Isabel M. Müller

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.

Co-expression and functional interaction of silicatein with galectin : Matrix-guided formation of siliceous spicules in the marine demosponge suberites domuncula

Sponges (phylum Porifera) of the class of Demospongiae are stabilized by a siliceous skeleton. It is composed of silica needles (spicules), which provide the morphogenetic scaffold of these metazoans. In the center of the spicules there is an axial filament that consists predominantly of silicatein, an enzyme that catalyzes the synthesis of biosilica. By differential display of transcripts we identified additional proteins involved in silica formation. Two genes were isolated from the marine demosponge Suberites domuncula; one codes for a galectin and the other for a fibrillar collagen. The galectin forms aggregates to which silicatein molecules bind. The extent of the silicatein-mediated silica formation strongly increased if associated with the galectin. By applying a new and mild extraction procedure that avoids hydrogen fluoride treatment, native axial filaments were extracted from spicules of S. domuncula. These filaments contained, in addition to silicatein, the galectin and a few other proteins. Immunogold electron microscopic studies underscored the role of these additional proteins, in particular that of galectin, in spiculogenesis. Galectin, in addition to silicatein, presumably forms in the axial canal as well as on the surface of the spicules an organized net-like matrix. In the extraspicular space most of these complexes are arranged concentrically around the spicules. Taken together, these additional proteins, working together with silicatein, may also be relevant for potential (nano)-biotechnological applications of silicatein in the formation of surface coatings. Finally, we propose a scheme that outlines the matrix (galectin/silicatein)-guided appositional growth of spicules through centripetal and centrifugal synthesis and deposition of biosilica.

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.

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.

Phylogenetic Position of the Hexactinellida Within the Phylum Porifera Based on the Amino Acid Sequence of the Protein Kinase C from Rhabdocalyptus dawsoni

Abstract. Recent analyses of genes encoding proteins typical for multicellularity, especially adhesion molecules and receptors, favor the conclusion that all metazoan phyla, including the phylum Porifera (sponges), are of monophyletic origin. However, none of these data includes cDNA encoding a protein from the sponge class Hexactinellida. We have now isolated and characterized the cDNA encoding a protein kinase C, belonging to the C subfamily (cPKC), from the hexactinellid sponge Rhabdocalyptus dawsoni. The two conserved regions, the regulatory part with the pseudosubstrate site, the two zinc fingers, and the C2 domain, as well as the catalytic domain were used for phylogenetic analyses. Sequence alignment and construction of a phylogenetic tree from the catalytic domains revealed that the yeast Saccharomyces cerevisiae and the protozoan Trypanosoma brucei are at the base of the tree, while the hexactinellid R. dawsoni branches off first among the metazoan sequences; the other two classes of the Porifera, the Calcarea (the sequence from Sycon raphanus was used) and the Demospongiae (sequences from Geodia cydonium and Suberites domuncula were used), branch off later. The statistically robust tree also shows that the two cPKC sequences from the higher invertebrates Drosophila melanogaster and Lytechinus pictus are most closely related to the calcareous sponge. This finding was also confirmed by comparing the regulatory part of the kinase gene. We suggest, that (i) within the phylum Porifera, the class Hexactinellida diverged first from a common ancestor to the Calcarea and the Demospongiae, which both appeared later, and (ii) the higher invertebrates are more closely related to the calcareous sponges.

Iron induces proliferation and morphogenesis in primmorphs from the marine sponge Suberites domuncula.

Dissociated cells from marine demosponges retain their proliferation capacity if they are allowed to form special aggregates, the primmorphs. On the basis of incorporation studies and septin gene expression, we show that Fe3+ ions are required for the proliferation of cells in primmorphs from Suberites domuncula. In parallel, Fe3+ induced the expression of ferritin and strongly stimulated the synthesis of spicules. This result is supported by the finding that the enzymatic activity of silicatein, converting organosilicon to silicic acid, depends on Fe3+. Moreover, the expression of a scavenger receptor molecule, possibly involved in the morphology of spicules, depends on the presence of Fe3+. We conclude that iron is an essential factor in proliferative and morphogenetic processes in primmorphs.

Origin of neuronal-like receptors in Metazoa: cloning of a metabotropic glutamate/GABA-like receptor from the marine sponge Geodia cydonium.

Abstract To date, no conclusive evidence has been presented for the existence of neuronal-like elements in Porifera (sponges). In the present study, isolated cells from the marine sponge Geodia cydonium are shown to react to the excitatory amino acid glutamate with an increase in the concentration of intracellular calcium[Ca2+]i. This effect can also be observed when the compounds L-quisqualic acid (L-QA) or L-(+)-2-amino-4-phosphonobutyric acid (L-AP-4) are used. The effect of L-QA and L-AP-4, both agonists for metabotropic glutamate receptors (mGluRs), can be abolished by the antagonist of group I mGluRs, (RS)-α-methyl-4-carboxyphenylglycine. These data suggest that sponge cells contain an mGluR-like protein. A cDNA encoding rat mGluR subtype 1 has been used to identify the complete nucleotide sequence of G. cydonium cDNA coding for a 528-amino-acid-long protein (59 kDa) that displays marked overall similarity to mGluRs and to γ-amino-butyric acid B receptors. The deduced sponge polypeptide, termed putative mGlu/GABA-like receptor, displays the highest similarity to the two families of metabotropic receptors within the transmembrane segment. The N-terminal part of the sponge sequence shows similarity to mGluR4 and mGluR5. These findings suggest that the earliest evolutionary metazoan phylum, the Porifera, possesses a sophisticated intercellular communication and signaling system, as seen in the neuronal network of higher Metazoa.

