Cell Tiss. Res. 192, 249-266 (1978)

Cell and Tissue Research 9 by Springer-Verlag 1978

Calcification of the Collagenous Axial Skeleton of Veretillum cynomorium Pall. (Cnidaria: Pennatulacea) Philip W. Ledger and Suzanne Franc Laboratoire d'Histologie, Universit6 Claude Bernard, Villeurbanne, France

Summary. The axial skeletal rod of Veretillum cynomorium consists of a fibrillar collagenous matrix calcified with calcite. The present paper describes ultrastructural and crystallographic details of its organization and deposition. At the inferior end of the rod is a calcification gradient between the noncalcified tip and the rest of the axis. Initial mineral deposits, which are sometimes associated with cell debris, give rise to calcitic nodules which enlarge by the radial growth of several lobes. These nodules fuse and form the core of the axis. Subsequent increase in diameter of the rod involves the radial development of irregular columns of calcite which arise from the peripheral nodules. Mineral surfaces exhibit a distinctive microarchitecture which can be related to the predominantly c-axis parallel growth of the calcite. Particular attention is paid to the relationship between mineral and matrix. The collagen fibrils, embedded in the calcite but never impregnated with it, are not responsible for the initial nucleation of mineral. The crystallographic orientation of the calcite also appears to be independent of these fibrils. Key words: Calcification - Calcite - Collagen - Pennatulid. R6sum6. L'axe squelettique de Veretillum cynomorium se compose d'une matrice fibrillaire de nature collag6ne, min6ralis6e par de la calcite. Le pr6sent article fournit des donn6es ultrastructurales et cristallographiques quant & l'organisation de cette structure et au d6p6t du min6ral dans la matrice. La pointe inf6rieure de l'axe constitue un gradient de calcification entre l'extr~mit~ non min6ralis~e et le corps enti~rement calcifi& Les d~p6ts initiaux de calcite, parfois associ6s fi des d6bris cellulaires, donnent naissance ~ des Sendoffprint requeststo: Dr. P. Ledger, Laboratoire d'Histologie,Universit+Claude Bernard, 43 Bvd 11 Novembre, 69621 Villeurbanne, France Acknowledgements:We wish to thank the personnel of the C.M.E.A.B.G. for their technical assistance, Dr. P. Bernier and Dr. K.M. Towe for their crystallographic advice and Mr. A. Bosch for his photographic work. P.W.L. is grateful to Prof. M. Pavans de Ceccatty for accepting him into the Laboratoire d'Histologieand to the Royal Society(U.K.) for financial support. This work was otherwise supported by C.N.R.S. (A.T.P. 651 2421, LA. 244)

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P.W. Ledger and S. Franc nodules qui s'accroissent de fagon rayonnre, par bourgeonnement de plusieurs lobes. Ces nodules fusionnent et forment le coeur de l'axe dont la croissance ult+rieure en diam&re, se r~alise par le drveloppement de colonnes irrrgulirres de calcite/t partir des nodules externes du coeur. Les surfaces minrralis+es prrsentent une microarchitecture qui peut ~tre relire aux axes cristallographiques de la calcite. Les relations entre le minrral et la matrice ont particulirrement retenues notre attention. Les fibres de collagrne, enrobres par la calcite et non imprrgnres par elle, n'interviennent jamais comme initiateur de nuclration du minrral. L'orientation cristallographique ult+rieure de la calcite est aussi totalement indrpendante de la matrice.

The axial skeletal rod o f Veretillum cynomorium was the subject o f an extensive study by Franc et al. (1974). These authors demonstrated that the organic phase of this calcified structure is at least partly collagenous in structure, as earlier X-ray diffraction studies (Marks et al., 1949) had shown for two other pennatulid species. Ultrastructural investigations of the fibrillar matrix showed that although some banded fibrils were present, the most abundant fibril type only rarely showed a transverse banding and therefore could not be unequivocally identified with the physicochemically identified collagen. However, the collagenous nature of these fibrils has since been confirmed by negative staining, which reveals a distinct banding in isolated fibrils, and by electron diffraction of frozen hydrated fibrils, which gives a diffraction pattern comparable to that o f vertebrate collagen (Ledger and Franc, in press; Ledger, Franc and Chanzy, in preparation). At the inferior tip of the skeletal rod mineral deposition apparently involves the formation and fusion of crystalline nodules (Franc, 1976). The rest of the axis is densely mineralized with calcium carbonate, which is present solely in the form of calcite (Franc et al., 1974). The axial rod o f Veretillum is the only authenticated example of a collagenous matrix calcified with calcite, as fibrillar collagen previously thought to be present in sponge and echinoderm skeletons (Travis et al., 1967) was undoubtedly extraskeletal in origin (Ledger and Jones, 1977; Wilbur, 1976). The axis is thus of immediate interest as a naturally occurring contrast to the collagen-hydroxyapatite association typical of vertebrate mineralized tissues. For this reason, and as a contribution to comparative studies of coelenterate calcification, it was decided to investigate the morphologic and crystallographic organization o f the mineral phase and its relationship to the organic matrix, both in the fully formed skeleton and in regions o f continuing calcification.

