Dev Genes Evol (2006) 216: 229–242DOI 10.1007/s00427-005-0047-2

ORIGINAL ARTICLE

Matthias Wiens . Sergey I. Belikov .Oxana V. Kaluzhnaya . Anatoli Krasko .Heinz C. Schröder . Sanja Perovic-Ottstadt .Werner E. G. Müller

Molecular control of serial module formation along the apical–basal axis in the sponge Lubomirskia baicalensis: silicateins,mannose-binding lectin and mago nashiReceived: 9 July 2005 / Accepted: 21 November 2005 / Published online: 28 December 2005# Springer-Verlag 2005

Abstract The freshwater sponge Lubomirskia baicalensis(from Lake Baikal) is characterized by a body plan com-posed of serial modules which are arranged along anapical–basal axis. In shallow water, the sponge occurs onlyencrusting, while in deeper environment (>3 m), this spe-cies forms branches and grows in an arborescent manner.Each module is stabilized by bundles of spined oxeas(amphioxeae spicules). The spicules are surrounded by anorganic matrix. cDNAs for structural proteins (silicateinand mannose-binding lectin (MBL)) as well as for oneregulatory protein (mago nashi) were isolated from L.baicalensis. Surprisingly the silicatein α molecule exists inseveral, at least four, isoforms (a1 to a4). Expression studiesrevealed that the steady-state levels of transcripts for thesilicateins, the mannose-binding lectin, and mago nashi are

highest at the top of the branches, while only very lowlevels are found in cells at the base. Based on in situhybridization studies, evidence is presented that the spiculeformation (1) starts and is completed inside of the bundles,and (2) occurs together with the mannose-binding lectinfrom the surfaces of the bundles. The data suggest that themodules are sequentially formed. It is speculated that theexpression of the silicateins and the mannose-binding lectinmight be (partially) controlled by mago nashi.

Keywords Sponges . Lubomirskia baicalensis .Silicateins . Cathepsins . Mannose-binding lectin .Mago nashi . Axis formation . Modules

Introduction

Prerequisites for the evolutionary transition to metazoanswere the appearance (1) of cell–cell and cell–matrix ad-hesion molecules (Müller 1997) and (2) of cell surfacereceptors, allowing outside-in signaling (reviewed in Mülleret al. 2004); these molecules are found in the phylogeneti-cally oldest taxon, the sponges (phylum Porifera). Patternformation, which is based on morphogenetic processes, be-came possible through controlled, differential spatial andtemporal expressions of genes that initiate or maintain alarge number of signaling pathways (reviewed in Müller2005). In addition, metazoans, in general, and sponges, inparticular, have acquired self–self and self–nonself recog-nition systems, including apoptotic pathways, which are thebasis for a potent immune defense and for the establishmentof an individuality (Müller andMüller 2003). Progress in theunderstanding of the basic genomic repertoire of the earliestMetazoa (Urmetazoa [Müller et al. 2004]), the sponges, waspossible by detailed molecular biological and cell biologicalstudies, performed primarily with the demosponge Suberitesdomuncula. It is obvious that some sponge species, such asthe freshwater sponge studied here (Lubomirskia baicalen-sis), already have a bauplan (body plan) which allows themto grow in an organized manner to sizes of up to 2 m.We are

Communicated by D.A. Weisblat

The sequences from Lubomirskia baicalensis have been deposited(EMBL/GenBank): the cDNA for silicatein-a2 (LBSILICAa2,accession number AJ968945), silicatein-a3 (LBSILICAa3,AJ968946), silicatein-a4 (LBSILICAa4, AJ968947), cathepsin L2(LBCATL2, AJ968949), mannose-binding lectin (LBMBL,AJ968948), mago nashi (LBMAGNA, AJ968950), and α-tubulin(LBTUB, AJ971711). The sequences for cathepsin L fromAphrocallistes vastus (AVCATL, AJ968951), Geodia cydonium(CATL_GEOCY, Y10527), and mago nashi fromSuberites domuncula were also deposited AM086401

M. Wiens . O. V. Kaluzhnaya . A. Krasko . H. C. Schröder .S. Perovic-Ottstadt . W. E. G. Müller (*)Institut für Physiologische Chemie,Abteilung Angewandte Molekularbiologie,Universität, Duesbergweg 6,D-55099 Mainz, Germanye-mail: wmueller@uni-mainz.deTel.: +49-6131-3925910Fax: +49-6131-3925243

S. I. Belikov . O. V. Kaluzhnaya . W. E. G. MüllerLimnological Institute of the Siberian Branch of RussianAcademy of Sciences, Ulan-Batorskaya 3,RUS-664033 Irkutsk, Russia

now beginning to understand the underlying genes/mole-cules governing body plan formation in sponges through theidentification and functional analyses of homeodomainmolecules (Wiens et al. 2003) and transcription factors insponges, e.g., Brachyury (Adell et al. 2003) that in trip-loblastic animals exists in organizer regions.

The Baikalian freshwater sponge L. baicalensis (size upto 1 m), which shows a pronounced branching architecturewith a highly ordered arrangement of spicules, is veryappropriate to study bauplan formation on. During growth,new modules are added to the tips of branches (Kaluzhnayaet al. 2005a,b). Such architecture was earlier described asradiate accretive pattern (Kaandorp 1994). Another char-acteristic feature of this species is the interdependencebetween depth of occurrence and growth form; it isencrusting in 2–4 m, branching/encrusting at 3–7 m andforming branches deeper than 10 m. Hence, this speciesoffers itself as an ideal model to study the expression of(potential) “marker” molecules governing the modular/segmental formation of the bauplan. In very recent studies(Kaluzhnaya et al. 2005a,b), it was demonstrated that theskeleton of the arborescently growing L. baicalensis showsa strictly ordered arrangement. The spicules (skeletalelements of the sponges) with sizes between 150 and220 μm form longitudinal bundles which originate fromthe apex of a previous module. The serial modules areseparated from each other by septate zones (annuli), fromwhich newly formed spicular bundles start. The spinedoxeas are held (cemented) together by an organic matrix,which has—in other sponge species—been assumed to becollagen or spongin (Garrone 1978).

As in all other demosponges, the skeletal spicules in L.baicalensis consist of polymeric biosilica. Only two ofthe three classes of Porifera, the Demospongiae and theHexactinellida, synthesize polymeric biosilica enzymati-cally by the enzyme silicatein (Sumerel and Morse 2003),while the third class Calcarea has a calcareous skeleton.Two isoforms of silicateins, the α- and β-forms havebeen cloned, first from the demosponge Tethya aurantium(Cha et al. 1999) and later also from S. domuncula(Krasko et al. 2000; Schröder et al. 2004). The product ofsilicatein, polymeric biosilica, provides the mechanicalsupport for the tissue and the sizes, shapes and arrange-ments of the siliceous spicules that determine the char-acteristic bauplan of each sponge species. As initiallyproposed (Schulze 1904) and now proven electron micro-scopically as well as immunologically (Müller et al.2005), the synthesis of spicules starts intracellularly invacuoles of the spicule-(biosilica)-forming sclerocytes. Inthese cells, the enzyme silicatein (Shimizu et al. 1998;Cha et al. 1999) forms an organic axial filament of ap-proximately 2 μm in diameter around which amorphoussilica layers are deposited. When the spicules reach acritical size, they are extruded from the sclerocytes intothe extracellular space where they acquire their final spe-cies-specific characteristic sizes and shapes through ap-positional growth (Müller et al. 2005). These observationsimply that the formation of spicules is under genetic (andperhaps also epigenetic) control. Also, the further extra-

cellular growth of the spicules is mediated by silicatein(Müller et al. 2005), indicating that the enzyme-releasingcells, the sclerocytes, are arranged in the body throughprecise movement (Uriz et al. 2003) and their positioningis—very likely—also under genetic control. The shape ofthe spicules is genetically influenced by morphogenesis-controlling genes as well as possibly epigeneticallythrough the concentration of silicic acid in the surround-ing aqueous milieu (Maldonado et al. 1999). Theformation of the spicules is very reminiscent of boneformation in the osteon (Parfitt 1994).

