SISTEMA VIAL Y DE TRANSPORTE

Werner E. G. Mu¨ller, Xiaohong Wang, Sergey I. Belikov, Wolfgang

Tremel, Ute Schloßmacher, Antonino Natoli, David Brandt, Alexandra Boreiko, Muhammad Nawaz Tahir, Isabel M. Mu¨ller, and Heinz C. Schro¨der

Abstract

Silica is a major constituent of sponge spicules in the classes Demospongiae and Hexactinellida. The spicules of these sponges are composed of hydrated, amor- phous, non-crystalline silica. In the case of the marine demosponge Suberites do- muncula, the initial secretion of spicules has been shown to occur in specialized cells, the sclerocytes, where silica is deposited around an organic filament. Subse- quently, the spicules are extruded and completed extracellularly within a galectin/ collagen lattice/scaffold. A major step in elucidating the formation of siliceous spicules on the molecular level was the finding that the ‘‘axial organic filament’’ of siliceous spicules is an enzyme, silicatein, which mediates the apposition of amorphous silica and hence the formation of spicules. The formation of siliceous spicules is certainly genetically controlled; this process initiates the morphogene- sis phase and involves, in addition to silicatein, galectin and collagen, other molecules. The aim of this chapter is to provide an understanding of spicule for- mation and to outline the application of the basic biological strategies of the con- trolled mineralization for nanobiotechnology.

Key words: sponges, Porifera, Suberites domuncula, spicules, biosilica, silica formation, biotechnology, nanobiotechnology.

4.1

Introduction

Since the times of Aristotle (384–322 bc) [1], sponges have occupied a distin- guished position among the animals because of their biomedical potential [2], their beauty [3], and their enigmatic evolutionary origin [4–6]. Difficulties in their systematic positioning and elucidation of their relationship to other multicellular organisms have resulted in their designation as ‘‘Zoophytes’’ or ‘‘Plant-animals’’ (a taxon placed between plants and animals), until they were finally recognized

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Handbook of Biomineralization. Edited by E. Ba¨uerlein

Copyright 8 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-31804-9

as genuine metazoans, which diverged first from the animal ancestor, the Urme- tazoa [7]. By then it had become clear that sponges are not ‘‘simple blobs’’ but rather contain and express a variety of metazoan-like transcription factors, and in turn form sophisticated tissue assemblies [8]. The sponges (phylum Porifera) were originally grouped into siliceous sponges and calcareous sponges [9], but after the discovery and/or appreciation of the glass sponges [10] they were re- allocated to three classes: Demospongiae, and Hexactinellida (both have a sili- ceous skeleton), and Calcarea (a calcareous skeleton) [11].

Examples are provided in Figure 4.1 of a hexactinellid (Hyalonema sieboldi; Fig. 4.1A), a demosponge (Suberites domuncula; Fig. 4.1F), and a calcarean sponge

Fig. 4.1 The three different classes of Porifera. (A) Hexactinellida:Hyalonema sieboldi. Lateral view of a specimen showing the body and the stalk (size: 500 mm). Specimens of this hexactinellid can be divided morphologically into the cylindrical upper body which is attached to 30 cm-long basal stalk spicules that fix the specimens to the substratum. (B) Earliest sponge in body preservation,Solactiniella plumata (Lowermost Cambrian Sansha section [Hunan, China]) (size: 40 mm). (C) A section

through the demospongeSpongites plicatus from the Jurassic (size: 30 mm). (D) The fossil freshwater spongeSpongilla guten- bergiana of the Middle Eocene (Lutetian) near Messel (Darmstadt, Germany); size: 3 mm. (E) The calcareous spongeClathrina coriacea; size: 10 mm. (F) Demospongiae: Suberites domuncula. This specimen lives on the hermit crabPagurites oculatus, which resides in shells of the molluskTruncu- lariopsis trunculus; size: 30 mm. 60 4 Formation of Siliceous Spicules in Demosponges: Example Suberites domuncula

