Wnt-signalling plays a critical role in animal development, and its misregulation results in serious human diseases, including cancer. While the Wnt pathway is well studied in eumetazoan models, little is known about the evolutionary origin of its components and their functions. Here, we have identified key machinery of the Wnt-β-catenin (canonical)-signalling pathway that is encoded in the Amphimedon queenslandica (Demospongiae; Porifera) genome, namely Wnt, Fzd, SFRP, Lrp5/6, Dvl, Axin, APC, GSK3, β-catenin, Tcf, and Groucho. Most of these genes are not detected in the choanoflagellate and other nonmetazoan eukaryotic genomes. In contrast, orthologues of some of key components of bilaterian Wnt-planar cell polarity and Wnt/Ca(2+) are absent from the Amphimedon genome, suggesting these pathways evolved after demosponge and eumetazoan lineages diverged. Sequence analysis of the identified proteins of the Wnt-β-catenin pathway has revealed the presence of most of the conserved motifs and domains responsible for protein-protein and protein-DNA interactions in vertebrates and insects. However, several protein-protein interaction domains appear to be absent from the Amphimedon Axin and APC proteins. These are also missing from their orthologues in the cnidarian Nematostella vectensis, suggesting that they are bilaterian novelties. All of the analyzed Wnt pathway genes are expressed in specific patterns during Amphimedon embryogenesis. Most are expressed in especially striking and highly dynamic patterns during formation of a simple organ-like larval structure, the pigment ring. Overall, our results indicate that the Wnt-β-catenin pathway was used in embryonic patterning in the last common ancestor of living metazoans. Subsequently, gene duplications and a possible increase in complexity of protein interactions have resulted in the precisely regulated Wnt pathway observed in extant bilaterian animals.

Recent interpretations of developmental gene expression patterns propose that the last common metazoan ancestor was segmented, although most animal phyla show no obvious signs of segmentation. Developmental studies of non-model system trochozoan taxa may shed light on this hypothesis by assessing possible cryptic segmentation patterns. In this paper, we present the first immunocytochemical data on the ontogeny of the nervous system and the musculature in the sipunculan Phascolion strombus. Myogenesis of the first anlagen of the body wall ring muscles occurs synchronously and not subsequently from anterior to posterior as in segmented spiralian taxa (i.e. annelids). The number of ring muscles remains constant during the initial stages of body axis elongation. In the anterior-posteriorly elongated larva, newly formed ring muscles originate along the entire body axis between existing myocytes, indicating that repeated muscle bands do not form from a posterior growth zone. During neurogenesis, the Phascolion larva expresses a non-metameric, paired, ventral nerve cord that fuses in the mid-body region in the late-stage elongated larva. Contrary to other trochozoans, Phascolion lacks any larval serotonergic structures. However, two to three FMRFamide-positive cells are found in the apical organ. In addition, late larvae show commissure-like neurones interconnecting the two ventral nerve cords, while early juveniles exhibit a third, medially placed FMRFamidergic ventral nerve. Although we did not find any indications for cryptic segmentation, certain neuro-developmental traits in Phascolion resemble the conditions found in polychaetes (including echiurans) and myzostomids and support a close relationship of Sipuncula and Annelida.

Gastropods are members of the Spiralia, a diverse group of invertebrates that share a common early developmental program, which includes spiral cleavage and a larval trochophore stage. The spiral cleavage program results in the division of the embryo into four quadrants. Specification of the dorsal (D) quadrant is intimately linked with body plan organization and in equally cleaving gastropods occurs when one of the vegetal macromeres makes contact with overlying micromeres and receives an inductive signal that activates a MAPK signaling cascade. Following the induction of the 3D macromere, the embryo begins to gastrulate and assumes a bilateral cleavage pattern. Here we inhibit MAPK activation in 3D with U0126 and examine its effect on the formation and patterning of the trochophore, using a suite of territory-specific markers. The head (pretrochal) region appears to maintain quadri-radial symmetry in U0126-treated embryos, supporting a role for MAPK signaling in 3D in establishing dorsoventral polarity in this region. Posterior (posttrochal) structures - larval musculature, shell and foot--fail to develop in MAPK inhibited trochophores. Inhibition of 3D specification by an alternative method--monensin treatment--yields similar abnormal trochophores. However, genes that are normally expressed in the ectodermal structures (shell and foot) are detected in U0126- and monensin-perturbed larvae in patterns that suggest that this region has latent dorsoventral polarity that is manifested even in the absence of D quadrant specification.

