Regulation of sclerocyte differentiation in the hydroxyurea treated sponge Ephydatia fluviatilis

Abstract. Freshwater sponges ( Ephydatia fluviatilis ) were raised in mineral medium containing hydroxyurea (HU) at a final concentration of 100 μg/ml. The spicules present in these sponges were counted daily. In HU-treated sponges, the regulation mechanisms of skeletogenesis remained functional despite the absence of an aquiferous system. Indeed, as in controls, the differentiation of sclerocytes from stem cells ceased when a critical number of spicules had been secreted. Stem cells again started to differentiate into sclerocytes when isolated from sponges that had completed their skeletogenesis. The number of spicules secreted was found to be an inverse function of silicate concentration. These results demonstrate that the regulation of skeletogenesis is not dependent on the differentiation of the aquiferous system.     

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.     

Oscarella lobularis (Homoscleromorpha,Porifera) Regeneration: Epithelial Morphogenesis and Metaplasia

Sponges are known to possess remarkable reconstitutive and regenerative abilities ranging from common wounding or body part regeneration to more impressive re-building of a functional body from dissociated cells. Among the four sponge classes, Homoscleromorpha is notably the only sponge group to possess morphologically distinct basement membrane and specialized cell-junctions, and is therefore considered to possess true epithelia. The consequence of this peculiar organization is the predominance of epithelial morphogenesis during ontogenesis of these sponges. In this work we reveal the underlying cellular mechanisms used during morphogenesis accompanying ectosome regeneration in the homoscleromorph sponge model: Oscarella lobularis. We identified three main sources of novel exopinacoderm during the processes of its regeneration and the restoration of functional peripheral parts of the aquiferous system in O. lobularis: (1) intact exopinacoderm surrounding the wound surface, (2) the endopinacoderm from peripheral exhalant and inhalant canals, and (3) the intact choanoderm found on the wound surface. The basic morphogenetic processes during regeneration are the spreading and fusion of epithelial sheets that merge into one continuous epithelium. Transdifferentiation of choanocytes into exopinacocytes is also present. Epithelial-mesenchymal transition is absent during regeneration. Moreover, we cannot reveal any other morphologically distinct pluripotent cells. In Oscarella, neither blastema formation nor local dedifferentiation and proliferation have been detected, which is probably due to the high morphogenetic plasticity of the tissue. Regeneration in O. lobularis goes through cell transdifferentiation and through the processes, when lost body parts are replaced by the remodeling of the remaining tissue. Morphogenesis during ectosome regeneration in O. lobularis is correlated with its true epithelial organization. Knowledge of the morphological basis of morphogenesis during Oscarella regeneration could have important implications for our understanding of the diversity and evolution of regeneration mechanisms in metazoans, and is a strong basis for future investigations with molecular-biological approaches.     

Phosphorylation-dependent regulation of skeletogenesis in sea urchin micromere-derived cells and embryos

Sea urchin embryo micromeres when isolated and cultured in vitro differentiate to produce spicules. Although several authors have used this model, almost nothing is known about the signaling pathways responsible for initiating skeletogenesis. In order to investigate the potential involvement of phosphorylation events in spiculogenesis, the effect of inhibitors of protein kinases and phosphatases on skeleton formation was studied. Results obtained using both cultured micromeres and embryos revealed that protein tyrosine kinase and phosphatase inhibitors blocked skeleton formation, but not serine/threonine phosphatase inhibitors. The inhibitors showed a dose-dependent effect and when removed from micromere or embryo culture, spicule formation resumed. Inhibition of tyrosine phosphatases resulted in an increase in the tyrosine phosphorylation level of two major proteins and a modest decrease in the expression of the mRNA coding for type I fibrillar collagen. These findings strongly suggest that tyrosine phosphorylation and dephosphorylation is required for micromere differentiation and for normal skeletogenesis during sea urchin embryo development.     

