Arthropod segmentation.

There is now compelling evidence that many arthropods pattern their segments using a clock-and-wavefront mechanism, analogous to that operating during vertebrate somitogenesis. In this Review, we discuss how the arthropod segmentation clock generates a repeating sequence of pair-rule gene expression, and how this is converted into a segment-polarity pattern by 'timing factor' wavefronts associated with axial extension. We argue that the gene regulatory network that patterns segments may be relatively conserved, although the timing of segmentation varies widely, and double-segment periodicity appears to have evolved at least twice. Finally, we describe how the repeated evolution of a simultaneous (Drosophila-like) mode of segmentation within holometabolan insects can be explained by heterochronic shifts in timing factor expression plus extensive pre-patterning of the pair-rule genes.

Experimental duplication of bilaterian body axes in spider embryos: Holm's organizer and self-regulation of embryonic fields.

Bilaterally symmetric body plans of vertebrates and arthropods are defined by a single set of two orthogonal axes, the anterior-posterior (or head-tail) and dorsal-ventral axes. In vertebrates, and especially amphibians, complete or partial doubling of the bilaterian body axes can be induced by two different types of embryological manipulations: transplantation of an organizer region or bi-sectioning of an embryo. Such axis doubling relies on the ability of embryonic fields to flexibly respond to the situation and self-regulate toward forming a whole body. This phenomenon has facilitated experimental efforts to investigate the mechanisms of vertebrate body axes formation. However, few studies have addressed the self-regulatory capabilities of embryonic fields associated with body axes formation in non-vertebrate bilaterians. The pioneer spider embryologist Åke Holm reported twinning of spider embryos induced by both types of embryological manipulations in 1952; yet, his experiments have not been replicated by other investigators, and access to spider or non-vertebrate twins has been limited. In this review, we provide a historical background on twinning experiments in spiders, and an overview of current twinning approaches in familiar spider species and related molecular studies. Moreover, we discuss the benefits of the spider model system for a deeper understanding of the ancestral mechanisms of body axes formation in arthropods, as well as in bilaterians.

A quantitative study of the diversity of stripe-forming processes in an arthropod cell-based field undergoing axis formation and growth.

One of the conserved traits of arthropod embryonic development is striped expression of homologs of Drosophila segment polarity genes, including hedgehog (hh). Although a diversity of stripe-forming processes is recognized among arthropod embryos, such varied stripe-forming processes have not been well characterized from cellular and quantitative perspectives. The spider Parasteatoda tepidariorum embryo, which has a hh-dependent mechanism of axis formation, offers a cell-based field where the stripes of Pt-hh (a hh homolog) expression dynamically develop in accordance with axis formation and growth, with the patterning processes varying among the regions of the field. In this study, using cell labeling, we mapped the future body subdivisions to the germ disc in the spider embryo and provided substantial evidence for the occurrence of kinetic waves of Pt-hh expression in the presumptive head and opisthosomal (or abdominal) regions of the embryonic field. Notably, combined with cell tracking, we showed that surface cells at and near the center of the germ disc persist in the posterior portion of the field from where Pt-hh stripes sequentially arise, suggesting the operation of ordered oscillations of Pt-hh expression. We then conducted a quantitative analysis of forming/formed Pt-hh stripes using serially timed fixation of sibling embryos. By utilizing length measurements that reflect the axis growth of the embryonic field, we reconstructed the pattern dynamics, which captured repeated splitting of Pt-hh stripes and oscillations of Pt-hh expression in the presumptive head and opisthosomal regions, respectively. In the intermediate thoracic region, three stripes of Pt-hh expression showed a late appearance, with the segmental units specified much earlier by another mechanism. Analyses provided quantitative estimates related to axis growth and stripe-splitting and oscillation events, including the periods of the patterning cycles. This work characterizes the diversity of stripe-forming processes in a cell-based field in a common spatiotemporal framework and highlights the contrasting dynamics of splitting versus oscillation. The cellular and quantitative data presented here provide the foundation for experimental, theoretical and evolutionary studies of cell-based pattern formation, especially body axis segmentation in arthropods.

Expression of NK genes that are not part of the NK cluster in the onychophoran Euperipatoides rowelli (Peripatopsidae).

