FGF signalling is involved in cumulus migration in the common house spider Parasteatoda tepidariorum.

Cell migration is a fundamental component during the development of most multicellular organisms. In the early spider embryo, the collective migration of signalling cells, known as the cumulus, is required to set the dorsoventral body axis. Here, we show that FGF signalling plays an important role during cumulus migration in the spider Parasteatoda tepidariorum. Spider embryos with reduced FGF signalling show reduced or absent cumulus migration and display dorsoventral patterning defects. Our study reveals that the transcription factor Ets4 regulates the expression of several FGF signalling components in the cumulus. In conjunction with a previous study, we show that the expression of fgf8 in the germ-disc is regulated via the Hedgehog signalling pathway. We also demonstrate that FGF signalling influences the BMP signalling pathway activity in the region around cumulus cells. Finally, we show that FGFR signalling might also influence cumulus migration in basally branching spiders and we propose that fgf8 might act as a chemo-attractant to guide cumulus cells towards the future dorsal pole of the spider embryo.

The T-box gene optomotor-blind organizes proximodistal leg patterning in the beetle Tribolium castaneum by repressing dorsal Dpp pathway activity.

Leg axis formation in Drosophila is organized by Wingless (Wg) and Decapentaplegic (Dpp) that control a number of downstream factors to pattern the dorsoventral (DV) and proximodistal (PD) axis. The T-box genes are important downstream factors mainly involved in dorsoventral leg axis formation. The ventral side is specified by H15 and midline, whereas optomotor-blind (omb) and Dorsocross (Doc1) are factors to specify dorsal cell fates. We show here that omb also organizes PD leg axis patterning in the beetle Tribolium castaneum. In the legs, Tc-omb is expressed along the dorsal side and represses ventral factors like wg and H15. Intriguingly, removing Tc-omb function leads to the activation of the Dpp pathway along the dorsal side of the legs, thus mimicking normal dpp expression in Drosophila. Dpp activity along the dorsal side leads to altered expression of proximal-distal patterning genes such as Distal-less (Dll) and dachshund (dac). Our results indicate a cell-autonomous activation of Dll and repression of dac by dpp. These findings are compatible with the cross-regulatory "cascade model" of proximal-distal leg imaginal disc patterning of Drosophila.

Expression of posterior Hox genes and opisthosomal appendage development in a mygalomorph spider.

Spiders represent an evolutionary successful group of chelicerate arthropods. The body of spiders is subdivided into two regions (tagmata). The anterior tagma, the prosoma, bears the head appendages and four pairs of walking legs. The segments of the posterior tagma, the opisthosoma, either lost their appendages during the course of evolution or their appendages were substantially modified to fulfill new tasks such as reproduction, gas exchange, and silk production. Previous work has shown that the homeotic Hox genes are involved in shaping the posterior appendages of spiders. In this paper, we investigate the expression of the posterior Hox genes in a tarantula that possesses some key differences of posterior appendages compared to true spiders, such as the lack of the anterior pair of spinnerets and a second set of book lungs instead of trachea. Based on the observed differences in posterior Hox gene expression in true spiders and tarantulas, we argue that subtle changes in the Hox gene expression of the Hox genes abdA and AbdB are possibly responsible for at least some of the morphological differences seen in true spiders versus tarantulas.

Lack of evidence for conserved parasegmental grooves in arthropods.

In the arthropod model species Drosophila melanogaster, a dipteran fly, segmentation of the anterior-posterior body axis is under control of a hierarchic gene cascade. Segmental boundaries that form morphological grooves are established posteriorly within the segmental expression domain of the segment-polarity gene (SPG) engrailed (en). More important for the development of the fly, however, are the parasegmental boundaries that are established at the interface of en expressing cells and anteriorly adjacent wingless (wg) expressing cells. In Drosophila, both segmental and transient parasegmental grooves form. The latter are positioned anterior to the expression of en. Although the function of the SPGs in establishing and maintaining segmental and parasegmental boundaries is highly conserved among arthropods, parasegmental grooves have only been reported for Drosophila, and a spider (Cupiennius salei). Here, we present new data on en expression, and re-evaluate published data, from four distantly related spiders, including Cupiennius, and a distantly related chelicerate, the harvestman Phalangium opilio. Gene expression analysis of en genes in these animals does not corroborate the presence of parasegmental grooves. Consequently, our data question the general presence of parasegmental grooves in arthropods.

Embryonic development and secondary axis induction in the Brazilian white knee tarantula Acanthoscurria geniculata, C. L. Koch, 1841 (Araneae; Mygalomorphae; Theraphosidae).