Efforts to understand the signals that induce in spongesthe differentiation of the toti/pluri-potent archaeocytes tothe other somatic cells, e.g. sclerocytes, were presentedrecently (Müller et al. 2003). The “terminal” differentiationto sclerocytes involves activation of genes coding for themesenchymal stem cell-like protein and for noggin; theirexpressions depend on morphogenetic inorganic elements,such as silicon and ferric iron. However, so far, nothing isknown about the expression patterns of the differentisoforms of silicateins in different regions of the spongeand in the proteinaceous layer which surrounds the spiculesand holds them in ascending/longitudinal and interconnect-ing/tangential bundles.

In the present paper, we study the different growth formsof L. baicalensis in relation to the architecture of theirspicule bundles. Different morphological characteristicscan be ascribed to genes that control skeleton formation,such as silicateins and a mannose-binding lectin (MBL);the respective genes/cDNAs have been identified. Surpris-ingly, we found that L. baicalensis contains four silicatein-α isoforms, which is more than what have been found inmarine sponges. In spite of intense screening, nosilicatein-β could be identified. From no other freshwatersponge have silicateins been isolated yet. The data obtainedshow for the first time that the expression level of thesilicatein gene correlates with the growth state of theanimal.

Porifera possess two predominant classes of lectins, thegalectins (Pfeifer et al. 1993; Hirabayashi et al. 2002) andthe mannose-binding lectins. Several proteins belonging tothe MBLwere identified in the marine sponge S. domuncula(to be published) and the fishes Takifugu rubripes, Tetraodonnigroviridis, and Lophiomus setigerus (Tsutsui et al. 2003).The motifs for MBL have been assigned a role in innateimmunity and adhesion (Klein 2005). Therefore, wescreened the L. baicalensis cDNA library for MBL; a se-quence was identified and the corresponding protein wasfound to be associated with spicule formation.

Finally, we studied the molecular basis for the (apical–basal) axis. In Protostomia and in Deuterostomia, magonashi is required to position gene transcripts that controlaxis formation (Boswell et al. 1991; Palacios 2002; Pozzoliet al. 2004). In the zebrafish, the mago nashi transcripts aretransported directionally by microtubules within the fer-tilized eggs and later, during the eight- to 16-cell stages,they accumulate in the central blastomeres (Pozzoli et al.2004). During these transport processes, the mago nashiprotein forms a complex with transcripts of the genes that

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polarize the egg or the embryo through interference withthe processing of primary transcripts or via intracellulartranslocation of the mRNAs (Palacios 2002). The sequencefor mago nashi was extracted from the cDNA library of L.baicalensis. Mago nashi is highly conserved but has nodistinct domain. By comparing the expression patterns ofthese genes (MBL and mago nashi) with the morphology ofL. baicalensis, it was approached to formulate for a spongeon genomic level a regulatory system that can explain theformation of the apical–basal axis.

Materials and methods

Chemicals, materials and enzymes

The sources of most chemicals and enzymes used weregiven earlier (Krasko et al. 2000).

Sponges and cDNA libraries

Specimens of L. baicalensis (Porifera, Demospongiae,Haplosclerida) were collected in Lake Baikal near thevillage Listvianka (Russia) from depths between 2 and12 m. In the more shallow zone (2 to 4 m), this spongegrows as approx. 2-cm-thick crusts of 2 to 50 cm indiameter without any branches (Fig. 1a). Slightly deeper,this species forms protrusions from the crusts (Fig. 1b);their cylindrical diameter is less than 2 cm. The outerappearance of the branches already shows that they are“segmentally” organized and the units (modules) arearranged on top of each other in longitudinal direction. Indepths of more than 6 m, some branches fuse laterally(Fig. 1c) and specimens grow to 50 to 80 cm in height.Deeper than 10 m, the sponges are rarely less than 80 cmhigh and the tendency to fuse and to form flattened, fan-shaped branches increases (Fig. 1d). In this habitat, thearborescent growth form dominates and only very seldom isa crust seen at the base of specimens.

Skeletons of sponges have been isolated by immersingthe specimens in 5% hypochloric acid. The cDNA libraryfrom L. baicalensis was prepared in TriplEx2 vector (BD,Palo Alto, CA). Where indicated, RNAwas isolated eitherfrom tissue of the top region of the branches (the topmodule), the middle region, or the base (see Fig. 1c).

Furthermore, RNAwas extracted from specimens of themarine sponge S. domuncula (Porifera, Demospongiae,Hadromerida) which were kept in aquaria in Mainz.

Microscopical analysis

Scanning electron microscopy (SEM) analysis of spiculeswas performed with a Zeiss DSM 962 digital scanningmicroscope (Zeiss, Aalen, Germany). The samples weremounted onto aluminum stubs (SEM-Stubs G031Z, Plano,Wetzlar, Germany) that had been covered with an adhesivecarbon tab (Leit-Tabs G3347, Plano). Then, the sampleswere sputtered with a 20-nm-thin layer of gold in argonplasma (Bal Tec Med 020 coating system, Bal Tec, Balzers,Liechtenstein). Digital light micrographs have been takenwith a VHX-100 microscope (Keyence, Neu-Isenburg,Germany), equipped with zoom lens 25×175.

Identification of the L. baicalensis silicateinand cathepsin cDNAs

Recently, the first cDNAs for silicatein and cathepsin hadbeen identified in L. baicalensis by polymerase chainreaction (PCR) (Kaluzhnaya et al. 2005b). The cDNAlibrary in TriplEx2 from this species was used. For theisolation of the cDNAs, a forward primer matching thecloning vector was chosen (5'-GTCTACCAGGCATTCGCTTCAT-3'), together with the degenerate reverse primer (5'-CCAGCTA/GTTC/TTTA/GACAAGCCAGTA-3') whichwas designed against the conserved region (around thethird amino acid [asparagine] of the catalytic triad) in knownsilicatein sequences. In L. baicalensis silicatein-α (accession

Fig. 1 Habitus of L. baicalensisat different depths of LakeBaikal. a Irregular crusts areformed in depths between 2 and4 m. b The arborescent growthpattern is seen below 4 m. Fromthe 2-cm-thick crusts, branchesemerge which show the modulararrangement of the tissue units.c Fusion of the branches startbelow 7 m and the bases of thespecimens are still embedded inthe crusts from where thebranches develop. d Finally, themorphology of the brancheschanges from cylindrical toflattened (photo taken at 12 m).Size bars are given

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number AJ872183), this region is present between nt955 andnt990 (corresponding to aa288 to aa299). The PCR product ofapproximately 1 kb was isolated, cloned into the pCRII-TOPO vector (Invitrogen, Carlsbad, CA, USA) andsequenced. PCR was carried out at an initial denaturationat 95°C for 4 min, followed by 30 amplification cycles at94°C for 30 s, 59°C for 45 s, 70°C for 1.5 min, and a finalextension step at 70°C for 10 min. The reaction mixture wasas described earlier (Wiens et al. 1998). Among the 25 clonesanalyzed, four isoforms of silicatein-α (now termedsilicatein-a1 to -a4) and two isoforms of cathepsin L(cathepsin L1 and L2) were found. The cDNAs obtainedwere completed by designing sequence-specific primers,together with TriplEx2 vector primers.

