Silicatein expression in the hexactinellid Crateromorpha meyeri: the lead marker gene restricted to siliceous sponges

The siliceous spicules of sponges (Porifera) are synthesized by the enzyme silicatein. This protein and its gene have been identified so far in the Demospongiae, e.g., Tethya aurantium and Suberites domuncula. In the Hexactinellida, the second class of siliceous sponges, the mechanism of synthesis of the largest bio-silica structures on Earth remains obscure. Here, we describe the morphology of the spicules (diactines and stauractines) of the hexactinellid Crateromorpha meyeri. These spicules are composed of silica lamellae concentrically arranged around a central axial canal and contain proteinaceous sheaths (within the siliceous mantel) and proteinaceous axial filaments (within the axial canal). The major protein in the spicules is a 24-kDa protein that strongly reacts with anti-silicatein antibodies in Western blots. Its cDNA has been successfully cloned; the deduced hexactinellid silicatein comprises, in addition to the characteristic catalytic triad amino acids Ser-His-Asn and the "conventional" serine cluster, a "hexactinellid C. meyeri-specific" Ser cluster. We show that anti-silicatein antibodies react specifically with the proteinaceous matrix of the C. meyeri spicules. The characterization of silicatein at the genetic level should contribute to an understanding of the molecular/biochemical mechanism of spiculogenesis in Hexactinellida. These data also indicate that silicatein is an autapomorphic molecule common to both classes of siliceous sponges.     

Analysis of the axial filament in spicules of the demosponge Geodia cydonium: different silicatein composition in microscleres (asters) and megascleres (oxeas and triaenes)

The skeleton of the siliceous sponges (Porifera: Hexactinellida and Demospongiae) is supported by spicules composed of bio-silica. In the axial canals of megascleres, harboring the axial filaments, three isoforms of the enzyme silicatein (-alpha, -beta and -gamma) have been identified until now, using the demosponges Tethya aurantium and Suberites domuncula. Here we describe the composition of the proteinaceous components of the axial filament from small spicules, the microscleres, in the demosponge Geodia cydonium that possesses megascleres and microscleres. The morphology of the different spicule types is described. Also in G. cydonium the synthesis of the spicules starts intracellularly and they are subsequently extruded to the extracellular space. In contrast to the composition of the silicateins in the megascleres (isoforms: -alpha, -beta and -gamma), the axial filaments of the microscleres contain only one form of silicatein, termed silicatein-alpha/beta, with a size of 25kDa. Silicatein-alpha/beta undergoes three phosphorylation steps. The gene encoding silicatein-alpha/beta was identified and found to comprise the same characteristic sites, described previously for silicateins-alpha or -beta. It is hypothesized, that the different composition of the axial filaments, with respect to silicateins, contributes to the morphology of the different types of spicules.     

Intra-epithelial spicules in a homosclerophorid sponge

Attempts to understand the intricacies of biosilicification in sponges are hampered by difficulties in isolating and culturing their sclerocytes, which are specialized cells that wander at low density within the sponge body, and which are considered as being solely responsible for the secretion of siliceous skeletal structures (spicules). By investigating the homosclerophorid Corticium candelabrum, traditionally included in the class Demospongiae, we show that two abundant cell types of the epithelia (pinacocytes), in addition to sclerocytes, contain spicules intracellularly. The small size of these intracellular spicules, together with the ultrastructure of their silica layers, indicates that their silicification is unfinished and supports the idea that they are produced "in situ" by the epithelial cells rather than being incorporated from the intercellular mesohyl. The origin of small spicules that also occur (though rarely) within the nucleus of sclerocytes and the cytoplasm of choanocytes is more uncertain. Not only the location, but also the structure of spicules are unconventional in this sponge. Cross-sectioned spicules show a subcircular axial filament externally enveloped by a silica layer, followed by two concentric extra-axial organic layers, each being in turn surrounded by a silica ring. We interpret this structural pattern as the result of a distinctive three-step process, consisting of an initial (axial) silicification wave around the axial filament and two subsequent (extra-axial) silicification waves. These findings indicate that the cellular mechanisms of spicule production vary across sponges and reveal the need for a careful re-examination of the hitherto monophyletic state attributed to biosilicification within the phylum Porifera.     