Tarantulas represent some of the heaviest and most famous spiders. However, there is little information about the embryonic development of these spiders or their relatives (infraorder Mygalomorphae) and time-lapse recording of the embryonic development is entirely missing. I here describe the complete development of the Brazilian white knee tarantula, Acanthoscurria geniculata, in fixed and live embryos. The establishment of the blastoderm, the formation, migration and signalling of the cumulus and the shape changes that occur in the segment addition zone are analysed in detail. In addition, I show that there might be differences in the contraction process of early embryos of different theraphosid spider species. A new embryonic reference transcriptome was generated for this study and was used to clone and analyse the expression of several important developmental genes. Finally, I show that embryos of A. geniculata are amenable to tissue transplantation and bead insertion experiments. Using these functional approaches, I induced axis duplication in embryos via cumulus transplantation and ectopic activation of BMP signalling. Overall, the mygalomorph spider A. geniculata is a useful laboratory system to analyse evolutionary developmental questions, and the availability of such a system will help understanding conserved and divergent aspects of spider/chelicerate development.

The house spider genome reveals an ancient whole-genome duplication during arachnid evolution.

BACKGROUND: The duplication of genes can occur through various mechanisms and is thought to make a major contribution to the evolutionary diversification of organisms. There is increasing evidence for a large-scale duplication of genes in some chelicerate lineages including two rounds of whole genome duplication (WGD) in horseshoe crabs. To investigate this further, we sequenced and analyzed the genome of the common house spider Parasteatoda tepidariorum. RESULTS: We found pervasive duplication of both coding and non-coding genes in this spider, including two clusters of Hox genes. Analysis of synteny conservation across the P. tepidariorum genome suggests that there has been an ancient WGD in spiders. Comparison with the genomes of other chelicerates, including that of the newly sequenced bark scorpion Centruroides sculpturatus, suggests that this event occurred in the common ancestor of spiders and scorpions, and is probably independent of the WGDs in horseshoe crabs. Furthermore, characterization of the sequence and expression of the Hox paralogs in P. tepidariorum suggests that many have been subject to neo-functionalization and/or sub-functionalization since their duplication. CONCLUSIONS: Our results reveal that spiders and scorpions are likely the descendants of a polyploid ancestor that lived more than 450 MYA. Given the extensive morphological diversity and ecological adaptations found among these animals, rivaling those of vertebrates, our study of the ancient WGD event in Arachnopulmonata provides a new comparative platform to explore common and divergent evolutionary outcomes of polyploidization events across eukaryotes.

Molecular control of gut formation in the spider Parasteatoda tepidariorum.

The development of a digestive system is an essential feature of bilaterians. Studies of the molecular control of gut formation in arthropods have been studied in detail in the fruit fly Drosophila melanogaster. However, little is known in other arthropods, especially in noninsect arthropods. To better understand the evolution of arthropod alimentary system, we investigate the molecular control of gut development in the spider Parasteatoda tepidariorum (Pt), the primary chelicerate model species for developmental studies. Orthologs of the ectodermal genes Pt-wingless (Pt-wg) and Pt-hedgehog (Pt-hh), of the endodermal genes, Pt-serpent (Pt-srp) and Pt-hepatocyte-nuclear factor-4 (Pt-hnf4) and of the mesodermal gene Pt-twist (Pt-twi) are expressed in the same germ layers during spider gut development as in D. melanogaster. Thus, our expression data suggest that the downstream molecular components involved in gut development in arthropods are conserved. However, Pt-forkhead (Pt-fkh) expression and function in spiders is considerably different from its D. melanogaster ortholog. Pt-fkh is expressed before gastrulation in a cell population that gives rise to endodermal and mesodermal precursors, suggesting a possible role for this factor in specification of both germ layers. To test this hypothesis, we knocked down Pt-fkh via RNA interference. Pt-fkh RNAi embryos not only fail to develop a proper gut, but also lack the mesodermal Pt-twi expressing cells. Thus, in spiders Pt-fkh specifies endodermal and mesodermal germ layers. We discuss the implications of these findings for the evolution and development of gut formation in Ecdysozoans.

Neofunctionalization of a Duplicate dachshund Gene Underlies the Evolution of a Novel Leg Segment in Arachnids.