Materials and Methods

Specimens of Veretillum cynomorium were obtained from the mediterranean coast of France, at Banyuls-sur-Mer, and maintained in a sea water aquarium. For transmission electron microscopy(TEM) freshlyexcisedskeletalrods and adherent tissue were immersedin a fixativecontaining3 ~ glutaraldehyde,0.3 M NaC1and 0.1 M Na cacodylate/HC1buffer at pH 7.8. In this mediumspecimenswerecarefullytrimmedof excesstissue and fixedfor a total of 1 h at 4~C. After thorough rinsingin a bufferedmedium(0.3 M NaC1and 0.2 M Na cacodylate/HC1at pH 7.8)

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material was post-fixed for 30 min at 4~ in a mixture containing 1 ~ osmium tetroxide, 0.43 M NaC1 and 0.1 M Na cacodylate/HCl at pH 7.8. After a rinse in distilled water, material was dehydrated in a graded series of ethanol and embedded in Epon. Thin sectionswere cut with a diamond knife and stained with uranyl acetate and lead citrate. Some material was block stained and decalcified by being left overnight in 2 ~o aqueous uranyl acetate, after fixation but before dehydration. For observation and electron diffraction of the mineral phase, sections were cut onto distilled water saturated with calcium carbonate and were generally left unstained. For scanning electron microscopy (SEM) whole skeletal rods were wiped clean of adherent tissue and cut into severalpieceswhich were fixedas for TEM. Dehydration time was extended, and in 70 ~oethanol the pieces were fractured by hand to expose the required surfaces.After absolute ethanol, specimenswere critical point dried from liquid CO2, mounted on stubs and sputter coated with gold. When only the mineral was being investigated, exposed organic material was removed by suspending unfixed skeletal rods for 20 min in a 10 ~ solution of commercialsodium hypochlorite saturated with calciumcarbonate. After a brief rinse in distilled water, rods were rapidly dehydrated and air dried from absolute ethanol. Some fractured specimenswere etched for 5-10 min in 2 ~oascorbic acid before the sodium hypochlorite treatment. For acid phosphatase histochemistry inferior tips of skeletal rods were pre-fixed for 30 min in glutaraldehyde, rinsed in buffer (both prepared as for TEM) and treated according to the method of Pasteels (1971). Incubation in the Na glycerophosphate medium was for 30min and controls were inhibited with 1 ~ sodium fluoride. The overgrowth of calcite crystals on piecesof skeletal rid, in order to determine the crystallographic orientation of the underlying mineral, was achieved by the method of Jones (1955). A saturated solution of calcium bicarbonate was prepared by bubbling CO2 through a suspension of calcium carbonate in warm distilled water. This mixture was filtered and pieces of sodium hypochlorite-treated axis were immersed in 50 ml of filtrate in a Petri dish. The time taken for a crop of suitable crystals to form was 23 h. The pieces of axis were then briefly rinsed in water, passed through absolute ethanol, and mounted and coated for SEM. Polarising optical microscopy was performed on 30 Ixm polished sections of Epon embedded material. All specimen preparation and electron microscopy was carried out at the 'Centre de Microscopie Electronique Appliqure ~i la Biologie et ~i la Grologie', Universit6 Claude Bernard.

Observations A t its inferior tip, the skeletal rod o f Veretillum is non-calcified (Fig. 1). This flexible tip m a i n l y consists o f collagen fibrils (Fig. 2), which r u n a p p r o x i m a t e l y parallel to the skeletal long axis a n d are generally more closely packed immediately u n d e r the limiting epithelium t h a n towards the centre of the axis. The collagen fibrils are e m b e d d e d in a ' g r a n u l o m i c r o f i b r i l l a r ' matrix which previously has been shown to have staining properties typical of glycoproteins ( F r a n c et al., 1974). A t the very centre o f the axis is usually f o u n d a whorl o f c o n v o l u t e d fibrils associated with dense material which m a y resemble the g r a n u l a r c o m p o n e n t of the interfibrillar matrix (Fig. 3). Variously distributed t h r o u g h o u t the non-calcified matrix are the vesicular a n d m e m b r a n o u s remains o f cell fragments, some o f which react positively to acid p h o s p h a t a s e electron-cytochemical tests (Fig. 4). The smallest, m o s t distal m i n e r a l deposits have straight sided profiles a n d / o r a n g u l a r projections a n d are often associated with the cell debris (Fig. 5). Larger m i n e r a l n o d u l e s show a r a d i a t i n g structure a n d are c o m p o s e d o f several m o r e or less distinct lobes (Figs. 6, 7). In decalcified material, the remains o f m e m b r a n o u s material or large a n g u l a r crystals can be seen at the centre o f nodules. The s u r r o u n d i n g lobes c o n t a i n irregular strands o f organic material (Fig. 6) and, when