BACKGROUND: NK genes are a group of homeobox transcription factors that are involved in various molecular pathways across bilaterians. They are typically divided into two subgroups, the NK cluster (NKC) and NK-linked genes (NKL). While the NKC genes have been studied in various bilaterians, corresponding data of many NKL genes are missing to date. To further investigate the ancestral roles of NK family genes, we analyzed the expression patterns of NKL genes in the onychophoran Euperipatoides rowelli. RESULTS: The NKL gene complement of E. rowelli comprises eight genes, including BarH, Bari, Emx, Hhex, Nedx, NK2.1, vax and NK2.2, of which only NK2.2 was studied previously. Our data for the remaining seven NKL genes revealed expression in different structures associated with the developing nervous system in embryos of E. rowelli. While NK2.1 and vax are expressed in distinct medial regions of the developing protocerebrum early in development, BarH, Bari, Emx, Hhex and Nedx are expressed in late developmental stages, after all major structures of the nervous system have been established. Furthermore, BarH and Nedx are expressed in distinct mesodermal domains in the developing limbs. CONCLUSIONS: Comparison of our expression data to those of other bilaterians revealed similar patterns of NK2.1, vax, BarH and Emx in various aspects of neural development, such as the formation of anterior neurosecretory cells mediated by a conserved molecular mechanism including NK2.1 and vax, and the development of the central and peripheral nervous system involving BarH and Emx. A conserved role in neural development has also been reported from NK2.2, suggesting that the NKL genes might have been primarily involved in neural development in the last common ancestor of bilaterians or at least nephrozoans (all bilaterians excluding xenacoelomorphs). The lack of comparative data for many of the remaining NKL genes, including Bari, Hhex and Nedx currently hampers further evolutionary conclusions. Hence, future studies should focus on the expression of these genes in other bilaterians, which would provide a basis for comparative studies and might help to better understand the role of NK genes in the diversification of bilaterians.

Embryonic expression patterns and phylogenetic analysis of panarthropod sox genes: insight into nervous system development, segmentation and gonadogenesis.

BACKGROUND: Sox (Sry-related high-mobility-group box) genes represent important factors in animal development. Relatively little, however, is known about the embryonic expression patterns and thus possible function(s) of Sox genes during ontogenesis in panarthropods (Arthropoda+Tardigrada+Onychophora). To date, studies have been restricted exclusively to higher insects, including the model system Drosophila melanogaster, with no comprehensive data available for any other arthropod group, or any tardigrade or onychophoran. RESULTS: This study provides a phylogenetic analysis of panarthropod Sox genes and presents the first comprehensive analysis of embryonic expression patterns in the flour beetle Tribolium castaneum (Hexapoda), the pill millipede Glomeris marginata (Myriapoda), and the velvet worm, Euperipatoides kanangrensis (Onychophora). 24 Sox genes were identified and investigated: 7 in Euperipatoides, 8 in Glomeris, and 9 in Tribolium. Each species possesses at least one ortholog of each of the five expected Sox gene families, B, C, D, E, and F, many of which are differentially expressed during ontogenesis. CONCLUSION: Sox gene expression (and potentially function) is highly conserved in arthropods and their closest relatives, the onychophorans. Sox B, C and D class genes appear to be crucial for nervous system development, while the Sox B genes Dichaete (D) and Sox21b likely play an additional conserved role in panarthropod segmentation. The Sox B gene Sox21a likely has a conserved function in foregut and Malpighian tubule development, at least in Hexapoda. The data further suggest that Sox D and E genes are involved in mesoderm differentiation, and that Sox E genes are involved in gonadal development. The new data expand our knowledge about the expression and implied function of Sox genes to Mandibulata (Myriapoda+Pancrustacea) and Panarthropoda (Arthropoda+Onychophora).

Embryonic expression of a Long Toll (Loto) gene in the onychophorans Euperipatoides kanangrensis and Cephalofovea clandestina.

Recent research has shown that Toll genes, and in particular a newly defined class of Toll genes, the so-called Long Toll Genes (Loto genes), are crucial factors in embryogenesis. In arthropods, they are involved in axis formation via a process called convergent extension (CE). A hallmark of Loto genes is their relatively (compared to other Toll genes) high number of leucine-rich repeat elements (LRRs) coupled with the fact that they are expressed in transverse stripes in all segments, or a subset of segments, patterns that are reminiscent of classical segmentation genes such as the pair-rule genes. Onychophorans represent a close outgroup to the arthropods; however, their embryonic development differs substantially. It is unclear if convergent extension contributes to onychophoran germ band formation and, if so, whether Loto genes are involved in governing this process. This study identifies a single onychophoran Toll gene from a sequenced embryonic transcriptome in two onychophoran species. The identified gene shows sequence and expression pattern characteristics of Loto genes. However, its expression pattern also comprises some general differences to arthropod Loto genes that are involved in CE.