深海六放海绵大骨针的结构与特性

在海绵动物(多孔动物)中,六放海绵和寻常海绵为硅质骨骼.生活在深海(1 000 m)中的六放海绵是最古老的海绵动物,其中间单根海绵和春氏单根海绵有长达3 m的骨针,是地球上最长的生物硅结构.利用电子显微技术观测, 这些直径达8 mm的巨大根须骨针具有同心层状结构,其横截面显示明显的构造分界:中间为含有轴丝的轴管,外围是一50-150 μm厚的轴筒,最外面为区状区(300-500层,每层厚度3-5 μm).生物化学研究显示其主要的蛋白质为35 kD大分子,另外,还检测到23-24 kD 多肽,可能是硅蛋白相关蛋白.依据现有的红血球凝聚活性,从骨针提取物中也检测到了凝集素.由电子探针获得其化学成分主要为Si,K和Na.此外,骨针的光传输实验表明,该巨大根须骨针用作光纤可传输600 nm至1 400 nm范围的光,而滤掉小于600 nm的光(类似高通滤波器)和大于1 400 nm 的红外光(类似低通滤波器).另外,从六放海绵的空囊泡沫海绵中分离出一个基因并确证了其推导的编码蛋白序列,该蛋白编码一个光裂合酶相关蛋白,蛋白相似性比较结果显示属于光裂合酶相关蛋白中多细胞动物隐色素一类.基于以上数据给出了六放海绵硅质骨针形成的示意图.另外,由单根海绵骨针可作为波导传输光/电和/或化学信号,推断在海绵动物中有类似神经系统的网络系统[动物学报 53(3):557-569,2007].     

Bioorganic/inorganic hybrid composition of sponge spicules: matrix of the giant spicules and of the comitalia of the deep sea hexactinellid Monorhaphis

The giant basal spicules of the siliceous sponges Monorhaphis chuni and Monorhaphis intermedia (Hexactinellida) represent the largest biosilica structures on earth (up to 3m long). Here we describe the construction (lamellar organization) of these spicules and of the comitalia and highlight their organic matrix in order to understand their mechanical properties. The spicules display three distinct regions built of biosilica: (i) the outer lamellar zone (radius: >300 microm), (ii) the bulky axial cylinder (radius: <75 microm), and (iii) the central axial canal (diameter: <2 microm) with its organic axial filament. The spicules are loosely covered with a collagen net which is regularly perforated by 7-10 microm large holes; the net can be silicified. The silica layers forming the lamellar zone are approximately 5 microm thick; the central axial cylinder appears to be composed of almost solid silica which becomes porous after etching with hydrofluoric acid (HF). Dissolution of a complete spicule discloses its complex structure with distinct lamellae in the outer zone (lamellar coating) and a more resistant central part (axial barrel). Rapidly after the release of the organic coating from the lamellar zone the protein layers disintegrate to form irregular clumps/aggregates. In contrast, the proteinaceous axial barrel, hidden in the siliceous axial cylinder, is set up by rope-like filaments. Biochemical analysis revealed that the (dominant) molecule of the lamellar coating is a 27-kDa protein which displays catalytic, proteolytic activity. High resolution electron microscopic analysis showed that this protein is arranged within the lamellae and stabilizes these surfaces by palisade-like pillars. The mechanical behavior of the spicules was analyzed by a 3-point bending assay, coupled with scanning electron microscopy. The load-extension curve of the spicule shows a biphasic breakage/cracking pattern. The outer lamellar zone cracks in several distinct steps showing high resistance in concert with comparably low elasticity, while the axial cylinder breaks with high elasticity and lower stiffness. The complex bioorganic/inorganic hybrid composition and structure of the Monorhaphis spicules might provide the blueprint for the synthesis of bio-inspired material, with unusual mechanical properties (strength, stiffness) without losing the exceptional properties of optical transmission.     