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se

4

Fig. 1. Diagram representing a longitudinal section through the inferior tip of the skeletal rod. The extremity consists of noncalcified, fibrillar matrix (ma, shaded). Mineral is first encountered as scattered initial deposits (id) and then as nodules (n). The main part of the axis consists of a core (c) of fused nodules surrounded by massive deposits of mineral (mi). Here, non-calcified matrix is represented only by the thin layer (se) beneath the secretory epithelium (ep). Chimney cell processes (ch) are present in both calcified and non-calcified parts of the axis. Approximately x 80

sectioned transversely, their g r o w i n g extremities are seen to consists o f a n e t w o r k o f t r i r a d i a t e structures (Fig. 8). In thin sections o f non-decalcified m a t e r i a l m u c h o f the m i n e r a l is often a r t e f a c t u a l l y shattered. Nevertheless, in certain well preserved areas it can be seen t h a t the m i n e r a l is n o t h o m o g e n e o u s b u t exhibits a s u b s t r u c t u r e suggestive o f elongate units o r stacks o f units (Fig. 10). These subunits, a term which is p r o v i s i o n a l l y used to refer to u l t r a s t r u c t u r e s whose m o r p h o l o g i c a n d crystallogr a p h i c identity will be discussed later, are parallel to one a n o t h e r a n d to the general direction o f g r o w t h o f the lobes. Selected area electron d i f f r a c t i o n o f such images i n v a r i a b l y shows the c r y s t a l l o g r a p h i c [001] axis (the c-axis o f calcite) to be in the p l a n e o f the section a n d parallel to the subunits (Fig. 10a).

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Fig. 2. Transverse section through the non-calcified tip. The fibrillar collagenous matrix (rna) contains cell debris (cd) and densely staining material (dm). Outside the epithelium (ep), with its chimney cell extensions (ch), is the fibrillar mesogloea (me). x 2,600 Fig. 3. Detail of the dense material (din), collagen fibrils ( f ) and interfibrillar matrix (/jr). x 34,000 Fig. 4. Reaction product of the acid phosphatase technique (e.g. arrow-heads) preferentially labels cell debris. Arrow indicates where the unit membrane is particularly well visible, confirming the cellular origin of the debris, f, collagen fibril, x 48,600 Fig. 5. The smallest mineral deposits (id) are often found associated with membranous, vesicular cell debris, x 32,000

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G r o w i n g nodules initially cause surrounding collagen fibrils to separate, but extension o f the mineral phase t h r o u g h the matrix soon surrounds the fibrils with calcite, although they never become impregnated with it (Fig. 9). The continued growth o f nodules results in their contact and partial or complete fusion (Figs. 11, 12), although smaller, separate nodules remain at the periphery o f the axis. Thus to some extent a radial gradient o f calcification can be identified, as well as the more obvious longitudinal one. However, the whole thickness o f the skeletal rod is not built up o f accreted nodules, but consists o f irregular columns o f mineral radiating out f r o m the n o d u l a r core (Fig. 13). These columns and their ultrastructural detail are m o s t easily seen on fracture surfaces which have been lightly etched (Figs. 15, 16). N o n - e t c h e d longitudinal surfaces (Fig. 14) do not reveal similar subunits, but the parallel collagen fibrils and the grooves left where fibrils have been torn away can give a false impression o f longitudinal mineral units. Cavities which contain extensions o f certain epithelial cells (the 'cellules ~ chemin6e' o f F r a n c et al., 1974) possess terminal expansions corresponding to the plateaux o f these cells. Some o f these plateaux are aligned parallel to the axis surface in an arrangement which is p r o b a b l y related to intermittent axis growth (i.e. is a growth ring phenomenon). The associated discontinuities in the skeleton (Fig. 14) are less m a r k e d on etched specimens, in which the mineral subunits are aligned across such interruptions. The latter thus p r o b a b l y represent concentrations o f matrix material rather than true discontinuities in the mineral phase. O n transverse fracture surfaces (Fig. 17) the collagen fibrils protrude, or leave holes where they have broken off within the mineral. In the massively calcified parts o f the axis the mineral phase extends to just under the secretory epithelium (which has been described in detail by F r a n c et al., 1974). The edge o f the mineral is irregular in section and consists o f numerous, sub-

