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Background: The interest in the NK genes, a group of conserved homeodomain encoding transcription factors, has increased significantly in the recent years, especially among arthropods and their closest relatives. One reason is that these genes have important and (often) conserved functions in development. Another reason is that some NK genes are clustered within the genomes of different species of arthropods and indeed animals in general. The fact that this clustering has been retained for hundreds of millions of years strongly implies a biological function, and it is believed that shared cis-regulatory elements (CREs) could act to constrain the clusters. Our knowledge about the expression and function of clustered NK genes, however, is still restricted to only a few well-investigated members of this group of genes, while others have only been studied in few model species, or have been completely neglected. Recently, genomic data have provided exciting new insights into the content and clustering of NK genes in non-model species, but corresponding gene expression data are still incomplete or lacking altogether.

Results: Here we present a comprehensive overview of the complement and embryonic expression pattern of NK genes in a variety of arthropod species and an onychophoran in order to gain further insight into conserved and divergent patterns of NK gene expression which we use to extrapolate the putative ancestral function(s) of these genes in panarthropods.

Conclusions: We report unexpected instances of expression-pattern swapping among closely-clustered NK genes that may result from CRE-shuffling. We also discuss and conclude on the clustering and order of NK genes in the last common ancestor of bilaterian animals. Finally, we come to the conclusion that NK genes are not just involved in mesoderm evolution but are found to have conserved functions in all germ layers.

Comparing head development among arthropods has helped identify ancestral aspects of brain patterning and structure in animals more generally. Most understanding of arthropod head patterning has been learned from insects and the myriapod Strigamia maritima. Chelicerates represent an outgroup to mandibulate arthropods and can provide a valuable perspective to arthropod evolution and development. We assayed the expression of key markers of head patterning and neurosecretory centres from mandibulates in the pre-cheliceral region of embryos of the spider Parasteatoda tepidariorum. We found that, like mandibulates, this spider likely has a pars intercerebralis, marked by six3.2 and visual system homeobox/chx. We also found some evidence for another neurosecretory centre, the pars lateralis, marked by six3.2 and fasciclin 2. Furthermore, we identified anterior-medial cells in the spider pre-cheliceral region that express six3.2, foxQ2 and collier1, suggesting they may be pioneer neurons. However, these spider cells do not appear to be equivalent to the central pioneer neuronal cells identified in S. maritima because they lack expression of other key markers. Taken together, our study of spider pre-cheliceral region patterning adds a new chelicerate perspective to understanding the development and evolution of the arthropod head.

Whole genome duplication (WGD) generates a new genetic material that can contribute to the evolution of developmental processes and phenotypic diversification. A WGD occurred in an ancestor of arachnopulmonates (spiders, scorpions, and their relatives), which provides an important independent comparison to WGDs in other animal lineages. After WGD, arachnopulmonates retained many duplicated copies (ohnologues) of developmental genes including clusters of homeobox genes, many of which have been inferred to have undergone subfunctionalization. However, there has been little systematic analysis of gene regulatory sequences and comparison of the expression of ohnologues versus their single-copy orthologues between arachnids. Here, we compare the regions of accessible chromatin and gene expression of ohnologues and single-copy genes during three embryonic stages between an arachnopulmonate arachnid, the spider Parasteatoda tepidariorum, and a nonarachnopulmonate arachnid, the harvestman Phalangium opilio. We found that the expression of each spider ohnologue was lower than their single-copy orthologues in the harvestman suggesting subfunctionalization. However, this was not reflected in a reduction in the number of peaks of accessible chromatin because both spider ohnologues and single-copy genes had more peaks than the orthologous harvestman genes. We also found that the number of peaks of accessible chromatin was higher in the late embryonic stage associated with activation of genes expressed later during embryogenesis in both species. Taken together, our study provides a genome-wide comparison of gene regulatory sequences and embryonic gene expression in arachnids and thus new insights into the impact of the arachnopulmonate WGD.

