Evelyn E. Schwager

The molecular machinery of germ line specification.

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. Mol. Reprod. Dev. 77: 3–18, 2010.

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.

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.

Duplicated Hox genes in the spider Cupiennius salei

BackgroundHox 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.ResultsWe 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.ConclusionThe 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.

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 orthodenticlepRNAi 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.

An ancestral regulatory network for posterior development in arthropods

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

BackgroundThe 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.ResultsWe 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.ConclusionsOur 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.

Hox gene expression in the harvestman Phalangium opilio reveals divergent patterning of the chelicerate opisthosoma

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.

hunchback functions as a segmentation gene in the spider Achaearanea tepidariorum.

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.

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.