Matthias Wiens

Improved Phylogenomic Taxon Sampling Noticeably Affects Nonbilaterian Relationships

Despite expanding data sets and advances in phylogenomic methods, deep-level metazoan relationships remain highly controversial. Recent phylogenomic analyses depart from classical concepts in recovering ctenophores as the earliest branching metazoan taxon and propose a sister-group relationship between sponges and cnidarians (e.g., Dunn CW, Hejnol A, Matus DQ, et al. (18 co-authors). 2008. Broad phylogenomic sampling improves resolution of the animal tree of life. Nature 452:745–749). Here, we argue that these results are artifacts stemming from insufficient taxon sampling and long-branch attraction (LBA). By increasing taxon sampling from previously unsampled nonbilaterians and using an identical gene set to that reported by Dunn et al., we recover monophyletic Porifera as the sister group to all other Metazoa. This suggests that the basal position of the fast-evolving Ctenophora proposed by Dunn et al. was due to LBA and that broad taxon sampling is of fundamental importance to metazoan phylogenomic analyses. Additionally, saturation in the Dunn et al. character set is comparatively high, possibly contributing to the poor support for some nonbilaterian nodes.

Deep metazoan phylogeny: When different genes tell different stories

Molecular phylogenetic analyses have produced a plethora of controversial hypotheses regarding the patterns of diversification of non-bilaterian animals. To unravel the causes for the patterns of extreme inconsistencies at the base of the metazoan tree of life, we constructed a novel supermatrix containing 122 genes, enriched with non-bilaterian taxa. Comparative analyses of this supermatrix and its two non-overlapping multi-gene partitions (including ribosomal and non-ribosomal genes) revealed conflicting phylogenetic signals. We show that the levels of saturation and long branch attraction artifacts in the two partitions correlate with gene sampling. The ribosomal gene partition exhibits significantly lower saturation levels than the non-ribosomal one. Additional systematic errors derive from significant variations in amino acid substitution patterns among the metazoan lineages that violate the stationarity assumption of evolutionary models frequently used to reconstruct phylogenies. By modifying gene sampling and the taxonomic composition of the outgroup, we were able to construct three different yet well-supported phylogenies. These results show that the accuracy of phylogenetic inference may be substantially improved by selecting genes that evolve slowly across the Metazoa and applying more realistic substitution models. Additional sequence-independent genomic markers are also necessary to assess the validity of the phylogenetic hypotheses.

Bauplan of urmetazoa: basis for genetic complexity of metazoa.

Sponges were first grouped to the animal-plants or plant-animals then to the Zoophyta or Mesozoa and finally to the Parazoa. Only after the application of molecular biological techniques was it possible to place the Porifera monophyletically with the other metazoan phyla, justifying a unification of all multicellular animals to only one kingdom, the Metazoa. The first strong support came from the discovery that cell-cell and cell-matrix adhesion molecules that were cloned from sponges and were subsequently expressed share a high DNA sequence and protein function similarity with the corresponding molecules of other metazoans. Besides these evolutionary novelties for Metazoa, sponges also have morphogens and transcription factors in common with other metazoan phyla. Surprisingly, even those elements exist in Porifera, which are characteristic for pattern and axis formation. Recent studies showed that epithelial layers of sponges are sealed against the extracellular milieu through tight-junction proteins. The cell culture system from sponges, the primmorphs, was suitable for understanding morphogenetic events. Finally, stem cell marker genes were isolated, which underscored that sponge cells have the capacity to differentiate. In the relatively short period of time, approximately 200 million years, the basic pathways had to be established that allowed the transition for multicellular organisms to a colonial system through the formation of adhesion molecules; based on the development of a complex immune system and the apoptotic machinery of an integrated system, the Urmetazoa, which evolved approximately 800 million years ago, could be reached. Hence, the Bauplan of the hypothetical Urmetazoa can now be constructed according to genomic regulatory systems similar to those found in higher Metazoa. These data caused a paradigmatic change; the Porifera are complex and simple but by far not primitive.

The role of biosilica in the osteoprotegerin/RANKL ratio in human osteoblast-like cells

Earlier studies have demonstrated that biosilica, synthesized by the enzyme silicatein, induces hydroxyapatite formation in osteoblast-like SaOS-2 cells. Here we study the effect of biosilica on the expressions of osteoprotegerin [OPG] and the receptor activator for NF-kappaB ligand [RANKL] in the SaOS-2 cell model. We show that during growth of SaOS-2 cells on biosiliceous matrices hydroxyapatite formation is induced, while syntheses of cartilaginous proteoglycans and sulfated glycosaminoglycans are down-regulated. Furthermore, quantitative real-time RT-PCR analysis revealed a strong time-depended increase in expression of OPG in biosilica exposed SaOS-2 cells while the steady-state expression level of RANKL remained unchanged. These results have been corroborated on the protein level by ELISA assays. Therefore, we propose that biosilica stimulated OPG synthesis in osteoblast-like cells counteracts those pathways that control RANKL expression and function (e.g. maturation of pre-osteoclasts and activation of osteoclasts). Hence, the data obtained in the present study reveal the considerable biomedical potential of biosilica for treatment and prophylaxis of osteoporotic disorders.