Mannose-binding lectin

This cDNAwas obtained by PCR with degenerate primersdirected against the conserved region in other D-mannose-binding lectins (Pfam_fs:B_lectin [pfam01453.11; http://myhits.isb-sib.ch) which reads DGNFVVY (aa32 to aa38,e.g., in the fish T. rubripes, accession number BAC57043;Tsutsui et al. 2003); the following forward primer wassuccessful: 5'-GAC/TGGIAAC/TTTC/TGTIGTITAC/T-3'.The reverse primer was designed against the TriplEx2vector. The PCR conditions to obtain the complete LBMBLclone were the same as described above (silicatein andcathepsin cDNAs).

Mago nashi cDNA

Themago nashi proteins in triploblastic animals display highsequence similarity especially at the N-terminal region.Therefore, a degenerate forward primerwas designed againstthe human mago nashi protein (NP_060518), specificallyagainst the segment aa21 to aa28 (primer: 5'-CAC/TGAA/GTTC/TCTIGAA/GTTC/TGAA/GTTC/T-3'). Togetherwith the vector primers, the complete sequence (LBMAG-NA) was obtained by PCR isolation and subsequent se-quencing. The cDNAwas isolated also from S. domuncula.

Sequence analyses

The sequences were analyzed using the programs http://www.ncbi.nlm.nih.gov/blast/blast.cgi and http://www.ncbi.nlm.nih.gov/BLAST/fasta.html. Multiple alignmentswere performed with CLUSTAL W Ver. 1.8 (Thompsonet al. 1994). Phylogenetic trees were constructed on thebasis of amino acid sequence alignments by neighbor-joining, as implemented in the “Neighbor” program fromthe PHYLIP package (Felsenstein 1993). The distancematrices were calculated using the Dayhoff PAM matrixmodel as described (Dayhoff et al. 1978). The degree ofsupport for internal branches was further assessed bybootstrapping (Felsenstein 1993). The graphic presenta-

tions of the alignments were prepared with Genedoc(http://biowww.net).

RNA preparation and Northern blot analysis

RNA was extracted from liquid-nitrogen-pulverized tissuewith TRIzol Reagent (GibcoBRL, Grand Island, NY) asdescribed (Grebenjuk et al. 2002), and then re-purifiedusing the SNAP Total RNA Isolation Kit (Invitrogen). Anamount of 5 μg of total RNA was electrophoresed andblotted onto a Hybond-N+ nylon membrane (Amersham,Little Chalfont, Buckinghamshire, UK). Hybridization wasperformed with the following probes: for silicateinLBSILICAa1 (nt121 to nt388), LBSILICAa2 (nt100 to nt397),LBSILICAa3 (nt16 to nt306), and LBSILICAa4 (nt105 tont366), for cathepsin L1 LBCATL1 (nt54 to nt350), for MBLLBMBL (nt50 to nt411) and for mago nashi LBMAGNA (nt28to nt514). As a reference, the housekeeping gene, α-tubulin(LBTUB), was used. This cDNAwas identified earlier as apart, and was now completed (accession numberAJ971711). From this cDNA, the probe ranging fromnt52 to nt279 was prepared. The probes were labeled withthe PCR-DIG-Probe-Synthesis Kit according to the“Instruction Manual” (Roche, Mannheim, Germany).After washing, DIG-labeled nucleic acid was detectedwith anti-DIG Fab fragments [conjugated to alkalinephosphatase, dilution of 1:10,000] and visualized bychemiluminescence technique using CDP according tothe instructions of the manufacturer (Roche). The screenswere scanned with the GS-525 Molecular Imager (Bio-Rad, Hercules, CA, USA).

In one series of experiments, total RNA was isolatedfrom different regions of a sponge, the top, the middle, andthe base. Amounts of 1 g of tissue each were used for theisolation of RNA which was subsequently quantitated,subjected to 1% agarose gel electrophoresis, and finallystained with ethidium bromide. In a parallel experiment,RNA was extracted from S. domuncula and processed inthe same way.

For the comparison of the expression of the LBMBLtranscripts, RNA was extracted from different animals.After purification, the RNA was used as template for thereverse transcription PCR (RT-PCR) as described (Schröderet al. 2003). cDNA was prepared using the SuperScriptreverse transcriptase and oligo (dT) as described in theprotocol (Invitrogen, Carlsbad, CA, USA). Then, RNAwasdigested with RNase and a cDNA segment was amplifiedwith the forward primer nt50–71 in combination with thereverse primer nt354–377. The size of the product wasapproximately 300 bp and was separated by electrophoresison agarose gel followed by staining with ethidium bromide.

In situ hybridization

In situ hybridization was performed with digoxigenin-labeled (DIG) ssDNA probes. Labeling was carried outwith the “PCR DIG Probe synthesis Kit” (Roche). Specific

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ASILCAa1_LUBAI ------------------MRSIILFSLASLAMAV--QYEFVEEWHLWKGQNNKRYASELEELERHAIWLSNKKYVEEHNARADAFGYT 68SILCAa2_LUBAI ------------------MRSILFLGLIGLAAAL--NCQFAEEWHSWKGSYGKSYASELEELERHSVWLSSRKYIEEHNAHSDVFGYS 68SILCAa3_LUBAI MGDRGPCTVKSLEGMLNVFIALGFLCLVELTYAAPDRYENLQEWSVWKGHHQRSYESQLQEMERHSIWVANKKYIEHHNANADLFGYT 88SILCAa4_LUBAI ------------------MKLILLLGLASISMAF--SYEFVEEWHLWKGQHQKSYMSGLEELERHSIWLSNKKYIEEHNAHSDDFGYT 68CATL1_LUBAI ------------------MKVLILLALVAAATAF----DFPEEWESWKKEHGKVYNSDREELTRHIIWQANRKYVDEHNAHAEKFGFT 66CATL2_LUBAI ------------------MKLLILCTLIAAVAAF----DFSKELRAWKAEHGKSYRNHKEEMLRHVTWQANKKYIDEHNQHAGVFGYT 66 SIGNAL {} SILCAa1_LUBAI LAMNHLGDLSAEEYVEQYLTNARGSNEHREMKAFLAPKGVQYAESIDWRTKGAVTSVKYQGQCGASYAFAATGALEGASALANDKQVT 156SILCAa2_LUBAI LAMNHFGDMSEVEFKDAFLTHEPGNYTSRGIATFKAPQGMKYVDSIDWRTKGAVTSVKNQGDCGASYAFAATGTMEGANALSNDKQVS 156SILCAa3_LUBAI LAMNGFGDLMSAEFTERYLTHKHSQ--RSGLQTFESPKGVTYADSLDWRTRGVVTSVQSQGQCGSSYAFAAAGALEGATALAADKLVA 174SILCAa4_LUBAI LAMNHFGDLSTEEYNAMYLTHDPGNYTHHGRKAFRTPKGVQYVDSIDWRTKGAVTSVKYQGQCGASYAFAATGALEGASALSNDKQVI 156CATL1_LUBAI VGMNQFADLESSEFGRLYNGYNNKPSMKKAQSKVFSTKVGDLPTSVDWRTKGFVTAIKNQGQCGSCWAFSAVAGLEGQHFNATGTLVS 154CATL2_LUBAI LKMNQFGDLENSEFKSLYNGYRMSNAPRKGKPFVPAARVQDLPASVDWSKKGWVTPVKNQGQCGSCWSFSATGSMEGQHFNATGTLMS 154 PRO [] # CT-Ser/Cys SILCAa1_LUBAI LSEQNIIDCSVPYGNHGCSGGDTYTAFKYVIDNGGIDTESSYSFKGKQSSCQYNNKTSGASATGVVSIGYG-SESDLLAAVATVGPVA 243SILCAa2_LUBAI LSEQNIIDCSVPYGNHGCSGGDTYTAIKYVVDNGGIDTESSYSFRGKQSSCQYNSKNSGASATGAVGIPYG-SESDLMAAVATVGPVA 243SILCAa3_LUBAI LSEQNIIDCSVPYGNHGCSGGDVYTAFKYVVDNGGIDTESSYPYKGKKSSCQYNSKNVGAISTGVVKIASG-SETDLLSAVASVGPIA 261SILCAa4_LUBAI LSEQNIIDCSVPYGNHGCSGGDTYTAMKYVIDNGGIDTESSYSFQGKQSSCQYSSKNSGASATGVISIASG-SETDLFAAVATVGPVA 243CATL1_LUBAI LSEQNLVDCSTAEGNQGCNGGLMDNAFQYVIKNGGIDTEASYPYKAVDQKCKFNAANVGSTCSGFSDILPHKSEAALQVAVAVVGPIS 242CATL2_LUBAI LSEQNLVDCSAAEGNHGCNGGLMDDAFEYVIKNNGIDTEASYPYRAVDSTCKFNTADVGATISGYVDVTKD-SESDLQVAVATIGPVS 241 SILCAa1_LUBAI VAVDANTNAFRFYQSGVFDSSSCSSTKLNHAMLVTGYGSYNGKDYWLVKNSWSKNWGDSGYILMVRNKYNQCGIASDALYPML 326SILCAa2_LUBAI VAVDANTNAFRFYQSGVFDSSTCSSTKLNHAMLVTGYGSYNGKDYWLVKNSWGKYWGDNGYIMMVRNKYNQCGIASDALYSML 326SILCAa3_LUBAI VAVDASVNAFMFYQSGVFDSSTCSTSKLNHAMLVTGYGSTNGKDYWLVKNSWGTGWGESGYIKMVRNKYNQCGIASDALYPML 344SILCAa4_LUBAI VAVDANTNAFRFYQSGVFDSSSCSNTKLNHAMLVTGYGSYNGKDYWLVKNSWSKNWGDNGYIMMVRNKYNQCGIATDALYPTL 326CATL1_LUBAI VAIDASHTSFQLYKSGVYSESACSQTSLDHGVTAVGYDSSSGVAYWIVKNSWGTTWGQAGYIWMSRNKNNQCGIATAASYPIVSK 327CATL2_LUBAI VAIDASHISFQFYSSGVYDPLICSSTNLDHGVLAVGYGTDGSKDYWLVKNSWGASWGMSGYIEMVRNHNNKCGIATSASYPVV 324

