Comparative analysis of neurogenesis in the myriapod Glomeris marginata (Diplopoda) suggests more similarities to chelicerates than to insects

Molecular data suggest that myriapods are a basal arthropod group and may even be the sister group of chelicerates. To find morphological indications for this relationship we have analysed neurogenesis in the myriapod Glomeris marginata (Diplopoda). We show here that groups of neural precursors, rather than single cells as in insects, invaginate from the ventral neuroectoderm in a manner similar to that in the spider: invaginating cell groups arise sequentially and at stereotyped positions in the ventral neuroectoderm of Glomeris, and all cells of the neurogenic region seem to enter the neural pathway. Furthermore, we have identified an achaete-scute, a Delta and a Notch homologue in GLOMERIS: The genes are expressed in a pattern similar to the spider homologues and show more sequence similarity to the chelicerates than to the insects. We conclude that the myriapod pattern of neural precursor formation is compatible with the possibility of a chelicerate-myriapod sister group relationship.     

Neurogenesis in myriapods and chelicerates and its importance for understanding arthropod relationships

Several alternative hypotheses on the relationships betweenthe major arthropod groups are still being discussed. We reexaminehere the chelicerate/myriapod relationship by comparing previouslypublished morphological data on neurogenesis in the euarthropodgroups and presenting data on an additional myriapod (Strigamiamaritima). Although there are differences in the formation ofneural precursors, most euarthropod species analyzed generateabout 30 single neural precursors (insects/crustaceans) or precursorgroups (chelicerates/myriapods) per hemisegment that are arrangedin a regular pattern. The genetic network involved in recruitmentand specification of neural precursors seems to be conservedamong euarthropods. Furthermore, we show here that neural precursoridentity seems to be achieved in a similar way. Besides theseconserved features we found 2 characters that distinguish insects/crustaceansfrom myriapods/chelicerates. First, in insects and crustaceansthe neuroectoderm gives rise to epidermal and neural cells,whereas in chelicerates and myriapods the central area of theneuroectoderm exclusively generates neural cells. Second, neuralcells arise by stem-cell-like divisions of neuroblasts in insectsand crustaceans, whereas groups of mainly postmitotic neuralprecursors are recruited for the neural fate in cheliceratesand myriapods. We discuss whether these characteristics representa sympleisiomorphy of myriapods and chelicerates that has beenlost in the more derived Pancrustacea or whether these characteristicsare a synapomorphy of myriapods and chelicerates, providingthe first morphological support for the Myriochelata group.     

The expression and function of the achaete-scute genes in Tribolium castaneum reveals conservation and variation in neural pattern formation and cell fate specification

The study of achaete-scute (ac/sc) genes has recently become a paradigm to understand the evolution and development of the arthropod nervous system. We describe the identification and characterization of the ac/sc genes in the coleopteran insect species Tribolium castaneum. We have identified two Tribolium ac/sc genes - achaete-scute homolog (Tc-ASH) a proneural gene and asense (Tc-ase) a neural precursor gene that reside in a gene complex. Focusing on the embryonic central nervous system we find that Tc-ASH is expressed in all neural precursors and the proneural clusters from which they segregate. Through RNAi and misexpression studies we show that Tc-ASH is necessary for neural precursor formation in Tribolium and sufficient for neural precursor formation in Drosophila. Comparison of the function of the Drosophila and Tribolium proneural ac/sc genes suggests that in the Drosophila lineage these genes have maintained their ancestral function in neural precursor formation and have acquired a new role in the fate specification of individual neural precursors. Furthermore, we find that Tc-ase is expressed in all neural precursors suggesting an important and conserved role for asense genes in insect nervous system development. Our analysis of the Tribolium ac/sc genes indicates significant plasticity in gene number, expression and function, and implicates these modifications in the evolution of arthropod neural development.     

Regulation of proneural gene expression and cell fate during neuroblast segregation in the Drosophila embryo.

