The Wnt and Delta-Notch signalling pathways interact to direct pair-rule gene expression via caudal during segment addition in the spider Parasteatoda tepidariorum.

In short-germ arthropods, posterior segments are added sequentially from a segment addition zone (SAZ) during embryogenesis. Studies in spiders such as Parasteatoda tepidariorum have provided insights into the gene regulatory network (GRN) underlying segment addition, and revealed that Wnt8 is required for dynamic Delta (Dl) expression associated with the formation of new segments. However, it remains unclear how these pathways interact during SAZ formation and segment addition. Here, we show that Delta-Notch signalling is required for Wnt8 expression in posterior SAZ cells, but represses the expression of this Wnt gene in anterior SAZ cells. We also found that these two signalling pathways are required for the expression of the spider orthologues of even-skipped (eve) and runt-1 (run-1), at least in part via caudal (cad). Moreover, it appears that dynamic expression of eve in this spider does not require a feedback loop with run-1, as is found in the pair-rule circuit of the beetle Tribolium Taken together, our results suggest that the development of posterior segments in Parasteatoda is directed by dynamic interactions between Wnt8 and Delta-Notch signalling that are read out by cad, which is necessary but probably not sufficient to regulate the expression of eve and run-1 Our study therefore provides new insights towards better understanding the evolution and developmental regulation of segmentation in other arthropods, including insects.

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.

Evolutionary crossroads in developmental biology: the spider Parasteatoda tepidariorum.

Spiders belong to the chelicerates, which is an arthropod group that branches basally from myriapods, crustaceans and insects. Spiders are thus useful models with which to investigate whether aspects of development are ancestral or derived with respect to the arthropod common ancestor. Moreover, they serve as an important reference point for comparison with the development of other metazoans. Therefore, studies of spider development have made a major contribution to advancing our understanding of the evolution of development. Much of this knowledge has come from studies of the common house spider, Parasteatoda tepidariorum. Here, we describe how the growing number of experimental tools and resources available to study Parasteatoda development have provided novel insights into the evolution of developmental regulation and have furthered our understanding of metazoan body plan 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.

Selection of distinct Hox-Extradenticle interaction modes fine-tunes Hox protein activity.

Hox genes encode transcription factors widely used for diversifying animal body plans in development and evolution. To achieve functional specificity, Hox proteins associate with PBC class proteins, Pre-B cell leukemia homeobox (Pbx) in vertebrates, and Extradenticle (Exd) in Drosophila, and were thought to use a unique hexapeptide-dependent generic mode of interaction. Recent findings, however, revealed the existence of an alternative, UbdA-dependent paralog-specific interaction mode providing diversity in Hox-PBC interactions. In this study, we investigated the basis for the selection of one of these two Hox-PBC interaction modes. Using naturally occurring variations and mutations in the Drosophila Ultrabithorax protein, we found that the linker region, a short domain separating the hexapeptide from the homeodomain, promotes an interaction mediated by the UbdA domain in a context-dependent manner. While using a UbdA-dependent interaction for the repression of the limb-promoting gene Distalless, interaction with Exd during segment-identity specification still relies on the hexapeptide motif. We further show that distinctly assembled Hox-PBC complexes display subtle but distinct repressive activities. These findings identify Hox-PBC interaction as a template for subtle regulation of Hox protein activity that may have played a major role in the diversification of Hox protein function in development and evolution.

Gene expression suggests conserved mechanisms patterning the heads of insects and myriapods.

Segmentation, i.e. the subdivision of the body into serially homologous units, is one of the hallmarks of the arthropods. Arthropod segmentation is best understood in the fly Drosophila melanogaster. But different from the situation in most arthropods in this species all segments are formed from the early blastoderm (so called long-germ developmental mode). In most other arthropods only the anterior segments are formed in a similar way (so called short-germ developmental mode). Posterior segments are added one at a time or in pairs of two from a posterior segment addition zone. The segmentation mechanisms are not universally conserved among arthropods and only little is known about the genetic patterning of the anterior segments. Here we present the expression patterns of the insect head patterning gene orthologs hunchback (hb), orthodenticle (otd), buttonhead-like (btdl), collier (col), cap-n-collar (cnc) and crocodile (croc), and the trunk gap gene Krüppel (Kr) in the myriapod Glomeris marginata. Conserved expression of these genes in insects and a myriapod suggests that the anterior segmentation system may be conserved in at least these two classes of arthropods. This finding implies that the anterior patterning mechanism already existed in the last common ancestor of insects and myriapods.

Expression of pair rule gene orthologs in the blastoderm of a myriapod: evidence for pair rule-like mechanisms?

