Cultivation of primmorphs from the marine sponge Suberites domuncula: morphogenetic potential of silicon and iron

Marine demosponges (phylum Porifera) are rich sources for potent bioactive compounds. With the establishment of the primmorph system from sponges, especially from Suberites domuncula, the technology to cultivate sponge cells in vitro improved considerably. This progress was possible after the elucidation that sponges are provided with characteristic metazoan cell adhesion receptors and extracellular matrix molecules which allow their cells a positioning in a complex organization pattern. This review summarizes recent data on the cultivation of sponges in aquaria and--with main emphasis--of primmorphs in vitro. It is outlined that silicon and Fe(+++) contribute substantially to the formation of larger primmorphs (size of 10 mm) as well as of a canal system in primmorphs; canals are probably required for an improved oxygen and food supply. We conclude that the primmorph system will facilitate a sustainable use of sponges in the production of bioactive compounds; it may furthermore allow new and hitherto not feasible insights into basic questions on the origin of Metazoa.     

Primmorphs from archaeocytes-dominant cell population of the sponge hymeniacidon perleve: improved cell proliferation and spiculogenesis

Marine sponges (Porifera) possess an extraordinary diversity of bioactive metabolites for new drug discovery and development. In vitro cultivation of sponge cells in a bioreactor system is very attractive for the sustainable production of sponge-derived bioactive metabolites; however, it is still a challenging task. The recent establishment of sponge primmorphs, multicellular aggregates from dissociated mixed-cell population (MCP), has been widely acknowledged to hold great promise for cultivation in vitro. Here we present a new method to establish an in vitro sponge primmorph culture from archaeocyte-dominant cell population (ADCP) enriched by a Ficoll gradient, rather than a mixed-cell population (MCP). Our rationale is based upon the totipotency (the ability of a cell to differentiate into other cell types) of archaeocyte cells and the different biological functions of various sponge cell types. A sponge, Hymeniacidon perleve collected from the China Yellow Sea was used as a model system for this investigation. Distinct dynamics of primmorph formation were observed while significant increases in DNA synthesis, cell proliferation (up to threefold), and cell growth (up to fourfold) were achieved. Furthermore, a time-dependent spiculogenesis was clearly demonstrated in our longterm culture, indicating high metabolic activity of primmorphs from the ADCP. This new method represents an important step forward to advance sponge cell culture in vitro that may lead to commercial exploitation of sponge-derived drugs.     

Optimizing the formation of in vitro sponge primmorphs from the Chinese sponge Stylotella agminata (Ridley)

The establishment and optimization of in vitro primmorph formation from a Chinese sponge, Stylotella agminata (Ridley), collected from the South China Sea, were investigated. Our aims were to identify the key factors affecting primmorph formation in this species and to optimize the technique for developing an in vitro primmorph culture system. The size of dissociated cells from S. agminata is relatively small, in the range between 5 and 10 microm. Round-shaped primmorphs of less than 100 microm were formed 3 days after transferring the dissociated cells into seawater containing Ca(2+) and Mg(2+). The effect of various cell dissociation conditions, inoculum cell density, concentration of antibiotics, pH, and temperature was further investigated upon the formation of primmorphs. The time required for primmorph formation, primmorph size distribution, and the proliferating capability were microscopically documented. Healthy sponge S. agminata, inoculum cell density and culture temperature play a critical role for the successful formation of primmorphs and that the microbial contamination will have to be controlled.     

Expression of one sponge Iroquois homeobox gene in primmorphs from Suberites domuncula during canal formation

Sponges (Porifera) represent the evolutionary oldest multicellular animals. They are provided with the basic molecules involved in cell-cell and cell-matrix interactions. We report here the isolation and characterization of a complementary DNA from the sponge Suberites domuncula coding for the sponge homeobox gene, SUBDOIRX-a. The deduced polypeptide with a predicted Mr of 44,375 possesses the highly conserved Iroquois-homeodomain. We applied in situ hybridization to localize Iroquois in the sponge. The expression of this gene is highest in cells adjacent to the canals of the sponge in the medulla region. To study the expression of Iroquois during development, the in vitro primmorph system from S. domuncula was used. During the formation of these three-dimensional aggregates composed of proliferating cells, the expression of Iroquois depends on ferric iron and water current. An increased expression in response to water current is paralleled with the formation of canal-like pores in the primmorphs. It is suggested that Iroquois expression is involved in the formation of the aquiferous system, the canals in sponges and the canal-like structures in primmorphs.     