Evolution and development of the arthropod head are heavily debated topics often referred to as "The arthropod head problem" (AHP) or the "Endless dispute". One aspect of the AHP concerns the evolutionary origin and homology of the most anterior head segments, the pre-gnathal segments (PGS), that are associated with the tripartite brain of arthropods. It has been suggested that the PGS could have evolved independently from the segments that build the rest of the arthropod body. One argument supporting this hypothesis is that the PGS are patterned by an initial transverse stripe of hedgehog (hh)-expression that splits once or twice, giving (in the case of double splitting) raise to the three PGS in distantly related arthropods such as the fly Drosophila melanogaster and the spider Parasteatoda tepidariorum. It has been implied that this splitting-event may recapitulate evolution of these segments, i.e. the splitting of an initial anterior body unit into three, or at least that the single splitting may represent a remnant of this process. In this paper, I show that two-fold splitting of an initial anterior hh-stripe is not conserved in chelicerates or even spiders. Instead, I find that a single splitting event correlated to the development of the most anterior two segments, the protocerebral and the deuterocerebral segment, is conserved among arthropods as a whole. There are, however, deviations from this pattern including a third or even fourth consecutive head segment, or even hh-splitting in more posterior segments.

Background

Jointed appendages represent one of the key innovations of arthropods, and thus understanding the development and evolution of these structures is important for the understanding of the evolutionary success of Arthropoda. In this paper, we analyze a cell cluster that was identified in a previous single-cell sequencing (SCS) experiment on embryos of the spider Parasteatoda tepidariorum. This cell cluster is characterized by marker genes that suggest a role in appendage patterning and joint development.

Results

We analyzed the expression profiles of these marker genes showing that they are expressed in the developing appendages and in a pattern that suggests a potential function during joint development. Several of the investigated genes represent new and unexpected factors such as dysfusion (dysf), spätzle3 (spz3), seven-up (svp). In order to study their evolutionary origin, we also investigated orthologs of the identified appendage-patterning genes in the harvestman Phalangium opilio, a distantly related chelicerate.

Conclusion

Our work highlights the usefulness of SCS experiments for the identification of potential new genetic factors that are involved in specific developmental processes. The current data provide potential new insights into the gene regulatory networks that underlie arthropod joint development.

Tardigrada is an ancient lineage of miniaturized animals. As an outgroup of the well-studied Arthropoda and Onychophora, studies of tardigrades hold the potential to reveal important insights into body plan evolution in Panarthropoda. Previous studies have revealed interesting facets of tardigrade development and genomics that suggest that a highly compact body plan is a derived condition of this lineage, rather than it representing an ancestral state of Panarthropoda. This conclusion was based on studies of several species from Eutardigrada. We review these studies and expand on them by analyzing the publicly available genome and transcriptome assemblies of Echiniscus testudo, a representative of Heterotardigrada. These new analyses allow us to phylogenetically reconstruct important features of genome evolution in Tardigrada. We use available data from tardigrades to interrogate several recent models of body plan evolution in Panarthropoda. Although anterior segments of panarthropods are highly diverse in terms of anatomy and development, both within individuals and between species, we conclude that a simple one-to-one alignment of anterior segments across Panarthropoda is the best available model of segmental homology. In addition to providing important insight into body plan diversification within Panarthropoda, we speculate that studies of tardigrades may reveal generalizable pathways to miniaturization.

Hox genes are known for their role in the specification of typical body plan features in animals. Evolutionary changes in Hox gene function are believed to be involved in the emergence of the diverse body plans we observe in animals today. Spiders share many body plan features with other arthropods, but also have numerous unique traits of their own. Studies of spider Hox genes have already provided insights into evolutionarily conserved and derived features of the spider body plan and their genetic basis. However, many aspects of Hox gene biology have been insufficiently studied in spiders so far. In this review, we highlight previous comparative studies of Hox genes in spiders and their significance for our understanding of the evolution of the spider body plan. We also identify aspects of Hox gene biology that need to be studied in greater detail. Many spider Hox genes have not been investigated beyond their mRNA expression patterns, and the role of Hox genes with apparently plesiomorphic or dual functions, like ftz and Hox3 is still unclear. Spiders have a duplicated Hox gene cluster, but possible sub-or neofunctionalisation of duplicates have not yet been studied systematically. Future research should therefore focus on these issues, in addition to the role of Polycomb and trithorax-mediated regulation, the identification of regulatory regions, cofactors or spider-specific target genes, and the significance of non-coding RNAs transcribed from within the Hox cluster and even from the antisense strand of particular Hox genes.