BACKGROUND: A hallmark of Drosophila segmentation is the stepwise subdivision of the body into smaller and smaller units, and finally into the segments. This is achieved by the function of the well-understood segmentation gene cascade. The first molecular sign of a segmented body appears with the action of the pair rule genes, which are expressed as transversal stripes in alternating segments. Drosophila development, however, is derived, and in most other arthropods only the anterior body is patterned (almost) simultaneously from a pre-existing field of cells; posterior segments are added sequentially from a posterior segment addition zone. A long-standing question is to what extent segmentation mechanisms known from Drosophila may be conserved in short-germ arthropods. Despite the derived developmental modes, it appears more likely that conserved mechanisms can be found in anterior patterning. RESULTS: Expression analysis of pair rule gene orthologs in the blastoderm of the pill millipede Glomeris marginata (Myriapoda: Diplopoda) suggests that these genes are generally involved in segmenting the anterior embryo. We find that the Glomeris pairberry-1 ( pby-1) gene is expressed in a pair rule pattern that is also found in insects and a chelicerate, the mite Tetraynchus urticae. Other Glomeris pair rule gene orthologs are expressed in double segment wide domains in the blastoderm, which at subsequent stages split into two stripes in adjacent segments. CONCLUSIONS: The expression patterns of the millipede pair rule gene orthologs resemble pair rule patterning in Drosophila and other insects, and thus represent evidence for the presence of an ancestral pair rule-like mechanism in myriapods. We discuss the possibilities that blastoderm patterning may be conserved in long-germ and short-germ arthropods, and that a posterior double segmental mechanism may be present in short-germ arthropods.

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.

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.

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. RESULTS: We find that col in myriapods is expressed at early developmental stages in the same anterior domain in the head, the parasegment 0, as in insects. Comparable early expression of col is not present in the anterior head of an onychophoran that serves as an out-group species closely related to the arthropods. CONCLUSIONS: Our findings suggest either that i) the function of col in head development has been conserved between insects and myriapods, and that these two classes of arthropods may be closely related supporting the traditional Atelocerata (or Tracheata) hypothesis; or ii) alternatively col function could have been lost in early head development in crustaceans, or may indeed have evolved convergently in insects and myriapods.

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.

Notch-mediated segmentation of the appendages is a molecular phylotypic trait of the arthropods.

Arthropod limbs are arguably the most diverse organs in the animal kingdom. Morphological diversity of the limbs is largely based on their segmentation, because this divides the limbs into modules that can evolve separately for new morphologies and functions. Limb segmentation also distinguishes the arthropods from related phyla (e.g. onychophorans) and thus forms an important evolutionary innovation in arthropods. Understanding the genetic basis of limb segmentation in arthropods can thus shed light onto the mechanisms of macroevolution and the origin of a character (articulated limbs) that defines a new phylum (arthropods). In the fly Drosophila limb segmentation and limb growth are controlled by the Notch signaling pathway. Here we show that the Notch pathway also controls limb segmentation and growth in the spider Cupiennius salei, a representative of the most basally branching arthropod group Chelicerata, and thus this function must trace from the last common ancestor of all arthropods. The similarities of Notch and Serrate function between Drosophila and Cupiennius are extensive and also extend to target genes like odd-skipped, nubbin, AP-2 and hairy related genes. Our data confirm that the jointed appendages, which are a morphological phylotypic trait of the arthropods and the basis for naming the phylum, have a common developmental genetic basis. Notch-mediated limb segmentation is thus a molecular phylotypic trait of the arthropods.

Probing the Drosophila retinal determination gene network in Tribolium (II): The Pax6 genes eyeless and twin of eyeless.

The Pax6 genes eyeless (ey) and twin of eyeless (toy) are upstream regulators in the retinal determination gene network (RDGN), which instructs the formation of the adult eye primordium in Drosophila. Most animals possess a singleton Pax6 ortholog, but the dependence of eye development on Pax6 is widely conserved. A rare exception is given by the larval eyes of Drosophila, which develop independently of ey and toy. To obtain insight into the origin of differential larval and adult eye regulation, we studied the function of toy and ey in the red flour beetle Tribolium castaneum. We find that single and combinatorial knockdown of toy and ey affect larval eye development strongly but adult eye development only mildly in this primitive hemimetabolous species. Compound eye-loss, however, was provoked when ey and toy were RNAi-silenced in combination with the early retinal gene dachshund (dac). We propose that these data reflect a role of Pax6 during regional specification in the developing head and that the subsequent maintenance and growth of the adult eye primordium is regulated partly by redundant and partly by specific functions of toy, ey and dac in Tribolium. The results from embryonic knockdown and comparative protein sequence analysis lead us further to conclude that Tribolium represents an ancestral state of redundant control by ey and toy.