The choanoderm of Sycettusa hastifera (Calcarea,Porifera) is able to generate new individuals

One of the main characteristics of sponges is their capacity for cell dedifferentiation. This capability can allow an impressive amount of asexual reproduction in these animals, because they are able to develop new individuals from just a few somatic cells. Studies of dedifferentiation, however, have focused mainly on sponges of the class Demospongiae. Therefore, we investigated here whether individuals of three different species of Calcarea are able to reconstitute new individuals following artificial fragmentation. We observed that fragmentation releases clumps of choanoderm able to initiate somatic embryogenesis. In Borojevia brasiliensis (asconoid aquiferous system, subclass Calcinea) and Paraleucilla magna (leuconoid aquiferous system, subclass Calcaronea), these clumps started to develop, but they did not pass through the first developmental phases. In Sycettusa hastifera (syconoid aquiferous system, subclass Calcaronea), the choanoderm was reorganized into primmorphs that fused to each other and formed an exopinacoderm. The first primmorphs’ spicules were triactines. Despite a large mortality rate, the primmorphs developed into olynthus stages. The somatic embryogenesis and the metamorphosis of the olynthus were similar to those observed during the sexual development of this and other calcareous sponge species. Our results show that in S. hastifera, and perhaps in other syconoid calcareous sponges, somatic embryogenesis occurs mainly from choanocytes, at least in vitro. However, primmorph development does not follow the same pattern observed in post‐metamorphic sexual development, as in that case diactines are always the first spicules to be synthesized in calcaronean species.     

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 myotrophin-like 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.     

Sulfated polysaccharides from marine sponges: conspicuous distribution among different cell types and involvement on formation of in vitro cell aggregates

Marine sponges (Porifera) display an ancestral type of cell-cell adhesion, based on carbohydrate-carbohydrate interaction. The aim of the present work was to investigate further details of this adhesion by using, as a model, the in vitro aggregation of dissociated sponge cells. Our results showed the participation of sulfated polysaccharides in this cell-cell interaction, as based on the following observations: (1) a variety of sponge cells contained similar sulfated polysaccharides as surface-associated molecules and as intracellular inclusions; (2) ³⁵S-sulfate metabolic labeling of dissociated sponge cells revealed that the majority (two thirds) of the total sulfated polysaccharide occurred as a cell-surface-associated molecule; (3) the aggregation process of dissociated sponge cells demanded the active de novo synthesis of sulfated polysaccharides, which ceased as cell aggregation reached a plateau; (4) the typical well-organized aggregates of sponge cells, known as primmorphs, contained three cell types showing sulfated polysaccharides on their cell surface; (5) collagen fibrils were also produced by the primmorphs in order to fill the extracellular spaces of their inner portion and the external layer surrounding their entire surface. Our data have thus clarified the relevance of sulfated polysaccharides in this system of in vitro sponge cell aggregation. The molecular basis of this system has practical relevance, since the culture of sponge cells is necessary for the production of molecules with biotechnological applications.     

Bacterial Community Dynamics in the Marine Sponge <Emphasis Type="Italic">Rhopaloeides odorabile</Emphasis> Under In Situ and Ex Situ Cultivation

