Search

Pathogenic variants in SF3B4, a component of the U2 snRNP complex important for branchpoint sequence recognition and splicing, are responsible for the acrofacial disorders Nager and Rodriguez Syndrome, also known as SF3B4 -related syndromes. Patients exhibit malformations in the head, face, limbs, vertebrae as well as the heart. To uncover the etiology of craniofacial malformations found in SF3B4 -related syndromes, mutant mouse lines with homozygous deletion of Sf3b4 in neural crest cells (NCC) were generated. Like in human patients, these embryos had craniofacial and cardiac malformations with variable expressivity and penetrance. The severity and survival of Sf3b4 NCC mutants was modified by the level of Sf3b4 in neighboring non-NCC. RNA sequencing analysis of heads of embryos prior to morphological abnormalities revealed significant changes in expression of genes forming the NCC regulatory network, as well as an increase in exon skipping. Additionally, several key histone modifiers involved in craniofacial and cardiac development showed increased exon skipping. Increased exon skipping was also associated with use of a more proximal branch point, as well as an enrichment in thymidine bases in the 50 bp around the branch points. We propose that decrease in Sf3b4 causes changes in the expression and splicing of transcripts required for proper craniofacial and cardiac development, leading to abnormalities., Competing Interests: Competing interests statement:The authors declare no competing interest.

Special AT-rich sequence binding protein 2 (Satb2) is a matrix attachment region (MAR) binding protein. Satb2 impacts skeletal development by regulating gene transcription required for osteogenic differentiation. Although its role as a high-order transcription factor is well supported, other roles for Satb2 in skeletal development remain unclear. In particular, the impact of dosage sensitivity (heterozygous mutations) and variance on phenotypic severity is still not well understood. To further investigate molecular and cellular mechanisms of Satb2-mediated skeletal defects, we used the CRISPR/Cas9 system to generate Satb2 mutations in MC3T3-E1 cells. Our data suggest that, in addition to its role in differentiation, Satb2 regulates progenitor proliferation. We also find that mutations in Satb2 cause chromatin defects including nuclear blebbing and donut-shaped nuclei. These defects may contribute to a slight increase in apoptosis in mutant cells, but apoptosis is insufficient to explain the proliferation defects. Satb2 expression exhibits population-level variation and is most highly expressed from late G1 to late G2. Based on these data, we hypothesize that Satb2 may regulate proliferation through two separate mechanisms. First, Satb2 may regulate the expression of genes necessary for cell cycle progression in pre-osteoblasts. Second, similar to other MAR-binding proteins, Satb2 may participate in DNA replication. We also hypothesize that variation in the severity or penetrance of Satb2-mediated proliferation defects is due to stochastic variation in Satb2 binding to DNA, which may be buffered in some genetic backgrounds. Further elucidation of the role of Satb2 in proliferation has potential impacts on our understanding of both skeletal defects and cancer., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Darwin's bark spider ( Caerostris darwini ) produces giant orb webs from dragline silk that can be twice as tough as other silks, making it the toughest biological material. This extreme toughness comes from increased extensibility relative to other draglines. We show C. darwini dragline-producing major ampullate (MA) glands highly express a novel silk gene transcript (MaSp4) encoding a protein that diverges markedly from closely related proteins and contains abundant proline, known to confer silk extensibility, in a unique GPGPQ amino acid motif. This suggests C. darwini evolved distinct proteins that may have increased its dragline's toughness, enabling giant webs. Caerostris darwini's MA spinning ducts also appear unusually long, potentially facilitating alignment of silk proteins into extremely tough fibers. Thus, a suite of novel traits from the level of genes to spinning physiology to silk biomechanics are associated with the unique ecology of Darwin's bark spider, presenting innovative designs for engineering biomaterials., Competing Interests: Competing interestsThe authors declare no competing interests.

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.

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., (© 2017 Wiley Periodicals, Inc.)