Novel photoreception system in sponges? Unique transmission properties of the stalk spicules from the hexactinellid Hyalonemasieboldi

Sponges (phylum Porifera) of the classes Hexactinellida and Demospongiae possess a skeleton composed of siliceous spicules, which are synthesized enzymatically. The longest spicules are found among the Hexactinellida, with the stalk spicules (length: 30 cm; diameter: 300 microm) of Hyalonema sieboldi as prominent examples. These spicules are constructed around a central axial filament, which is formed by approximately 40 siliceous layers. The stratified spicules function as optical glass fibers with unique properties. If free-spaced coupled with a white light source (WLS), the entire fiber is illuminated. Special features of the light transmission: (i) only wavelengths between 615 and 1310 nm can pass through the fibers and (ii) light below wavelengths of 615 nm and above 1310 nm is completely cut-off. The transmission efficiency is around 60% (measured at 1080-1100 nm [length of the fiber: 5 cm]). The spicules acts as sharp high- and low-pass filters, suggesting that these silica-based fibers might be involved in a photoreception system. This assumption is supported by the finding that sponges are provided with a bioluminescent system. It is hypothesized that the spicules/siliceous fibers might be involved in a photoreception system in these animals.     

Bio-sintering processes in hexactinellid sponges: Fusion of bio-silica in giant basal spicules from Monorhaphis chuni

The two sponge classes, Hexactinellida and Demospongiae, comprise a skeleton that is composed of siliceous skeletal elements (spicules). Spicule growth proceeds by appositional layering of lamellae that consist of silica nanoparticles, which are synthesized via the sponge-specific enzyme silicatein. While in demosponges during maturation the lamellae consolidate to a solid rod, the lamellar organization of hexactinellid spicules largely persists. However, the innermost lamellae, near the spicule core, can also fuse to a solid axial cylinder. Similar to the fusion of siliceous nanoparticles and lamella, in several hexactinellid species individual spicules unify during sintering-like processes. Here, we study the different stages of a process that we termed bio-sintering, within the giant basal spicule (GBS) of Monorhaphis chuni. During this study, a major GBS protein component (27 kDa) was isolated and analyzed by MALDI-TOF-MS. The sequences were used to isolate and clone the encoding cDNA via degenerate primer PCR. Bioinformatic analyses revealed a significant sequence homology to silicatein. In addition, the native GBS protein was able to mediate bio-silica synthesis in vitro. We conclude that the syntheses of bio-silica in M. chuni, and the subsequent fusion of nanoparticles to lamellae, and finally to spicules, are enzymatically-driven by a silicatein-like protein. In addition, evidence is now presented that in hexactinellids those fusions involve sintering-like processes.     

Silicateins, silicatein interactors and cellular interplay in sponge skeletogenesis: formation of glass fiber-like spicules

Biomineralization processes are characterized by controlled deposition of inorganic polymers/minerals mediated by functional groups linked to organic templates. One metazoan taxon, the siliceous sponges, has utilized these principles and even gained the ability to form these polymers/minerals by an enzymatic mechanism using silicateins. Silicateins are the dominant protein species present in the axial canal of the skeletal elements of the siliceous sponges, the spicules, where they form the axial filament. Silicateins also represent a major part of the organic components of the silica lamellae, which are cylindrically arranged around the axial canal. With the demosponge Suberites domuncula as a model, quantitative enzymatic studies revealed that both the native and the recombinant enzyme display in vitro the same biosilica-forming activity as the enzyme involved in spicule formation in vivo. Monomeric silicatein molecules assemble into filaments via fractal intermediates, which are stabilized by the silicatein-interacting protein silintaphin-1. Besides the silicateins, a silica-degrading enzyme silicase acting as a catabolic enzyme has been identified. Growth of spicules proceeds in vivo in two directions: first, by axial growth, a process that is controlled by evagination of cell protrusions and mediated by the axial filament-associated silicateins; and second, by appositional growth, which is driven by the extraspicular silicateins, a process that provides the spicules with their final size and morphology. This radial layer-by-layer accretion is directed by organic cylinders that are formed around the growing spicule and consist of galectin and silicatein. The cellular interplay that controls the morphogenetic processes during spiculogenesis is outlined.     