The acquisition of a novel function, or neofunctionalization, protects duplicated genes from redundancy and subsequent loss, and is a major force that drives adaptive evolution. Neofunctionalization has been inferred for many duplicated genes based on differences in regulation between the parental gene and its duplicate. However, only few studies actually link the new function of a duplicated gene to a novel morphological or physiological character of the organism. Here we show that the duplication of dachshund (dac) in arachnids (spiders and allies) is linked with the evolution of a novel leg segment, the patella. We have studied dac genes in two distantly related spider species, the entelegyne spider Parasteatoda tepidariorum and the haplogyne spider Pholcus phalangioides. Both species possess two paralogous dac genes that duplicated before the split between entelegyne and haplogyne spiders. In contrast to the evolutionarily highly conserved dac1, its duplicate dac2 is strongly expressed in the patella leg segment during embryogenesis in both species. Using parental RNA interference in P. tepidariorum we show that dac2 is required for the development of the patella segment. If dac2 function is impaired, then the patella is fused with the tibia into a single leg segment. Thus, removing the function of dac2 experimentally reverts P. tepidariorum leg morphology into a stage before the duplication of dac and the evolution of the patella segment. Our results indicate that the origin of the patella is the result of the duplication and subsequent neofunctionalization of dac in the arachnid lineage.

Expression and function of the zinc finger transcription factor Sp6-9 in the spider Parasteatoda tepidariorum.

Zinc finger transcription factors of the Sp6-9 group are evolutionarily conserved in all metazoans and have important functions in, e.g., limb formation and heart development. The function of Sp6-9-related genes has been studied in a number of vertebrates and invertebrates, but data from chelicerates (spiders and allies) was lacking so far. We have isolated the ortholog of Sp6-9 from the common house spider Parasteatoda tepidariorum and the cellar spider Pholcus phalangioides. We show that the Sp6-9 gene in these spider species is expressed in the developing appendages thus suggesting a conserved role in limb formation. Indeed, RNAi with Sp6-9 in P. tepidariorum leads not only to strong limb defects, but also to the loss of body segments and head defects in more strongly affected animals. Together with a new expression domain in the early embryo, these data suggest that Sp6-9 has a dual role P. tepidariorum. The early role in head and body segment formation is not known from other arthropods, but the role in limb formation is evolutionarily highly conserved.

Formation of the germ-disc in spider embryos by a condensation-like mechanism.

BACKGROUND: Determination of the embryonic body axes is a crucial developmental process in all animals. The establishment of the embryonic axes of spiders has been best studied in the common-house-spider Parasteatoda tepidariorum. Here, anteroposterior (AP) polarity arises during germ disc formation; the centre of the germ-disc marks the future posterior pole, and the rim of the disc the future anterior pole of the spider embryo. The centre of the germ disc is also needed for the formation of the cumulus, a group of migratory cells needed to establish dorsoventral (DV) polarity. Thus, both body axes depend on proper germ disc formation and patterning. However, these processes have not been fully analysed at the cellular and molecular level. RESULTS: Here I present new techniques to stain the cell membranes/outlines in live and fixed spider embryos. I show that the germ-disc is formed from a regular and contiguous blastoderm and that co-ordinated cell shape changes, rather than migration of single cells, are required to drive germ-disc formation in P. tepidariorum embryos. Furthermore, I show that the rate of cell divisions within the embryonic and extra-embryonic region is not involved in the rapid establishment of the germ-disc. Finally, I show that the process of germ-disc formation is dependent on the initiation of zygotic transcription. CONCLUSIONS: The presented data provide new insights in to the formation of the germ-disc in spider embryos. The establishment of the germ-disc in Parasteatoda embryos is a highly dynamic process that involves wide scale cell-shape changes. While most of the blastodermal cells become cuboidal to form the dense germ-disc, the remaining blastodermal cells stay squamous and develop into huge extra-embryonic, yolk rich cells. In addition, this study shows that the onset of zygotic transcription is needed to establish the germ-disc itself, and that the mid-blastula transition of Parasteatoda tepidariorum embryos is prior to any overt axis establishment.

Regressive evolution of the arthropod tritocerebral segment linked to functional divergence of the Hox gene labial.

The intercalary segment is a limbless version of the tritocerebral segment and is present in the head of all insects, whereas other extant arthropods have retained limbs on their tritocerebral segment (e.g. the pedipalp limbs in spiders). The evolutionary origin of limb loss on the intercalary segment has puzzled zoologists for over a century. Here we show that an intercalary segment-like phenotype can be created in spiders by interfering with the function of the Hox gene labial. This links the origin of the intercalary segment to a functional change in labial. We show that in the spider Parasteatoda tepidariorum the labial gene has two functions: one function in head tissue maintenance that is conserved between spiders and insects, and a second function in pedipalp limb promotion and specification, which is only present in spiders. These results imply that labial was originally crucial for limb formation on the tritocerebral segment, but that it has lost this particular subfunction in the insect ancestor, resulting in limb loss on the intercalary segment. Such loss of a subfunction is away to avoid adverse pleiotropic effects normally associated with mutations in developmental genes, and may thus be a common mechanism to accelerate regressive evolution.