Cultivation of sponges is being explored to supply biomaterial for the pharmaceutical and cosmetics industries. This study assesses the impact of various cultivation methods on the microbial community within the sponge Rhopaloeides odorabile during: (1) in situ cultivation under natural environmental conditions, (2) ex situ cultivation in small flow-through aquaria and (3) ex situ cultivation in large mesocosm systems. Principal components analysis of denaturing gradient gel electrophoresis profiles indicated a stable microbial community in sponges cultured in situ (grown in the wild) and in sponges cultured ex situ in small flow-through aquaria over 12 weeks. In contrast, a shift in the microbial community was detected in sponges cultivated ex situ in large mesocosm aquaria for 12 months. This shift included (1) a loss of some stable microbial inhabitants, including members of the Poribacteria, Chloroflexi and Acidobacteria and (2) the addition of new microbes not detected in the wild sponges. Many of these acquired bacteria had highest similarity to known sponge-associated microbes, indicating that the sponge may be capable of actively selecting its microbial community. Alternatively, long-term ex situ cultivation may cause a shift in the dominant microbes that facilitates the growth of the more rare species. The microbial community composition varied between sponges cultivated in mesocosm aquaria with different nutrient concentrations and seawater chemistry, suggesting that these variables play a role in structuring the sponge-associated microbes. The high growth and symbiont stability in R. odorabile cultured in situ confirm that this is the preferred method of aquaculture for this species at this time.     

Sponge cell aggregation: checkpoints in development indicate a high level of organismal complexity

Studies of regeneration provide insight across many scales of animal biology from the processes of cellular communication to the ecology of whole populations. Sponges are highly regenerative animals, with studies showing adults can both recover large portions of their body after predation or damage due to storms, and even reform whole individuals, via an aggregation stage, from dissociated tissues. While sponges are clearly highly regenerative, few studies actually show dissociated cells forming functional individuals. As sponges often serve as model organisms for studying the development and function of traits in metazoans, determining the universality and mechanics of their regeneration potential is important. We tested the capacity of members of seven sponge species from temperate freshwater and marine environments, from a range of taxonomic positions, and with different habits, to form functional sponges after dissociation. Development to a functional sponge progressed through a series of checkpoints: the sorting of cells and removal of debris; adhesion to a substrate and differentiation of cells; organization of cells into tissues; and regionalization of tissues. Two of the seven species tested, Spongilla lacustris and Haliclona cf. permollis, progressed through all four checkpoints, while the remaining five species progressed to various levels of development before aggregates disintegrated. Our findings highlight three important conclusions: (1) The ability of aggregates to differentiate into functional sponges is not as widespread as previously thought; (2) The species‐specific ability of aggregates to develop to functional sponges appears to be an adaptive trait; and (3) The progression of development in aggregates through checkpoints, which in later development involves formation of tissues and regionalization of tissues, highlights the complexity of the sponge body plan and suggests fundamental rules in development shared across metazoans.     

Influence of rocky substrata on three-dimensional sponge cells model development

Many marine and freshwater organisms are rocky bottom dwellers, and the mineralogical composition of the substratum is known to potentially condition their biology and ecology. In this work, we propose the use of 3D sponge cellular aggregates, called primmorphs, as suitable models for a multidisciplinary study of the mechanisms which regulate the biological responses triggered by the contact with different inorganic substrata. In our experiments, primmorphs obtained from the marine sponge Petrosia ficiformis (Poiret, 1789) were reared on calcium carbonate or on quartzitic substrata, respectively, and their morphological development was described. In parallel, the quantitative expression levels of two genes, silicatein and heat shock protein 70 (HSP70), were evaluated. The first gene is strictly correlated to spiculogenesis and sponge growth, while the second is an important indicator of stress. The results achieved with this in vitro model clearly demonstrate that quartzitic substrata determine the increase of silicatein gene expression, a lower expression of HSP70 gene, and a remarkable difference in primmorphs morphology compared to the analogous samples grown on marble.     

Molecular Cloning of Silicatein Gene from Marine Sponge <Emphasis Type="Italic">Petrosia ficiformis</Emphasis> (Porifera,Demospongiae) and Development of Primmorphs as a Model for Biosilicification Studies

In some sponges peculiar proteins called silicateins catalyze silica polymerization in ordered structures, and their study is of high interest for possible biotechnological applications in the nanostructure industry. In this work we describe the isolation and the molecular characterization of silicatein from spicules of Petrosia ficiformis, a common Mediterranean sponge, and the development of a cellular model (primmorphs) suitable for in vitro studies of silicatein gene regulation. The spicule of P. ficiformis contains an axial filament composed of 2 insoluble proteins, of 30 and 23 kDa. The 23-kDa protein was characterized, and the full-length cDNA was cloned. The putative amino acid sequence has high homology with previously described silicateins from other sponge species and also is very similar to cathepsins, a cystein protease family. Finally, P. ficiformis primmorphs express the silicatein gene, suggesting that they should be a good model for biosilicification studies.     

