Acorn worms, also known as enteropneust (literally, ‘gut-breathing’) hemichordates, are marine invertebrates that share features with echinoderms and chordates. Together, these three phyla comprise the deuterostomes. Here we report the draft genome sequences of two acorn worms, Saccoglossus kowalevskii and Ptychodera flava. By comparing them with diverse bilaterian genomes, we identify shared traits that were probably inherited from the last common deuterostome ancestor, and then explore evolutionary trajectories leading from this ancestor to hemichordates, echinoderms and chordates. The hemichordate genomes exhibit extensive conserved synteny with amphioxus and other bilaterians, and deeply conserved non-coding sequences that are candidates for conserved gene-regulatory elements. Notably, hemichordates possess a deuterostome-specific genomic cluster of four ordered transcription factor genes, the expression of which is associated with the development of pharyngeal ‘gill’ slits, the foremost morphological innovation of early deuterostomes, and is probably central to their filter-feeding lifestyle. Comparative analysis reveals numerous deuterostome-specific gene novelties, including genes found in deuterostomes and marine microbes, but not other animals. The putative functions of these genes can be linked to physiological, metabolic and developmental specializations of the filter-feeding ancestor.

*Fibroblast growth factor (FGF) signalling regulates essential developmental processes in vertebrates and invertebrates, but its role during early metazoan evolution remains obscure. Here, we analyse the function of FGF signalling in a non-bilaterian animal, the sea anemone Nematostella vectensis. We identified the complete set of FGF ligands and FGF receptors, of which two paralogous FGFs (NvFGFa1 and NvFGFa2) and one FGF receptor (NvFGFRa) are specifically coexpressed in the developing apical organ, a sensory structure located at the aboral pole of ciliated larvae from various phyla. Morpholino-mediated knockdown experiments reveal that NvFGFa1 and NvFGFRa are required for the formation of the apical organ, whereas NvFGFa2 counteracts NvFGFRa signalling to prevent precocious and ectopic apical organ development. Marker gene expression analysis shows that FGF signalling regulates local patterning in the aboral region. Furthermore, NvFGFa1 activates its own expression and that of the antagonistic NvFGFa2, thereby establishing positive-and negative-feedback loops. Finally, we show that loss of the apical organ upon NvFGFa1 knockdown blocks metamorphosis into polyps. We propose that the control of the development of sensory structures at the apical pole of ciliated larvae is an ancestral function of FGF signalling.

A protocol was established to reproducibly induce spawning in the basal cnidarian Nematostella vectensis (Anthozoa). We found that a combination of feeding regime, dark-light cycle and temperature shift synergistically induced gametogenesis in adult polyps. Females lay between 100-600 eggs. This procedure led reproducibly to the production of thousands of eggs over the course of more than 1 year in weekly cycles. Gametes are released in a time window of about 2 h resulting in predictable and fairly synchronized development. We also present a method for in vitro fertilization allowing manipulation of early embryos. These methods as well as the simple culture conditions could provide important prerequisites for the use of Nematostella as a model system for the development of a basal Metazoa.

The winged helix transcription factor Forkhead and the zinc finger transcription factor Snail are crucially involved in germ layer formation in Bilateria. Here, we isolated and characterized a homolog of forkhead/HNF3 (FoxA/group 1) and of snail from a diploblast, the sea anemone Nematostella vectensis. We show that Nematostella forkhead expression starts during late Blastula stage in a ring of cells that demarcate the blastopore margin during early gastrulation, thereby marking the boundary between ectodermal and endodermal tissue. snail, by contrast, is expressed in a complementary pattern in the center of forkhead-expressing cells marking the presumptive endodermal cells fated to ingress during gastrulation. In a significant portion of early gastrulating embryos, forkhead is expressed asymmetrically around the blastopore. While snail-expressing cells form the endodermal cell mass, forkhead marks the pharynx anlage throughout embryonic and larval development. In the primary polyp, forkhead remains expressed in the pharynx. The detailed analysis of forkhead and snail expression during Nematostella embryonic and larval development further suggests that endoderm formation results from epithelial invagination, mesenchymal immigration, and reorganization of the endodermal epithelial layer, that is, by epithelial-mesenchymal transitions (EMT) in combination with extensive morphogenetic movements. snail also governs EMT at different processes during embryonic development in Bilateria. Our data indicate that the function of snail in Diploblasts is to regulate motility and cell adhesion, supporting that the triggering of changes in cell behavior is the ancestral role of snail in Metazoa.