Background: Orb-web weaving spiders and their relatives use multiple types of task-specific silks. The majority of spider silk studies have focused on the ultra-tough dragline silk synthesized in major ampullate glands, but other silk types have impressive material properties. For instance, minor ampullate silks of orb-web weaving spiders are as tough as draglines, due to their higher extensibility despite lower strength. Differences in material properties between silk types result from differences in their component proteins, particularly members of the spidroin (spider fibroin) gene family. However, the extent to which variation in material properties within a single silk type can be explained by variation in spidroin sequences is unknown. Here, we compare the minor ampullate spidroins (MiSp) of orb-weavers and cobweb weavers. Orb-web weavers use minor ampullate silk to form the auxiliary spiral of the orb-web while cobweb weavers use it to wrap prey, suggesting that selection pressures on minor ampullate spidroins (MiSp) may differ between the two groups., Results: We report complete or nearly complete MiSp sequences from five cobweb weaving spider species and measure material properties of minor ampullate silks in a subset of these species. We also compare MiSp sequences and silk properties of our cobweb weavers to published data for orb-web weavers. We demonstrate that all our cobweb weavers possess multiple MiSp loci and that one locus is more highly expressed in at least two species. We also find that the proportion of β-spiral-forming amino acid motifs in MiSp positively correlates with minor ampullate silk extensibility across orb-web and cobweb weavers., Conclusions: MiSp sequences vary dramatically within and among spider species, and have likely been subject to multiple rounds of gene duplication and concerted evolution, which have contributed to the diverse material properties of minor ampullate silks. Our sequences also provide templates for recombinant silk proteins with tailored properties.

Background: Black widow spiders are infamous for their neurotoxic venom, which can cause extreme and long-lasting pain. This unusual venom is dominated by latrotoxins and latrodectins, two protein families virtually unknown outside of the black widow genus Latrodectus, that are difficult to study given the paucity of spider genomes. Using tissue-, sex- and stage-specific expression data, we analyzed the recently sequenced genome of the house spider (Parasteatoda tepidariorum), a close relative of black widows, to investigate latrotoxin and latrodectin diversity, expression and evolution., Results: We discovered at least 47 latrotoxin genes in the house spider genome, many of which are tandem-arrayed. Latrotoxins vary extensively in predicted structural domains and expression, implying their significant functional diversification. Phylogenetic analyses show latrotoxins have substantially duplicated after the Latrodectus/Parasteatoda split and that they are also related to proteins found in endosymbiotic bacteria. Latrodectin genes are less numerous than latrotoxins, but analyses show their recruitment for venom function from neuropeptide hormone genes following duplication, inversion and domain truncation. While latrodectins and other peptides are highly expressed in house spider and black widow venom glands, latrotoxins account for a far smaller percentage of house spider venom gland expression., Conclusions: The house spider genome sequence provides novel insights into the evolution of venom toxins once considered unique to black widows. Our results greatly expand the size of the latrotoxin gene family, reinforce its narrow phylogenetic distribution, and provide additional evidence for the lateral transfer of latrotoxins between spiders and bacterial endosymbionts. Moreover, we strengthen the evidence for the evolution of latrodectin venom genes from the ecdysozoan Ion Transport Peptide (ITP)/Crustacean Hyperglycemic Hormone (CHH) neuropeptide superfamily. The lower expression of latrotoxins in house spiders relative to black widows, along with the absence of a vertebrate-targeting α-latrotoxin gene in the house spider genome, may account for the extreme potency of black widow venom.

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., (© 2016. Published by The Company of Biologists Ltd.)

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.