Conserved versus derived patterns of controlled cell death during the embryonic development of two species of Onychophora (velvet worms).

BACKGROUND: Apoptosis is involved in various developmental processes, including cell migration and tissue and organ formation. Some of these processes are conserved across metazoans, while others are specific to particular taxa. Although the patterns of apoptosis have been investigated in arthropods, no corresponding data are available from one of their closest relatives, the Onychophora (velvet worms). RESULTS: We analyzed the patterns of apoptosis in embryos of two onychophoran species: the lecithotrophic/matrotrophic viviparous peripatopsid Euperipatoides rowelli, and the placentotrophic viviparous peripatid Principapillatus hitoyensis. Our data show that apoptosis occurs early in development and might be responsible for the degeneration of extra-embryonic tissues. Moreover, apoptosis might be involved in the morphogenesis of the ventral and preventral organs in both species and occurs additionally in the placental stalk of P. hitoyensis. CONCLUSIONS: Despite the different developmental modes in these onychophoran species, our data suggest that patterns of apoptosis are conserved among onychophorans. While apoptosis in the dorsal extra-embryonic tissue might contribute to dorsal closure-a process also known from arthropods-the involvement of apoptosis in ventral closure might be unique to onychophorans. Apoptosis in the placental stalk of P. hitoyensis is most likely a derived feature of the placentotrophic onychophorans. Developmental Dynamics 246:403-416, 2017. © 2017 Wiley Periodicals, Inc.

An anterior medial cell population with an apical-organ-like transcriptional profile that pioneers the central nervous system in the centipede Strigamia maritima.

The apical plate of primary marine larvae is characterized by a common set of transcription factors comprising six3, rx, hbn, nk2.1 and FoxQ2. It harbours the apical organ, a neural and ciliary structure with neurosecretory properties. Recent studies in lophotrochozoans have found that apical organ cells form the anterior tip of the developing central nervous system. We identify an anterior medial tissue in the embryonic centipede head that shares the transcriptional profile of the apical plate of marine larvae, including nested domains of FoxQ2 and six3 expression. This domain gives rise to an anterior medial population of neural precursors distinct from those arising within the segmental neuroectoderm. These medial cells do not express achaete scute homologue in proneural clusters, but express collier, a marker for post mitotic cells committed to a neural fate, while they are still situated in the surface ectodermal layer. They then sink under the surface to form a compact cell cluster. Once internalized these cells extend axons that pioneer the primary axonal scaffold of the central nervous system. The same cells express phc2, a neural specific prohormone convertase, which suggests that they form an early active neurosecretory centre. Some also express markers of hypothalamic neurons, including otp, vtn and vax1. These medial neurosecretory cells of the centipede are distinct from those of the pars intercerebralis, the anterior neurosecretory part of the insect brain. The pars intercerebralis derives from vsx positive placodal-like invagination sites. In the centipede, vsx expressing invaginating ectoderm is situated bilaterally adjacent to the medial pioneer cell population. Hence the pars intercerebralis is present in both insect and centipede brains, whereas no prominent anterior medial cluster of pioneer neurons is present in insects. These observations suggest that the arthropod brain retained ancestrally an anterior medial population of neurosecretory cells homologous to those of the apical plate in other invertebrate phyla, but that this cell population has been lost or greatly reduced in insects.

Germ cells of the centipede Strigamia maritima are specified early in embryonic development.

We provide the first systematic description of germ cell development with molecular markers in a myriapod, the centipede Strigamia maritima. By examining the expression of Strigamia vasa and nanos orthologues, we find that the primordial germ cells are specified from at least the blastoderm stage. This is a much earlier embryonic stage than previously described for centipedes, or any other member of the Myriapoda. Using these genes as markers, and taking advantage of the developmental synchrony of Strigamia embryos within single clutches, we are able to track the development of the germ cells throughout embryogenesis. We find that the germ cells accumulate at the blastopore; that the cells do not internalize through the hindgut, but rather through the closing blastopore; and that the cells undergo a long-range migration to the embryonic gonad. This is the first evidence for primordial germ cells displaying these behaviours in any myriapod. The myriapods are a phylogenetically important group in the arthropod radiation for which relatively little developmental data is currently available. Our study provides valuable comparative data that complements the growing number of studies in insects, crustaceans and chelicerates, and is important for the correct reconstruction of ancestral states and a fuller understanding of how germ cell development has evolved in different arthropod lineages.