Comparative studies on two potential methods for the biotechnological production of sponge biomass.

The production of marine sponge biomass is one of the main outstanding goals of marine biotechnology. Due to the increased number of sponge secondary metabolites of economical value the interest in sponge cultivation increased over the last years, too. Therefore, we examined cultivation properties of 11 Mediterranean sponge species. Two methodologies were tested: functional fragment culture and multicell reaggregate culture. The in vitro cultivation of sponge fragments without further dissociation and reaggregation is a method formerly not reported. Reaggregates and functional fragments are promising attempts for culture system development. A broad spectrum of reaggregation properties was found among the species tested. In three species multicell aggregate cultures could be maintained for several months: Petrosia ficiformis, Suberites domuncula and Acanthella acuta. Our results indicate that cellular aggregates or fragments of sponges can be valuable tools in the development of methods for biotechnological production of sponge biomass. Further focus on nutritional demands and the biochemical status of the cells in these kind of cellular associations are needed in order to obtain functional aggregates and fragments.     

Cultivation of Marine Sponges

There is increasing interest in biotechnological production of marine sponge biomass owing to the discovery of many commercially important secondary metabolites in this group of animals. In this article, different approaches to producing sponge biomass are reviewed, and several factors that possibly influence culture success are evaluated. In situ sponge aquacultures, based on old methods for producing commercial bath sponges, are still the easiest and least expensive way to obtain sponge biomass in bulk. However, success of cultivation with this method strongly depends on the unpredictable and often suboptimal natural environment. Hence, a better-defined production system would be desirable. Some progress has been made with culturing sponges in semicontrolled systems, but these still use unfiltered natural seawater. Cultivation of sponges under completely controlled conditions has remained a problem. When designing an in vitro cultivation method, it is important to determine both qualitatively and quantitatively the nutritional demands of the species that is to be cultured. An adequate supply of food seems to be the key to successful sponge culture. Recently, some progress has been made with sponge cell cultures. The advantage of cell cultures is that they are completely controlled and can easily be manipulated for optimal production of the target metabolites. However, this technique is still in its infancy: a continuous cell line has yet to be established. Axenic cultures of sponge aggregates (primmorphs) may provide an alternative to cell culture. Some sponge metabolites are, in fact, produced by endosymbiotic bacteria or algae that live in the sponge tissue. Only a few of these endosymbionts have been cultivated so far. The biotechnology for the production of sponge metabolites needs further development. Research efforts should be continued to enable commercial exploitation of this valuable natural resource in the near future. Received November 5, 1998; accepted June 20, 1999.     

Effects of dissolved oxygen levels on survival and growth in vitro of <Emphasis Type="Italic">Haliclona pigmentifera</Emphasis> (Demospongiae)

In vitro sponge cultures are considered as legitimate alternatives for utilizing marine sponges (Porifera) to yield bioactive molecules. Optimization of culture methodologies for enhancing sponge survival is in progress for the identification of the factors regulating sponge survival in vitro. Dissolved oxygen (DO) is an essential factor promoting sponge survival. However, the effects of variable DO levels on the in vitro survival responses of sponges are not fully understood. Hence, we have investigated the effects of variable DO levels on the survival of the marine sponge, Haliclona pigmentifera (Demospongiae), with no external nutritional supplementation in closed type incubator chambers. Our results indicate that, under hypoxic conditions (1.5-2.0 ppm DO), H. pigmentifera with intact ectodermal layers and subtle oscula show adherent growth for 42±3 days. Sponges with prominent oscula, foreign material, and damaged pinacoderm exhibit poor survival under similar conditions. Complete mortality occurs within 2 days under anoxia (<0.3 ppm DO), and survival for a few days has been observed at >4.0 ppm DO without adhesion. Cellular differences between the outer and inner zones and collagen-like extracellular matrix have been identified in adherent sponges. Based on the hypothesis that hypoxia-inducible factor1-α (HIF-1α) is a ubiquitous protein promoting hypoxic survival in animals, we have detected, by Western blot, a protein band corresponding to human HIF-1α-like protein from sponges exposed to hypoxia and to hypoxia-mimicking agents. We thus report, for the first time, adhesive growth and a protein band corresponding to human HIF-1α-like protein in sponges surviving hypoxia in vitro. This work was supported by the “Task Force Network Programme (CMM-004)” of the Council of Scientific and Industrial Research (CSIR), Government of India and by the Department of Ocean Development. CSIR is also acknowledged for providing a Senior Research Fellowship to V.G.G.     