Fig. 4.2 Phylogenetic position of the Porifera between the Urmetazoa and the Urbilateria. The major evolutionary novelties which must be attributed to the Urmetazoa are those molecules which mediate apoptosis and control morphogenesis, the immune molecules, and primarily the cell adhesion molecules. The siliceous sponges with the two classes Hexactinellida and Demo- spongiae emerged first, and finally the Calcarea, which possess a calcareous skeleton, appeared. The Archaeocyatha, which are sponge-related animals with a calcareous skeleton, became extinct. The Calcarea are very likely a sister group of the Cnidaria. From the latter phylum the Ctenophora evolved which comprise not only an oral/aboral polarity but also a biradial

symmetry. Finally, the Urbilateria emerged from which the Protostomia and the Deuterostomia originated. It is very likely that the Urmetazoa emerged between the two major ‘‘snowball earth events’’, the Sturtian glaciation (710–680 MYA) and the Varanger- Marinoan ice ages (605–585 MYA). In the two poriferan classes, Hexactinellida and Demospongiae, the skeleton is composed of amorphous and hydrated silica, while the spicules of Calcarea are composed of calcium carbonate. The latter biomineral is also prevalent in Protostomia and also in Deuterostomia. In vertebrates, the bones are composed of calcium phosphate (apatite). The autapomorphic character of the Demospongiae is the spicule-synthesizing enzyme, silicatein.

Fig. 4.3 Siliceous spicules. (A) The first description of spicules from a species belonging to genusGeodia [21]. Highlighted are the spheres (asters [between t and c]) and also the spines (protriaenes and oxeas [between c and g]). (B) The first description of siliceous spicule formation in sponges in the freshwater spongeSpongilla fluviatilis [22]; this starts intracellularly, within the ‘‘Schw€aarmsporen’’. (C) Other cells have also been suggested to form spicules, especially those which have a large nucleus and nucleolus [22]. (D) Schematic outline of the spicule formation, based on studies with the hexactinellidsHyalonema and Monorhaphis [23]. In the center of the spicules the axial filament (A) exists around, which the first layer of siphons (silica) is formed. The next

organic layers, termed ‘‘spiculin lamellae’’ (Sp), are located beside this layer. The spicule-forming sclerocytes, which were originally termed ‘‘epithelium-like cells’’ (E), surround the spicule. (E) Section through an entire demosponge (Craniella schmidtii), showing embryos within the parent [27]. This small specimen with a diameter of 5 mm is traversed by 0.5 mm-long protriaenes (spicules; sp), while the embryo (em) comprises only small oxeas (not shown). (F)Euplectella marshalli, showing the mature floricome, complex spicules, which are surrounded by scleroblasts [61]; the spicules may be up to 300 mm in size. (G) Fossil spicules fromS. plumata; the spicules are 0.5 to 5 mm in length.

(Clathrina coriacea; Fig. 4.1E). Sponges were united to the phylum Porifera, for the characteristic and distinct pores on the surface of the animals [12, 13].

Sponges represent the evolutionarily oldest, still extant taxon – which testifies to the developmental level of animals living in the Neo-Proterozoic Eon (some 1000 to 520 million years ago; MYA); hence they can be termed ‘‘living fossils’’ [14]. Based on the sequence data of informative genes, which code for structural and functional proteins, it had been calculated that the sponges diverged from the common metazoan ancestor approximately 650 MYA [15]. This calculation is in close accordance with fossil records, and implies that the sponges evolved between the two glaciations, Sturtian and Varanger-Marinoan (Fig. 4.2). The pri- mordial Earth’s surface comprised initially insoluble silicates, carbonates, and also phosphates. During the cycle of silicate weathering and carbonate precipita- tion, which occurred prior to or simultaneously with the glaciations, a dissolution of surface rocks composed of insoluble silicates [CaSiO3] resulted in the forma- tion of soluble calcium carbonate [CaCO3] and soluble silica [SiO2], under the consumption of atmospheric CO2[16, 17].