Fig. 6. TEM of a nodule in a transverse section of a uranyl acetate decalcified skeletal rod. The large crystal spaces at the centre (ce) are surrounded by several radiating lobes (/). Fibrils (f) are present around and within the nodule./f, interfibrillar matrix, x 12,000

Fig. 7. SEM of a nodule exposed by splitting the axis longitudinally. Note its lobed aspect and the slight separation of the surrounding fibrils. Interfibrillar matrix is poorly preserved and is represented only by the irregular strands between the collagen fibrils, x 6,200 Fig. 8. Seen in transverse section, the extremities of nodule lobes consist of an interlocking network of mineral (here decalcified by uranyl acetate). The arrow indicates where the triradial aspect is particularly well shown.f, collagen fibril; tf, interfibrillar matrix, x 14,000 Fig. 9. Extension of the mineral phase (m/) occurs only in the interfibrillar matrix (/f). Collagen fibrils (f), seen here in transverse section, become embedded in mineral but not impregnated. Although not decalcified during fixation, the mineral has been slightly dissolved during staining of the thin section. x 14,400 Fig. 10 and 10a. Non-decalcified unstained section passing longitudinally through a nodule lobe. Compare the mineral subunits with the mineral spaces in lobes of decalcified nodules (Fig. 6). Selected area electron diffration of the area within the circle gave the diagram in Fig. 10a. Spots were indexed with reference to a gold standard. The [001] axis (white arrow) coincides with the orientation of the mineral units (black arrow). The correct orientation of the diffraction diagram was determined by reference to a M o O 3 standard. Fig. 10. x 25,000

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Fig. 11. SEM of a region of partially fused nodules (n). The fracture has mainly passed between the nodules and thus exposes their surfaces. In this and the following figures depicting longitudinal surfaces the double headed arrow indicates the long axis of the skeletal rod; f, collagen fibrils, x 1,000 Fig. 12. SEM of an etched fracture surface passing through nodules that are completely fused together. In some nodules the lobed structure is evident, and in one of them the larger, central crystals can be seen (ce). Channels are left where collagen fibrils have been removed by NaOC1 treatment, x 1,500

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Fig. 13. Diagram of part of the main body of the skeletal rod fractured so as to display transverse (T) and longitudinal (L) surfaces. One of the latter (L') is represented as having been etched to show the mineral columns arising out of the core of fused nodules (c). Around the skeletal rod is the layer of non-calcified matrix (se) and the epithelium (ep), from which chimney cell processes (ch) extend to various depths. These layers are not shown over part of the mineral surface (s) on which imprints of chimney cell plateaux are thus visible. Approximately x 80

parallel projections (Fig. 18). Between mineral and epithelium is a layer of organic material which is usually finely granular, although sometimes parts o f it a p p e a r finely fibrillar, a condition perhaps related to the f o r m a t i o n of the collagen fibrils simultaneous with deposition o f the mineral phase. The subepithelial layer also contains small a m o u n t s o f cell debris which becomes incorporated into the axis, but is never itself associated with de n o v o calcite deposition. T r e a t m e n t o f axes with sodium hypochlorite removes all superficial organic m a t t e r and reveals the underlying mineral growth surfaces (Fig. 19). As this process causes completely isolated nodules to fall away, the remaining axis tip consists o f partially fused nodules penetrated by clefts and chimney cell spaces (Fig. 20). This merges into a zone o f m o r e closely interlocking mineral masses and then into a surface typical o f the rest o f the axis, which is m a r k e d by n u m e r o u s depressions, each corresponding to the previous position o f a chimney cell process (Fig. 21). All these surfaces exhibit a microarchitecture consisting o f triangular or triradiate projections whose axes o f triradial s y m m e t r y are always approximately perpendicular to the surface o f the skeletal rod (Figs. 20, 22). These mineral