=====Ser== # CT-His # CT-Asn +++++++++++++

B

378521

503999

1000

999917

990

1000

890899

651

475

391

PAPAIN_ARATH

CATL_CAEEL

CATL_HUMAN

CATL_DROME

SILCAb_SUBDO

SILCAa3_LUBAI

SILCAa4_LUBAI

SILCAa2_LUBAI

SILCAa1_LUBAI

SILCAb_TETHYA

SILCAa_SUBDO

SILCAa_TETHYA

CATL_SUBDO

CATL_GEOCY

CATL2_LUBAI

CATL1_ LUBAI

CATL_APHRVAS

SILICATEINS

Fig. 2 Silicateins and cathepsins from L. baicalensis. a The fourdeduced silicatein sequences of the isoform silicatein-α [-α1, -α2, -α3,and -α4] from L. baicalensis (SILICAa1_LUBAI, accession numberAJ872183; SILICAa2_LUBAI, AJ968945; SILICAa3_LUBAI,AJ968946; SILICAa4_LUBAI, AJ968947) and the two cathepsin Lsequences (CATL1_LUBAI, AJ96849; CATL1_LUBAI, AJ968951)were aligned. Residues conserved (similar or related with respect totheir physico-chemical properties) in all sequences are shown inwhite on black and those in at least two sequences in black on gray.The characteristic sites in the sequences are marked: the catalytictriad (CT) amino acids, Ser (#) in silicateins and Cys in cathepsin, aswell as His (#) and Asn (#), and the processing site for theconversion of the proenzyme to the mature enzyme ([] PRO). Theserine cluster ([∼Ser∼]) and the cleavage site of the signal peptideare indicated ({}). The region towards which the primer wasdesigned is highlighted (+++). b The slanted phylogenetic tree was

constructed after the alignment of these sequences together withsilicatein-á from S. domuncula (SILICAa_SUBDO; CAC03737.1),from Tethya aurantia [T. aurantium] (SILICAa_TETYA,AAD23951), and with the β-isoenzymes from S. domuncula(SILICAb_SUBDO, AJ547635.1) and T. aurantia (SILICAb_TETYA, AF098670), as well as with the cathepsin L sequencesfrom sponges S. domuncula (CATL_SUBDO, AJ784224), G.cydonium (CATL_GEOCY, Y10527), and A. vastus (CATL_APHRVAS, AJ968951) as well as from higher Metazoa: human(CATL_HUMAN, X12451), D. melanogaster (CATL_DROME,S67481) and from C. elegans (CATL_CAEEL, NP_507199.1), andthe related papain-like cysteine peptidase XBCP3 from A. thaliana(PAPAIN_ARATH, AAK71314) [outgroup]. The numbers at thenodes are an indication of the level of confidence for the branches asdetermined by bootstrap analysis [1,000 bootstrap replicates]. Thetree has been rooted with the plant enzyme as an outgroup

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DNA oligonucleotide probes were constructed: the silica-teins LBSILICAa1 (nt121 to nt388), LBSILICAa2 (nt100 tont397), and the mannose-binding lectin LBMBL (nt50 tont411). The hybridization method was described previously(Polak and McGee 1998) with modifications (Perović et al.2003). Eight-micrometer-thick cryosections (on “Silane-prep slides”) were fixed with paraformaldehyde andwashed with PBS at room temperature. The sectionswere incubated with 1 μg/ml of proteinase K for 30 min atroom temperature. After treatment, the sections were fixedagain with paraformaldehyde and then hybridized withDIG-labeled antisense and sense ssDNA probes. Senseprobes were always used in parallel as negative controls inthe experiments. After blocking, the sections were in-cubated for 1 h at 37°C with an anti-DIG antibody con-jugated with alkaline phosphatase. After two washes, thedye reagent NBT/X-Phosphate in a Tris buffer (100 mM

Tris/HCl, pH 9.5; 100 mM NaCl and 50 mM MgCl2) wasused to visualize the signals.

Analytical method

RNAwas quantitated according to the method described byMoffatt and Khorana (1961).