Aspects of dorso-ventral and proximo-distal limb patterning in onychophorans.

Onychophorans (velvet worms) are closely related to the arthropods, but their limb morphology represents a stage before arthropodization (i.e., the segmentation of the limbs). We investigated the expression of onychophoran homologs of genes that are involved in dorso-ventral (DV) and proximo-distal (PD) limb patterning in arthropods. We find that the two onychophoran optomotor-blind (omb) genes, omb-1 and omb-2, are both expressed in conserved patterns in the dorsal ectoderm of the limbs, including the onychophoran antennae (the frontal appendages). Surprisingly, the expression of decapentaplegic (dpp), which acts upstream of omb in Drosophila, is partially reversed in onychophoran limbs compared to its expression in arthropods. A conserved feature of dpp expression in arthropods and onychophorans, however, is the prominent expression of dpp in the tips of developing limbs, which, therefore, may represent the ancestral pattern. The expression patterns of wingless (wg) and H15 are very diverged in onychophorans. The wg gene is only expressed in the limb tips and the single H15 gene is expressed in a few dorsal limb cells, but not on the ventral side. The expression of wg and dpp at the limb tips is one of the three possible alternatives predicted by the topology model of arthropod limb patterning and is, thus, compatible with a conserved function of wg and dpp in the patterning of the PD axis. On the other hand, DV limb gene expression is less conserved, and the specification of ventral fate appears to involve neither wg nor H15 expression.

Functional analysis of centipede development supports roles for Wnt genes in posterior development and segment generation.

The genes of the Wnt family play important and highly conserved roles in posterior growth and development in a wide range of animal taxa. Wnt genes also operate in arthropod segmentation, and there has been much recent debate regarding the relationship between arthropod and vertebrate segmentation mechanisms. Due to its phylogenetic position, body form, and possession of many (11) Wnt genes, the centipede Strigamia maritima is a useful system with which to examine these issues. This study takes a functional approach based on treatment with lithium chloride, which causes ubiquitous activation of canonical Wnt signalling. This is the first functional developmental study performed in any of the 15,000 species of the arthropod subphylum Myriapoda. The expression of all 11 Wnt genes in Strigamia was analyzed in relation to posterior development. Three of these genes, Wnt11, Wnt5, and WntA, were strongly expressed in the posterior region and, thus, may play important roles in posterior developmental processes. In support of this hypothesis, LiCl treatment of S. maritima embryos was observed to produce posterior developmental defects and perturbations in AbdB and Delta expression. The effects of LiCl differ depending on the developmental stage treated, with more severe effects elicited by treatment during germband formation than by treatment at later stages. These results support a role for Wnt signalling in conferring posterior identity in Strigamia. In addition, data from this study are consistent with the hypothesis of segmentation based on a "clock and wavefront" mechanism operating in this species.

Evolving specialization of the arthropod nervous system.

The diverse array of body plans possessed by arthropods is created by generating variations upon a design of repeated segments formed during development, using a relatively small "toolbox" of conserved patterning genes. These attributes make the arthropod body plan a valuable model for elucidating how changes in development create diversity of form. As increasingly specialized segments and appendages evolved in arthropods, the nervous systems of these animals also evolved to control the function of these structures. Although there is a remarkable degree of conservation in neural development both between individual segments in any given species and between the nervous systems of different arthropod groups, the differences that do exist are informative for inferring general principles about the holistic evolution of body plans. This review describes developmental processes controlling neural segmentation and regionalization, highlighting segmentation mechanisms that create both ectodermal and neural segments, as well as recent studies of the role of Hox genes in generating regional specification within the central nervous system. We argue that this system generates a modular design that allows the nervous system to evolve in concert with the body segments and their associated appendages. This information will be useful in future studies of macroevolutionary changes in arthropod body plans, especially in understanding how these transformations can be made in a way that retains the function of appendages during evolutionary transitions in morphology.

The centipede Strigamia maritima possesses a large complement of Wnt genes with diverse expression patterns.