Middle and Late Cambrian sponge spicules from Hunan, China

Abundant and well-preserved assemblages of disarticulated sponge spicules occur in Middle and Late Cambrian platform carbonates of western Hunan, China. Assemblages recovered from 11 stratigraphic horizons include calcisponges, demosponges, and hexactinellids. Hexactinellida, in particular, are both abundant and diverse in Upper Cambrian carbonates. Comparison with spicule assemblages from Australia indicates that many of these taxa have long stratigraphic ranges, limiting their use in correlation. The morphological diversity of these spicules exceeds that known for living siliceous sponges, supporting the observation that during the Cambrian radiation, sponges, like other metazoans, evolved a variety of architectural forms not observed in later periods. Like conodonts, individual sponges can produce more than one spicule form; thus, an "apparatus genus" concept based on multiple co-occurring elements may eventually prove useful in the biostratigraphic and paleobiological interpretation of disarticulated sponge spicules. Four distinctive forms are recognized as new taxa: Australispongia sinensis new genus and species, Flosculus gracilis new genus and species, Pinnatispongia bengtsoni new genus and species, and Nabaviella paibiensis new species.     

Dynamics of spicule production in the marine sponge <Emphasis Type="Italic">Hymeniacidon perlevis</Emphasis> during in vitro cell culture and seasonal development in the field

To characterize the formation of silica spicules, the dynamics of spiculogenesis of an intertidal marine sponge Hymeniacidon perlevis (Montagu 1818) (Porifera: Demospongiae) were investigated by measuring the gene expression of silicatein (the enzyme responsible for spicule silicification) and the dimensional changes of spicules during the developmental process of individual sponges and in cell cultures of primmorphs of archaeocyte-dominant cell populations. The different developmental stages of spicules were documented by time-lapse microscopy and observed by transmission electron microscopy during a 1-month culture period. During its annual life cycle, H. perlevis has four different developmental stages: dormancy, resuscitation, bloom, and decline. Field-grown individual sponge samples at different stages were collected over 7 months (March to September 2005). The dimensions of the silica spicules from these samples were microscopically measured and statistically analyzed. This analysis and the material properties of the spicules allowed them to be classified into four groups representing the different developmental stages of spiculogenesis. Silicatein expression in the bloom stage was more than 100 times higher than that in the other stages and was correlated with the spicule developmental stage. The trend of spicule formation in field-grown sponges was consistent with the trend in cell culture. A new parameter, the maturation degree (MD) of spicules (defined as the ratio of actual to theoretical silica deposition of mature spicules), was introduced to quantify spicule development. Silica spiculogenesis during H. perlevis development was delineated by comparing MD and silicatein expression.     

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.     