Results

Diversity of silicatein-α and cathepsin L genes

As outlined under Materials and methods, the differentisoforms of silicatein were identified by PCR techniqueusing one degenerate primer binding to the cDNA coding

AMBL_LUBAI MSSTNTLYAGQALVPGGKLVSTNGKYVLIYQKDGNLVGYAASTPFWASGPQTASPLMAVM 60LECs_TAKRU -MSINVLEKGSELKRGDSVLSKNSKWIALFQHDGNFVVYR-TEPVWASDTSGMDPTRLCM 58LECi_TAKRU -MSVTVLENGSELKRGDSVLSKNSQWIALFQHDGNFVVYR-TEPVWASDTSGMDPTRLCM 58LEC_TETNIG -MSINVLDKGTEWKKGDFVLSKNGEWKAVFQEDGNFVVYG-WQPVWASDTGGLDPTRLCM 58LEC_LOPSET -MSRNYMSKNDELRAGDYLMSNNGEWKAVFQDNGNFVIYG-WTPTWASNTCEVGGYRLCL 58 [MBL~~~~~~~~~~~~~~~MBL] [MBLMBL_LUBAI QADGNFVLYDVKMNQYWASNTGGVGNPPNFKIFMQDDRNIVIYDNDHKPTFASNTDTPGN 120LECs_TAKRU QGDCNLVMYNDEDKPRWHTNTS-KGGCKTCVLSLTDEGKLVLEKDGHQ-LWNSDRDHGMK 116LECi_TAKRU QEDCNLVMYNDEDKPRWHTNTS-KGGRNTCVLSLTDEGKLVLKKDCQQ-LWNSDRDHGMK 116LEC_TETNIG QADCNLVMYNPEEKPRWHTNTA-KGSCDTCTLYLTDQGKLVLKKNGNE-IWNSDHNHGMK 116LEC_LOPSET QQDCNLVMYDVGGKAVWHTNSS-KSAPNMCRLQLTDKGSLVVYREGES-IWKSED----- 111 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~MBL]

ARK3_ARATH

CG15327_DROME

LEC_LOPSET

LEC_TETNIG

LECs_TAKRUB

LECi_TAKRUB

1000

1000

1000

MBL_LUBAI

878

878

B

Fig. 3 Mannose-binding lectin (MBL). a The L. baicalensis MBL(MBL_LUBAI) is aligned with the skin mucus lectin from the fishT. rubripes (LECs_TAKRUB, BAC57043; Tsutsui et al. 2003), theintestine mucus lectin from T. rubripes (LECi_TAKRUB,BAC57044; Tsutsui et al. 2003), the putative lectin from T.xnigroviridis (LEC_TETNIG, CAG10253), and the fish skinmucus lectin L. setigerus (LEC_LOPSET, BAD90685). The motifs

for D-mannose-binding lectins (MBL) are marked. b The slantedphylogenetic tree was constructed using the alignment of thesesequences together with the next closest, but distantly related,sequences from D. melanogaster (the putative protein CG15327-PA [CG15327_DROME, AAF46280]) and A. thaliana (the ARK3product protein [ARK3_ARATH, AAB33487])

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for the third amino acid of the catalytic triad of the papainsuperfamily of the cysteine proteases. Besides the fourisoforms of silicatein, two cathepsins were also identified.All silicatein sequences share the highest sequence sim-ilarity to the silicatein-α forms (termed here silicatein-a),and the two cathepsins to cathepsin L.

Silicatein-a1 The open reading frame (ORF) of 1,116-nt-long sequence LBSILICAa1 ranges from nt60–62 tont1038–1040(stop). Northern blot analysis revealed a tran-script size of 1.2 kb (see below), indicating that thecomplete sequence was identified. The deduced proteintermed SILCAa1_LUBAI comprises 326 aa (Fig. 2a),corresponding to a calculated size of 35,686 Da. The aminoacids of the cysteine proteases catalytic triad, Ser (aa124),His (aa273), and Asn (aa293), are found at positions whichmatch those described for the other silicateins (Shimizuet al. 1998; Schröder et al. 2005). The serine cluster (Shimizuet al. 1998) is present between aa258 and aa268; the predictedsignal peptide (aa14 and aa15) as well as the cleavage site forthe conversion of the proenzyme to the mature enzyme(cleavage site between aa110 and aa111) are indicated inFig. 2a.

Silicatein-a2 The cDNA for LBSILICAa2 is 1,196 nts longand has the ORF between nt72–74 to nt1050–1052(stop); thededuced protein SILICAa2_LUBAI has a size of 35,422 Daand a transcript size of 1.3 kb. The characteristic sites areidentical to those found in SILICAa1_LUBAI.

Silicatein-a3 The cDNA, LBSILICAa3, with 1,134 nts,comprises the longest ORF (nt25–27 to nt1157–1059(stop);SILICAa3_LUBAI) with 344 amino acids, correspondingto 37,344 Da. Interestingly, this protein has a longer signalpeptide, spanning the region aa1 to aa33.

Silicatein-a4 This 1,199-nts-long cDNA, LBSILICAa4,has the ORF within nt69–71 and nt1047–1049(stop) coding forthe 326-aa-long deduced protein SILICAa4_LUBAI; thecalculated size is 35,799 Da.

In as much as for the identification of the silicateins inthe cDNA library a degenerate primer was used which wasdesigned against the conserved region coding for thesilicateins and cathepsins L, one further cathepsin L cDNAwas also found.

Cathepsin L1 The first L. baicalensis cathepsin L sequence(AJ877019) already described is now termed LBCATL1(Fig. 2a). In contrast to the silicateins, cathepsins L havecysteine as the first amino acid in the catalytic triad insteadof serine.

Cathepsin L2 The second cDNA identified (LBCATL2) with1,114 nts has its ORF between nt56–58 to nt1028–1030(stop); thededuced size is 35,449 Da.

Phylogenetic analysis was performed with the newsilicateins from L. baicalensis as well as with the silicateinsα and β from the marine sponges (Demospongiae) S.domuncula and T. aurantium together with proteins similar

to the silicateins, the two cathepsins L from L. baicalensisas well as those from S. domuncula and another marinesponge, Geodia cydonium, and the cathepsins from human,Caenorhabditis elegans andDrosophila melanogaster. Thetree was rooted with the ancestor of the cathepsin/silicateinenzyme family (Mort 2002), the papain cysteine peptidasefrom Arabidopsis thaliana (Fig. 2b). The slanted treeshows that the cathepsin L sequences branch off first, whilethe silicateins evolved from a common ancestor with thecathepsins L. The tree also indicates that the cathepsin Lfrom the other class of Porifera, the hexactinellidAphrocallistes vastus, likewise derived from a commonancestor of all metazoan cathepsins L.

L. baicalensis mannose-binding lectin

The complete cDNA sequence of the mannose-bindinglectin (LBMBL), 548 nts long, was obtained as describedunder Materials and methods. The protein (MBL_LUBAI)was deduced from the ORF, which ranges from nt49–51 tont409–411(stop); the size was calculated to be 12,961 Da. Thecharacteristic motifs for D-mannose-binding lectins arefound also in the sponge protein (Fig. 3a). The spongeMBL shares highest sequence similarity to the relatedmolecules from the fishes T. rubripes (skin and intestine

A

BSubDo

topmiddle

base

28S23S18S

16S

a b c d

500

bp

200100

300

1 2 3 4 5 65 m 10 m

LuBai:

Fig. 4 Mannose-binding lectin. a Separation of total RNA from L.baicalensis by agarose gel electrophoresis. Samples from RNAwereextracted from three different regions: top (top module; lane a) andmiddle region (lane b) as well as from the base (basal module; lanec). Aliquots, corresponding to 50 mg of tissue, were loaded onto thegel. After size fractionation, the gel was stained with ethidiumbromide. In parallel, an RNA sample from the marine sponge S.domuncula was analyzed (lane d). b Expression of LBMBL indifferent specimens of L. baicalensis. Specimens (six accordinglynumbered animals]) were collected from depths of 5 to 10 m andused for extraction of RNA. Then, RT-PCR (using specific primerstowards LBMBL) was applied and the products separated byagarose gel electrophoresis. After staining of the products withethidium bromide, the expected sizes of the fragments (300 bp) wereidentified

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mucus lectin, Tsutsui et al. 2003), T. nigroviridis, and L.setigerus (Fig. 3b).

To prove that this molecule (mannose-binding lectin)exists in all sponge specimens, the LBMBL transcripts wereprobed in six different animals (Fig. 4b). RNA was ex-tracted and the transcripts were identified by RT-PCR. In allspecimens, the expected PCR product of a size of 300 bpwas identified; samples from animals collected in depths of5 m (animal 1–3) and 10 m (animal 4–6) were used.

