in the annelid tree, taking into account the various processes taking place in the life histories of these animals, including embryogenesis, post-embryonic development, regeneration and asexual reproduction. As an example of the variations that occur at the cellular and genetic level in annelid segment formation, I discuss the processes of teloblastic growth or posterior addition in key groups in the annelid tree. I propose a comprehensive definition for the teloblasts, stem cells that are responsible for sequential segment addition. There are a diversity of different mechanisms used in annelids to produce segments depending on the species, the developmental time and also the life history processes of the worm. A major goal for the future will be to reconstitute an ancestral process (or several ancestral processes) in the ancestor of the whole clade. This in turn will provide key insights in the current debate on ancestral bilaterian segmentation.

Posterior elongation in the annelid Platynereis dumerilii involves stem cells molecularly related to primordial germ cells.

Like most bilaterian animals, the annelid Platynereis dumerilii generates the majority of its body axis in an anterior to posterior temporal progression with new segments added sequentially. This process relies on a posterior subterminal proliferative body region, known as the "segment addition zone" (SAZ). We explored some of the molecular and cellular aspects of posterior elongation in Platynereis, in particular to test the hypothesis that the SAZ contains a specific set of stem cells dedicated to posterior elongation. We cloned and characterized the developmental expression patterns of orthologs of 17 genes known to be involved in the formation, behavior, or maintenance of stem cells in other metazoan models. These genes encode RNA-binding proteins (e.g., tudor, musashi, pumilio) or transcription factors (e.g., myc, id, runx) widely conserved in eumetazoans. Most of these genes are expressed both in the migrating primordial germ cells and in overlapping ring-like patterns in the SAZ, similar to some previously analyzed genes (piwi, vasa). The SAZ patterns are coincident with the expression of proliferation markers cyclin B and PCNA. EdU pulse and chase experiments suggest that new segments are produced through many rounds of divisions from small populations of teloblast-like posterior stem cells. The shared molecular signature between primordial germ cells and posterior stem cells in Platynereis thus corresponds to an ancestral "stemness" program.

Extensive chordate and annelid macrosynteny reveals ancestral homeobox gene organization.

Genes with the homeobox motif are crucial in developmental biology and widely implicated in the evolution of development. The Antennapedia (ANTP)-class is one of the two major classes of animal homeobox genes, and includes the Hox genes, renowned for their role in patterning the anterior-posterior axis of animals. The origin and evolution of the ANTP-class genes are a matter of some debate. A principal guiding hypothesis has been the existence of an ancient gene Mega-cluster deep in animal ancestry. This hypothesis was largely established from linkage data from chordates, and the Mega-cluster hypothesis remains to be seriously tested in protostomes. We have thus mapped ANTP-class homeobox genes to the chromosome level in a lophotrochozoan protostome. Our comparison of gene organization in Platynereis dumerilii and chordates indicates that the Mega-cluster, if it did exist, had already been broken up onto four chromosomes by the time of the protostome-deuterostome ancestor (PDA). These results not only elucidate an aspect of the genome organization of the PDA but also reveal high levels of macrosynteny between P. dumerilii and chordates. This implies a very low rate of interchromosomal genome rearrangement in the lineages leading to P. dumerilii and the chordate ancestor since the time of the PDA.

Conservation, loss, and redeployment of Wnt ligands in protostomes: implications for understanding the evolution of segment formation.

BACKGROUND: The Wnt genes encode secreted glycoprotein ligands that regulate a wide range of developmental processes, including axis elongation and segmentation. There are thirteen subfamilies of Wnt genes in metazoans and this gene diversity appeared early in animal evolution. The loss of Wnt subfamilies appears to be common in insects, but little is known about the Wnt repertoire in other arthropods, and moreover the expression and function of these genes have only been investigated in a few protostomes outside the relatively Wnt-poor model species Drosophila melanogaster and Caenorhabditis elegans. To investigate the evolution of this important gene family more broadly in protostomes, we surveyed the Wnt gene diversity in the crustacean Daphnia pulex, the chelicerates Ixodes scapularis and Achaearanea tepidariorum, the myriapod Glomeris marginata and the annelid Platynereis dumerilii. We also characterised Wnt gene expression in the latter three species, and further investigated expression of these genes in the beetle Tribolium castaneum. RESULTS: We found that Daphnia and Platynereis both contain twelve Wnt subfamilies demonstrating that the common ancestors of arthropods, ecdysozoans and protostomes possessed all members of all Wnt subfamilies except Wnt3. Furthermore, although there is striking loss of Wnt genes in insects, other arthropods have maintained greater Wnt gene diversity. The expression of many Wnt genes overlap in segmentally reiterated patterns and in the segment addition zone, and while these patterns can be relatively conserved among arthropods and the annelid, there have also been changes in the expression of some Wnt genes in the course of protostome evolution. Nevertheless, our results strongly support the parasegment as the primary segmental unit in arthropods, and suggest further similarities between segmental and parasegmental regulation by Wnt genes in annelids and arthropods respectively. CONCLUSIONS: Despite frequent losses of Wnt gene subfamilies in lineages such as insects, nematodes and leeches, most protostomes have probably maintained much of their ancestral repertoire of twelve Wnt genes. The maintenance of a large set of these ligands could be in part due to their combinatorial activity in various tissues rather than functional redundancy. The activity of such Wnt 'landscapes' as opposed to the function of individual ligands could explain the patterns of conservation and redeployment of these genes in important developmental processes across metazoans. This requires further analysis of the expression and function of these genes in a wider range of taxa.