Divergent role of the Hox gene Antennapedia in spiders is responsible for the convergent evolution of abdominal limb repression.

Evolution often results in morphologically similar solutions in different organisms, a phenomenon known as convergence. However, there is little knowledge of the processes that lead to convergence at the genetic level. The genes of the Hox cluster control morphology in animals. They may also be central to the convergence of morphological traits, but whether morphological similarities also require similar changes in Hox gene function is disputed. In arthropods, body subdivision into a region with locomotory appendages ("thorax") and a region with reduced appendages ("abdomen") has evolved convergently in several groups, e.g., spiders and insects. In insects, legs develop in the expression domain of the Hox gene Antennapedia (Antp), whereas the Hox genes Ultrabithorax (Ubx) and abdominal-A mediate leg repression in the abdomen. Here, we show that, unlike Antp in insects, the Antp gene in the spider Achaearanea tepidariorum represses legs in the first segment of the abdomen (opisthosoma), and that Antp and Ubx are redundant in the following segment. The down-regulation of Antp in A. tepidariorum leads to a striking 10-legged phenotype. We present evidence from ectopic expression of the spider Antp gene in Drosophila embryos and imaginal tissue that this unique function of Antp is not due to changes in the Antp protein, but likely due to divergent evolution of cofactors, Hox collaborators or target genes in spiders and flies. Our results illustrate an interesting example of convergent evolution of abdominal leg repression in arthropods by altering the role of distinct Hox genes at different levels of their action.

Novel function of Distal-less as a gap gene during spider segmentation.

Despite many aspects of the regulation of segmentation being conserved among arthropods, the evolution of novel gene functions has played an important role in the evolution of developmental regulation and the emergence of new segmental structures. Moreover the study of such novel gene functions can be informative with respect to the patterns and direction of evolutionary changes in developmental programs. The homeobox gene Distal-less (Dll) is known for its conserved function in appendage development in metazoans. In arthropods, Dll is required for the specification of distal appendage structures. Here we describe a novel and unexpected role of Dll in the spider Achaearanea tepidariorum. We detect At-Dll transcripts not only in the appendages, but unexpectedly also in an anterior domain during early development, prior to the specification of the limb primordia. A similar early Dll domain is present in the distantly related spider Pholcus phalangioides. In A. tepidariorum this early At-Dll expression is required for head segmentation. RNA interference results in spiders that lack either the first or the first and the second walking leg segments. The early At-Dll expression is also required for the activation of the segment polarity genes engrailed and hedgehog in this region. Our work identifies the Distal-less gene as a novel factor in anterior spider segmentation with a gap gene-like function. This novel role of Dll is interesting because Dll expression is reduced in this region in crustaceans and the homologous insect segment, the mandible segment, does not express Dll and does not require this gene for patterning. We therefore discuss the possible implications of our results for understanding the evolution and diversification of the mandible segment.

Single-cell sequencing suggests a conserved function of Hedgehog-signalling in spider eye development.

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.

An atlas of spider development at single-cell resolution provides new insights into arthropod embryogenesis.

Spiders are a diverse order of chelicerates that diverged from other arthropods over 500 million years ago. Research on spider embryogenesis, particularly studies using the common house spider Parasteatoda tepidariorum, has made important contributions to understanding the evolution of animal development, including axis formation, segmentation, and patterning. However, we lack knowledge about the cells that build spider embryos, their gene expression profiles and fate. Single-cell transcriptomic analyses have been revolutionary in describing these complex landscapes of cellular genetics in a range of animals. Therefore, we carried out single-cell RNA sequencing of P. tepidariorum embryos at stages 7, 8 and 9, which encompass the establishment and patterning of the body plan, and initial differentiation of many tissues and organs. We identified 20 cell clusters, from 18.5 k cells, which were marked by many developmental toolkit genes, as well as a plethora of genes not previously investigated. We found differences in the cell cycle transcriptional signatures, suggestive of different proliferation dynamics, which related to distinctions between endodermal and some mesodermal clusters, compared with ectodermal clusters. We identified many Hox genes as markers of cell clusters, and Hox gene ohnologs were often present in different clusters. This provided additional evidence of sub- and/or neo-functionalisation of these important developmental genes after the whole genome duplication in an arachnopulmonate ancestor (spiders, scorpions, and related orders). We also examined the spatial expression of marker genes for each cluster to generate a comprehensive cell atlas of these embryonic stages. This revealed new insights into the cellular basis and genetic regulation of head patterning, hematopoiesis, limb development, gut development, and posterior segmentation. This atlas will serve as a platform for future analysis of spider cell specification and fate, and studying the evolution of these processes among animals at cellular resolution.