L. baicalensis mago nashi

The mago nashi sequence was identified by screening thecDNA library with a degenerate primer. The completecDNA (LBMAGNA) is 649 nts long and comprises oneORF between nt95–97 and nt533–535(stop) coding for 148 aa(Fig. 5a). The calculated size of the protein (MAGNA_LUBAI) is 17,107 Da. No characteristic domain has beenassigned to mago nashi. The sequence similarity to therelated metazoan mago nashi sequences is high; thehighest score is found in the human protein (NP_060518),with an expect value of (“Expect value [E]”; Coligan etal. 2000) 4e−67.

For a phylogenetic analysis, the mago nashi sequencefrom L. baicalensis was aligned with the S. domuncula

mago nashi (cDNA was isolated in this study) as well aswith the proteins from D. melanogaster (the mago-nashi-related Y14 core protein), C. elegans (ce-Mago protein),Saccharomyces cerevisiae (transcription initiation proteinSPT6), and from A. thaliana (mago-nashi-like protein).The slanted tree shows that the two sponge mago nashis areclosely related and form one branch with the insect andhuman sequences (Fig. 5b).

Growth forms of L. baicalensis

The outer appearance/habitus of the specimens dependsstrongly on the depth where they grow. In very shallowwater, the specimens occur only as crusts (Fig. 1a). Deeperthan 5 m, cylindrical branches emerge from the crusts(Fig. 1b) which start to fuse (Fig. 1c), forming flattened,fan-like branches.

Longitudinal cross-sections through the skeleton of L.baicalensis (Fig. 6a) show the pronounced apical–basalorganization of the branches into modules of approxi-mately 1 cm in longitudinal extension. The modularorganization is determined by the arrangement of spiculebundles; the ascending, longitudinal bundles of onemodule originate from the annulus, which is formed by adense fusiform arrangement of the bundles. This annular

A MAGNA_LUBAI --MAGEFYVRYYVGHKGKFGHEFLEFEFRPDGRLRYANNSNYKNDTMIRKEVTVSPLVMDELKKIIDDSEVLKE 72MAGNA_HUMAN MAVASDFYLRYYVGHKGKFGHEFLEFEFRPDGKLRYANNSNYKNDVMIRKEAYVHKSVMEELKRIIDDSEITKE 74 MAGNA_LUBAI DDKQWPVPDRVGRQELEIVMGDSHISFTTTKIGSLLDCKNSKDEEGLRVFYYLVQDLKCFVFSLIGLHFKIKPI 146MAGNA_HUMAN DDALWPPPDRVGRQELEIVIGDEHISFTTSKIGSLIDVNQSKDPEGLRVFYYLVQDLKCLVFSLIGLHFKIKPI 148

MAGNAl_ARATH

SPT6_YEAST

MAGNA_CAEEL

663

MAGNA_HUMAN

MAGNA_DROME

997

MAGNA_LUBAI

MAGNA_SUBDO

910

373

373

B

Fig. 5 Mago-nashi protein from L. baicalensis (MAGNA_LUBAI).a This deduced protein is aligned with the corresponding proteinfrom human (MAGNA_HUMAN; NP_060518). b After alignment,the slanted phylogenetic tree was constructed from these sequencestogether with the S.domuncula mago nashi protein (MAGNA_SUBDO, AM086401), the mago-nashi-related Y14 core protein

from D. melanogaster (MAGNA_DROME, 1RK8_B), the ce-Magoprotein from C. elegans (MAGNA_CAEEL, AAB66721), thetranscription initiation protein SPT6 from S. cerevisiae (SPT6_YEAST, P23615), as well as the mago-nashi-like protein from A.thaliana (MAGNAl_ARATH, AAM65009)

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zone is concavely curved in longitudinal direction towardsthe tip of the branch. It is remarkable that the curvature issteeper in the middle of a branch (Fig. 6c) while at the tip(Fig. 6b) or the base of a branch (Fig. 6d), the vaults areflatter. This architecture is especially obvious in the basalregion of the branches (Fig. 6d). As mentioned, the archesare less pronounced at the end of the first module, whilealready the second and from thereon towards the tip of thebranches, all modules are delimited by dome-like annuloidzones.

Arrangement of the spicules within the bundles

Digital light microscopical analysis shows (Fig. 7) that theannuli, which separate the modules, are composed of aramified network of longitudinal and traverse bundles

which confer a bright appearance to the section (Fig. 7a).At higher magnification, it becomes obvious that the annuliare formed, besides of longitudinal bundles, also bytraverse bundles (Fig. 7b–d). This network is also seen atthe top of the branches (Fig. 6b).

The spicules of L. baicalensis are megascleres with sizesof up to 180×10 μm that have a uniform shape; they are

Fig. 6 Axial and apical–basal orientation of the modules along thebranches of L. baicalensis. The branches develop either from thenon-structured crusts or as shown in one specimen. a From anotherbranch which had lost its vertical orientation by changing thesubstratum onto which the specimen grows. b A cross-sectionthrough a tip of a branch reveals that the top module (mo) originatesfrom a less bent arch, the annulus (an), compared to the previousones. It becomes obvious that the articulated appearance of thebranches is due to the dense arrangement of the spicule bundles inthe annuloid zone. c Occasionally, in the middle part of thebranches, ramifications occur which originate from the preformedarches (>) of a module. d Base of the branch. The first basal module(1) of a branch is only slightly bent while the second (2) and third(3) modules are delimited by very pronounced annuloid (annular)zones. Size bars are given

Fig. 7 Microscopical analysis of the skeleton. Digital lightmicrographs through a longitudinal section of a branch. a Thelower magnification shows the two modules (mo) which areseparated from each other by an annulus (an). b–d At highermagnification, it is seen that the longitudinal bundles (lo) arefortified by traverse bundles (tr). The size bars are given

Fig. 8 Scanning electron microscopy analysis of sections throughL. baicalensis. a At the annulus, the transition from the ramifiedlongitudinal bundles of the lower module (l-mo) to the upperbundles in the newly formed module (u-mo) is seen. b At highermagnification, the branching of one longitudinal bundle (lo) to atraverse bundle (tr) can be observed. The spicules are surrounded bya proteinaceous layer. c At the top (to) of the branches, the spiculesare not embedded into a protein matrix. d Free spicules, spinedoxeas, are shown. Size bars are given

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oxeas and in the adult form are covered with spines(Fig. 8d). Within the animal, they are embedded in anorganic matrix through which they are fixed in an orderedskeleton (Fig. 8a and b); thus, bundles are formed whichrun ascending and longitudinal towards the annuli(Fig. 8a). Tangential bundles cross-link the longitudinalbundles at the end of a module (Fig. 8b); thus, a highlyordered arrangement of the bundles is established. Eachmodule is delimited by the annuloid zones (Fig. 8a). At thebeginning of a new module, close to the annular zone of theprevious, the bundles are hardly interconnected by cross-braces, while at the border to the following module traversebraces are formed. At the top of a branch, the spicules

protrude freely into the environment and are not embeddedin an organic matrix (Fig. 8c).