The Drosophila embryonic central nervous system develops from sets of progenitor neuroblasts which segregate from the neuroectoderm during early embryogenesis. Cells within this region can follow either the neural or epidermal developmental pathway, a decision guided by two opposing classes of genes. The proneural genes, including the members of the achaete-scute complex (AS-C), promote neurogenesis, while the neurogenic genes prevent neurogenesis and facilitate epidermal development. To understand the role that proneural gene expression and regulation play in the choice between neurogenesis and epidermogenesis, we examined the temporal and spatial expression pattern of the achaete (ac) regulatory protein in normal and neurogenic mutant embryos. The ac protein is first expressed in a repeating pattern of four ectodermal cell clusters per hemisegment. Even though 5-7 cells initially express ac in each cluster, only one, the neuroblast, continues to express ac. The repression of ac in the remaining cells of the cluster requires zygotic neurogenic gene function. In embryos lacking any one of five genes, the restriction of ac expression to single cells does not occur; instead, all cells of each cluster continue to express ac, enlarge, delaminate and become neuroblasts. It appears that one key function of the neurogenic genes is to silence proneural gene expression within the nonsegregating cells of the initial ectodermal clusters, thereby permitting epidermal development.     

Pax3/7 genes reveal conservation and divergence in the arthropod segmentation hierarchy

Several features of Pax3/7 gene expression are shared among distantly related insects, including pair-rule, segment polarity, and neural patterns. Recent data from arachnids imply that roles in segmentation and neurogenesis are likely to be played by Pax3/7 genes in all arthropods. To further investigate Pax3/7 genes in non-insect arthropods, we isolated two monoclonal antibodies that recognize the products of Pax3/7 genes in a wide range of taxa, allowing us to quickly survey Pax3/7 expression in all four major arthropod groups. Epitope analysis reveals that these antibodies react to a small subset of Paired-class homeodomains, which includes the products of all known Pax3/7 genes. Using these antibodies, we find that Pax3/7 genes in crustaceans are expressed in an early broad and, in one case, dynamic domain followed by segmental stripes, while myriapods and chelicerates exhibit segmental stripes that form early in the posterior-most part of the germ band. This suggests that Pax3/7 genes acquired their role in segmentation deep within, or perhaps prior to, the arthropod lineage. However, we do not detect evidence of pair-rule patterning in either myriapods or chelicerates, suggesting that the early pair-rule expression pattern of Pax3/7 genes in insects may have been acquired within the crustacean-hexapod lineage.     

Neurogenesis in the chilopod <Emphasis Type="Italic">Lithobius forficatus</Emphasis> suggests more similarities to chelicerates than to insects

In a recent comparative study on neurogenesis in the diplopod Glomeris marginata we have shown that the millipede and the spider share several features that cannot be found in homologous form in insects and crustaceans. The most distinctive difference is that groups of neural precursors are singled out from the neuroectoderm of the spider and the diplopod, rather than individual cells (i.e. neuroblasts) as in insects or crustacean. This observation constitutes the first morphological indication for a close myriapod/chelicerate relationship that has otherwise only been suggested by molecular phylogenetic analysis. To see whether the pattern of neurogenesis described for the diplopod is representative for myriapods, we analysed neurogenesis in the basal chilopod Lithobius forficatus. We show here that groups of cells invaginate from the chilopod neuroectoderm at strikingly similar positions as the invaginating cell groups of the diplopod and the spider. Furthermore, the expression patterns of the proneural and neurogenic genes reveal more similarities to the chelicerate and the diplopod than to insects. Thus, chelicerates and myriapods share the developmental mechanism for neurogenesis, either because they are true sister groups, or because this reflects the ancestral state of neurogenesis in arthropods.Edited by P. Simpson     

Neurogenesis in the water flea Daphnia magna (Crustacea, Branchiopoda) suggests different mechanisms of neuroblast formation in insects and crustaceans