Ultrastructural evidence of extruding exocytosis of residual bodies in the freshwater sponge Ephydatia fluviatilis

Exocytosis of residual bodies by choanocytes, archeocytes and endopinacocytes lining the aquiferous system of Ephydatia fluviatilis has been demonstrated using calibrated latex beads and Escherichia coli as tracers. In passing into the mesohyl or the lumen of the exhalant aquiferous canals, beads, and altered bacteria were enveloped by the plasma membrane of the cell containing them. The membrane constricted at a neck region to form extruding vacuoles. This process appeared first in choanocytes and later in other cell types. The occurrence of these buds increased with the length of incubation time, as did the number of particles they contained. Acid phosphatase activity was frequently associated with the particles budding from the cell membrane, confirming that this process followed digestive activity. Membranous vacuoles were recovered from the external medium and observed by TEM and those adhering to the substratum were seen by SEM. These observations proved that vacuoles were released from the sponges. This membrane-consuming mechanism of exoctyosis implies intense membrane replacement in the digestive cells of the sponge.     

Regeneration of amphioxus oral cirri and its skeletal rods: implications for the origin of the vertebrate skeleton

The oral cirri of amphioxus function as the first filter during feeding by eliminating unwanted large or noxious particulates. In this study, we were able to regenerate cirri following artificial amputation. This is the first firm observation of regeneration in amphioxus. Using this regeneration system, we studied skeletogenesis of the cellular skeleton of amphioxus oral cirri. During regeneration, the skeletal cells showed expression of fibrillar collagen and SoxE genes. These observations suggest that an evolutionarily conserved genetic regulatory system is involved in amphioxus cirrus and vertebrate cartilage skeletogenesis. In addition, Runx and SPARC/osteonectin expression were observed in regenerating cirral skeletal cells, indicating that cirral skeletogenesis is similar to vertebrate osteogenesis. We propose that the common ancestors of chordates possessed a genetic regulatory system that was the prototype of chondrogenesis and osteogenesis in vertebrates. Genome duplications caused divergence of this genetic regulatory system resulting in the emergence of cartilage and mineralized bone. The development of the vertebrate skeleton is an example of the functional segregation and subsequent recruitment of unique genetic materials that may account for the evolutionary diversification of novel cell types.     

A bioreaction–diffusion model for growth of marine sponge explants in bioreactors

Marine sponges are sources of high-value bioactives. Engineering aspects of in vitro culture of sponges from cuttings (explants) are poorly understood. This work develops a diffusion-controlled growth model for sponge explants. The model assumes that the explant growth is controlled by diffusive transport of at least some nutrients from the surrounding medium into the explant that generally has a poorly developed aquiferous system for internal irrigation during early stages of growth. Growth is assumed to obey Monod-type kinetics. The model is shown to satisfactorily explain the measured growth behavior of the marine sponge Crambe crambe in two different growth media. In addition, the model is generally consistent with published data for growth of explants of the sponges Disidea avara and Hemimycale columella. The model predicted that nutrient concentration profiles for nutrients, such as dissolved oxygen within the explant, are consistent with data published by independent researchers. In view of the proposed model’s ability to explain available data for growth of several species of sponge explants, diffusive transport does play a controlling role in explant growth at least until a fully developed aquiferous system has become established. According to the model and experimental observations, the instantaneous growth rate depends on the size of the explant and all those factors that influence the diffusion of critical nutrients within the explant. Growth follows a hyperbolic profile that is consistent with the Monod kinetics.     

Evolution of the bone gene regulatory network

Current fossil, embryological and genetic data shed light on the evolution of the gene regulatory network (GRN) governing bone formation. The key proteins and genes involved in skeletogenesis are well accepted. We discuss when these essential components of the GRN evolved and propose that the Runx genes, master regulators of skeletogenesis, functioned in early cartilages well before they were co-opted to function in the making of bone. Two rounds of whole genome duplication, together with additional tandem gene duplications, created a genetic substrate for segregation of one GRN into several networks regulating the related tissues of cartilage, bone, enamel, and dentin. During this segregation, Runx2 assumed its position at the top of the bone GRN, and Sox9 was excluded from bone, retaining its ancient role in cartilage.     