Hedgehog signaling regulates segment formation in the annelid Platynereis.

Annelids and arthropods share a similar segmented organization of the body whose evolutionary origin remains unclear. The Hedgehog signaling pathway, prominent in arthropod embryonic segment patterning, has not been shown to have a similar function outside arthropods. We show that the ligand Hedgehog, the receptor Patched, and the transcription factor Gli are all expressed in striped patterns before the morphological appearance of segments in the annelid Platynereis dumerilii. Treatments with small molecules antagonistic to Hedgehog signaling disrupt segment formation. Platynereis Hedgehog is not necessary to establish early segment patterns but is required to maintain them. The molecular similarity of segment patterning functions of the Hedgehog pathway in an annelid and in arthropods supports a common origin of segmentation in protostomes.

Features of the ancestral bilaterian inferred from Platynereis dumerilii ParaHox genes.

BACKGROUND: The ParaHox gene cluster is the evolutionary sister to the Hox cluster. Whilst the role of the Hox cluster in patterning the anterior-posterior axis of bilaterian animals is well established, and the organisation of vertebrate Hox clusters is intimately linked to gene regulation, much less is known about the more recently discovered ParaHox cluster. ParaHox gene clustering, and its relationship to expression, has only been described in deuterostomes. Conventional protostome models (Drosophila melanogaster and Caenorhabditis elegans) are secondarily derived with respect to ParaHox genes, suffering gene loss and cluster break-up. RESULTS: We provide the first evidence for ParaHox gene clustering from a less-derived protostome animal, the annelid Platynereis dumerilii. Clustering of these genes is thus not a sole preserve of the deuterostome lineage within Bilateria. This protostome ParaHox cluster is not entirely intact however, with Pdu-Cdx being on the opposite end of the same chromosome arm from Pdu-Gsx and Pdu-Xlox. From the genomic sequence around the P. dumerilii ParaHox genes the neighbouring genes are identified, compared with other taxa, and the ancestral arrangement deduced. CONCLUSION: We relate the organisation of the ParaHox genes to their expression, and from comparisons with other taxa hypothesise that a relatively complex pattern of ParaHox gene expression existed in the protostome-deuterostome ancestor, which was secondarily simplified along several invertebrate lineages. Detailed comparisons of the gene content around the ParaHox genes enables the reconstruction of the genome surrounding the ParaHox cluster of the protostome-deuterostome ancestor, which existed over 550 million years ago.

Insights into the evolution of the snail superfamily from metazoan wide molecular phylogenies and expression data in annelids.

BACKGROUND: An important issue concerning the evolution of duplicated genes is to understand why paralogous genes are retained in a genome even though the most likely fate for a redundant duplicated gene is nonfunctionalization and thereby its elimination. Here we study a complex superfamily generated by gene duplications, the snail related genes that play key roles during animal development. We investigate the evolutionary history of these genes by genomic, phylogenetic, and expression data studies. RESULTS: We systematically retrieved the full complement of snail related genes in several sequenced genomes. Through phylogenetic analysis, we found that the snail superfamily is composed of three ancestral families, snail, scratchA and scratchB. Analyses of the organization of the encoded proteins point out specific molecular signatures, indicative of functional specificities for Snail, ScratchA and ScratchB proteins. We also report the presence of two snail genes in the annelid Platynereis dumerilii, which have distinct expression patterns in the developing mesoderm, nervous system, and foregut. The combined expression of these two genes is identical to that of two independently duplicated snail genes in another annelid, Capitella spI, but different aspects of the expression patterns are differentially shared among paralogs of Platynereis and Capitella. CONCLUSION: Our study indicates that the snail and scratchB families have expanded through multiple independent gene duplications in the different bilaterian lineages, and highlights potential functional diversifications of Snail and ScratchB proteins following duplications, as, in several instances, paralogous proteins in a given species show different domain organizations. Comparisons of the expression pattern domains of the two Platynereis and Capitella snail paralogs provide evidence for independent subfunctionalization events which have occurred in these two species. We propose that the snail related genes may be especially prone to subfunctionalization, and this would explain why the snail superfamily underwent so many independent duplications leading to maintenance of functional paralogs.