Differential expression

Total RNATotal RNAwas extracted and used for Northernblot experiments. From the top of the animals, 0.9 mg ofRNA per gram of wet tissue was extracted; the yield in themiddle part was 1.9 mg/g and in the base region 0.3 mg/g.As shown in Fig. 4a (lanes b–d), separation of total RNAfrom L. baicalensis results in two main fractions, cor-responding to 28S rRNA and 18S rRNA, reflecting the

Fig. 10 Expression ofsilicatein-α1 and -α2, as well asof MBL in tissue from a topmodule. The tissue was sec-tioned (cryosections) and sub-jected to in situ hybridizationusing the following antisenseprobes: for silicatein-α1(SILICAa1) (a, d, g) and silica-tein-α2 (SILICAa2) (b, e, h) aswell as for the mannose-bindinglectin (MBL) (c, f). i In onecontrol, the slices were reactedwith a sense probe for silica-tein-α1 (SILICAa1(s)). Thebundles (bu) are composed ofseveral spicules (sp)

MBL

silicatein:

a1

a2

a3

a4

kb

1.2

1.3

1.2

1.2

cathepsin L

mago nashi

tubulin

0.6

0.7

1.2

1.5

kb

topmiddle

base topmiddle

basetopmiddle

base

5 m 10 m

Fig. 9 Northern blot analysis of RNA, isolated from differentregions of the animal: from the top of the branches, the middle part,and the base. RNA was prepared and equal amounts (5 μg) wereloaded onto the gel. The RNA was separated by size and, after blottransfer, finally hybridized with the probes for L. baicalensissilicateins, LBSILICAa1 (a1), LBSILICAa2 (a2), LBSILICAa3 (a3),

and LBSILICAa4 (a4), for mannose-binding lectin LBMBL and formago nashi (LBMAGNA), as well as for cathepsin L1 (L1). Thehousekeeping gene, á-tubulin, was used as a control to show that thesame amounts of RNAwere loaded onto the gels. For the expressionstudy of LBMBL, LBMAGNA, and of cathepsin L1, animals fromdepths of 5 and 10 m had been analyzed

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sizes of the sponge rRNAs. No bands of bacterial 23S or16S rRNA could be detected by this method. Hence, wehave no reasons to assume that the different geneexpression levels are due to interference with a symbiont.In comparison, RNA isolated from S. domuncula con-tained also a small fraction of bacterial rRNA (Fig. 4a,lane a). The signals of the rRNA bands, starting from 1 gof tissue, were highest in tissue from the middle part of theanimal (lane c), while lower levels were detected insamples taken from the upper part of the specimen (laneb); very low were the levels from the base (lane d).According to the staining pattern, the quality of the L.baicalensis RNA was sufficient to perform Northernblotting.

Northern blot analysis RNA was isolated from differentregions of one branch of an animal collected in a depth of8 m, from the top, the middle and the base, and equalamounts of RNAwere size fractionated and hybridized withprobes for silicatein-a1, -a2, -a3 and -a4, as well as forMBL, mago nashi, and cathepsin L, as described underMaterials and methods. As shown in Fig. 9, the steady-statelevel of transcription of all four LBSILICAa genes washighest in the top region of the branches, which representsthe growing zone of the animal. While for LBSILICAa1(15% in comparison to the level in the top tissue [set to100%]), LBSILICAa3 (10%), and LBSILICAa4 (35%)lower levels of expression could be detected in tissuefrom the middle zone of the branches; no transcripts couldbe detected for the LBSILICAa2 gene. Almost parallel withthe expression levels for the silicatein genes is that for the

mannose-binding lectin LBMBL; the largest amount is seenin RNA extracted from the top of a branch (set to 100%),while lower levels are seen in the middle region (15%) andthe base (8%) (Fig. 9). This result has been obtained fromthree different specimens. Animals collected from depths of5 and 10 m had been studied; the level of expression wasalmost identical in these two samples. Also, the steady-statelevel for mago nashi decreases from the top to the base ofthe animals (Fig. 9). In contrast, the expression level forcathepsin L1 was different; the highest level was in the baseof the branch and lowest in the top region. To ensure thatalways the same amount of RNAwas applied onto the gel,the RNAwas hybridized with a probe of the housekeepinggene, α-tubulin. In a further series of Northern blot studies,RNA from the base of animals, collected at 4, 6 to 8 and12 m depth, was prepared and subjected to hybridizationwith LBSILICAa1 to LBSILICAa4, as well as to LBMBLand mago nashi. In no case was any expression of thesegenes seen (data not shown).

In situ hybridization analysis

Cryosections have been prepared through tissue from a topmodule. They were reacted with antisense probes forsilicatein-α1 LBSILICAa1 and -α2 LBSILICAa2, as well asfor the mannose-binding lectin LBMBL. Hybridizationanalysis revealed that the two silicatein genes (-a1 and -a2)were highly expressed in cells around the bundlescomposed of spicules (Fig. 10a,b,d,e,g, and h). The MBLprobe displayed the same expression pattern; one to three

adult

apical-basal axis

paired HP

LIM HP

egg

1

2

4

3

module

module

module

base

top

module

serial modules

cathepsin L

silicateins

MBL

mago nashi

larva

ccos

ca

ca

Fig. 11 Schematic view of pattern formation in Porifera. Duringembryogenesis, the fertilized egg develops to a larva with a centralcavity (ca). During and after settlement of the larva, first choanocytechambers (cc) are formed and the body cavity opens up to theexternal milieu through an oscule (os). The growth of the adultproceeds along the apical–basal axis in a compartmental manner.Next to the photo of a branch, a schematic outline is given. The datasuggest that the growth of those sponges which display anarborescent morphology, like the freshwater sponge L. baicalensis,

proceeds by addition of serial modules along the body axis. Theexpression levels of those genes that code for structural proteins (thefour silicateins and the MBL) as well as the mago nashi follow anapical–basal gradient; while the expressions of the catabolic enzymecathepsin L and the housekeeping gene tubulin follows an oppositedirection or remains constant. In addition, it is proposed that thebody axis is controlled by paired-class and LIM-class homeopro-teins (HP)

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cells layers which surround the bundles hybridized with theantisense MBL probe (Fig. 10c and f).

It is notable that, also within the bundles, the silicateingene is expressed. As illustrated for silicatein-a1, positivereacting cells are found at the surface of the bundles as wellas within those between the spicules (Fig. 10d,e, and h). Incontrast, LBMBL is only expressed around the bundles(Fig. 10f). In rare cases also, single spicules, not arrangedin bundles, were found that are surrounded by cells thatreact with the antisense LBSILICAa1 probe. In controls, thesense probes did not display any significant staining, asshown for silicatein-α1 (LBSILICAa1; Fig. 10i).

Discussion

In triploblastic animals, in Protostomia and in Deutero-stomia, segmentation (almost simultaneous formation ofvery similar functional units along a body axis [in insects])or somite formation (sequential formation of the unitsalong a body axis [in vertebrates]) are characteristicfeatures that are under control of homeotic gene clusters(reviewed in Müller 2005). Despite great efforts withmolecular biological techniques, the search for geneclusters of homeotic genes in siliceous sponges (Hexacti-nellida and Demospongiae) was not yet successful. Hence,the search for mechanisms which might control axisformation in sponges was broadened. It could be demon-strated that in S. domuncula transcription factors, e.g.Brachyury (Adell et al. 2003) or homeodomain molecules[LIM/homeobox protein] (Wiens et al. 2003), exist whichcontrol morphogenetic events during tissue formation.Interestingly, in triploblastic animals, such factors had beenidentified in organizer regions (Grunz 2004). Expressionstudies revealed that in S. domuncula, they are highlyexpressed in the oscule region (Müller 2005). On thegrounds of these findings, it was supposed that the polarity/axis formation in sponges is under genetic control oftranscription factors; an expression gradient along theapical–basal axis exists also for other organizer-specificgenes, e.g. for frizzled receptor, noggin or Iroquois (Mülleret al. 2004; Müller 2005).