Innate Immune Defense of the Sponge Suberites domuncula against Bacteria Involves a MyD88-dependent Signaling Pathway INDUCTION OF A PERFORIN-LIKE MOLECULE

Sponges (phylum Porifera) are the phylogenetically oldest metazoa; as filter feeders, they are abundantly exposed to marine microorganisms. Here we present data indicating that the demosponge Suberites domuncula is provided with a recognition system for Gram-negative bacteria. The lipopolysaccharide (LPS)-interacting protein was identified as a receptor on the sponge cell surface, which recognizes the bacterial endotoxin LPS. The cDNA was isolated, and the protein (Mr 49,937) was expressed. During binding to LPS, the protein dimerizes and interacts with MyD88, which was also identified and cloned. The sponge MyD88 (Mr 28,441) is composed of two protein interaction domains, a Toll/interleukin-1 receptor domain (found in MyD88 and in Toll-like receptors) and a death domain (present in MyD88 and interleukin-1 receptor-associated kinase). Northern blot experiments and in situ hybridization studies showed that after LPS treatment, the level of the LPS-interacting protein remains unchanged, whereas MyD88 is strongly up-regulated. A perforin-like molecule (Mr 74,171), the macrophage-expressed protein, was identified as an executing molecule of this pathway. This gene is highly expressed after LPS treatment, especially at the surfaces of the animals. The recombinant protein possesses biological activity and eliminates Gram-negative bacteria; it is inactive against Gram-positive bacteria. These data indicate that S. domuncula is provided with an innate immune system against Gram-negative bacteria; the ligand LPS (a pathogen-associated molecular pattern) is recognized by the pattern recognition receptor (LPS-interacting protein), which interacts with MyD88. A signal transduction is established, which results in an elevated expression of MyD88 as well as of the macrophage-expressed protein as an executing protein.

Sponge spicules as blueprints for the biofabrication of inorganic–organic composites and biomaterials

While most forms of multicellular life have developed a calcium-based skeleton, a few specialized organisms complement their body plan with silica. However, of all recent animals, only sponges (phylum Porifera) are able to polymerize silica enzymatically mediated in order to generate massive siliceous skeletal elements (spicules) during a unique reaction, at ambient temperature and pressure. During this biomineralization process (i.e., biosilicification) hydrated, amorphous silica is deposited within highly specialized sponge cells, ultimately resulting in structures that range in size from micrometers to meters. Spicules lend structural stability to the sponge body, deter predators, and transmit light similar to optic fibers. This peculiar phenomenon has been comprehensively studied in recent years and in several approaches, the molecular background was explored to create tools that might be employed for novel bioinspired biotechnological and biomedical applications. Thus, it was discovered that spiculogenesis is mediated by the enzyme silicatein and starts intracellularly. The resulting silica nanoparticles fuse and subsequently form concentric lamellar layers around a central protein filament, consisting of silicatein and the scaffold protein silintaphin-1. Once the growing spicule is extruded into the extracellular space, it obtains final size and shape. Again, this process is mediated by silicatein and silintaphin-1, in combination with other molecules such as galectin and collagen. The molecular toolbox generated so far allows the fabrication of novel micro- and nanostructured composites, contributing to the economical and sustainable synthesis of biomaterials with unique characteristics. In this context, first bioinspired approaches implement recombinant silicatein and silintaphin-1 for applications in the field of biomedicine (biosilica-mediated regeneration of tooth and bone defects) or micro-optics (in vitro synthesis of light waveguides) with promising results.

Inorganic polymeric phosphate/polyphosphate as an inducer of alkaline phosphatase and a modulator of intracellular Ca2+ level in osteoblasts (SaOS-2 cells) in vitro

Inorganic polymeric phosphate is a physiological polymer that accumulates in bone cells. In the present study osteoblast-like SaOS-2 cells were exposed to this polymer, complexed in a 2:1 stoichiometric ratio with Ca(2+), polyP (Ca(2+) salt). At a concentration of 100 μM, polyP (Ca(2+) salt) caused a strong increase in the activity of the alkaline phosphatase and also an induction of the steady-state expression of the gene encoding this enzyme. Comparative experiments showed that polyP (Ca(2+) salt) can efficiently replace β-glycerophosphate in the in vitro hydroxyapatite (HA) biomineralization assay. In the presence of polyP (Ca(2+) salt) the cells extensively form HA crystallites, which remain intimately associated with or covered by the plasma membrane. Only the tips of the crystallites are directly exposed to the extracellular space. Element mapping by scanning electron microscopy/energy-dispersive X-ray spectroscopy coupled to a silicon drift detector supported the finding that organic material was dispersed within the crystallites. Finally, polyP (Ca(2+) salt) was found to cause an increase in the intracellular Ca(2+) level, while polyP, as well as inorganic phosphate (P(i)) or Ca(2+) alone, had no effect at the concentrations used. These findings are compatible with the assumption that polyP (Ca(2+) salt) is locally, on the surface of the SaOS-2 cells, hydrolyzed to P(i) and Ca(2+). We conclude that the inorganic polymer polyP (Ca(2+) salt) in concert with a second inorganic, and physiologically occurring, polymer, biosilica, activates osteoblasts and impairs the maturation of osteoclasts.