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projections form arrays, up to several hundred p,m2 in area, within which they have a common orientation (Fig. 19). When polished sections are examined in the polarizing microscope individual columns can be distinguished by slight differences in their optical behaviour. In a complete rotation of the microscope stage, columns of a longitudinally sectioned skeletal rod show four positions of brightness and four positions of extinction. The extinction is not, however, instantaneous but spreads over each column as a wave. Despite this slight inhomogeneity, insertion of the quartz wedge suggests that the mean extinction parallel to the long axis of each column corresponds to the crystallographic c-axis of calcite. This conclusion is supported by the tendency for transversely sectioned columns (in sub-tangential sections of the skeletal rod) to show complete extinction at all points of rotation. Further information was gained by the technique of artificially growing calcite crystals onto sodium hypochlorite-cleaned surfaces of the axis. The rationale of this method is that the crystals, showing identifiable faces, grow in crystalline continuity with the underlying biomineral and thereby reveal the orientation of the crystallographic axes of the latter (Jones, 1955; Towe et al., 1977). Crystals deposited on column growth surfaces invariably display three faces directed away from the rod (Fig. 22). Occasional well-formed crystals reveal these faces to be attributable to the { 1011 } calcite rhombohedron, and their orientation confirms the column-parallel disposition of the crystallographic c-axis of the skeletal calcite. In addition, the orientation of non-contiguous crystals present upon single arrays of the mineral projections show that within each array the crystallographic a-axes have a particular orientation. This varies between adjacent arrays and is not related to the morphologic axes of the skeletal rod. Crystals deposited on fracture surfaces revealing longitudinal sections of columns have a form compatible with a c-axis perpendicular view (Fig. 23). Their aspect is constant across a single column but varies between adjacent columns because of the different orientation of the crystallographic a-axes. Using the edges of well developed faces as guides, it can be seen that the c-axes of deposited crystals are approximately parallel to the mineral subunits revealed by etching. Thus, just as these units are slightly fanned out across a column, so the c-axis deviates from its mean, column parallel orientation, consistent with the wavy extinction seen in polarized light.

Fig. 14, Longitudinal fracture surface of a fully mineralized part of the axis showing several chimney cell extensions (ch). Grooves are left where collagen fibrils have been torn away (arrows). x 1,100 Fig. 15. After etching, surfaces with the same orientation as Fig. 14, show mineral columns (co) radiating out from the axis core (c). s, outer surface of the skeletal rod. x 400 Fig. 16. Detail of two adjacent columns. The horizontal channels (arrows) were occupied by collagen fibrils. Perpendicular to this are the mineral structures revealed by etching, which have a different orientation in the two columns, x 2,800 Fig. 17, Transverse fracture surface. The collagen fibrils protrude from the mineral (arrow) or leave holes where they have been torn out. x 2,500

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Fig. 18, TEM of a transverse section at the periphery of a uranyl acetate decalcified skeletal rod. The column growth surface is separated from the epithelium (ep) by a layer of non-calcified matrix (se). The mineral space (m0 contains irregular strands of included matrix and collagen fibrils (f). Other fibrils ( f ') are as yet incompletely surrounded by mineral. Some cell debris is present in the subepithelial space immediately below the plateau of a chimney cell (p). Around the axis, outside the epithelium, is the mesogloea (me). x 16,000 Fig, 19, SEM of the axis surface after removal of superficial organic material with NaOCI. Slight etching has accentuated the surface structures. Their different orientations in the several arrays are emphasized by the superimposed symbols. The unequal length of the structures in some arrays can be attributed to deviation of their axes of symmetry from a strictly surface-perpendicular orientation. • 2,100

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Fig. 20, SEM stereopair o f the axis surface cleaned of organic material but not etched. In this region, towards the tip, the surface is highly irregular. Mineral projections are absent from depressions (ch) which contained the chimney cells. Tilt angle, 8 ~ x 1,000 Fig. 21. Surfaces typical o f the rest of the axis are less irregular. Channels at the bottom of chimney cell depressions (ch) indicate the previous position o f collagen fibrils. • 800

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Fig. 22. A column growth surface, cleaned with NaOC1 but not etched, with artificially overgrown calcite crystals. Those crystals growing on a single array of mineral projections have a common orientation (compare all those marked *). Crystals on adjacent arrays have different orientations, but bear a constant relationship to their underlying mineral projections, x 3,900 Fig. 23. Crystals on a fracture surface perpendicular to the surface of the skeletal rod show that the structures revealed by etching (cf. Fig. 16) are parallel to the c-axis o f the calcite. Comparing the orientation of the crystals (e.g. those two outlined) indicates how the orientation of the c-axis varies across the single column, x 4,100