Early sponge evolution: A review and phylogenetic framework

Sponges are one of the critical groups in understanding the early evolution of animals. Traditional views of these relationships are currently being challenged by molecular data, but the debate has so far made little use of recent palaeontological advances that provide an independent perspective on deep sponge evolution. This review summarises the available information, particularly where the fossil record reveals extinct character combinations that directly impinge on our understanding of high-level relationships and evolutionary origins. An evolutionary outline is proposed that includes the major early fossil groups, combining the fossil record with molecular phylogenetics. The key points are as follows. (1) Crown-group sponge classes are difficult to recognise in the fossil record, with the exception of demosponges, the origins of which are now becoming clear. (2) Hexactine spicules were present in the stem lineages of Hexactinellida, Demospongiae, Silicea and probably also Calcarea and Porifera; this spicule type is not diagnostic of hexactinellids in the fossil record. (3) Reticulosans form the stem lineage of Silicea, and probably also Porifera. (4) At least some early-branching groups possessed biminerallic spicules of silica (with axial filament) combined with an outer layer of calcite secreted within an organic sheath. (5) Spicules are homologous within Silicea, but also between Silicea and Calcarea, and perhaps with Homoscleromorpha. (6) The last common ancestor of extant sponges was probably a thin-walled, hexactine-bearing sponge with biminerallic spicules. (7) The stem group of sponges included tetraradially-symmetric taxa that grade morphologically into Cambrian fossils described as ctenophores. (8) The protomonaxonid sponges are an early-branching group, probably derived from the poriferan stem lineage, and include the problematic chancelloriids as derived members of the piraniid lineage. (9) There are no definite records of Precambrian sponges: isolated hexactine-like spicules may instead be derived from radiolarians. Early sponges had mineralised skeletons and thus should have a good preservation potential: the lack of sponge fossils in Precambrian strata may be due to genuine absence of sponges. (10) In contrast to molecular clock and biomarker evidence, the fossil record indicates a basal Cambrian diversification of the main sponge lineages, and a clear relationship to ctenophore-like ancestors. Overall, the early sponge fossil record reveals a diverse suite of extinct and surprising character combinations that illustrate the origins of the major lineages; however, there are still unanswered questions that require further detailed studies of the morphology, mineralogy and structure of early sponges.     

Spicule structure and affinities of the Late Ordovician hexactinellid‐like sponge Cyathophycus loydelli from the Llanfawr Mudstones Lagerstätte,Wales

Exceptionally well‐preserved specimens of the reticulosan sponge Cyathophycus loydelli from the Sandbian (Late Ordovician) Llanfawr Mudstones Formation of Llandrindod, Waes, UK, have been examined using scanning electron microscopy (SEM). The specimens include exquisitely detailed pyritized spicules, and granular pyritization of surrounding soft tissues. Spicules frequently show axial canals of diameter similar to those of modern siliceous sponges, with hexagonal symmetry typical of modern demosponges rather than hexactinellids. In one case, the axial filament is also preserved. The largest spicules (ray diameter >20 μm) show a complex structure, with a laminar external region similar to that of the extant hexactinellid Monorhaphis. Some spicules preserve sub‐micron detail of the spicule surface, resembling the reticulate collagenous sheath of Monorhaphis. The hexagonal symmetry of the canal confirms that at least some Reticulosa are not crown‐group hexactinellids, but stem‐group Hexactinellida or Demospongea, or stem‐group Silicea. This suggests that a square canal is a sufficient diagnostic feature of total‐group Hexactinellida, but that hexagonal canals were more widely distributed among Early Silicea and were probably not restricted to demosponges. Alternatively, comparison with the structure of modern verongiid fibres suggests that these may be homologous with the outer layers of Cyathophycus spicules, and Cyathophycus may instead be a stem‐group demosponge. The preserved detail of the surface layer shows that pyritization can preserve certain material with extraordinarily fine resolution.     

Spicules of hexactinellid sponges (Hexactinellida: Porifera) as natural composite materials

This paper reviews studies on the hexactinellid glass sponges (Hexactinellida: Porifera) that have organic silica spicules. According to its physical properties (microdensity, Young’s modulus, and light transmission), the material of the spicules is similar to amorphous silica; however, sponge spicules are birefringent, which suggests that they have a highly ordered crystal-like nature. Mineralized remnants of siliceous spicules composed of chemically inert materials are preserved in sedimentary rocks and provide evidence of the ecological state of the ancient biosphere. Sponges occur in waters with low temperatures; therefore, they grow very slowly and live for hundreds of years. The organic silica spicules exhibit the capacity for triboluminescence. The generated light emission may be used by symbiotic bacteria on the spicule surface.     

