In Situ Hybridization Techniques in the Homoscleromorph Sponge Oscarella lobularis.

The Porifera are one of the best candidates as the sister group to all other metazoans. Studies on this phylum are therefore expected to shed light on the origin and early evolution of key animal features. Transcriptomic or genomic data acquired during the last 10 years have highlighted the conservation of most of the main genes and pathways involved in the development of the other metazoans. The next step is to determine how similar genetic tool boxes can result in widely dissimilar body plan organization, dynamics, and life histories. To answer these questions, three main axes of research are necessary: (1) conducting more gene expression studies; (2) developing knockdown protocols; and (3) reinterpreting sponge cell biology using modern tools. In this chapter we focus on the in situ hybridization (ISH) technique, needed to establish the spatiotemporal expression of genes, both on whole mount individuals and paraffin sections, and at different stages of development (adults, embryos, larvae, buds) of the homoscleromorph sponge Oscarella lobularis.

Animal multicellularity and polarity without Wnt signaling.

Acquisition of multicellularity is a central event in the evolution of Eukaryota. Strikingly, animal multicellularity coincides with the emergence of three intercellular communication pathways - Notch, TGF-ß and Wnt - all considered as hallmarks of metazoan development. By investigating Oopsacas minuta and Aphrocallistes vastus, we show here that the emergence of a syncytium and plugged junctions in glass sponges coincides with the loss of essential components of the Wnt signaling (i.e. Wntless, Wnt ligands and Disheveled), whereas core components of the TGF-ß and Notch modules appear unaffected. This suggests that Wnt signaling is not essential for cell differentiation, polarity and morphogenesis in glass sponges. Beyond providing a comparative study of key developmental toolkits, we define here the first case of a metazoan phylum that maintained a level of complexity similar to its relatives despite molecular degeneration of Wnt pathways.

The Conservation of the Germline Multipotency Program, from Sponges to Vertebrates: A Stepping Stone to Understanding the Somatic and Germline Origins.

The germline definition in metazoans was first based on few bilaterian models. As a result, gene function interpretations were often based on phenotypes observed in those models and led to the definition of a set of genes, considered as specific of the germline, named the "germline core". However, some of these genes were shown to also be involved in somatic stem cells, thus leading to the notion of germline multipotency program (GMP). Because Porifera and Ctenophora are currently the best candidates to be the sister-group to all other animals, the comparative analysis of gene contents and functions between these phyla, Cnidaria and Bilateria is expected to provide clues on early animal evolution and on the links between somatic and germ lineages. Our present bioinformatic analyses at the metazoan scale show that a set of 18 GMP genes was already present in the last common ancestor of metazoans and indicate more precisely the evolution of some of them in the animal lineage. The expression patterns and levels of 11 of these genes in the homoscleromorph sponge Oscarella lobularis show that they are expressed throughout their life cycle, in pluri/multipotent progenitors, during gametogenesis, embryogenesis and during wound healing. This new study in a nonbilaterian species reinforces the hypothesis of an ancestral multipotency program.

Retracing the path of planar cell polarity.

BACKGROUND: The Planar Cell Polarity pathway (PCP) has been described as the main feature involved in patterning cell orientation in bilaterian tissues. Recently, a similar phenomenon was revealed in cnidarians, in which the inhibition of this pathway results in the absence of cilia orientation in larvae, consequently proving the functional conservation of PCP signaling between Cnidaria and Bilateria. Nevertheless, despite the growing accumulation of databases concerning basal lineages of metazoans, very few information concerning the existence of PCP components have been gathered outside of Bilateria and Cnidaria. Thus, the origin of this module or its prevalence in early emerging metazoans has yet to be elucidated. RESULTS: The present study addresses this question by investigating the genomes and transcriptomes from all poriferan lineages in addition to Trichoplax (Placozoa) and Mnemiopsis (Ctenophora) genomes for the presence of the core components of this pathway. Our results confirm that several PCP components are metazoan innovations. In addition, we show that all members of the PCP pathway, including a bona fide Strabismus ortholog (Van gogh), are retrieved only in one sponge lineage (Homoscleromorpha) out of four. This highly suggests that the full PCP pathway dates back at least to the emergence of homoscleromorph sponges. Consequently, several secondary gene losses would have occurred in the three other poriferan lineages including Amphimedon queenslandica (Demospongiae). Several proteins were not retrieved either in placozoans or ctenophores leading us to discuss the difficulties to predict orthologous proteins in basally branching animals. Finally, we reveal how the study of multigene families may be helpful to unravel the relationships at the base of the metazoan tree. CONCLUSION: The PCP pathway antedates the radiation of Porifera and may have arisen in the last common ancestor of animals. Oscarella species now appear as key organisms to understand the ancestral function of PCP signaling and its potential links with Wnt pathways.

ROCK inhibition abolishes the establishment of the aquiferous system in Ephydatia muelleri (Porifera, Demospongiae).

The Rho associated coiled-coil protein kinase (ROCK) plays crucial roles in development across bilaterian animals. The fact that the Rho/Rock pathway is required to initiate epithelial morphogenesis and thus to establish body plans in bilaterians makes this conserved signaling pathway key for studying the molecular mechanisms that may control early development of basally branching metazoans. The purpose of this study was to evaluate whether or not the main components of this signaling pathway exist in sponges, and if present, to investigate the possible role of the regulatory network in an early branching non-bilaterian species by evaluating ROCK function during Ephydatia muelleri development. Molecular phylogenetic analyses and protein domain predictions revealed the existence of Rho/Rock components in all studied poriferan lineages. Binding assays revealed that both Y-27632 and GSK429286A are capable of inhibiting Em-ROCK activity in vitro. Treatment with both drugs leads to impairment of growth and formation of the basal pinacoderm layer in the developing sponge. Furthermore, inhibition of Em-Rock prevents the establishment of a functional aquiferous system, including the absence of an osculum. In contrast, no effect of ROCK inhibition was observed in juvenile sponges that already possess a fully developed and functional aquiferous system. Thus, the Rho/Rock pathway appears to be essential for the proper development of the freshwater sponge, and may play a role in various cell behaviors (e.g. cell proliferation, cell adhesion and cell motility). Taken together, these data are consistent with an ancestral function of Rho/Rock signaling in playing roles in early developmental processes and may provide a new framework to study the interaction between Wnt signaling and the Rho/Rock pathway.

Insights into Frizzled evolution and new perspectives.

The Frizzled proteins (FZDs) are a family of trans-membrane receptors that play pivotal roles in Wnt pathways and thus in animal development. Based on evaluation of the Amphimedon queenslandica genome, it has been proposed that two Fzd genes may have been present before the split between demosponges and other animals. The major purpose of this study is to go deeper into the evolution of this family of proteins by evaluating an extended set of available data from bilaterians, cnidarians, and different basally branching animal lineages (Ctenophora, Placozoa, Porifera). The present study provides evidence that the last common ancestor of metazoans did possess two Fzd genes, and that the last common ancestor of cnidarians and bilaterians may have possessed four Fzd. Furthermore, amino acid analyses revealed an accurate diagnostic motif for these four FZD subfamilies facilitating the assignation of Frizzled paralogs to each subfamily. By highlighting conserved amino acids for each FZD subfamily, our study could also provide a framework for further research on the precise mechanisms that have driven FZD neo-functionalization.