Studies in vertebrate and invertebrate model organisms on the molecular basis of primordial germ cell (PGC) specification have revealed that metazoans can specify their germ line either early in development by maternally transmitted cytoplasmic factors (inheritance), or later in development by signaling factors from neighboring tissues (induction). Regardless of the mode of PGC specification, once animal germ cells are specified, they invariably express a number of highly conserved genes. These include vasa and piwi, which can play essential roles in any or all of PGC specification, development, or gametogenesis. Although the arthropods are the most speciose animal phylum, to date there have been no functional studies of conserved germ line genes in species of the most basally branching arthropod clade, the chelicerates (which includes spiders, scorpions, and horseshoe crabs). Here we present the first such study by using molecular and functional tools to examine germ line development and the roles of vasa and piwi orthologues in the common house spider Parasteatoda (formerly Achaearanea) tepidariorum. We use transcript and protein expression patterns of Pt-vasa and Pt-piwi to show that primordial germ cells (PGCs) in the spider arise during late embryogenesis. Neither Pt-vasa nor Pt-piwi gene products are localized asymmetrically to any embryonic region before PGCs emerge as paired segmental clusters in opisthosomal segments 2-6 at late germ band stages. RNA interference studies reveal that both genes are required maternally for egg laying, mitotic progression in early embryos, and embryonic survival. Our results add to the growing body of evidence that vasa and piwi can play important roles in somatic development, and provide evidence for a previously hypothesized conserved role for vasa in cell cycle progression., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The segmental architecture of the arthropod head is one of the most controversial topics in the evolutionary developmental biology of arthropods. The deutocerebral (second) segment of the head is putatively homologous across Arthropoda, as inferred from the segmental distribution of the tripartite brain and the absence of Hox gene expression of this anterior-most, appendage-bearing segment. While this homology statement implies a putative common mechanism for differentiation of deutocerebral appendages across arthropods, experimental data for deutocerebral appendage fate specification are limited to winged insects. Mandibulates (hexapods, crustaceans and myriapods) bear a characteristic pair of antennae on the deutocerebral segment, whereas chelicerates (e.g. spiders, scorpions, harvestmen) bear the eponymous chelicerae. In such hexapods as the fruit fly, Drosophila melanogaster, and the cricket, Gryllus bimaculatus, cephalic appendages are differentiated from the thoracic appendages (legs) by the activity of the appendage patterning gene homothorax (hth). Here we show that embryonic RNA interference against hth in the harvestman Phalangium opilio results in homeonotic chelicera-to-leg transformations, and also in some cases pedipalp-to-leg transformations. In more strongly affected embryos, adjacent appendages undergo fusion and/or truncation, and legs display proximal defects, suggesting conservation of additional functions of hth in patterning the antero-posterior and proximo-distal appendage axes. Expression signal of anterior Hox genes labial, proboscipedia and Deformed is diminished, but not absent, in hth RNAi embryos, consistent with results previously obtained with the insect G. bimaculatus. Our results substantiate a deep homology across arthropods of the mechanism whereby cephalic appendages are differentiated from locomotory appendages., (© 2015 The Author(s) Published by the Royal Society. All rights reserved.)

The evolutionary success of the largest animal phylum, Arthropoda, has been attributed to tagmatization, the coordinated evolution of adjacent metameres to form morphologically and functionally distinct segmental regions called tagmata. Specification of regional identity is regulated by the Hox genes, of which 10 are inferred to be present in the ancestor of arthropods. With six different posterior segmental identities divided into two tagmata, the bauplan of scorpions is the most heteronomous within Chelicerata. Expression domains of the anterior eight Hox genes are conserved in previously surveyed chelicerates, but it is unknown how Hox genes regionalize the three tagmata of scorpions. Here, we show that the scorpion Centruroides sculpturatus has two paralogues of all Hox genes except Hox3, suggesting cluster and/or whole genome duplication in this arachnid order. Embryonic anterior expression domain boundaries of each of the last four pairs of Hox genes (two paralogues each of Antp, Ubx, abd-A and Abd-B) are unique and distinguish segmental groups, such as pectines, book lungs and the characteristic tail, while maintaining spatial collinearity. These distinct expression domains suggest neofunctionalization of Hox gene paralogues subsequent to duplication. Our data reconcile previous understanding of Hox gene function across arthropods with the extreme heteronomy of scorpions., (© 2014 The Author(s) Published by the Royal Society. All rights reserved.)