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Discussion

The present description of the skeletal rod of Veretillum is in general agreement with the basic morphology of the axes of some fossil pennatulids described by Bayer (1955). Apart from the work of Franc et al. (1974) there have been no recent detailed studies of pennatulid axes. Chia and Crawford (1977) observed what they considered to be an early stage of axis formation, within endodermal cells of the primary polyp of Ptilosareus gurneyi, but as this structure was entirely lost during preparation it is impossible to compare it to the fully formed axis of Veretillum. The radial arrangement of mineral columns has also been described in the skeletons of other octocorals, namely certain holaxonian gorgonians (Bayer, 1955; Goldberg, 1976). However, Lowenstam (1964) has shown that in that group the axial skeletons are not necessarily calcitic but may be composed of aragonite, or even a mixture of both morphs. Also, general ultrastructural observations made on holaxonian gorgonian skeletons by Goldberg (1976) do not immediately suggest an organization identical to that of the axis of Veretillum. Nevertheless, considering the possible homology of the pennatulid axis with the holaxonian gorgonian skeleton (Delage and H6rouard, 1901), the collagenous content of the skeletons of both groups (Franc et al., 1974; Goldberg, 1974; Marks et al., 1949) and the similarity of Veretillum 'chimney cells' (Franc et al., 1974) to the 'plate cells' of the epithelium surrounding the gorgonian axis (Bouligand, 1968), it would not be surprising if more extensive comparative studies do reveal points of similarity. A gradient of mineral density such as that found in the tip of the skeletal axis is not necessarily a manifestation of a continuous process of calcification. However, as this gradient has been found in numerous, differently sized skeletal rods, and as each structure along the gradient can be related to smaller (earlier) and larger (later) structures, there is justification for interpreting the morphologic gradient in terms of the temporal sequence of events involved in the process of calcification. The initial crystal-like deposits are not pre-formed within the epithelium surrounding the axis so they presumably arise by precipitation from inorganic ions secreted into the general compartment of the axis. Subsequent development of nodules occurs by the radial growth of the lobes. Their component subunits are consistently parallel to the direction of growth and are unrelated to the direction of sectioning; they are thus considered to be intrinsic features of the mineral, not sectioning artefacts. In addition, diffraction diagrams obtained from nodule lobes (Fig. 10a) show very little arcing of the diffraction spots. Thus, if each lobe is polycrystalline, as their ultrastructural appearance would suggest, then the crystallites show a very high preferred orientation. The lobed form of nodules and their growth around a single-crystal core are features that are also found in calcitic aggregates that can be formed in vitro, in gelled media, under specific physicochemical conditions (McCauley and Roy, 1974). This suggests that the formation of nodules around initial deposits can be considered as a predominantly physicochemical process, requiring little or no immediate biological control. The radiating columns that grow out from the nodular core can be considered as greatly extended nodule lobes. This is shown by the similarity between the triradiate surface structures of columns and lobes, by their predominantly c-axis parallel extension and by the common presence of c-axis parallel subunits. This transition

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from lobe to column, and the orientation of columns perpendicular to the skeletal rod, can be explained in terms of interaction and competition between adjacent growing lobes: within the axis core, nodule growth would be limited by space and/or by the supply of mineral ions. Some lobes of peripheral nodules would, however, be free to continue growing more or less radially. As their c-axis parallel growth continued, so obliquely oriented lobes would tend to grow against, and be suppressed by, more perpendicular ones. Eventually only those perpendicular lobes would survive and continue growing as columns. The triradial and triangular projections of lobe and column growth surfaces are clearly manifestations of the trigonal symmetry of calcite. The artificially overgrown calcite crystals indicate that each array of projections has a common set of a-axes and that each apparently corresponds to the growth surface of a single column. The long range crystallographic order that this represents is somewhat contrary to the incomplete optical and ultrastructural homogeneity of columns otherwise observed. Thus, while the latter characteristics may suggest that each column is a highly oriented polycrystalline aggregate that merely simulates the single crystal state, an alternative possibility should also be considered: that the column itself represents the single crystal unit, but occurs in an imperfect state because of an abundance of lattice defects. Now, as Towe (1972) has discussed, it is technically and conceptually difficult to define a 'single crystal', particularly with respect to biomineralized structures, let alone to determine what morphologic unit can be considered as such. Moreover, the distinction proposed above concerns the skeletal product, but is really of importance only in connexion with the process of calcification. In Veretillum, this appears not to involve the repeated formation of discrete crystallites. Rather, the triradial projections, in morphologic and crystallographic continuity with the underlying skeleton, more closely resemble three dimensional dendritic processes which grow into, and displace, the supporting organic matrix. As mineral filled in between the bases of the projections, so would organic material become trapped, thereby accounting for the irregular strands seen within the mineral space in decalcified preparations. It is known that foreign material included within a crystal lattice can be an important source of lattice defects (Brice, 1973). The repeated inclusion of organic material within the skeletal calcite would thereby favour the formation of such dislocations, and their accumulated effect could account for the observed polycrystalline-like features of the skeletal product. In this way the subunits seen in TEM (Fig. 10) would not be discrete units, being incompletely separated by the discontinuous strands of included organic material; those substructures revealed by etching (Fig. 16) would represent dendritic skeletons denuded of their interjacent calcite, similar to the dendritic 'microcrystals' revealed by the etching of certain foraminifer skeletons (Bellemo, 1974). Considering one of the original reasons for our interest in the axial skeleton of Veretillum, we note particularly the apparent absence of interactions between the calcite and the collagen fibrils, the latter apparently being involved neither in the initiation of mineralization nor in the subsequent control of that process. In effect, the calcite forms a distinct phase extending between the fibrils and containing them within a system of interconnecting 'tunnels', a disposition well illustrated on transverse sections of the axis (Fig. 17). The obvious contrast to make is with