Dynamic gene expression is required for anterior regionalization in a spider.

Patterning of a multicellular embryo requires precise spatiotemporal control of gene expression during development. The gradient of the morphogen bicoid regulates anterior regionalization in the syncytial blastoderm of Drosophila. However many arthropod embryos develop from a cellular blastoderm that does not allow the formation of transcription factor gradients. Here we show that correct anterior development of the cellularized embryo of the spider Achaearanea tepidariorum requires an anterior-to-posterior wave of dynamic gene expression for positioning the stripes of hairy, hedgehog, and orthodenticle expression. Surprisingly, this dynamic repositioning of the expression of these segmentation genes is blocked in orthodenticle(pRNAi) embryos and no anterior structures are specified in those embryos. Our data suggest that dynamic gene expression across a field of cells is required for anterior regionalization in spiders and provides an explanation for the problem of how positional values for anterior segmentation genes are specified via a morphogen-independent mechanism across a field of cells.

Extraembryonic tissue in chelicerates: a review and outlook.

The formation of extraembryonic membranes (EEMs) contributes to the proper development of many animals. In arthropods, the formation and function of EEMs have been studied best in insects. Regarding the development of extraembryonic tissue in chelicerates (spiders and relatives), most information is available for spiders (Araneae). Especially two populations of cells have been considered to represent EEMs in spiders. The first of these potential EEMs develops shortly after egg deposition, opposite to a radially symmetrical germ disc that forms in one hemisphere of the egg and encloses the yolk. The second tissue, which has been described as being extraembryonic is the so-called dorsal field, which is required to cover the dorsal part of the developing spider germ rudiment before proper dorsal closure. In this review, we summarize the current knowledge regarding the formation of potential extraembryonic structures in the Chelicerata. We describe the early embryogenesis of spiders and other chelicerates, with a special focus on the formation of the potential extraembryonic tissues. This article is part of the theme issue 'Extraembryonic tissues: exploring concepts, definitions and functions across the animal kingdom'.

Evolutionary plasticity of collier function in head development of diverse arthropods.

The insect intercalary segment represents a small and appendage-less head segment that is homologous to the second antennal segment of Crustacea and the pedipalpal segment in Chelicerata, which are generally referred to as "tritocerebral segment." In Drosophila, the gene collier (col) has an important role for the formation of the intercalary segment. Here we show that in the beetle Tribolium castaneum col is required for the activation of the segment polarity genes hedgehog (hh), engrailed (en) and wingless (wg) in the intercalary segment, and is a regulatory target of the intercalary segment specific Hox gene labial (lab). Loss of Tc col function leads to increased cell death in the intercalary segment. In the milkweed bug Oncopeltus fasciatus, the loss of col function has a more severe effect in lacking the intercalary segment and also affecting the adjacent mandibular and antennal segments. By contrast, col is not expressed early in the second antennal segment in the crustacean Parhyale hawaiensis or in the pedipalpal segment of the spider Achaearanea tepidariorum. This suggests that the early expression of col in a stripe and its role in tritocerebral segment development is insect-specific and might correlate with the appendage-less morphology of the intercalary segment.

Wnt8 is required for growth-zone establishment and development of opisthosomal segments in a spider.

The Wnt genes encode secreted glycoprotein ligands that regulate many developmental processes from axis formation to tissue regeneration [1]. In bilaterians, there are at least 12 subfamilies of Wnt genes [2]. Wnt3 and Wnt8 are required for somitogenesis in vertebrates [3-7] and are thought to be involved in posterior specification in deuterostomes in general [8]. Although TCF and beta-catenin have been implicated in the posterior patterning of some short-germ insects [9, 10], the specific Wnt ligands required for posterior specification in insects and other protostomes remained unknown. Here we investigated the function of Wnt8 in a chelicerate, the common house spider Achaearanea tepidariorum[11]. Knockdown of Wnt8 in Achaearanea via parental RNAi caused misregulation of Delta, hairy, twist, and caudal and resulted in failure to properly establish a posterior growth zone and truncation of the opisthosoma (abdomen). In embryos with the most severe phenotypes, the entire opisthosoma was missing. Our results suggest that in the spider, Wnt8 is required for posterior development through the specification and maintenance of growth-zone cells. Furthermore, we propose that Wnt8, caudal, and Delta/Notch may be parts of an ancient genetic regulatory network that could have been required for posterior specification in the last common ancestor of protostomes and deuterostomes.