Phylogeny and evolution of glass sponges (porifera, hexactinellida)

Reconstructing the phylogeny of sponges (Porifera) is one of the remaining challenges to resolve the metazoan Tree of Life and is a prerequisite for understanding early animal evolution. Molecular phylogenetic analyses for two of the three extant classes of the phylum, Demospongiae and Calcarea, are largely incongruent with traditional classifications, most likely because of a paucity of informative morphological characters and high levels of homoplasy. For the third class, Hexactinellida (glass sponges)--predominantly deep-sea inhabitants with unusual morphology and biology--we present the first molecular phylogeny, along with a cladistic analysis of morphological characters. We collected 18S, 28S, and mitochondrial 16S ribosomal DNA sequences of 34 glass sponge species from 27 genera, 9 families, and 3 orders and conducted partitioned Bayesian analyses using RNA secondary structure-specific substitution models (paired-sites models) for stem regions. Bayes factor comparisons of different paired-sites models against each other and conventional (independent-sites) models revealed a significantly better fit of the former but, contrary to previous predictions, the least parameter-rich of the tested paired-sites models provided the best fit to our data. In contrast to Demospongiae and Calcarea, our rDNA phylogeny agrees well with the traditional classification and a previously proposed phylogenetic system, which we ascribe to a more informative morphology in Hexactinellida. We find high support for a close relationship of glass sponges and Demospongiae sensu stricto, though the latter may be paraphyletic with respect to Hexactinellida. Homoscleromorpha appears to be the sister group of Calcarea. Contrary to most previous findings from rDNA, we recover Porifera as monophyletic, although support for this clade is low under paired-sites models.     

Silica deposition in Demosponges: spiculogenesis in Crambe crambe

Transmission electron-microscopy images coupled with dispersive X-ray analysis of the species Crambe crambe have provided information on the process of silica deposition in Demosponges. Sclerocytes (megasclerocytes) lie close to spicules or surround them at different stages of growth by means of long thin enveloping pseudopodia. Axial filaments occur free in the mesohyl, in close contact with sclerocytes, and are triangular in cross section, with an internal silicified core. The unit-type membrane surrounding the growing spicule coalesces with the plasmalemma. The axial filament of a growing spicule and that of a mature spicule contain 50%-70% Si and 30%-40% Si relative to that contained in the spicule wall, respectively. The extracellular space between the sclerocyte and the growing spicule contains 50%-65%. Mitochondria, vesicles and dense inclusions of sclerocytes exhibit less than 10%. The cytoplasm close to the growing spicule and that far from the growing spicule contain up to 50% and less than 10%, respectively. No Si has been detected in other parts of the sponge. The megascleres are formed extracellularly. Once the axial filament is extruded to the mesohyl, silicification is accomplished in an extracellular space formed by the enveloping pseudopodia of the sclerocyte. Si deposition starts at regularly distributed sites along the axial filament; this may be related to the highly hydroxylated zones of the silicatein-alpha protein. Si is concentrated in the cytoplasm of the sclerocyte close to the plasmalemma that surrounds the growing spicules. Orthosilicic acid seems to be pumped, both from the mesohyl to the sclerocyte and from the sclerocyte to the extracellular pocket containing the growing spicule, via the plasmalemma.     

Plasmodesmata: intercellular tunnels facilitating transport of macromolecules in plants