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mineralized tissues of vertebrates, in which the collagen fibrils are impregnated with calcium phosphate. Although the active/passive nature of this impregnation is still in dispute (Urist, 1976), Landis et al. (1977) have pointed out that since each intrafibrillar deposit is a discrete particle of mineral, then the regions of the fibril where they occur must contain sites which induce the heterogeneous nucleation of calcium phosphate. The occurrence of a similarly intimate association between mineral and matrix in Veretillum might conceivably be precluded by the absence of sufficiently small mineral units, or an unusual subfibrillar organization of its collagen. However, a more useful way of considering this mineral-matrix association is in terms of the differing patterns of mineral deposition. Thus, the advance of a mineralization front in vertebrate tissues consists predominantly of the multiplication of crystal units, each of which has an independent locus of nucleation (Landis et al., 1977). On the other hand, the advance of a column growth surface in the skeleton of Veretillum consists of the extension of a single crystalline phase and is a physicochemical phenomenon that does not rely on a matrix capable of repeatedly inducing heterogeneous nucleation. The only aspect of the latter in Veretillum may be the formation of the initial deposits, some of which depend on the presence of cell derived debris. Although this debris may be functionally analogous to the matrix vesicles of some vertebrate calcifying tissues (Anderson, 1976), the mineral-debris association appears not to be obligate. Thus, although the cell debris can provide a microenvironment favourable for calcite precipitation, this process may occur in a quite non-specific way. For example, precipitation may simply be more common there by virtue of localized differences in ionic concentration, or in pH, that it might provide. Such an effect could be accentuated by enzymatic action, for example by the acid phosphatase shown to be present. The virtual negation of the role of fibrillar and interfibrillar matrix components in the control of mineral deposition should not be misconstrued; it is not intended to signify that the matrix is necessarily completely inactive with respect to the mineral phase. For example, Kitano et al. (1969) have shown that the presence of organic molecules (including chondroitin sulphate and'glycoprotein') can affect the composition and morph of calcium carbonates precipitated in vitro. A less specific but equally important effect is that noted by Morse et al. (1932): that gelled media tend to favour the (in vitro) formation of spherulites and related aggregates in general. The point to be made is that the absence of specific epitactic or compartmental roles does not lessen the potential importance of an organic phase, the only requirement of an organic matrix being that it acts as an intermediary between the biologic action of the skeletogenic cells and the wholly physicochemical growth of the mineral phase. In conclusion, the skeleton of Veretillum represents a far more intimate calcitecollagen association than that described for any other invertebrate. Indeed, the collagen occupies the very extracellular compartment in which crystallization of the calcite occurs. Nevertheless, despite this physical proximity, it has been shown that even though the skeletal rod is a calcified collagenous tissue, it is not a calcified collagen as such. This development in the exploitation of the ubiquitous collagen appears not to occur until far higher in the phylogenetic scale when it involves mineralization by calcium phosphates, in the bone, dentine and tendon of vertebrate animals.