Background: Early during onychophoran development and prior to the formation of the germ band, a posterior tissue thickening forms the posterior pit. Anterior to this thickening forms a groove, the embryonic slit, that marks the anterior-posterior orientation of the developing embryo. This slit is by some authors considered the blastopore, and thus the origin of the endoderm, while others argue that the posterior pit represents the blastopore. This controversy is of evolutionary significance because if the slit represents the blastopore, then this would support the amphistomy hypothesis that suggests that a slit-like blastopore in the bilaterian ancestor evolved into protostomy and deuterostomy.

Results: In this paper, we summarize our current knowledge about endoderm and mesoderm development in onychophorans and provide additional data on early endoderm- and mesoderm-determining marker genes such as Blimp, Mox, and the T-box genes.

Conclusion: We come to the conclusion that the endoderm of onychophorans forms prior to the development of the embryonic slit, and thus that the slit is not the primary origin of the endoderm. It is thus unlikely that the embryonic slit represents the blastopore. We suggest instead that the posterior pit indeed represents the lips of the blastopore, and that the embryonic slit (and surrounding tissue) represents a morphologically superficial archenteron-like structure. We conclude further that both endoderm and mesoderm development are under control of conserved gene regulatory networks, and that many of the features found in arthropods including the model Drosophila melanogaster are likely derived.

Background

The common house spider Parasteatoda tepidariorum represents an emerging new model organism of arthropod evolutionary and developmental (EvoDevo) studies. Recent technical advances have resulted in the first single-cell sequencing (SCS) data on this species allowing deeper insights to be gained into its early development, but mid-to-late stage embryos were not included in these pioneering studies.

Results

Therefore, we performed SCS on mid-to-late stage embryos of Parasteatoda and characterized resulting cell clusters by means of in-silico analysis (comparison of key markers of each cluster with previously published information on these genes). In-silico prediction of the nature of each cluster was then tested/verified by means of additional in-situ hybridization experiments with additional markers of each cluster.

Conclusions

Our data show that SCS data reliably group cells with similar genetic fingerprints into more or less distinct clusters, and thus allows identification of developing cell types on a broader level, such as the distinction of ectodermal, mesodermal and endodermal cell lineages, as well as the identification of distinct developing tissues such as subtypes of nervous tissue cells, the developing heart, or the ventral sulcus (VS). In comparison with recent other SCS studies on the same species, our data represent later developmental stages, and thus provide insights into different stages of developing cell types and tissues such as differentiating neurons and the VS that are only present at these later stages.

Background

Spiders evolved different types of eyes, a pair of primary eyes that are usually forward pointing, and three pairs of secondary eyes that are typically situated more posterior and lateral on the spider’s head. The best understanding of arthropod eye development comes from the vinegar fly Drosophila melanogaster, the main arthropod model organism, that also evolved different types of eyes, the larval eyes and the ocelli and compound eyes of the imago. The gene regulatory networks that underlie eye development in this species are well investigated revealing a conserved core network, but also show several differences between the different types of eyes. Recent candidate gene approaches identified a number of conserved genes in arthropod eye development, but also revealed crucial differences including the apparent lack of some key factors in some groups of arthropods, including spiders.

Results

Here, we re-analysed our published scRNA sequencing data and found potential key regulators of spider eye development that were previously overlooked. Unlike earlier research on this topic, our new data suggest that Hedgehog (Hh)-signalling is involved in eye development in the spider Parasteatoda tepidariorum. By investigating embryonic gene expression in representatives of all main groups of spiders, we demonstrate that this involvement is conserved in spiders. Additionally, we identified genes that are expressed in the developing eyes of spiders, but that have not been studied in this context before.

Conclusion

Our data show that single-cell sequencing represents a powerful method to gain deeper insight into gene regulatory networks that underlie the development of lineage-specific organs such as the derived set of eyes in spiders. Overall, we gained deeper insight into spider eye development, as well as the evolution of arthropod visual system formation.