A role for Wnt/beta-catenin signaling in axial patterning has been demonstrated in animals as basal as cnidarians, while roles in axial patterning for retinoic acid (RA) probably evolved in the deuterostomes and may be chordate-specific. In vertebrates, these two pathways interact both directly and indirectly. To investigate the evolutionary origins of interactions between these two pathways, we manipulated Wnt/beta-catenin and RA signaling in the basal chordate amphioxus during the gastrula stage, which is the RA-sensitive period for anterior/posterior (A/P) patterning. The results show that Wnt/beta-catenin and RA signaling have distinctly different roles in patterning the A/P axis of the amphioxus gastrula. Wnt/beta-catenin specifies the identity of the ends of the embryo (high Wnt = posterior; low Wnt = anterior) but not intervening positions. Thus, upregulation of Wnt/beta-catenin signaling induces ectopic expression of posterior markers at the anterior tip of the embryo. In contrast, RA specifies position along the A/P axis, but not the identity of the ends of the embryo-increased RA signaling strongly affects the domains of Hox expression along the A/P axis but has little or no effect on the expression of either anterior or posterior markers. Although the two pathways may both influence such things as specification of neuronal identity, interactions between them in A/P patterning appear to be minimal.

Previous studies of vertebrate development have shown that retinoic acid (RA) signaling at the gastrula stage strongly influences anterior-posterior (A-P) patterning of the neurula and later stages. However, much less is known about the more immediate effects of RA signaling on gene transcription and developmental patterning at the gastrula stage. To investigate the targets of RA signaling during the gastrula stage, we used the basal chordate amphioxus, in which gastrulation involves very minimal tissue movements. First, we determined the effect of altered RA signaling on expression of 42 genes (encoding transcription factors and components of major signaling cascades) known to be expressed in restricted domains along the A-P axis during the gastrula and early neurula stage. Of these 42 genes, the expression domains during gastrulation of only four (Hox1, Hox3, HNF3-1 and Wnt3) were spatially altered by exposure of the embryos to excess RA or to the RA antagonist BMS009. Moreover, blocking protein synthesis with puromycin before adding RA or BMS009 showed that only three of these genes (Hox1, Hox3 and HNF3-1) are direct RA targets at the gastrula stage. From these results we conclude that in the amphioxus gastrula RA signaling primarily acts via regulation of Hox transcription to establish positional identities along the A-P axis and that Hox1, Hox3, HNF3-1 and Wnt3 constitute a basal module of RA action during chordate gastrulation.