In L. baicalensis, the apical–basal axis of the branches isexceptionally well developed. The appearance of L.baicalensis changes depending on the environment; closeto the surface, in depths to 3 m, only encrusting specimensoccur in Lake Baikal. In water deeper than 5 m, the growthpattern is arboreal, branches protrude from a variabledeveloped crust. The encrusting form is generally notthicker than 12 mm, and composed of only one module,which does not grow vertically with serial patterning. Asoutlined in the Introduction, one module is stabilized bylongitudinal main bundles of spicules. Now, we show thatthe annuli which separate the modules sequentially areformed by cross-connections of these longitudinal bundles.The connecting bundles extend in a traverse orientationfrom the main bundles with a regular periodicity ofapproximately 8–12 mm. This morphological description

already suggests that a module represents one 8–12 mmstrong growth unit within a branch of a sponge.

In the focus of the present study was the elucidation ofgenes that encode structural proteins, influencing size andthe delimitation of a module. The basic structural elementswhich keep the organization of the skeleton are the spicules.Therefore, we performed experiments which focused on thedifferential expression of the silicatein genes. In addition,the expression of MBL has been determined; this gene istranscribed/translated to a protein that forms the organiccoats around the spicule bundles.

From marine sponges, two (or perhaps three) groups ofsilicateins (silicatein-α, -β, and perhaps -γ) are known(Cha et al. 1999; Krasko et al. 2000; Schröder et al. 2004).The experiments summarized here present the surprisingfinding that the freshwater sponge L. baicalensis containsat least four isoforms of silicatein-α (silicatein-a1 to a4).The silicatein α isoforms display all characteristic featuresof the silicateins identified in marine sponges (Sumerel andMorse 2003), the catalytic triad, and the Ser-rich region.Interestingly, as concluded from alignments and phyloge-netic trees, the four silicateins are phylogenetically veryrelated, suggesting their emergence by gene duplication.Studies are in progress to elucidate on genomic level ifthese genes are arranged in clusters. L. baicalensis mightcontain even more silicatein genes that are similarlyexpressed as isoforms 1–4. Data available indicate thatmarine sponges do not possess such a diversity/polymor-phism of silicatein genes.

The cDNA for MBL isolated from L. baicalensisshowed high sequence similarity to the sequences fromfishes (T. rubripes, T. nigroviridis, and L. setigerus; Tsutsuiet al. 2003). The motifs for D-mannose-binding lectins arealso present in the sponge lectin.

In addition, we identified the mago nashi gene andstudied its expression pattern, as this protein is known to beinvolved in the transport of mRNAs that are involved inaxis formation (Palacios 2002); furthermore, it was alsoclaimed that this protein functions as a regulator for controlof proliferation (Zhao et al. 1998). The deduced proteinfrom L. baicalensis shares highest sequence similarity tohuman mago nashi. The rational to select the mago nashigene came from recent evidence that cells from spongesalso have a polarity, based on the formation of structures,which are reminiscent of tight junctions, e.g., the scaffoldprotein MAGI and the tetraspan receptor tetraspanin (Adellet al. 2004). In S. domuncula, these are expressed espe-cially in the epithelial layer.

With these molecular tools at hand, it was possible totackle the question if the expression levels of genesencoding structural proteins of the spicule bundles, thesilicateins, MBL, as well as the “regulatory” protein magonashi, differ along the apical–basal axis. Therefore, tissuesamples from the top (tip of the branch), the middle zone,and the base of a branch were taken. The crucial samplesfrom the top and the base of a branch were only 1-cm thickand comprised only one module. The transcripts for thestructural proteins, silicateins and MBL, occurred in high

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abundance in the top module of the branch. No transcriptsfor the silicateins could be detected in the lowest (first)module, and only a small fraction of MBL transcripts wasdetected in the base of the branch. It is interesting to notethat in parallel with the decrease from top to base in thesteady-state level of silicatein and MBL transcripts, theexpression of mago nashi also drops. Future studies shouldaddress the question if mago nashi is involved in axisformation, resulting in the construction of serial modulesalong the apical–basal axis. To demonstrate that thisexpression gradient is not a reflection of a mere generalreduction in overall gene expression capacity, the abun-dance of the transcripts for cathepsin L, the closest relatedprotein to silicateins, and tubulin was determined. TheseNorthern blot studies showed that tubulin was expressedevenly in all three zones, while the level of the transcript forcathepsin L even increased in the basal module.

The outcome of the experiments presented here issummarized schematically in Fig. 11. In triploblasts anddiploblasts, body plan formation starts by polarization ofthe embryo. The subsequent development is followed indiploblasts only to the stage of serial module patterning,while in triploblasts the development to the adult includescompartmental and subsequent segmental patterning(Müller 2005). It has been shown that in sponges, afterfertilization of the eggs, the zygote increases in size anddevelops flagellae. Depending on the taxon, morphologi-cally slightly different types of larvae (amphiblastulae,parenchymellae, or coeloblastulae) are formed and releasedfrom the maternal body (Leys 2003). During this phase, theembryo polarizes, a process which is primarily morpholog-ically obvious in the localization of the cilia and theformation of the body cavity (Leys and Degnan 2002).Then, gastrulation takes place, driven by an asymmetric andtangentially arranged cleavage. The epithelium within thebody cavity invaginates under formation of the choanocytechamber. The embryo becomes sessile and the youngsponge forms an oscule, through which the body cavityopens to the external milieu (Weissenfels 1989); with thisprocess, the sponge establishes an apical–basal axis(Fig. 11). In L. baicalensis, with its pronounced morpho-logical organization into serial modules, the module at theapical pole contains the highest level of transcripts for thestructural proteins, silicateins and MBL. The apical–basalgradients of expression are paralleled by the level of magonashi mRNA. Our findings indicate that gradients ofexpression from the apical to the basal part of the animalexist and suggest further that the expression of these genesallows the establishment of the body plan. It will be a taskfor the future to study if expression of silicatein and MBP iscontrolled by a region-specific differential splicing processmediated by mago nashi.

The expression studies (Northern blotting) were sup-ported by in situ hybridization analysis, which demonstrat-ed that the cells surrounding the spicule bundles, and alsocells within the bundles, contain increased levels ofsilicatein-a1 and -a2 transcripts. This implies that thecells in the organic matrix on the surface and in the centerof the bundles produce the silica-forming enzyme silica-

tein, and are very likely involved in spicule formation.Additionally, the cells in the organic mantel around thebundles produce MBL. We postulate that spicule formationstarts also in L. baicalensis intracellularly in the sclerocytesalong the axial filament, as it is known from S. domuncula(Müller et al. 2005). The completion of spicule formationby appositional deposition of silica proceeds also in concertwith the mannose-binding lectin.

Based on the morphological and gene expression data,future work can now focus on the identification of thosetranscription factors that control the expression of thestructural proteins, the silicateins and the mannose-bindinglectin. The major outcome of the present study is theelucidation that the apical–basal axis of the branches of thearborescently growing freshwater sponge L. baicalensismight be genetically controlled through the expressionlevel of structural proteins. The data may suggest thatsponges as the basal metazoans, which are perhaps lackingclusters of homeobox genes, form the serial modulesthrough a repetitive expression program controlling spiculebundle formation. At present, we postulate that among thetranscription factors involved in this process are the paired-class (Hoshiyama et al. 1998) and LIM-class homeopro-teins (Wiens et al. 2003).

Acknowledgements This work was supported by grants from theDeutsche Forschungsgemeinschaft, the Bundesministerium fürBildung und Forschung, Germany [project: Center of ExcellenceBIOTECmarin], the International Human Frontier Science Program,the WTZ Germany–Russia (German–Russian cooperation throughthe BMBF), and a grant from the Presidium of the Russian Academyof Science (no. 25.5) and from RFBR (no. 03-04-4985).

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