Parasteatoda tepidariorum is an increasingly popular model for the study of spider development and the evolution of development more broadly. However, fully understanding the regulation and evolution of P. tepidariorum development in comparison to other animals requires a genomic perspective. Although research on P. tepidariorum has provided major new insights, gene analysis to date has been limited to candidate gene approaches. Furthermore, the few available EST collections are based on embryonic transcripts, which have not been systematically annotated and are unlikely to contain transcripts specific to post-embryonic stages of development. We therefore generated cDNA from pooled embryos representing all described embryonic stages, as well as post-embryonic stages including nymphs, larvae and adults, and using Illumina HiSeq technology obtained a total of 625,076,514 100-bp paired end reads. We combined these data with 24,360 ESTs available in GenBank, and 1,040,006 reads newly generated from 454 pyrosequencing of a mixed-stage embryo cDNA library. The combined sequence data were assembled using a custom de novo assembly strategy designed to optimize assembly product length, number of predicted transcripts, and proportion of raw reads incorporated into the assembly. The de novo assembly generated 446,427 contigs with an N50 of 1,875 bp. These sequences obtained 62,799 unique BLAST hits against the NCBI non-redundant protein data base, including putative orthologs to 8,917 Drosophila melanogaster genes based on best reciprocal BLAST hit identity compared with the D. melanogaster proteome. Finally, we explored the utility of the transcriptome for RNA-Seq studies, and showed that this resource can be used as a mapping scaffold to detect differential gene expression in different cDNA libraries. This resource will therefore provide a platform for future genomic, gene expression and functional approaches using P. tepidariorum.

Background: The monophyly of Mandibulata - the division of arthropods uniting pancrustaceans and myriapods - is consistent with several morphological characters, such as the presence of sensory appendages called antennae and the eponymous biting appendage, the mandible. Functional studies have demonstrated that the patterning of the mandible requires the activity of the Hox gene Deformed and the transcription factor cap-n-collar (cnc) in at least two holometabolous insects: the fruit fly Drosophila melanogaster and the beetle Tribolium castaneum. Expression patterns of cnc from two non-holometabolous insects and a millipede have suggested conservation of the labral and mandibular domains within Mandibulata. However, the activity of cnc is unknown in crustaceans and chelicerates, precluding understanding of a complete scenario for the evolution of patterning of this appendage within arthropods. To redress these lacunae, here we investigate the gene expression of the ortholog of cnc in Parhyale hawaiensis, a malacostracan crustacean, and two chelicerates: the harvestman Phalangium opilio, and the scorpion Centruroides sculpturatus., Results: In the crustacean P. hawaiensis, the segmental expression of Ph-cnc is the same as that reported previously in hexapods and myriapods, with two distinct head domains in the labrum and the mandibular segment. In contrast, Po-cnc and Cs-cnc expression is not enriched in the labrum of either chelicerate, but instead is expressed at comparable levels in all appendages. In further contrast to mandibulate orthologs, the expression domain of Po-cnc posterior to the labrum is not confined within the expression domain of Po-Dfd., Conclusions: Expression data from two chelicerate outgroup taxa suggest that the signature two-domain head expression pattern of cnc evolved at the base of Mandibulata. The observation of the archetypal labral and mandibular segment domains in a crustacean exemplar supports the synapomorphic nature of mandibulate cnc expression. The broader expression of Po-cnc with respect to Po-Dfd in chelicerates further suggests that the regulation of cnc by Dfd was also acquired at the base of Mandibulata. To test this hypothesis, future studies examining panarthropod cnc evolution should investigate expression of the cnc ortholog in arthropod outgroups, such as Onychophora and Tardigrada.

The discovery of genetic mechanisms that can transform a morphological structure from a plesiomorphic (=primitive) state to an apomorphic (=derived) one is a cardinal objective of evolutionary developmental biology. However, this objective is often impeded for many lineages of interest by limitations in taxonomic sampling, genomic resources, or functional genetic methods. In order to investigate the evolution of appendage morphology within Chelicerata, the putative sister group of the remaining arthropods, we developed an RNA interference (RNAi) protocol for the harvestman Phalangium opilio. We silenced the leg gap genes Distal-less (Dll) and dachshund (dac) in the harvestman via zygotic injections of double-stranded RNA (dsRNA), and used in situ hybridization to confirm RNAi efficacy. Consistent with the conserved roles of these genes in patterning the proximo-distal axis of arthropod appendages, we observed that embryos injected with Dll dsRNA lacked distal parts of appendages and appendage-like structures, such as the labrum, the chelicerae, the pedipalps, and the walking legs, whereas embryos injected with dac dsRNA lacked the medial podomeres femur and patella in the pedipalps and walking legs. In addition, we detected a role for these genes in patterning structures that do not occur in well-established chelicerate models (spiders and mites). Dll RNAi additionally results in loss of the preoral chamber, which is formed from pedipalpal and leg coxapophyses, and the ocularium, a dorsal outgrowth bearing the eyes. In one case, we observed that an embryo injected with dac dsRNA lacked the proximal segment of the chelicera, a plesiomorphic podomere that expresses dac in wild-type embryos. This may support the hypothesis that loss of the cheliceral dac domain underlies the transition to the two-segmented chelicera of derived arachnids., (© 2013 Wiley Periodicals, Inc.)