Nutrient utilisation by shallow water temperate sponges in New Zealand

Major nutrients such as phosphate, nitrate, ammonium and silicate, are involved in the metabolic processes of marine organisms. Sponges take up and produce inorganic nutrients and the extent at which they affect the budgets available for other organisms has received little attention. For this reason, we investigated nutrient fluxes for several sponge species in order to estimate whether sponges were net producers or consumers of nutrients from the water column, and how these patterns changed over time. Nutrient fluxes were examined on the south coast of Wellington, New Zealand. For the nutrient analysis (nitrate, nitrite, ammonium, phosphate and silicate), water samples were collected in situ from the inhalant and exhalant water of different sponge species. Samples were analysed both in a multi-species survey and over a two-year period for three other species to determine any temporal changes in fluxes. Our results yielded significant differences in nutrient concentrations between the inhalant and exhalant water for some of the species, but there was no clear pattern associated with the time of year. The levels of dissolved inorganic nutrients in the ambient water varied considerably over the 2-year study period. It is possible that a lack of a clear pattern of nutrient uptake/release of nutrients in some of the study species, and the fact that not all species showed significant uptake/release at different times of the year, may be related to high levels of temporal and spatial variation in the ambient nutrient availability, as well as other temporal fluctuations in parameters, such as water temperature, sponge size, and concentration of food in the water column. Finally, we believe that the activity of specific microbial communities associated with these sponges may be important in explaining the fluxes we have reported.     

Symbiosis of Mycale (Mycale) vansoesti sp. nov. (Porifera, Demospongiae) with a coralline alga from North Sulawesi (Indonesia)

Abstract. The symbiotic association between the new sponge species Mycale vansoesti and the coralline alga Amphiroa sp. from the Bunaken Marine Park (North Sulawesi, Indonesia) is described. The alga completely pervades the sponge. The color of the sponge ectosome is white, both on the external surface and on the atrial wall, but where the alga is present the sponge takes on the light pink color of the alga. The sponge spicular complement is characterized by mycalostyles, anisochelae of two types, sigmas (often "C" shaped), and extremely abundant toxas organized in bundles forming toxadragma. In the association, the sponge shows very low silicate value, and consequently the alga represents the main skeleton of the sponge. On the other hand, the sponge affects the morphology of the alga, leading to a cylindrical shape, with thalli running parallel to the sponge surface. This association seems to be obligate for the sponge, as we found no sponges of this species living in isolation.     

Commitment and response to inductive signals of primary mesenchyme cells of the sea urchin embryo

In the sea urchin embryo, primary mesenchyme cells (PMC) are committed to produce the larval skeleton, although their behavior and skeleton production are influenced by signals from the embryonic environment. Results from our recent studies showed that perturbation of skeleton development, by interfering with ectoderm-extracellular matrix (ECM) interactions, is linked to a reduction in the gene expression of a transforming growth factor (TGF)-beta growth factor, Pl-univin, suggesting a reduction in the blastocoelic amounts of the protein and its putative involvement in signaling events. In the present study, we examined PMC competence to respond to environmental signals in a validated skeleton perturbation model in Paracentrotus lividus. We found that injection of blastocoelic fluid (BcF), obtained from normal embryos, into the blastocoelic cavity of skeleton-defective embryos rescues skeleton development. In addition, PMC from skeleton-defective embryos transplanted into normal or PMC-less blastula embryos are able to position in correct regions of the blastocoel and to engage spicule elongation and patterning. Taken together, these results demonstrate that PMC commitment to direct skeletogenesis is maintained in skeleton perturbed embryos and confirm the role played by inductive signals in regulating skeleton growth and shape.     