The Hexactinellida are the oldest group of sponges in the fossil records of the Sansha section in Hunan (Early Cambrian; China) [18], where more or less com- pletely preserved sponge fossils, such as Solactiniella plumata (Fig. 4.1B) have been found. This fossil is especially noteworthy as it shows, besides the unusual body preservation, also very intact siliceous spicules (skeletal elements). An ap- proximately 40-mm-sized specimen comprises spicules that are 0.5 to 5 mm long and have a diameter of 0.1 mm (Fig. 4.3G); some of these spicules may be broken and present the open axial canals. A section through a fossil demosponge Spongites plicatus (Jurassic) found near Oppeln (Poland) [19] shows the character- istic complex aquiferous canal system by which the sponges pump and filter large volumes of water through their tissues (Fig. 4.1C). Very rich fauna of fossil fresh- water sponges have been discovered in the oil-shales of the Messel pit, near Darmstadt (Germany). There, Spongilla gutenbergiana from the Middle Eocene (Lutetian; some 50 MYA) has been found (Fig. 4.1D) [20]; most of this sponge’s spicules are between 160 and 230 mm in length.

4.2

Early Descriptions

The first reports of sponge spicules were published in 1753 by Donati [21], who isolated them from a species belonging to the genus Geodia (Alcyonium) (Fig. 4.3A). Distinct cytological studies on spicule formation were first published in 1856 by Lieberku¨hn [22], who used the freshwater sponge Spongilla fluviatilis (De- mospongiae) as a model. These studies provided the earliest detailed analyses of the development and differentiation of fertilized eggs, together with the differen- tiation stages of somatic sponge cells from the ‘‘Schwa¨rmsporen’’ to the spicule- forming sclerocytes (Fig. 4.3B,C). Precise descriptions of the structure and also

the growth of spicules of the glass sponges Hyalonema and Monorhaphis were later provided by Schulze (1904) [23]. The spicules of Monorhaphis can reach lengths of 70 cm, and may be up to 8 mm thick. Schulze proposed that the growth started from axial organic filaments and suggested that thickening of the spicules proceeded by fairly regular apposition of lamellar silica layers under the formation of centric rings (siphons). Schulze also described the presence of organic layers located between the concentric lamellae (Fig. 4.3D); during a later phase of growth the spicules become surrounded by sclerocytes, which are the spicule-forming cells.

The spicules function as the main stabilizing inorganic elements in the body in demosponges (Fig. 4.3E) and hexactinellids (Fig. 4.3F). The inorganic material which constitutes the spicules of the Demospongiae and also the Hexactinellida is silica. The first report on the composition of the spicules dates back to 1789, when Bindheim reported that these skeletal elements are formed from ‘‘Kiese- lerde’’ (siliceous earth) [24]. Later, Gray (1826) mentioned that the ‘‘. . . fusiformes transparent spiculae are composed of glass, which are cemented together by a cartilage-like material’’ [25]. In 1864, Bowerbank noted also organic material be- sides silica within the spicules [26]. Detailed analyses of siliceous spicules were reported later, and revealed that silicic acid contributes more than 92% of the mass of a spicule [27, 28]. Sollas, in 1888, presented especially comprehensive re- sults on the physical and chemical properties of the siliceous spicules [27]; for ex- ample, he reported that they have a refractive index of 1.5 and that colloidal silica contains about 10% of water and has the general formula of (SiO2)2a5
H2O.

4.3

Structural Features of the Sponge Body Plan

Sponges – which are sessile filter-feeding organisms – form the oldest yet still ex- tant metazoan phylum. They are characterized by a simple body plan [6, 14], their body being composed of an epithelial layer which surrounds a mesohyl compart- ment that is reticulated in a highly organized manner by a canal system. The main structural and functional novelties, which evolved during the major evolu- tionary transitions to the Porifera and Cnidaria, are summarized in Figure 4.2. The autapomorphic character of the sponges, the spicules, represents an amazing assortment of highly complex and morphologically diverse and filigree skeletal elements.