In the basal chordate amphioxus (Branchiostoma), somites extend the full length of the body. The anteriormost somites segment during the gastrula and neurula stages from dorsolateral grooves of the archenteron. The remaining ones pinch off, one at a time, from the tail bud. These posterior somites appear to be homologous to those of vertebrates, even though the latter pinch off from the anterior end of bands of presomitic mesoderm rather than directly from the tail bud. To gain insights into the evolution of mesodermal segmentation in chordates, we determined the expression of ten genes in nascent amphioxus somites. Five (Uncx4.1, NeuroD/atonal-related, IrxA, Pcdhdelta2-17/18, and Hey1) are expressed in stripes in the dorsolateral mesoderm at the gastrula stage and in the tail bud while three (Paraxis, Lcx, and Axin) are expressed in the posterior mesendoderm at the gastrula and neurula stages and in the tail bud at later stages. Expression of two genes (Pbx and OligA) suggests roles in the anterior somites that may be unrelated to initial segmentation. Together with previous data, our results indicate that, with the exception that Engrailed is only segmentally expressed in the anterior somites, the genetic mechanisms controlling formation of both the anterior and posterior somites are probably largely identical. Thus, the fundamental pathways for mesodermal segmentation involving Notch-Delta, Wnt/beta-catenin, and Fgf signaling were already in place in the common ancestor of amphioxus and vertebrates although budding of somites from bands of presomitic mesoderm exhibiting waves of expression of Notch, Wnt, and Fgf target genes was likely a vertebrate novelty. Given the conservation of segmentation gene expression between amphioxus and vertebrate somites, we propose that the clock mechanism may have been established in the basal chordate, while the wavefront evolved later in the vertebrate lineage.

The development of gymnolaemate Ectoprocta includes a larval stage of either the coronate or the cyphonautes type. Herein, we provide the first description of the larval neural anatomy of a coronate larva using immunocytochemical methods. We used antibodies against the neurotransmitters serotonin and FMRFamide and followed the fate of immunoreactive cells through metamorphosis. The larval serotonergic nervous system of Triphyllozoon mucronatum consists of an apical commissure, one pair of lateral axons, a coronate nerve net, an internal nerve mesh, and one pair of axons innervating the frontal organ. FMRFamide is only found in the larval commissure and in the lateral axons. The entire serotonergic and FMRFamidergic nervous system is lost during metamorphosis and the adult neural structures form independent of the larval ones. In the postlarval zooid, both neurotransmitters are detected in the cerebral commissure, in cell bodies located at the base of the lophophore, and in neurites connecting these somata to the cerebral commissure. These findings differ significantly from that observed in other lophotrochozoans, where certain larval neural features are either incorporated in the adult nervous system and/or have inductive functions during its ontogeny. The occurrence of a larval commissure and the lack of a serotonergic or FMRFamidergic apical organ in T. mucronatum are unique among lophotrochozoan larvae, which usually have a distinct apical organ containing serotonergic cells. Our data show that the larval neuroanatomy and the processes that underlie the reorganization of larval organ systems during metamorphosis may vary much more among lophotrochozoan taxa than previously thought.

BackgroundThe molecular mechanisms underlying the development of the unusual echinoderm pentameral body plan and their likeness to mechanisms underlying the development of the bilateral plans of other deuterostomes are of interest in tracing body plan evolution. In this first study of the spatial expression of genes associated with Nodal and BMP2/4 signalling during the transition to pentamery in sea urchins, we investigate Heliocidaris erythrogramma, a species that provides access to the developing adult rudiment within days of fertilization.Results BMP2/4, and the putative downstream genes, Six1/2, Eya, Tbx2/3 and Msx were expressed in the earliest morphological manifestation of pentamery during development, the five hydrocoele lobes. The formation of the vestibular ectoderm, the specialized region overlying the left coelom that forms adult ectoderm, involved the expression of putative Nodal target genes Chordin, Gsc and BMP2/4 and putative BMP2/4 target genes Dlx, Msx and Tbx. The expression of Nodal, Lefty and Pitx2 in the right ectoderm, and Pitx2 in the right coelom, was as previously observed in other sea urchins.ConclusionThat genes associated with Nodal and BMP2/4 signalling are expressed in the hydrocoele lobes, indicates that they have a role in the developmental transition to pentamery, contributing to our understanding of how the most unusual body plan in the Bilateria may have evolved. We suggest that the Nodal and BMP2/4 signalling cascades might have been duplicated or split during the evolution to pentamery.Electronic supplementary materialThe online version of this article (doi:10.1186/s12861-017-0145-1) contains supplementary material, which is available to authorized users.