The major structural and enzymatically active protein in spicules from siliceous sponges, e.g., for Suberites domuncula studied here, is silicatein. Silicatein has been established to be the key enzyme that catalyzes the formation of biosilica, a polymer that represents the inorganic scaffold for the spicule. In the present study, it is shown, by application of high-resolution transmission and scanning transmission electron microscopy that, during the initial phase of spicule synthesis, nanofibrils with a diameter of around 10 nm are formed that comprise bundles of between 10 and 20 nanofibrils. In intracellular vacuoles, silicasomes, the nanofibrils form polar structures with a pointed tip and a blunt end. In a time-dependent manner, these nanofibrillar bundles become embedded into a Si-rich matrix, indicative for the formation of biosilica via silicatein molecules that form the nanofibrils. These biosilicified nanofibrillar bundles become extruded from the intracellular space, where they are located in the silicasomes, to the extracellular environment by an evagination process, during which a cellular protrusion forms the axial canal in the growing spicule. The nanofibrillar bundles condense and progressively form the axial filament that becomes localized in the extracellular space. It is concluded that the silicatein-composing nanofibrils act not only as enzymatic silica bio-condensing platforms but also as a structure-giving guidance for the growing spicule.     

Formation of giant spicules in the deep-sea hexactinellid <Emphasis Type="Italic">Monorhaphis chuni</Emphasis> (Schulze 1904): electron-microscopic and biochemical studies

The siliceous sponge Monorhaphis chuni (Hexactinellida) synthesizes the largest biosilica structures on earth (3 m). Scanning electron microscopy has shown that these spicules are regularly composed of concentrically arranged lamellae (width: 3–10 μm). Between 400 and 600 lamellae have been counted in one giant basal spicule. An axial canal (diameter: ~2 μm) is located in the center of the spicules; it harbors the axial filament and is surrounded by an axial cylinder (100–150 μm) of electron-dense homogeneous silica. During dissolution of the spicules with hydrofluoric acid, the axial filament is first released followed by the release of a proteinaceous tubule. Two major proteins (150 kDa and 35 kDa) have been visualized, together with a 24-kDa protein that cross-reacts with antibodies against silicatein. The spicules are surrounded by a collagen net, and the existence of a hexactinellidan collagen gene has been demonstrated by cloning it from Aphrocallistes vastus. During the axial growth of the spicules, silicatein or the silicatein-related protein is proposed to become associated with the surface of the spicules and to be finally internalized through the apical opening to associate with the axial filament. Based on the data gathered here, we suggest that, in the Hexactinellida, the growth of the spicules is mediated by silicatein or by a silicatein-related protein, with the orientation of biosilica deposition being controlled by lectin and collagen. Carsten Eckert was previously with the Museum für Naturkunde, Invalidenstrasse 43, 10115 Berlin, Germany. The collagen sequence from Aphrocallistes vastus reported here, viz., [COL_APHRO] APHVACOL (accession number AM411124), has been deposited in the EMBL/GenBank data base. This work was supported by grants from the European Commission, the Deutsche Forschungsgemeinschaft, the Bundesministerium für Bildung und Forschung Germany (project: Center of Excellence BIOTECmarin), the National Natural Science Foundation of China (grant no. 50402023), and the International Human Frontier Science Program.     

Evagination of cells controls bio-silica formation and maturation during spicule formation in sponges

The enzymatic-silicatein mediated formation of the skeletal elements, the spicules of siliceous sponges starts intracellularly and is completed extracellularly. With Suberites domuncula we show that the axial growth of the spicules proceeds in three phases: (I) formation of an axial canal; (II) evagination of a cell process into the axial canal, and (III) assembly of the axial filament composed of silicatein. During these phases the core part of the spicule is synthesized. Silicatein and its substrate silicate are stored in silicasomes, found both inside and outside of the cellular extension within the axial canal, as well as all around the spicule. The membranes of the silicasomes are interspersed by pores of ≈ 2 nm that are likely associated with aquaporin channels which are implicated in the hardening of the initial bio-silica products formed by silicatein. We can summarize the sequence of events that govern spicule formation as follows: differential GENETIC READOUT (of silicatein) → FRACTAL ASSOCIATION of the silicateins → EVAGINATION of cells by hydro-mechanical forces into the axial canal → and finally PROCESSIVE BIO-SILICA POLYCONDENSATION around the axial canal. We termed this process, occurring sequentially or in parallel, BIO-INORGANIC SELF-ORGANIZATION.