oskar is the only gene in the animal kingdom necessary and sufficient for specifying functional germ cells. However, oskar has only been identified in holometabolous ("higher") insects that specify their germline using specialized cytoplasm called germ plasm. Here we show that oskar evolved before the divergence of higher insects and provide evidence that its germline role is a recent evolutionary innovation. We identify an oskar ortholog in a basally branching insect, the cricket Gryllus bimaculatus. In contrast to Drosophila oskar, Gb-oskar is not required for germ cell formation or axial patterning. Instead, Gb-oskar is expressed in neuroblasts of the brain and CNS and is required for neural development. Taken together with reports of a neural role for Drosophila oskar, our data demonstrate that oskar arose nearly 50 million years earlier in insect evolution than previously thought, where it may have played an ancestral neural role, and was co-opted to its well-known essential germline role in holometabolous insects., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The proximo-distal axis of the arthropod leg is patterned by mutually antagonistic developmental expression domains of the genes extradenticle, homothorax, dachshund, and Distal-less. In the deutocerebral appendages (the antennae) of insects and crustaceans, the expression domain of dachshund is frequently either absent or, if present, is not required to pattern medial segments. By contrast, the dachshund domain is entirely absent in the deutocerebral appendages of spiders, the chelicerae. It is unknown whether absence of dachshund expression in the spider chelicera is associated with the two-segmented morphology of this appendage, or whether all chelicerates lack the dachshund domain in their chelicerae. We investigated gene expression in the harvestman Phalangium opilio, which bears the plesiomorphic three-segmented chelicera observed in "primitive" chelicerate orders. Consistent with patterns reported in spiders, in the harvestman chelicera homothorax, extradenticle, and Distal-less have broadly overlapping developmental domains, in contrast with mutually exclusive domains in the legs and pedipalps. However, unlike in spiders, the harvestman chelicera bears a distinct expression domain of dachshund in the proximal segment, the podomere that is putatively lost in derived arachnids. These data suggest that a tripartite proximo-distal domain structure is ancestral to all arthropod appendages, including deutocerebral appendages. As a corollary, these data also provide an intriguing putative genetic mechanism for the diversity of arachnid chelicerae: loss of developmental domains along the proximo-distal axis., (© 2012 Wiley Periodicals, Inc.)

Among chelicerates, Hox gene expression has only been investigated in representatives of two arachnid orders to date: Acari (mites and ticks) and Araneae (spiders). Limited data are available for the "primitive" arachnid orders, such as Scorpiones (scorpions) and Opiliones (harvestmen). Here, we present the first data on Hox gene expression in the harvestman Phalangium opilio. Ten Hox genes of this species were obtained from a de novo assembled developmental transcriptome using the Illumina GAII platform. All 10 genes are expressed in characteristic Hox-like expression patterns, and the expression of the anterior and central Hox genes is similar to those of other chelicerates. However, intriguingly, the three posteriormost genes-Ultrabithorax, abdominal-A, and Abdominal-B-share an identical anterior expression boundary in the second opisthosomal segment, and their expression domains extend through the opisthosoma to the posterior growth zone. The overlap in expression domains of the posterior Hox genes is correlated with the absence of opisthosomal organs posterior to the tubular tracheae, which occur on the second opisthosomal segment. Together with the staggered profile of posterior Hox genes in spiders, these data suggest the involvement of abdominal-A and Abdominal-B in the evolution of heteronomous patterning of the chelicerate opisthosoma, providing a mechanism that helps explain the morphological diversity of chelicerates., (© 2012 Wiley Periodicals, Inc.)

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.

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.