The genes of the Wnt family play important roles in the development of many animals. In the arthropods, these genes are known to have multiple functions, including roles in posterior development and segmentation. Despite this, secondary loss of Wnt genes is common among the Arthropoda. Unlike many arthropods, Strigamia maritima, a geophilomorph centipede, possesses a large complement of Wnt ligands, with 11 Wnt genes present. In this study, the expression of each of these genes was examined across a range of stages during embryonic development. The expression of Wnt genes in Strigamia displays much variability. Most Wnt genes are expressed in segmental stripes in the trunk; near the proctodeum; and in the head region. However, despite this overall broad similarity, there are many differences between the various Wnt genes in their exact patterns of expression. These data should be considered in the context of different hypotheses regarding the functional relationships between the Wnt genes and the degree of redundancy present in this system. The findings of this study are consistent with one particular model of Wnt activity, the combinatorial model, whereby the combination of Wnt ligands present in a particular region defines its identity. These findings should also be useful in attempts to reconstruct the evolutionary history of Wnt signaling in arthropods.

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.

Opening a can of centipedes: new insights into mechanisms of body segmentation.

The search for a common developmental genetic mechanism of body segmentation appears to become more difficult, and more interesting, as new segmented organisms are added to the roster. Recent work in this journal by Brena and Akam on segmentation of the geophilomorph centipede Strigamia maritima, an arthropod distantly related to the standard insect models, contains developmental and evolutionary surprises that highlight the importance of a wider sampling of phyla.See research article: http://www.biomedcentral.com/1741-7007/11/112.

An analysis of segmentation dynamics throughout embryogenesis in the centipede Strigamia maritima.

BACKGROUND: Most segmented animals add segments sequentially as the animal grows. In vertebrates, segment patterning depends on oscillations of gene expression coordinated as travelling waves in the posterior, unsegmented mesoderm. Recently, waves of segmentation gene expression have been clearly documented in insects. However, it remains unclear whether cyclic gene activity is widespread across arthropods, and possibly ancestral among segmented animals. Previous studies have suggested that a segmentation oscillator may exist in Strigamia, an arthropod only distantly related to insects, but further evidence is needed to document this. RESULTS: Using the genes even skipped and Delta as representative of genes involved in segment patterning in insects and in vertebrates, respectively, we have carried out a detailed analysis of the spatio-temporal dynamics of gene expression throughout the process of segment patterning in Strigamia. We show that a segmentation clock is involved in segment formation: most segments are generated by cycles of dynamic gene activity that generate a pattern of double segment periodicity, which is only later resolved to the definitive single segment pattern. However, not all segments are generated by this process. The most posterior segments are added individually from a localized sub-terminal area of the embryo, without prior pair-rule patterning. CONCLUSIONS: Our data suggest that dynamic patterning of gene expression may be widespread among the arthropods, but that a single network of segmentation genes can generate either oscillatory behavior at pair-rule periodicity or direct single segment patterning, at different stages of embryogenesis.

Single-minded and the evolution of the ventral midline in arthropods.

In insects and crustaceans, ventral midline cells are present that subdivide the CNS into bilateral symmetric halves. In both arthropod groups unpaired midline neurons and glial cells have been identified that contribute to the embryonic patterning mechanisms. In the fruitfly Drosophila melanogaster, for example, the midline cells are involved in neural cell fate specification along the dorso-ventral axis but also in axonal pathfinding and organisation of the axonal scaffold. Both in insects and malacostracan crustaceans, the bHLH-PAS transcription factor single-minded is the master regulator of ventral midline development and homology has been suggested for individual midline precursors in these groups. The conserved arrangement of the axonal scaffold as well as the regular pattern of neural precursors in all euarthropod groups raises the question whether the ventral midline system is conserved in this phylum. In the remaining euarthropod groups, the chelicerates and myriapods, a single-minded homologue has been identified in the spider Achaearanea tepidariorum (chelicerate), however, the gene is not expressed in the ventral midline but in the median area of the ventral neuroectoderm. Here we show that At-sim is not required for ventral midline development. Furthermore, we identify sim homologues in representatives of arthropods that have not yet been analysed: the myriapod Strigamia maritima and a representative of an outgroup to the euarthropods, the onychophoran Euperipatoides kanangrensis. We compare the expression patterns to the A. tepidariorum sim homologue expression and furthermore analyse the nature of the arthropod midline cells. Our data suggest that in arthropods unpaired midline precursors evolved from the bilateral median domain of the ventral neuroectoderm in the last common ancestor of Mandibulata (insects, crustaceans, myriapods). We hypothesize that sim was expressed in this domain and recruited to ventral midline development. Subsequently, sim function has evolved in parallel to the evolution of midline cell function in the individual Mandibulata lineages.