Emergence and disappearance of an immune molecule, an antimicrobial lectin, in basal metazoa: a tachylectin-related protein in the sponge suberites domuncula

Sponges (phylum Porifera) represent the evolutionarily oldest metazoans that comprise already a complex immune system and are related to the crown taxa of the protostomians and the deuterostomians. Here, we demonstrate the existence of a tachylectin-related protein in the demosponge Suberites domuncula, termed Suberites lectin. The MAPK pathway was activated in response to lipopolysaccharide treatment of the three-dimensional cell aggregates, the primmorphs; this process was abolished by the monosaccharide d-GlcNAc. The cDNA encoding the S. domuncula lectin was identified and cloned; it comprises 238 amino acids (26 kDa) in the open reading frame. The deduced protein has one potential transmembrane region, three characteristic Cys residues, and six internal tandem repeats; it shares the highest sequence similarity with lectins from the horseshoe crab Tachypleus trunculus. The steady-state level of expression of the Suberites lectin rises in primmorphs in response to lipopolysaccharide, an effect that was prevented by co-incubation with d-GlcNAc. The natural sponge lectin was purified by affinity chromatography; it has a size of 27 kDa and displays antibacterial activity against the Gram-negative bacteria Escherichia coli and the Gram-positive bacteria Staphylococcus aureus. The putative protein, deduced from the cloned gene, is identical/similar to the purified natural protein, as demonstrated by immunological cross-reactivity with specific antibodies. We conclude that the S. domuncula lectin acts as an antibacterial molecule involved in immune defense against bacterial invaders.

Origin of the metazoan immune system: identification of the molecules and their functions in sponges.

Abstract During the evolutionary transition to Metazoa, cell-cell- as well as cell-matrix recognition molecules have been formed, which made a further step in evolution possible, the establishment of an immune system. Sponges [Porifera] represent the oldest still extant metazoan phylum and consequently testify to major features of the common metazoan ancestor, the Urmetazoa. Most studies with respect to evolution and phylogeny in sponges have been performed with the marine demosponges Suberites domuncula and Geodia cydonium. These animals possess effective defense systems against microbes and parasites which involve engulfment of bacteria into specific cells, but also signal transduction pathways which actively kill bacteria. Among those is the LPS-mediated pathway, with the stress-responsive kinases. In addition, sponges are provided with an interferon-related system, with the (2–5)A synthetase as controlling enzyme. Transplantation studies have been performed on tissue, as well as at the cellular level (“mixed sponge cell reaction assay”) which demonstrate the complex molecular strategy by which sponges respond to allogeneic- and/or autogeneic signals. Among the molecules involved in histo(in)compatibility response of sponges, cytokines e.g., the allograft inflammatory factor 1, have been identified which control rejection of allografts. Furthermore, transcription factors, with Tcf-like factor as an example, have been identified which very likely control gene expression during histocompatibility reactions. The immune reactions in sponges can be modulated by FK506, a drug which has been successfully used as immunosuppressant in humans. One further surprising finding is the fact that G. cydonium has several molecules containing polymorphic Ig-like domains of the variable type. It is concluded that the successful evolutionary transition to the Metazoa, with the sponges as the oldest still extant phylum, and the subsequent rapid radiation into the other metazoan phyla, became possible because of the acquisition of modular molecules, involved in cell adhesion and the immune system.

Stimulation of protein (collagen) synthesis in sponge cells by a cardiac myotrophin-related molecule from Suberites domuncula

The body wall of sponges (Porifera), the lowest metazoan phylum, is formed by two epithelial cell layers of exopinacocytes and endopinacocytes, both of which are associated with collagen fibrils. Here we show that a myotrophinlike polypeptide from the sponge Suberites domuncula causes the expression of collagen in cells from the same sponge in vitro. The cDNA of the sponge myotrophin was isolated; the potential open reading frame of 360 nt encodes a 120 aa long protein (Mr of 12,837). The sequence SUBDOMYOL shares high similarity with the known metazoan myotrophin sequences. The expression of SUBDOMYOL is low in single cells but high after formation of primmorph aggregates as well as in intact animals. Recombinant myotrophin was found to stimulate protein synthesis by fivefold, as analyzed by incorporation studies using [3H] lysine. In addition, it is shown that after incubation of single cells with myotrophin, the primmorphs show an unusual elongated, oval‐shaped appearance. It is demonstrated that in the presence of recombinant myotrophin, the cells up‐regulate the expression of the collagen gene. The cDNA for S. domuncula collagen was isolated; the deduced aa sequence shows that the collagenous internal domain is rather short, with only 24 G‐x‐y collagen triplets. We conclude that the sponge myotrophin causes in homologous cells the same/similar effect as the cardiac myotrophin in mammalian cells, where it is involved in initiation of cardial ventricular hypertrophy. We assume that an understanding of sponge molecular cell biology will also contribute to a further elucidation of human diseases, here of the cardiovascular system.—Schröder, H. C., Krasko, A., Batel, R., Skorokhod, A., Pahler, S., Kruse, M., Müller, I. M., Müller, W. E. G. Stimulation of protein (collagen) synthesis in sponge cells by a cardiac myotrophin‐related molecule from Suberites domuncula. FASEB J. 14, 2022–2031 (2000)