Silica transport in the demosponge Suberites domuncula: fluorescence emission analysis using the PDMPO probe and cloning of a potential transporter

Silicon is, besides oxygen, the most abundant element on earth. Only two taxa use this element as a major constituent of their skeleton, namely sponges (phylum Porifera) and unicellular diatoms. Results from combined cytobiological and molecularbiological techniques suggest that, in the demosponge Suberites domuncula, silicic acid is taken up by a transporter. Incubation of cells with the fluorescent silica tracer PDMPO [2-(4-pyridyl)-5-[[4-(2-dimethylaminoethylaminocarbamoyl)methoxy]phenyl]-oxazole] showed a response to silicic acid by an increase in fluorescence; this process is temperature-dependent and can be blocked by DIDS (4,4-di-isothiocyanatostilbene-2,2-disulphonic acid). The putative NBC (Na+/HCO3-) transporter was identified, cloned and analysed. The deduced protein comprises all signatures characteristic of those molecules, and phylogenetic analysis also classifies it to the NBC transporter family. This cDNA was used to demonstrate that the expression of the gene is strongly up-regulated after treatment of cells with silicic acid. In situ hybridization demonstrated that the expression of the sponge transporter occurs in those cells that are located adjacent to the spicules (the skeletal element of the animal) or in areas in which spicule formation occurs. We conclude that this transporter is involved in silica uptake and have therefore termed it the NBCSA [Na+/HCO3-[Si(OH)4]] co-transporter.

Retinoid X receptor and retinoic acid response in the marine sponge Suberites domuncula.

SUMMARY To date no nuclear receptors have been identified or cloned from the phylogenetically oldest metazoan phylum, the Porifera (sponges). We show that retinoic acid causes tissue regression in intact individuals of the demosponge Suberites domuncula and in primmorphs, special three-dimensional cell aggregates. Primmorphs were cultivated on a galectin/poly-L-lysine matrix in order to induce canal formation. In the presence of 1 or 50 μmol l–1 retinoic acid these canals undergo regression, a process that is reversible. We also cloned the cDNA from S. domuncula encoding the retinoid X receptor (RXR), which displays the two motifs of nuclear hormone receptors, the ligand-binding and the DNA-binding domains, and performed phylogenetic analyses of this receptor. RXR expression undergoes strong upregulation in response to treatment with retinoic acid, whereas the expression of the sponge caspase is not increased. The gene encoding the LIM homeodomain protein was found to be strongly upregulated in response to retinoic acid treatment. These data indicate that the RXR and its ligand retinoic acid play a role in the control of morphogenetic events in sponges.

Molecular evolution of apoptotic pathways: cloning of key domains from sponges (Bcl-2 homology domains and death domains) and their phylogenetic relationships.

Abstract. Cells from metazoan organisms are eliminated in a variety of physiological and pathophysiological processes by apoptosis. In this report, we describe the cloning and characterization of molecules from the marine sponges Geodia cydonium and Suberites domuncula, whose domains show a high similarity to those that are found in molecules of the vertebrate Bcl-2 superfamily and of the death receptors. The Bcl-2 proteins contain up to four Bcl-2 homology regions (BH). Two Bcl-2-related molecules have been identified from sponges that are provided with two of those regions, BH1 and BH2, and are termed Bcl-2 homology proteins (BHP). The G. cydonium molecule, BHP1_GC, has a putative size of 28,164, while the related sequence from S. domuncula, BHP1_SD, has a Mr of 24,187. Phylogenetic analyses of the entire two sponge BHPs revealed a high similarity to members of the mammalian Bcl-2 superfamilies and to the Caenorhabditis elegans Ced-9. When the two domains, BH1 and BH2, are analyzed separately, again the highest similarity was found to the members of the Bcl-2 superfamily, but a clearly lower relationship to the C. elegans BH1 and BH2 domains in Ced-9. In unrooted phylogenetic trees the sponge BH1 and BH2 are grouped among the mammalian sequences and are only distantly related to the C. elegans BH domains. The analysis of the gene structure of the G. cydonium BHP showed that the single intron present is located within the BH2 domain at the same position as in C. elegans and rat Bcl-xL. In addition, a sponge molecule comprising two death domains has been characterized from G. cydonium. The two death domains of the potential proapoptotic molecule GC_DD2, Mr 24,970, share a high similarity with the Fas-FADD/MORT1 domains. A death domain-containing molecule has not been identified in the C. elegans genome. The phylogenetic analysis revealed that the sponge domain originated from an ankyrin building block from which the mammalian Fas-FADD/MORT1 evolved. It is suggested that the apoptotic pathways that involve members of the Bcl-2 superfamily and of the death receptors are already present in the lowest metazoan phylum, the Porifera.