Marine-Based Cultivation of <Emphasis Type="Italic">Diacarnus</Emphasis> Sponges and the Bacterial Community Composition of Wild and Maricultured Sponges and Their Larvae

Marine organisms including sponges (Porifera) contain many structurally diverse bioactive compounds, frequently in a low concentration that hampers their commercial production. Two solutions to this problem are: culturing sponge explants for harvesting the desired compound and cultivation of sponge-associated bacteria. These bacteria (often considered the source of the desired compounds) include the Actinobacteria, from which many novel drugs were developed. In a long-term experiment (lasting 767 days), we evaluated the culture amenability of the sponge Diacarnus erythraenus in a mariculture system, placed at 10- and 20-m depths. The growth and survival rates of sponge fragments were monitored. Wild and maricultured sponges from both depths and their larvae were sampled at different time intervals for denaturing gradient gel electrophoresis (DGGE) profiling of the bacterial community residing within them. 16S rRNA gene sequences of both cultured bacterial isolates and clone libraries of unculturable bacteria were composed and compared, focusing on Actinobacteria. Sponges from both depths did not differ significantly either in mean growth rates (percent weight change year⁻¹ ± S.E.) (64.5% ± 21% at 10 m and 79.3% ± 19.1% at 20 m) or in seasonal growth rates. Survival was also very similar (72% at 10 m and 70% at 20 m). There were 88 isolates identified from adults and 40 from their larvae. The isolates and clone libraries showed diverse bacterial communities. The DGGE profiles of wild and maricultured sponges differed only slightly, without a significant effect of depths or dates of sampling. This long-term experiment suggests that D. erythraenus probably remained healthy and indicates its mariculture suitability.     

Antiangiogenic, Antimicrobial, and Cytotoxic Potential of Sponge-Associated Bacteria

The bacteria associated with marine invertebrates are a rich source of bioactive metabolites. In the present study bacteria associated with the sponge Suberites domuncula and its primmorphs (3-dimensional aggregates containing proliferating cells) were isolated and cultured. These bacteria were extracted, and the extracts were assayed for antiangiogenic, hemolytic, antimicrobial, and cytotoxic activities. Our studies revealed that extract obtained from the bacterium (PB2) isolated from sponge primmorphs is a potent angiogenesis inhibitor. In the chick chorio-allantoic membrane (CAM) assay, it showed 50% activity at 5 μg ml⁻¹ and 100% activity at 10 and 20 μg ml⁻¹ concentrations. Extracts obtained from 5 bacterial strains isolated from sponge and its primmorphs showed hemolytic activity. The sponge-associated bacteria belonging to the α subdivision of Proteobacteria and the primmorph-associated bacterium identified as a possible novel Pseudomonas sp. displayed remarkable antimicrobial activity. It is important to note that these bacterial extracts were strongly active against multidrug-resistant clinical strains such as Staphylococcus aureus and Staphylococcus epidermidis, isolated from hospital patients. The bacterial extracts having antimicrobial activity also showed cytotoxicity against HeLa and PC12 cells. In summary, this investigation explores the importance of sponge-associated bacteria as a valuable resource for the discovery of novel bioactive molecules.     