In 1907, Wilson introduced the sponges as a biological system to experimental biology, and later this became a traditional model to study both cell–cell as well as cell–matrix adhesion [29]. A completely new approach to a further understanding of the body plan organization in sponges was opened following the application of molecular biological techniques, mainly by cloning of their cDNAs. Our studies have focused especially on molecules (genes and proteins) which have been iden- tified in the marine demosponges S. domuncula and Geodia cydonium, as well

as the freshwater sponge Lubomirskia baicalensis, an endemic sponge of the Lake Baikal. With the isolation of a galectin sequence as the first cell–cell adhesion molecule, and the integrin sequence as the first cell–matrix adhesion receptor from sponges, it became clear that sponges contain molecules closely related to those known to promote adhesion also in Protostomia and Deuterostomia. Col- lagen is the dominant molecule present in the extracellular matrix of sponges, which functions as a cell–matrix adhesion molecule. The corresponding gene was cloned from the freshwater sponge Ephydatia muelleri and from the marine sponge S. domuncula. Cell surface-spanning receptors – in particular the single-pass as well as the seven-pass transmembrane receptor proteins – serve as receivers for extracellular signaling molecules. The first identified sponge 1-transmembrane receptor which represents an autapomorphic character for Metazoa, was the receptor tyrosine kinase. In G. cydonium the metabotropic glutamate/GABA-like receptor was characterized as a 7-transmembrane receptor. Besides cell surface-associated molecules, receptors and their interacting li- gands, molecules involved in morphogenesis are of prime interest for the under- standing of body plan formation. During the development of animals, a set of genes – most of which are transcription factors and responsible for cell fate and pattern determination – are expressed.

4.4

Cells Involved in Spicule Formation

Until recently it has not been possible to define cell types in sponges in a strict manner. Elucidation of the details of the molecular markers also allows the distinction of some steps during the differentiation of stem cells to the sclerocyte lineage – the cells that are involved in the formation of spicules. As with any other metazoan, fertilized eggs also develop in sponges during a series of cell divisions to morulae, blastulae, and perhaps even to gastrulae [30]. Recently, the first report was made of using molecular markers to determine the restriction of gene expression during embryogenesis in a sponge [31]. The results indicated that, in oocytes, morulae and blastulae/larvae from S. domuncula, distinct genes are expressed, among them a sponge-specific receptor tyrosine kinase (RTKvs). In addition, the sex-determining protein FEM1 and the sperm-associated antigen-related protein are highly expressed; in adult animals the expression levels of these genes are very low (Fig. 4.4).

At present, studies on embryonic stem cells in sponges are restricted by the lack of techniques to induce mass production of embryos under controlled condi- tions. As a substitution, a three-dimensional (3-D) cell culture system has been established for S. domuncula [32] whereby, under suitable conditions, dissociated single cells form special types of cell aggregates, known as ‘‘primmorphs’’. These contain cells of high proliferation and differentiation capacity that can be consid-

ered operationally as embryonic cells (archaeocytes), as they express the gene en- coding the sponge-specific RTKvs (Fig. 4.4). In a second step, a homeobox gene, Iroquois, is up-regulated in primmorphs that are cultivated in a stronger water current [33]. This transcription factor is expressed in the cells which form the ca- nal system. The further phase to differentiation to sclerocytes is induced by addi- tion of the morphogenetic inorganic elements silicon and ferric iron [34]. First, the mesenchymal stem cell-like protein and noggin are expressed [35], and sub- sequently the key enzymes and functional proteins, silicatein and collagen (see Section 4.5) can be identified in the primmorphs (Fig. 4.4). The formation of spi- cules then commences [34, 36]. Generally, spicule formation is a rapid process;

Fig. 4.4 Sequential expression of stem cell marker genes inS. domuncula. In prim- morphs, as well as in germ cells, high expres- sion of two genes – the sponge-specific receptor tyrosine kinase (RTKvs) and the embryonic development protein (EED) – can be identified. These genes could be considered as markers for totipotent stem cells. On exposure of the primmorphs to a

water current, the transcription factorIroquois is expressed, primarily in epithelial cells. Subsequently, the gene forNoggin and later those genes forsilicatein and collagen, are translated. Early drawings of a larva of Aplysilla sulfurea (above; Delage 1892) [5] and an electron micrograph of a spicule from G. cydonium (a sterraster) are given as an underlay of the bars.

for example, in the freshwater sponge E. fluviatilis the 100 to 300 mm-long spi- cules are synthesized under optimal conditions within 40 h [37].