We investigated the early development of the sea anemone Nematostella vectensis, an emerging model system of the Cnidaria. Early cleavage stages are characterized by substantial variability from embryo to embryo, yet invariably lead to the formation of a coeloblastula. The coeloblastula undergoes a series of unusual broad invaginations-evaginations which can be blocked by cell cycle inhibitors suggesting a causal link of the invagination cycles to the synchronized cell divisions. Blastula invagination cycles stop as cell divisions become asynchronous. Marking experiments show a clear correspondence of the animal-vegetal axis of the egg to the oral-aboral axis of the embryo. The animal pole gives rise to the concave side of the blastula and later to the blastopore of the gastrula, and hence the oral pole of the future polyp. Asymmetric distribution of granules in the unfertilized egg suggest an animal-vegetal asymmetry in the egg in addition to the localized position of the pronucleus. To determine whether this asymmetry reflects asymmetrically distributed determinants along the animal-vegetal axis, we carried out blastomere isolations and embryonic divisions at various stages. Our data strongly indicate that normal primary polyps develop only if cellular material from the animal hemisphere is included, whereas the vegetal hemisphere alone is incapable to differentiate an oral pole. Molecular marker analysis suggests that also the correct patterning of the aboral pole depends on signals from the oral half. This suggests that in Nematostella embryos the animal hemisphere contains organizing activity to form a normal polyp.

BackgroundThe Fox gene family is a large family of transcription factors that arose early in organismal evolution dating back to at least the common ancestor of metazoans and fungi. They are key components of many gene regulatory networks essential for embryonic development. Although much is known about the role of Fox genes during vertebrate development, comprehensive comparative studies outside vertebrates are sparse. We have characterized the Fox transcription factor gene family from the genome of the enteropneust hemichordate Saccoglossus kowalevskii, including phylogenetic analysis, genomic organization, and expression analysis during early development. Hemichordates are a sister group to echinoderms, closely related to chordates and are a key group for tracing the evolution of gene regulatory mechanisms likely to have been important in the diversification of the deuterostome phyla.ResultsOf the 22 Fox gene families that were likely present in the last common ancestor of all deuterostomes, S. kowalevskii has a single ortholog of each group except FoxH, which we were unable to detect, and FoxQ2, which has three paralogs. A phylogenetic analysis of the FoxQ2 family identified an ancestral duplication in the FoxQ2 lineage at the base of the bilaterians. The expression analyses of all 23 Fox genes of S. kowalevskii provide insights into the evolution of components of the regulatory networks for the development of pharyngeal gill slits (foxC, foxL1, and foxI), mesoderm patterning (foxD, foxF, foxG), hindgut development (foxD, foxI), cilia formation (foxJ1), and patterning of the embryonic apical territory (foxQ2).ConclusionsComparisons of our results with data from echinoderms, chordates, and other bilaterians help to develop hypotheses about the developmental roles of Fox genes that likely characterized ancestral deuterostomes and bilaterians, and more recent clade-specific innovations.

The Wnt family of secreted proteins has been proposed to play a conserved role in early specification of the bilaterian anteroposterior (A/P) axis. This hypothesis is based predominantly on data from vertebrate embryogenesis as well as planarian regeneration and homeostasis, indicating that canonical Wnt (cWnt) signaling endows cells with positional information along the A/P axis. Outside of these phyla, there is strong support for a conserved role of cWnt signaling in the repression of anterior fates, but little comparative support for a conserved role in promotion of posterior fates. We further test the hypothesis by investigating the role of cWnt signaling during early patterning along the A/P axis of the hemichordate Saccoglossus kowalevskii. We have cloned and investigated the expression of the complete Wnt ligand and Frizzled receptor complement of S. kowalevskii during early development along with many secreted Wnt modifiers. Eleven of the 13 Wnt ligands are ectodermally expressed in overlapping domains, predominantly in the posterior, and Wnt antagonists are localized predominantly to the anterior ectoderm in a pattern reminiscent of their distribution in vertebrate embryos. Overexpression and knockdown experiments, in combination with embryological manipulations, establish the importance of cWnt signaling for repression of anterior fates and activation of mid-axial ectodermal fates during the early development of S. kowalevskii. However, surprisingly, terminal posterior fates, defined by posterior Hox genes, are unresponsive to manipulation of cWnt levels during the early establishment of the A/P axis at late blastula and early gastrula. We establish experimental support for a conserved role of Wnt signaling in the early specification of the A/P axis during deuterostome body plan diversification, and further build support for an ancestral role of this pathway in early evolution of the bilaterian A/P axis. We find strong support for a role of PLOS Biology | https://doi.org/10.1371/journal.pbio

Hemichordate worms possess ciliated gills on their trunk, and the homology of these structures with the pharyngeal gill slits of chordates has long been a topic of debate in the fields of evolutionary biology and comparative anatomy. Here, we show conservation of transcription factor expression between the developing pharyngeal gill pores of the hemichordate Saccoglossus kowalevskii and the pharyngeal gill slit precursors (i.e. pharyngeal endodermal outpockets) of vertebrates. Transcription factors that are expressed in the pharyngeal endoderm, ectoderm and mesenchyme of vertebrates are expressed exclusively in the pharyngeal endoderm of S. kowalevskii. The pharyngeal arches and tongue bars of S. kowalevskii lack Tbx1-expressing mesoderm, and are supported solely by an acellular collagenous endoskeleton and by compartments of the trunk coelom. Our findings suggest that hemichordate and vertebrate gills are homologous as simple endodermal outpockets from the foregut, and that much vertebrate pharyngeal complexity arose coincident with the incorporation of cranial paraxial mesoderm and neural crest-derived mesenchyme within pharyngeal arches along the chordate and vertebrate stems, respectively.