Phylogenetic diversity of bacteria associated with the endemic freshwater sponge Lubomirskia baicalensis

In this study, for the first time the diversity of bacteria associated with the endemic freshwater sponge Lubomirskia baicalensis collected from the Sousern Basin of Lake Baikal was investigated employing cultivation-independent approaches. In total, 102 bacterial 16S rRNA clones were screened using restriction fragment length polymorphism (RFLP) and 30 were selected for sequencing. BLASTN and phylogenetic analysis based on near full length 16S rDNA sequences showed that 22 operational taxonomic units (OTUs) were clustered in six known phyla: Actinobacteria (8 OTUs), alpha-Proteobacteria (4 OTUs), beta-Proteobacteria (4 OTUs), Verrucomicrobia (4 OTUs), Nitrospiracea (1 OTU) and Bacteroidetes (1 OTU). Remarkably all phylotypes were affiliated to uncultured microorganisms, however, all alpha-Proteobacteria sequences were closely related to bacteria derived from the freshwater sponge Spongilla lacustris. Our results reveal a high diversity in the L. baicalensis bacterial community and provide an insight into microbial ecology and diversity within freshwater sponges inhabiting the ancient Lake Baikal ecosystem.     

Purification and in vitro cultivation of archaeocytes (stem cells) of the marine sponge <Emphasis Type="Italic">Hymeniacidon perleve</Emphasis> (Demospongiae)

Marine sponges (Porifera) are the best source of marine bioactive metabolites for drug discovery and development, although the sustainable production of most sponge-derived metabolites remains a difficult task. In vitro cultivation of sponge cells in bioreactors has been proposed as a promising technology. However, no continuous cell line has as yet been developed. Archaeocytes are considered to be toti/multipotent stem cells in sponges and, when purified, may allow the development of continuous sponge cell lines. As a prerequisite, we have developed a novel four-step protocol for the purification of archaeocytes from a marine sponge, Hymeniacidon perleve: (1) differential centrifugation to separate large sponge cells including archaeocytes; (2) selective agglomeration in low-Ca²⁺/Mg²⁺ artificial seawater in which living archaeocytes form small loose aggregates with some pinacocytes and collencytes; (3) differential adherence to remove anchorage-dependent pinacocytes, collencytes and other mesohyl cells; (4) Ficoll-Vrografin density gradient centrifugation to purify archaeocytes. The final purity of archaeocytes is greater than 80%. The proliferation potential of the archaeocytes has been demonstrated by high levels of BrdU incorporation, PCNA expression and telomerase activity. In 4-day primary cultures, the purified archaeocytes show a 2.5-fold increase in total cell number. This study opens an important avenue towards developing sponge cell cultures for the commercial exploitation of sponge-derived drugs. The authors are grateful for the financial support of the Chinese Academy of Sciences under the “100 Talent Project”, the “Innovation Fund” from the Dalian Institute of Chemical Physics, the “Hi-Tech Research and Development Program of China” (2001AA620404), and the European Commission (project: Silicon Biotechnology).     

Origin of metazoan stem cell system in sponges: first approach to establish the model (Suberites domuncula)

It is established that Porifera (sponges) represent the earliest phylum which branched off from the common ancestor of all multicellular animals, the Urmetazoa. In the present study, the hypothesis is tested if, during this transition, pluripotent stem cells were formed which are provided-similar to the totipotent cells (archaeocytes/germ cells)-with a self-renewal capacity. As a model system, primmorphs from the sponge Suberites domuncula were used. These 3D-cell aggregates were cultivated in medium (RPMI 1640/seawater) either lacking silicate and ferric iron or in medium which was supplemented with these 'morphogenetic' factors. As molecular markers for the potential existence of stem cells in primmorphs, two genes which encode proteins found in stem cells of higher metazoan species, were cloned from S. domuncula. First, the noggin gene, which is present in the Spemann organizer of amphibians and whose translation product acts during the formation of dorsal mesoderm derivatives. The second gene encodes the mesenchymal stem cell-like protein. Both cDNAs were used to study their expression in primmorphs in dependence on the incubation conditions. It was found that noggin expression is strongly upregulated in primmorphs kept in the presence of silicate and ferric iron, while the expression of the mesenchymal stem cell-like protein was downregulated. These data are discussed with respect to the existence of stem cells in sponges.