Tribolium Wnts: evidence for a larger repertoire in insects with overlapping expression patterns that suggest multiple redundant functions in embryogenesis.

Wingless (wg)/Wnt family genes encode secreted glycoproteins that function as signalling molecules in the development of vertebrates as well as invertebrates. In a survey of Wnt family genes in the newly sequenced Tribolium genome, we found a total of nine Wnt genes. In addition to wg or Wnt1, Tribolium contains orthologs of the vertebrate Wnt5-7 and Wnt9-11 genes. As in Drosophila, Wnt1, Wnt6 and Wnt10 are clustered in the genome. Comparative genomics indicates that Wnt9 is also a conserved member of this cluster in several insects for which genome sequence is available. One of the Tribolium Wnt genes appears to be a member of the WntA family, members of which have been identified in Anopheles and other invertebrates but not in Drosophila or vertebrates. Careful phylogenetic examination suggests an Apis Wnt gene, previously identified as a Wnt4 homolog, is also a member of the WntA family. The ninth Tribolium Wnt gene is related to the diverged Drosophila WntD gene, both of which phylogenetically group with Wnt8 genes. Some of the Tribolium Wnt genes display multiple overlapping expression patterns, suggesting that they may be functionally redundant in segmentation, brain, appendage and hindgut development. In contrast, the unique expression patterns of Wnt5, Wnt7 and Wnt11 in developing appendages likely indicate novel functions.

Complementary striped expression patterns of NK homeobox genes during segment formation in the annelid Platynereis.

NK genes are related pan-metazoan homeobox genes. In the fruitfly, NK genes are clustered and involved in patterning various mesodermal derivatives during embryogenesis. It was therefore suggested that the NK cluster emerged in evolution as an ancestral mesodermal patterning cluster. To test this hypothesis, we cloned and analysed the expression patterns of the homologues of NK cluster genes Msx, NK4, NK3, Lbx, Tlx, NK1 and NK5 in the marine annelid Platynereis dumerilii, a representative of trochozoans, the third great branch of bilaterian animals alongside deuterostomes and ecdysozoans. We found that most of these genes are involved, as they are in the fly, in the specification of distinct mesodermal derivatives, notably subsets of muscle precursors. The expression of the homologue of NK4/tinman in the pulsatile dorsal vessel of Platynereis strongly supports the hypothesis that the vertebrate heart derived from a dorsal vessel relocated to a ventral position by D/V axis inversion in a chordate ancestor. Additionally and more surprisingly, NK4, Lbx, Msx, Tlx and NK1 orthologues are expressed in complementary sets of stripes in the ectoderm and/or mesoderm of forming segments, suggesting an involvement in the segment formation process. A potentially ancient role of the NK cluster genes in segment formation, unsuspected from vertebrate and fruitfly studies so far, now deserves to be investigated in other bilaterian species, especially non-insect arthropods and onychophorans.

Molecular architecture of annelid nerve cord supports common origin of nervous system centralization in bilateria.

To elucidate the evolutionary origin of nervous system centralization, we investigated the molecular architecture of the trunk nervous system in the annelid Platynereis dumerilii. Annelids belong to Bilateria, an evolutionary lineage of bilateral animals that also includes vertebrates and insects. Comparing nervous system development in annelids to that of other bilaterians could provide valuable information about the common ancestor of all Bilateria. We find that the Platynereis neuroectoderm is subdivided into longitudinal progenitor domains by partially overlapping expression regions of nk and pax genes. These domains match corresponding domains in the vertebrate neural tube and give rise to conserved neural cell types. As in vertebrates, neural patterning genes are sensitive to Bmp signaling. Our data indicate that this mediolateral architecture was present in the last common bilaterian ancestor and thus support a common origin of nervous system centralization in Bilateria.

Caudal and even-skipped in the annelid Platynereis dumerilii and the ancestry of posterior growth.

In order to address the question of the conservation of posterior growth mechanisms in bilaterians, we have studied the expression patterns of the orthologues of the genes caudal, even-skipped, and brachyury in the annelid Platynereis dumerilii. Annelids belong to the still poorly studied third large branch of the bilaterians, the lophotrochozoans, and have anatomic and developmental characteristics, such as a segmented body plan, indirect development through a microscopic ciliated larva, and building of the trunk through posterior addition, which are all hypothesized by some authors (including us) to be present already in Urbilateria, the last common ancestor of bilaterians. All three genes are shown to be likely involved in the building of the anteroposterior axis around the slit-like amphistomous blastopore as well as in the patterning of the terminal anus-bearing piece of the body (the pygidium). In addition, caudal and even-skipped are likely involved in the posterior addition of segments. Together with the emerging results on the conservation of segmentation genes, these results reinforce the hypothesis that Urbilateria had a segmented trunk developing through posterior addition.