Germ cells occupy a unique position in animal reproduction, development, and evolution. In sexually reproducing animals, only they can produce gametes and contribute genetically to subsequent generations. Nonetheless, germ line specification during embryogenesis is conceptually the same as the specification of any somatic cell type: germ cells must activate a specific gene regulatory network in order to differentiate and go through gametogenesis. While many genes with critical roles in the germ line have been characterized with respect to expression pattern and genetic interactions, it is the molecular interactions of the relevant gene products that are ultimately responsible for germ cell differentiation. This review summarizes the current state of knowledge on the molecular functions and biochemical connections between germ line gene products. We find that homologous genes often interact physically with the same conserved molecular partners across the metazoans. We also point out cases of nonhomologous genes from different species whose gene products play analogous biological roles in the germ line. We suggest a preliminary molecular definition of an ancestral "pluripotency module" that could have been modified during metazoan evolution to become specific to the germ line.

Background: In insects, the gap gene hunchback (hb) is required for the formation of a set of adjacent segments through the regulation of downstream target genes of the pair rule and segment-polarity classes. In addition, hb is a major regulator of Hox genes and it has been suggested that this is the ancestral role of hb in insects or perhaps even arthropods. To date, however, hb function has been analyzed only in insects., Results: Here we show that hb acts as a segmentation gene during anterior patterning of a noninsect arthropod, the spider Achaearanea tepidariorum. The leg-bearing segments L1, L2, and L4 are missing after downregulation of At-hb via RNAi. At-hb is required for the correct organization of target genes in this region of the embryo, suggesting that At-hb acts as a gap gene in the spider. In contrast to insects, hb does not control Hox gene expression in the spider. Furthermore, analysis of twist expression in At-hb knockdown embryos demonstrates that hb is not required for initiating the segmental organization of the mesoderm in the affected region, but only for its maintenance., Conclusions: Our findings suggest that hb might have had a segmentation gene function in the arthropod ancestor and contradicts the suggestion that the control of Hox genes is the ancestral role of hb. Anterior spider segmentation thus utilizes a Drosophila-like genetic mode, whereas a vertebrate-like mechanism involving Wnt8 and Notch/Delta signaling is used to pattern posterior segments. These data support the hypothesis that short-germ arthropods employ two distinct mechanisms to segment their anterior and posterior body parts.

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.

A number of recent studies have investigated posterior development in several different arthropods. As previously found in spiders, it has been discovered that Delta-Notch signaling is required for the development of posterior segments in an insect, the cockroach Periplaneta americana. Furthermore analysis of Wnt8 function in the spider Achaearanea tepidariorum and the beetle Tribolium castaneum demonstrates that this Wnt ligand is required for the establishment of the growth zone and development of posterior segments in both these arthropods. Taken together these studies provide an interesting insight into the architecture of the genetic network that regulated posterior development in the common ancestor of the arthropods.

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.

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., ((c) 2008 Wiley Periodicals, Inc.)

Background: Hox genes are expressed in specific domains along the anterior posterior body axis and define the regional identity. In most animals these genes are organized in a single cluster in the genome and the order of the genes in the cluster is correlated with the anterior to posterior expression of the genes in the embryo. The conserved order of the various Hox gene orthologs in the cluster among most bilaterians implies that such a Hox cluster was present in their last common ancestor. Vertebrates are the only metazoans so far that have been shown to contain duplicated Hox clusters, while all other bilaterians seem to possess only a single cluster., Results: We here show that at least three Hox genes of the spider Cupiennius salei are present as two copies in this spider. In addition to the previously described duplicated Ultrabithorax gene, we here present sequence and expression data of a second Deformed gene, and of two Sex comb reduced genes. In addition, we describe the sequence and expression of the Cupiennius proboscipedia gene. The spider Cupiennius salei is the first chelicerate for which orthologs of all ten classes of arthropod Hox genes have been described. The posterior expression boundary of all anterior Hox genes is at the tagma border of the prosoma and opisthosoma, while the posterior boundary of the posterior Hox genes is at the posterior end of the embryo., Conclusion: The presence of at least three duplicated Hox genes points to a major duplication event in the lineage to this spider, perhaps even of the complete Hox cluster as has taken place in the lineage to the vertebrates. The combined data of all Cupiennius Hox genes reveal the existence of two distinct posterior expression boundaries that correspond to morphological tagmata boundaries.