A Novel Filtering Mutualism between a Sponge Host and Its Endosymbiotic Bivalves

Sponges, porous filter-feeding organisms consisting of vast canal systems, provide unique substrates for diverse symbiotic organisms. The Spongia (Spongia) sp. massive sponge is obligately inhabited by the host-specific endosymbiotic bivalve Vulsella vulsella, which benefits from this symbiosis by receiving protection from predators. However, whether the host sponge gains any benefit from this association is unclear. Considering that the bivalves exhale filtered water into the sponge body rather than the ambient environment, the sponge is hypothesized to utilize water exhaled by the bivalves to circulate water around its body more efficiently. We tested this hypothesis by observing the sponge aquiferous structure and comparing the pumping rates of sponges and bivalves. Observations of water currents and the sponge aquiferous structure revealed that the sponge had a unique canal system enabling it to inhale water exhaled from bivalves, indicating that the host sponge adapted morphologically to receive water from the bivalves. In addition, the volume of water circulating in the sponge body was dramatically increased by the water exhaled from bivalves. Therefore, this sponge-bivalve association can be regarded as a novel mutualism in which two filter-feeding symbionts promote mutual filtering rates. This symbiotic association should be called a “filtering mutualism”.     

Life habits and functional morphology of the sediment infaunal spongesOceanapia oleracea andOceanapia peltata (Porifera,Haplosclerida)

Summary In the Santa Marta area of the northern coast of Colombia two species of sponges have been found living within the sediment. The only connection these sponges have with the open water consists of a number of protruding, tubule-like siphons. Through field observations and aquarium experiments, the life habits and the function of the water current system ofOceanapia oleracea andO. peltata have been studied. As an adaptation to life embedded in sediment, both species possess inhalant siphons which draw water from above the sediment surface and duct it to the central body. The inhalant system shows a most unusual separation and concentration of inhalant pores at the tips of the inhalant ducts. The exhalant water leaves the sponge through separate ducts at the opposite side of the central body. Based on the observations onOceanapia, the water flow model forDisyringa proposed by Fry and Fry (1979) is reconsidered.     

Oxygen dynamics in choanosomal sponge explants

Oxygen microprofiles were measured over the boundary layer and into the tissue of 10-day-old cultivated tissue fragments (explants of 2-4 cm³) from the choanosome of the cold-water sponge Geodia barretti with oxygen-sensitive Clark-type microelectrodes. At this time of cultivation, the surface tissue and the aquiferous system of the explants is regenerating, which makes oxygen and nutrient supply by pumping activity impossible. Oxygen profiles showed a parabolic shape, indicating oxygen flux over a diffusive boundary layer and into the tissue. Oxygen was always depleted only 1 mm below the sponge surface, leaving the major part of the explants anoxic. Diffusive oxygen flux into the explant was calculated from three oxygen profiles using Fick's first law of diffusion and revealed 9 μmol O₂ cm^-3 day^-1, which is in the lower range of in situ oxygen consumption of whole sponges. The ability of G. barretti to handle continuous tissue anoxia enables choanosomal explants to survive the critical first weeks of cultivation without a functional aquiferous system, when oxygen is supplied to the sponge explant by molecular diffusion over its surface.     

Wnt signaling and induction in the sponge aquiferous system: evidence for an ancient origin of the organizer

The importance of polarity-the possession of a primary body axis-is evident in the functional features of animals, such as feeding, and therefore must have arisen simultaneously with the evolution of multicellular animal body plans. Sponges are thought to represent the most ancient extant lineage of multicellular animals and whereas adult sponges do not possess obvious polarity, they are useful study organisms in which to examine the origin and evolution of body polarity. We tested the effect of pharmacological agents known to disrupt the polarity of a wide variety of animals on sponge organization during development. Lithium chloride and alsterpaullone, which mimic canonical Wnt signaling in other animals, caused formation of ectopic oscula and disrupted the ability of the sponge to feed. Transplanted oscula were able to attach to and induce canal reorganization in host sponges suggesting that the osulum has inductive capabilities. This work suggests that canonical Wnt signaling is responsible for setting up the aquiferous system, which acts as an organizing center polarizing the sponge.     