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References Anderson, H.C.: Matrix vesicles of cartilage and bone. In: The biochemistry and physiology of bone, 2nd ed., Vol. IV (G.H. Bourne, ed.), pp. 135-157. New York: Academic Press 1976 Bayer, F.M.: Contributions to the nomenclature, systematics, and morphology of the Octocorallia. Proc. U.S. natn. Mus. 105, 207-220 (1955) Bellemo, S.: Ultrastructures in recent radial and granular calcareous foraminifera. Bull. geol. Instn. Univ. Upsala, N.S. 4, 117-122 (1974) Bouligand, Y.: Sur une catrgorie de cellules trrs particuli+res chez les Gorgones (Coelentrrrs Octocoralliaires). Vie et Milieu 19, 59%9 (1968) Brice, J.C.: The growth of crystals from liquids. Amsterdam: North Holland Pub. Co. 1973 Chia, F.S., Crawford, B.: Comparative fine structural studies of planulae and primary polyps of identical age of the sea pen Ptilosarcus gurneyi. J. Morph. 151, 131-158 (1977) Delage, Y., Hrrouard, E.: Trait~ de Zoologic concrete. II. Les Coelent~rrs (Schleicher, ed.). Paris 1901 Franc, S., Huc, A., Chassagne, G.: Etude ultrastructurale et physicochimique de raxe squelettique de Veretillum cynomorium Pall. (Cnidaire, Anthozoaire): Cellules, calcite, colla#ne. J. Microscopic 21, 93-110 (1974) Franc, S.: Ph6nom~ne de nucleation au cours de la calcification chez un Invert6br6 marin (Cnidaire). J. Microscopie Biol. Cell. 27, 11a (1976) Goldberg, W.M.: Evidence of a sclerotized collagen from the skeleton of a gorgonian coral. Comp. Biochem. Physiol. 49B, 525-529 (1974) Goldberg, W.M.: Comparative study of the chemistry and structure of gorgonian and antipatharian coral skeletons. Mar. Biol. 35, 253-267 (1976) Jones, W.C.: Crystalline properties of spicules of Leucosolenia complicata. Quart. J. micr. Sci. 96, 129149 (1955) Kitano, Y., Kanamori, N., Tokuyama, A.: Effects of organic matter on solubilities and crystal form of carbonates. Amer. Zool. 9, 681%88 (1969) Landis, W.I., Paine, M.C., Glimcher, MJ.: Electron microscopic observations of bone tissue prepared anhydrously in organic solvents. J. Ultrastruct. Res. 59, 1-30 (1977) Ledger, P.W., Franc, S.: Polymorphic collagen in an invertebrate (Cnidaria: Octocorallia). Coll. Intern. C.N.R.S. (Paris), (in press) Ledger, P.W., Jones, W.C.: Spicule formation in the calcareous sponge Sycon ciliatum. Cell Tiss. Res. 181, 553 567 (1977) Lowenstam, H.A.: Coexisting calcites and aragonites from skeletal carbonates of marine organisms and their strontium and magnesium contents. In: Recent researches in the fields of hydrosphere, atmosphere and nuclear geochemistry (T. Miyake and T. Koyama, eds.). Tokyo: Maruzen Co. 1964 Marks, M.H., Bear, R.S., Blake, C.H.: X-ray diffraction evidence of collagen-type protein fibres in the Echinodermata, Coelenterata and Porifera. J. exp. Zool. 111, 55-78 (1949) McCauley, J.W., Roy, R.: Controlled nucleation and crystal growth of various CaCO3 phases by the silica gel technique. Amer. Miner. 59, 947-963 (1974) Morse, H.W., Warren, C.H., Donnay, J.D.H.: Artificial spherulites and related aggregates. Amer. J. Sci. 23, 421-439 (1932) Pasteels, J.J.: Phosphatase acide et polarit6 golgienne dans les cellules absorbantes de la branchie de Mytilus edulis. Etude au microscope 61ectronique. Histochemie 28, 296-304 (1971) Towe, K.M.: Invertebrate shell structure and the organic matrix concept. Biomineralization 4, 1-14 (1972) Towe, K.M., Berthold, W.-U., Appleman, D.E.: The crystallography ofPatellina corrugata Williamson: a-axis preferred orientation. J. Foraminiferal Res. 7, 58%1 (1977) Travis, D.F., Francois, C.J., Bonar, L.C., Glimcher, M.J.: Comparative studies of the organic matrices of invertebrate mineralized tissues. J. Ultrastruct. Res. 18, 51%550 (1967) Urist, M.R.: Biochemistry of calcification. In: The biochemistry and physiology of bone, 2nd ed., Vol. IV (G.H. Bourne, ed.), pp. 1-59. New York: Academic Press 1976 Wilbur, K.M.: Recent studies of invertebrate mineralization. In: The mechanisms of mineralization in the invertebrates and plants (N. Watabe and K.M. Wilbur, eds.), pp. 79 108. Univ. of South Carolina Press 1976 Accepted April 17, 1978

Calcification of the collagenous axial skeleton of Veretillum cynomorium pall. (Cnidaria: Pennatulacea).

Cell Tiss. Res. 192, 249-266 (1978) Cell and Tissue Research 9 by Springer-Verlag 1978 Calcification of the Collagenous Axial Skeleton of Veretillum...
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