4.5

Anabolic Enzyme for the Synthesis of Silica: Silicatein

A major breakthrough in our understanding of spicule formation occurred fol- lowing the discovery of the key enzyme involved in spiculogenesis. The research group led by Morse discovered that the organic filament in the central canal of spicules is composed of a cathepsin L-related enzyme, which they termed silica- tein [38, 39]. The group then cloned two of the proposed three isoforms of silica- teins, the a- and b-forms, from the marine demosponge Tethya aurantium [39]. In subsequent years these molecules were also cloned from other sponges, in- cluding the marine sponge S. domuncula and the freshwater sponge L. baicalensis [34, 36, 40, 41, 42].

Silicatein exists in the axial canal (diameter ca. 1 mm) of spicules as an axial fil- ament. This can be freed from the silica mantel with hydrofluoric acid (HF), and subsequently visualized by staining with Coomassie brilliant blue [43]. The silica material around the axial filament of the spicules is in some geographic regions (e.g., on the bottom of Lake Baikal) amazingly resistant to dissolution, and pro- tects the proteinaceous axial filament against disintegration. Sediments obtained from the Baikal Drilling Project and known to be between 2 and 3 million years old were extensively studied and found to contain fossil spicules from the ances- tral species of L. baicalensis [44]. From these fossil spicules, organic filaments dating back 2 million years can be released by HF treatment and stained with Coomassie brilliant blue (Fig. 4.5A). All spicules from recent specimens of L. baicalensis also contain an axial filament consisting of silicatein (Fig. 4.5B and C). The S. domuncula cDNA encoding silicatein has been isolated and character- ized [36]; the 1169 bp-long sequence has an open reading frame of 993 nucleo- tides (nt), and the predicted translation product of 330 amino acids (Fig. 4.6) has a calculated Mrof 36 306. As described above, silicatein is a new member of the cathepsin subfamily [35, 36, 38]. The three amino acids Cys, His and Asn – which form the catalytic triad of cysteine proteases – are present in the sponge cathepsin at the characteristic sites: Cys125, His164and Asn184. Furthermore, the silicatein sequence comprises one cluster of characteristic hydroxy amino acids (serine) (Fig. 4.6).

Phylogenetic analysis was performed with the silicateins from the demo- sponges L. baicalensis, Tethya aurantium, S. domuncula, S. lacustris and E. flu- viatilis, as well as with the cathepsins L from these sponges and the glass sponge Aphrocallistes vastus; analyses were also conducted with the papain cysteine pepti- dase from the plant Arabidopsis thaliana as an outgroup. The polypeptides were aligned and a slanted tree was calculated which showed that the cathepsin L se- quences form the basic branches from which the silicatein sequences originate.

This suggests that the silicateins derive from a common ancestor of the cathepsin L sequences from the marine Hexactinellida Aphrocallistes vastus and the marine demosponges, here from S. domuncula. The tree indicates also that the silicateins from the cosmopolitan species S. lacustris and E. fluviatilis form the basal branch from which the polypeptides of the endemic Lake Baikal sponge, L. baicalensis, emerge (Fig. 4.6). This analysis underscores also that the freshwater sponges

Fig. 4.5 Dissolution of the polymerized silica of the spicules with HF in time-lapse images. For this series of experiments the freshwater spongeLubomirskia baicalensis was used. The spicules were treated with HF to dissolve the siliceous material of the spicules. Within 2 min, optical time-lapse micrographs were taken. Initially, the surfaces of the partially broken spicule samples still show spines. With time, the silica is dissolved, showing first a perforation of the silica mantel at the sites where spines had protruded. Finally, the silica is completely dissolved leaving behind only the organic axial filament (af ), which was stained with Coomassie brilliant blue. (A: a–f ) Fossil spicules (1 MYA) were treated with HF. The release of one axial filament (af ) is shown. In addition to the siliceous spicule, the siliceous diatoms can also be

seen at the start of the dissolution process.