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

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

Cupiennius salei and Achaearanea tepidariorum: Spider models for investigating evolution and development.

The spiders Cupiennius salei and Achaearanea tepidariorum are firmly established laboratory models that have already contributed greatly to answering evolutionary developmental questions. Here we appraise why these animals are such useful models from phylogeny, natural history and embryogenesis to the tools available for their manipulation. We then review recent studies of axis formation, segmentation, appendage development and neurogenesis in these spiders and how this has contributed to understanding the evolution of these processes. Furthermore, we discuss the potential of comparisons of silk production between Cupiennius and Achaearanea to investigate the origins and diversification of this evolutionary innovation. We suggest that further comparisons between these two spiders and other chelicerates will prove useful for understanding the evolution of development in metazoans.

Hox genes and the body plans of chelicerates and pycnogonids.

Hox genes are found in all metazoan phyla and are involved in specifying identity along the anterior-posterior body axis. In arthropods, ten different classes of Hox genes can be distinguished, which are expressed in a typical staggered array along the anterior-posterior axis of the embryo in characteristically stable domains. These features have been used to align segments between different arthropod groups and in this way have contributed to solving longstanding zoological questions. In this chapter I summarize Hox gene data from chelicerates, including the enigmatic pycnogonids (sea spiders) and how these data have helped us to understand the body plans of different arthropod taxa.

Diverged and conserved aspects of heart formation in a spider.

Heart development exhibits some striking similarities between vertebrates and arthropods, for example in both cases the heart develops as a linear tube from mesodermal cells. Furthermore, the underlying molecular pathways exhibit a significant number of similarities between vertebrates and the fruit fly Drosophila, suggesting a common origin of heart development in the last common ancestor of flies and vertebrates. However, there is hardly any molecular data from other animals. Here we show that many of the key genes are also active in heart development in the spider Cupiennius salei. Spiders belong to the chelicerates and are distantly related to insects with respect to the other arthropods. The tinman/Nkx2.5 ortholog is the first gene to be specifically expressed in the presumptive spider heart, like in flies and vertebrates. We also show that tinman is expressed in a similar way in the beetle Tribolium castaneum. Taken together this demonstrates that tinman has a conserved role in the specification of the arthropod heart. In addition, we analyzed the expression of other heart genes (decapentaplegic, Wnt5, H15, even-skipped, and Mef2 ) in Cupiennius. The expression of these genes suggests that the genetic pathway of heart development may be largely conserved among arthropods. However, a major difference is seen in the earlier expression of the even-skipped gene in the developing spider heart compared with Drosophila, implying that the role of even-skipped in heart formation might have changed during arthropod evolution. The most striking finding, however, is that in addition to the dorsal tissue of the fourth walking leg segment and the opisthosomal segments, we discovered tinman-expressing cells that arise from a position dorsal to the cephalic lobe and that contribute to the anterior dorsal vessel. In contrast to the posterior heart tissue, these cells do not express the other heart genes. The spider heart thus is composed of two distinct populations of cells.

The T-box genes H15 and optomotor-blind in the spiders Cupiennius salei, Tegenaria atrica and Achaearanea tepidariorum and the dorsoventral axis of arthropod appendages.

Dorsoventral axis formation in the legs of the fly Drosophila melanogaster requires the T-box genes optomotor-blind (omb) and H15. Evolutionary conservation of the patterning functions of these genes is unclear, because data on H15 expression in the spider Cupiennius salei did not support a general role of H15 in ventral fate specification. However, H15 has a paralogous gene, midline (mid) in Drosophila and H15 duplicates are also present in Cupiennius and the millipede Glomeris marginata. H15 therefore seems to have been subject to gene duplication opening the possibility that the previous account on Cupiennius has overlooked one or several paralogs. We have studied omb- and H15-related genes in two additional spider species, Tegenaria atrica and Achearanea tepidariorum and show that in both species one of the H15 genes belongs to a third group of spider H15 genes that has an expression pattern very similar to the H15 pattern in Drosophila. The expression patterns of all omb-related genes are also very similar to the omb expression pattern in Drosophila. These data suggest that the dorsoventral patterning functions of omb and H15 are conserved in the arthropods and that the previous conclusions were based on an incomplete data set in Cupiennius. Our results emphasize the importance of a broad taxon sampling in comparative studies.

Arthropod segmentation: why centipedes are odd.

Recent work has revealed a double segmental periodicity of gene expression in the centipede, a potential molecular explanation for the observation that this arthropod always has an odd number of trunk segments. Is this an oddity of centipedes, or might it mean that double segmental pair-rule patterning dates back to the Ur-arthropod?