Problems of Coloniality,Modularity, and Individuality in Sponges and Special Features of Their Morphogeneses During Growth and Asexual Reproduction

A comparative analysis of the organization of sponges has been carried out to clarify problems of their coloniality, individuality, and modularity. The morphological, physiological, morphogenetic, and immunological aspects of the problem have been analyzed. The followers of the hypothesis of colonial organization of sponges interpret the process of “new zooid” formation as an “incomplete asexual reproduction.” A comparative analysis of morphogeneses in sponges during growth processes and asexual reproduction has clearly shown them to be different. A rearrangement (remodeling) of structures accompanied by disorganization and reorganization of tissues in neighboring elements of aquiferous system is the basis of growth. Migration of polypotent and secretory cells into the core of bud development is the major mechanism of budding. The formation of new aquiferous units (aquiferous modules) does not represent an “incomplete asexual reproduction.” Thus, the terms “colony” and “zooid” cannot be applied to the sponges. A morphologically separate sponge, irrespective of its level of organization (ascon, sycon, or leucon) and the number of oscula (aquiferous modules) should be considered as an individual.     

P16 is an essential regulator of skeletogenesis in the sea urchin embryo

The primary mesenchyme cells (PMCs) of the sea urchin embryo undergo a dramatic sequence of morphogenetic behaviors that culminates in the formation of the larval endoskeleton. Recent studies have identified components of a gene regulatory network that underlies PMC specification and differentiation. In previous work, we identified novel gene products expressed specifically by PMCs (Illies, M.R., Peeler, M.T., Dechtiaruk, A.M., Ettensohn, C.A., 2002. Identification and developmental expression of new biomineralization proteins in the sea urchin, Strongylocentrotus purpuratus. Dev. Genes Evol. 212, 419-431). Here, we show that one of these gene products, P16, plays an essential role in skeletogenesis. P16 is not required for PMC specification, ingression, migration, or fusion, but is essential for skeletal rod elongation. We have compared the predicted sequences of P16 from two species and show that this small, acidic protein is highly conserved in both structure and function. The predicted amino acid sequence of P16 and the subcellular localization of a GFP-tagged form of the protein suggest that P16 is enriched in the plasma membrane. It may function to receive signals required for skeletogenesis or may play a more direct role in the deposition of biomineral. Finally, we place P16 downstream of Alx1 in the PMC gene network, thereby linking the network to a specific “effector” protein involved in biomineralization.     

Study of larval and adult skeletogenic cells in developing sea urchin larvae

The larval skeleton of sea urchin embryos is formed by primary mesenchyme cells (PMCs). Thereafter, the larvae start feeding and additional arms develop. An adult rudiment that contains spines, tube feet, tests, and other parts of the adult body is formed in the eight-armed larva. The cellular mechanism of the later skeletogenesis and the lineage of the adult skeletogenic cells are not known. In this study, the morphogenesis of larval and adult skeletons during larval development of the sea urchin Hemicentrotus pulcherrimus was investigated by immunostaining cells with PMC-specific monoclonal antibodies, which are useful markers of skeletogenic cells. All spicules and the associated cells in the later larvae were stained with the antibodies. We could observe the initiation of skeletal morphogenesis at each developmental stage and visualize the cellular basis of skeleton formation in whole-mount embryos that possessed an intact morphology. There were some similarities between PMCs and the later skeletogenic cells. Both had a rounded shape with some filopodia, and the antigen expression started just before overt spicule formation. In the later-stage embryos, cells with filopodia and faint antigen expression were observed migrating in the blastocoel or aggregating in the presumptive location of new skeletogenesis.