Within euarthropods, the morphological and molecular mechanisms of early nervous system development have been analysed in insects and several representatives of chelicerates and myriapods, while data on crustaceans are fragmentary. Neural stem cells (neuroblasts) generate the nervous system in insects and in higher crustaceans (malacostracans); in the remaining euarthropod groups, the chelicerates (e.g. spiders) and myriapods (e.g. millipedes), neuroblasts are missing. In the latter taxa, groups of neural precursors segregate from the neuroectoderm and directly differentiate into neurons and glial cells. In all euarthropod groups, achaete–scute homologues are required for neuroblast/neural precursor group formation. In the insects Drosophila melanogaster and Tribolium castaneum achaete–scute homologues are initially expressed in clusters of cells (proneural clusters) in the neuroepithelium but expression becomes restricted to the future neuroblast. Subsequently genes such as snail and prospero are expressed in the neuroblasts which are required for asymmetric division and differentiation. In contrast to insects, malacostracan neuroblasts do not segregate into the embryo but remain in the outer neuroepithelium, similar to vertebrate neural stem cells. It has been suggested that neuroblasts are present in another crustacean group, the branchiopods, and that they also remain in the neuroepithelium. This raises the questions how the molecular mechanisms of neuroblast selection have been modified during crustacean and insect evolution and if the segregation or the maintenance of neuroblasts in the neuroepithelium represents the ancestral state. Here we take advantage of the recently published Daphnia pulex (branchiopod) genome and identify genes in Daphnia magna that are known to be required for the selection and asymmetric division of neuroblasts in the fruit fly D. melanogaster. We unambiguously identify neuroblasts in D. magna by molecular marker gene expression and division pattern. We show for the first time that branchiopod neuroblasts divide in the same pattern as insect and malacostracan neuroblasts. Furthermore, in contrast to D. melanogaster, neuroblasts are not selected from proneural clusters in the branchiopod. Snail rather than ASH is the first gene to be expressed in the nascent neuroblasts suggesting that ASH is not required for the selection of neuroblasts as in D. melanogaster. The prolonged expression of ASH in D. magna furthermore suggests that it is involved in the maintenance of the neuroblasts in the neuroepithelium. Based on these and additional data from various representatives of arthropods we conclude that the selection of neural precursors from proneural clusters as well as the segregation of neural precursors represents the ancestral state of neurogenesis in arthropods. We discuss that the derived characters of malacostracans and branchiopods – the absence of neuroblast segregation and proneural clusters – might be used to support or reject the possible groupings of paraphyletic crustaceans.     

Hox genes and the phylogeny of the arthropods

The arthropods are the most speciose, and among the most morphologically diverse, of the animal phyla. Their evolution has been the subject of intense research for well over a century, yet the relationships among the four extant arthropod subphyla - chelicerates, crustaceans, hexapods, and myriapods - are still not fully resolved. Morphological taxonomies have often placed hexapods and myriapods together (the Atelocerata) [1, 2], but recent molecular studies have generally supported a hexapod/crustacean clade [2-9]. A cluster of regulatory genes, the Hox genes, control segment identity in arthropods, and comparisons of the sequences and functions of Hox genes can reveal evolutionary relationships [10]. We used Hox gene sequences from a range of arthropod taxa, including new data from a basal hexapod and a myriapod, to estimate a phylogeny of the arthropods. Our data support the hypothesis that insects and crustaceans form a single clade within the arthropods to the exclusion of myriapods. They also suggest that myriapods are more closely allied to the chelicerates than to this insect/crustacean clade.     

Neurogenesis in the spider: new insights from comparative analysis of morphological processes and gene expression patterns

While there is a detailed understanding of neurogenesis in insects and partially also in crustaceans, little is known about neurogenesis in chelicerates. In the spider Cupiennius salei Keyserling, 1877 (Chelicerata, Arachnida, Araneae) invaginating cell groups arise sequentially and in a stereotyped pattern comparable to the formation of neuroblasts in Drosophila melanogaster Meigen, 1830 (Insecta, Diptera, Cyclorrhapha, Drosophilidae). In addition, functional analysis revealed that in the spider homologues of the D. melanogaster proneural and neurogenic genes control the recruitment and singling out of neural precursors like in D. melanogaster. Although groups of cells, rather than individual cells, are singled out from the spider neuroectoderm which can thus not be homologized with the insect neuroblasts, similar genes seem to confer neural identity to the neural precursor cells of the spider. We show here that the pan-neural genes snail and the neural identity gene Krüppel are expressed in neural precursors in a heterogenous spatio-temporal pattern that is comparable to the pattern in D. melanogaster. Our data suggest that the early genetic network involved in recruitment and specification of neural precursors is conserved among insects and chelicerates.     