The embryonic development of the centipede Strigamia maritima.

The geophilomorph centipede Strigamia maritima is an emerging model for studies of development and evolution among the myriapods. A draft genome sequence has recently been completed, making it also an important reference for comparative genomics, and for studies of myriapod physiology more generally. Here we present the first detailed description of myriapod development using modern techniques. We describe a timeline for embryonic development, with a detailed staging system based on photographs of live eggs and fixed embryos. We show that the early, cleavage and nuclear migration, stages of development are remarkably prolonged, accounting for nearly half of the total developmental period (approx 22 of 48 days at 13 °C). Towards the end of this period, cleavage cells migrate to the egg periphery to generate a uniform blastoderm. Asymmetry quickly becomes apparent as cells in the anterior half of the egg condense ventrally to form the presumptive head. Five anterior segments, the mandibular to the first leg-bearing segment (1st LBS) become clearly visible through the chorion almost simultaneously. Then, after a short pause, the next 35 leg-bearing segments appear at a uniform rate of 1 segment every 3.2 h (at 13 °C). Segment addition then slows to a halt with 40-45 LBS, shortly before the dramatic movements of germ band flexure, when the left and right halves of the embryo separate and the embryo folds deeply into the yolk. After flexure, segment morphogenesis and organogenesis proceed for a further 10 days, before the egg hatches. The last few leg-bearing segments are added during this period, much more slowly, at a rate of 1-2 segments/day. The last leg-bearing segment is fully defined only after apolysis of the embryonic cuticle, so that at hatching the embryo displays the final adult number of leg-bearing segments (typically 47-49 in our population).

Developmental abnormalities in Glomeris marginata (Villers 1789) (Myriapoda: Diplopoda): implications for body axis determination in a myriapod.

Abnormally developing embryos (ADEs) of the common pill millipede Glomeris marginata have been investigated by means of nuclear staining and mRNA in situ hybridization. It showed that all ADEs represent cases of Duplicitas posterior, which means that the posterior body pole is duplicated. The severity of the duplication ranges from duplicated posterior trunk segments in one specimen to an almost completely duplicated specimen that only shares the very anterior head region. Remarkably, none of the encountered ADEs represents a case of Duplicitas anterior (duplicated anterior pole) or a case of Duplicitas cruciata (cruciate duplication with two anterior and two posterior poles). This observation is discussed in the light of earlier reports on G. marginata ADEs that claim to have found these abnormalities. The lack of any other axial abnormality aside from D. posterior implies that early axis determination in G. marginata, and possibly myriapods in general, underlies the developmental mechanisms that prevent the formation of any other type of axial duplication. It is proposed that the formation of D. posterior-type embryos could be caused by the formation of two instead of only one posterior cumulus early during development.

Expression patterns of neural genes in Euperipatoides kanangrensis suggest divergent evolution of onychophoran and euarthropod neurogenesis.

One of the controversial debates on euarthropod relationships centers on the question as to whether insects, crustaceans, and myriapods (Mandibulata) share a common ancestor or whether myriapods group with the chelicerates (Myriochelata). The debate was stimulated recently by studies in chelicerates and myriapods that show that neural precursor groups (NPGs) segregate from the neuroectoderm generating the nervous system, whereas in insects and crustaceans the nervous tissue is produced by stem cells. Do the shared neural characters of myriapods and chelicerates represent derived characters that support the Myriochelata grouping? Or do they rather reflect the ancestral pattern? Analyses of neurogenesis in a group closely related to euarthropods, the onychophorans, show that, similar to insects and crustaceans, single neural precursors are formed in the neuroectoderm, potentially supporting the Myriochelata hypothesis. Here we show that the nature and the selection of onychophoran neural precursors are distinct from euarthropods. The onychophoran nervous system is generated by the massive irregular segregation of single neural precursors, contrasting with the limited number and stereotyped arrangement of NPGs/stem cells in euarthropods. Furthermore, neural genes do not show the spatiotemporal pattern that sets up the precise position of neural precursors as in euarthropods. We conclude that neurogenesis in onychophorans largely does not reflect the ancestral pattern of euarthropod neurogenesis, but shows a mixture of derived characters and ancestral characters that have been modified in the euarthropod lineage. Based on these data and additional evidence, we suggest an evolutionary sequence of arthropod neurogenesis that is in line with the Mandibulata hypothesis.