Velvet worm development links myriapods with chelicerates

Despite the advent of modern molecular and computational methods, the phylogeny of the four major arthropod groups (Chelicerata, Myriapoda, Crustacea and Hexapoda, including the insects) remains enigmatic. One particular challenge is the position of myriapods as either the closest relatives to chelicerates (Paradoxopoda/Myriochelata hypothesis), or to crustaceans and hexapods (Mandibulata hypothesis). While neither hypothesis receives conclusive support from molecular analyses, most morphological studies favour the Mandibulata concept, with the mandible being the most prominent feature of this group. Although no morphological evidence was initially available to support the Paradoxopoda hypothesis, a putative synapomorphy of chelicerates and myriapods has recently been put forward based on studies of neurogenesis. However, this and other morphological characters remain of limited use for phylogenetic systematics owing to the lack of data from an appropriate outgroup. Here, we show that several embryonic characters are synapomorphies uniting the chelicerates and myriapods, as revealed by an outgroup comparison with the Onychophora or velvet worms. Our findings, thus provide, to our knowledge, first morphological/embryological support for the monophyly of the Paradoxopoda and suggest that the mandible might have evolved twice within the arthropods.     

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

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

Nematostella vectensis achaete-scute homolog NvashA regulates embryonic ectodermal neurogenesis and represents an ancient component of the metazoan neural specification pathway

achaete-scute homologs (ash) regulate neural development in all bilaterian model animals indicating that they represent a component of the ancestral neurogenic pathway. We test this by investigating four ash genes during development of a basal metazoan, the cnidarian sea anemone Nematostella vectensis. Spatiotemporal expression of ash genes in the early embryo and larval stages suggests that they regulate neurogenesis. More specifically, NvashA is co-expressed with neural genes in the embryonic ectoderm. Knockdown of NvashA results in decreased expression of eight neural markers, including the six novel neural targets identified here. Conversely, overexpression of NvashA induces increased expression of all eight genes, but only within their normal axial domains. Overexpression of NvashB-D differentially increases expression of NvashA targets. The expression patterns and differential ability of ash genes to regulate neural gene expression reveals surprising molecular complexity in these 'simple' animals. These data suggest that achaete-scute homologs functioned in the ancestral metazoan neurogenic pathway and provide a foundation to investigate further the evolution of neurogenesis and the origin of complex central nervous systems.     

The ‘Ventral Organs’ of Pycnogonida (Arthropoda) Are Neurogenic Niches of Late Embryonic and Post-Embryonic Nervous System Development

Early neurogenesis in arthropods has been in the focus of numerous studies, its cellular basis, spatio-temporal dynamics and underlying genetic network being by now comparably well characterized for representatives of chelicerates, myriapods, hexapods and crustaceans. By contrast, neurogenesis during late embryonic and/or post-embryonic development has received less attention, especially in myriapods and chelicerates. Here, we apply (i) immunolabeling, (ii) histology and (iii) scanning electron microscopy to study post-embryonic ventral nerve cord development in Pseudopallene sp., a representative of the sea spiders (Pycnogonida), the presumable sister group of the remaining chelicerates. During early post-embryonic development, large neural stem cells give rise to additional ganglion cell material in segmentally paired invaginations in the ventral ectoderm. These ectodermal cell regions – traditionally designated as ‘ventral organs’ – detach from the surface into the interior and persist as apical cell clusters on the ventral ganglion side. Each cluster is a post-embryonic neurogenic niche that features a tiny central cavity and initially still houses larger neural stem cells. The cluster stays connected to the underlying ganglionic somata cortex via an anterior and a posterior cell stream. Cell proliferation remains restricted to the cluster and streams, and migration of newly produced cells along the streams seems to account for increasing ganglion cell numbers in the cortex. The pycnogonid cluster-stream-systems show striking similarities to the life-long neurogenic system of decapod crustaceans, and due to their close vicinity to glomerulus-like neuropils, we consider their possible involvement in post-embryonic (perhaps even adult) replenishment of olfactory neurons – as in decapods. An instance of a potentially similar post-embryonic/adult neurogenic system in the arthropod outgroup Onychophora is discussed. Additionally, we document two transient posterior ganglia in the ventral nerve cord of Pseudopallene sp. and evaluate this finding in light of the often discussed reduction of a segmented ‘opisthosoma’ during pycnogonid evolution.     

Expression of collier in the premandibular segment of myriapods: support for the traditional Atelocerata concept or a case of convergence?

Background  

A recent study on expression and function of the ortholog of the Drosophila collier (col) gene in various arthropods including insects, crustaceans and chelicerates suggested a de novo function of col in the development of the appendage-less intercalary segment of insects. However, this assumption was made on the background of the now widely-accepted Pancrustacea hypothesis that hexapods represent an in-group of the crustaceans. It was therefore assumed that the expression of col in myriapods would reflect the ancestral state like in crustaceans and chelicerates, i.e. absence from the premandibular/intercalary segment and hence no function in its formation.     

Expression of the decapentaplegic ortholog in embryos of the onychophoran Euperipatoides rowelli

The gene decapentaplegic (dpp) and its homologs are essential for establishing the dorsoventral body axis in arthropods and vertebrates. However, the expression of dpp is not uniform among different arthropod groups. While this gene is expressed along the dorsal body region in insects, its expression occurs in a mesenchymal group of cells called cumulus in the early spider embryo. A cumulus-like structure has also been reported from centipedes, suggesting that it might be either an ancestral feature of arthropods or a derived feature (=synapomorphy) uniting the chelicerates and myriapods. To decide between these two alternatives, we analysed the expression patterns of a dpp ortholog in a representative of one of the closest arthropod relatives, the onychophoran Euperipatoides rowelli. Our data revealed unique expression patterns in the early mesoderm anlagen of the antennal segment and in the dorsal and ventral extra-embryonic tissue, suggesting a divergent role of dpp in these tissues in Onychophora. In contrast, the expression of dpp in the dorsal limb portions resembles that in arthropods, except that it occurs in the mesoderm rather than in the ectoderm of the onychophoran limbs. A careful inspection of embryos of E. rowelli revealed no cumulus-like accumulation of dpp expressing cells at any developmental stage, suggesting that this feature is either a derived feature of chelicerates or a synapomorphy uniting the chelicerates and myriapods.     

Expression of the decapentaplegic ortholog in embryos of the onychophoran Euperipatoides rowelli

The gene decapentaplegic (dpp) and its homologs are essential for establishing the dorsoventral body axis in arthropods and vertebrates. However, the expression of dpp is not uniform among different arthropod groups. While this gene is expressed along the dorsal body region in insects, its expression occurs in a mesenchymal group of cells called cumulus in the early spider embryo. A cumulus-like structure has also been reported from centipedes, suggesting that it might be either an ancestral feature of arthropods or a derived feature (=synapomorphy) uniting the chelicerates and myriapods. To decide between these two alternatives, we analysed the expression patterns of a dpp ortholog in a representative of one of the closest arthropod relatives, the onychophoran Euperipatoides rowelli. Our data revealed unique expression patterns in the early mesoderm anlagen of the antennal segment and in the dorsal and ventral extra-embryonic tissue, suggesting a divergent role of dpp in these tissues in Onychophora. In contrast, the expression of dpp in the dorsal limb portions resembles that in arthropods, except that it occurs in the mesoderm rather than in the ectoderm of the onychophoran limbs. A careful inspection of embryos of E. rowelli revealed no cumulus-like accumulation of dpp expressing cells at any developmental stage, suggesting that this feature is either a derived feature of chelicerates or a synapomorphy uniting the chelicerates and myriapods.     

Chelicerate Hox genes and the homology of arthropod segments

Genes of the homeotic complex (HOM-C) in insects and vertebrates are required for the specification of segments along the antero-posterior axis. Multiple paralogues of the Hox genes in the horseshoe crab Limulus poliphemus have been used as evidence for HOM-C duplications in the Chelicerata. We addressed this possibility through a limited PCR survey to sample the homeoboxes of two spider species, Steatoda triangulosa and Achaearanea tepidariorum. The survey did not provide evidence for multiple Hox clusters although we have found apparent duplicate copies of proboscipedia ( pb ) and Deformed ( Dfd   ). In addition, we have cloned larger cDNA fragments of pb, zerknullt ( zen / Hox3 ) and Dfd. These fragments allowed the determination of mRNA distribution by in situ hybridization. Our results are similar to the previously published expression patterns of Hox genes from another spider and an oribatid mite. Previous studies compared spider/mite Hox gene expression patterns with those of insects and argued for a pattern of segmental homology based on the assumption that the co-linear anterior boundaries of the Hox domains can be used as markers. To test this assumption we performed a comparative analysis of the expression patterns for UBX/ABD-A in chelicerates, myriapods, crustaceans, and insects. We conclude that the anterior boundary can be and is changed considerably during arthropod evolution and, therefore, Hox expression patterns should not be used as the sole criterion for identifying homology in different classes of arthropods.