Ultrastructure and growth of the sea urchin tooth

Calc. Tiss. Res. 14, 211--228 (1974) �9 by Springer-Verlag 1974

Uhrastructure and Growth of the Sea Urchin Tooth

E r n s t K n i p r a t h

Lehrstuhl ffir Zellmorphologie, Abteilung fiir Biologic, Ruhr-Universi tBt Bochum

Received April 25, accepted Ju ly 30, 1973

Individual sea urchin teeth consist of many elements, each secreted by a syncytium formed for the purpose. The numerous syncytia of each tooth take up secondary connection with one another in the vicinity of needles and prisms. The elements of the pr imary tooth skeleton are surrounded by cytoplasm and are therefore intracellular. Following the origin of a syneytium in the plumula, a new tooth element sheath originates in the form of a vesicle, which develops a unified crystallization cavity in the shape of the future tooth element. During the early growth of the sheath, calcium carbonate crystallization begins within the sheath. An inner coating of the sheath functions as a crystallization matrix, and fur ther growth of calcium carbonate takes place centripetally. Collagen does not take par t in mineralization. Neither an axial thread nor other organic material inside the hardened mineral was found.

Key words: Echinoid - - Tooth - - Odontoblast - - Matrix - - Mineralization.

Les dents individuelles d'oursin sont compos6es de nombreux 616ments, s6er6t6s chacun par un syncytium. Les nombreux syncytiums de ehaque dent pr6sentent des liaisons secon- daires entre eux, dans la region des aiguilles et prismes. Les 616ments du squelette de la dent primaire sont entour6s de cytoplasme et sont, par suite, intracellulaires. Faisant suite

l 'appari t ion du syncytium dans la plumula, une nouvelle enveloppe d'616ment dentaire apparai t sous la forme d 'une v6sicule, qui pr6sente une cavit6 unie de cristallisation ayan t la forme d 'un 616ment dentaire. Pendan t le d6but de la croissance de la gaine, la cristallisation de carbonate de calcium commence dans l 'enveloppe. Un rev~tement interne de cette derni6re sert de matrice de cristallisation et la croissance ult6rieure de carbonate de calcium se d6veloppe en direction centrip~te. Le collag~ne ne participe pas ~ l a min6ralisation. Ni un fi lament axi l, ni un mat6riel organique n 'on t 6t6 observ6s s l ' int6rieur du min6ral.

Der einzelne Seeigelzahn besteht aus sehr vielen Zahnelementen. Jedes dieser Zahn- elemente wird yon einem ad hoc gebildeten Syncyt ium ausgeschieden. Die entsprechend zahlreichen Syncytien eines Zahnes t re ten im Bereich der Nadeln und Prismen sekundgr miteinander in Verbindung. Zumindest das hier untersuchte primBre Zahnskelet t ist allseits von Zellplasma umgeben, also intrazellul~r. Nach der Neubildung eines Syncytiums in der Plumula des Zahnes wird in Form eines Vesikels eine neue Zahnelementhiille angelegt. Diese ttiille w~ichst stetig weiter und bildet einen einheitlichen Kristall isationsraum in Form des spBteren Zahnelementes. Bereits wi~hrend des friihen Wachstums der Zahnelementhfille be- girmt innerhalb dieser die Kristall isation des Kalkes auf einem inneren Belag der ttfille als Matrix. ]:)as weitere Wachs tum des Kalkes in der H/ille ist zentripetal. Kollagen ist an der Mineralisation nicht beteiligt. Ein , ,Achsenfaden" oder anderes organisches Material innerhalb des festen Kalkes wurde nicht gefunden.

Introduction

T h e c a l c i f i c a t i o n of t h e e x c l u s i v e l y m e s o d e r m a l p o r t i o n s of t h e sea u r c h i n

s k e l e t o n is d i f f i cu l t t o s t u d y . T h e f i r m l y a r t i c u l a t e d she l l is m a d e u p of a n a l m o s t

u n s e c t i o n a b l e s y s t e m of s m a l l g i r de r - l i ke s t r u c t u r e s ( t he t r a b e c u l a e ) of sclero-

For reprints: Dr. E. Knipra th , Lehrstuhl ffir Zellmorphologie der Ruhr-Universi t / i t Bochum, 463 Bochum, Postfach 2148 - Geb~ude N D F 05, Federal Republic of Germany.

212 E. Kniprath

blasts distributed in the remaining spaces, and of other cells of mesodermal origin. Consequently, studies of this material undertaken with the light microscope [8, 17] and biochemical [3, 9, 24] or electron microscopical [4, 20, 30, 31] methods, which have been carried out specifically in search of an organic skeletal matrix, are rare.

A large part of our present knowledge of skeletal development is derived from old observations on sea urchin larvae. The first worker in this field, Selenka, determined in 1879 that at least the primordium of the tri-radiate spicule is an intracellular formation. As growth proceeds, additional cells become annexed. Semon (1887) reported that the most advanced skeletal component is also surrounded by a thin membrane. He could not decide whether this membrane is a degenerated cell or a development of the original cell. The findings of ThSel (1892) extend further. He at tr ibuted skeletal formation to a "pseudopodial p lasm" which arises from the fusion of pseudopodia from at least three pr imary mesen- ehyme cells [similarly 1, 2, 6, 18, 21-23, 32-34]. The growth of the skeleton takes place within a membrane-bound space inside this fusion product, which, since cell boundaries are indeed absent, must be termed a syncytinm (yon Ubisch, 1937; Gibbins et al., 1969).

The teeth of the already metamorphosed sea urchin are more amenable to examination as a calcifying model than is the shell. They grow rapidly (1 to 1,5 mm per week) from a growth center, the plumula (Holland, 1965; M~rkel, 1969). In addition, they do not form a compact system similar to tha t of the trabeculae, but rather are composed, at least in their younger portions, of tooth elements which lie free in the tissue (see below). In the present article, the interrelationship between the calcifying mesenchyme, organic matrix and inorganic part of the skeleton already reported by Pilkington (1969) for the urchin spine and by Gib- bins et al. (1969) for the larval skeleton will be considered. The teeth of Para- centrotus lividus were chosen because their structure is already well known through the reports of Prenant (1926) and M~rkel (1969).

Material and Methods

Lanterns from Paracentrotus lividus obtained in Banyuls, France (diameter of entire animals about 10 ram), were fixed in 5% glutarMdehyde in 0.5 molar sodium cacodylate (pH 7) immediately after collection, and individual teeth were post-fixed in sodium cacodylate buffered (pH 7.2) 2% osmium tetroxide. Some of the material was decalcified with 0.2 N nitric acid either prior to fixation with osmium tetroxide or in 70 % ethanol during dehydra- tion. Embedding followed in styrol methacrylate and sectioning was carried out with a diamond knife.

Results

The Anatomy o/ the Growing Tooth

Since the construction of the sea urchin tooth is complicated, a general clari- fication is necessary (Figs. 1-3). Par t of the following description has been taken from M&rkel [11-16]. Each individual tooth of Paraeentrotus is set into a pocket which extends well over the plumula, the growth center of the tooth. The pocket is made up of a single-layered, shallow epithelium which is set off from the tooth cavity by means of a basal membrane (Fig. 4). The tooth itself is built up of two rows of alternating tooth elements. These elements, which are at least in the

Fig. 1. Lateral view of a tooth of Paracentrotus lividus. D distal end of the tooth, K keel, X - - X ~ see Fig. 3

Fig. 2. Diagrammatic structure of a vertical section of the plumula (corresponding to the rectangle in :Fig. 1). Odontoblasts are not drawn. E epithelium of the tooth pocket, L lamellae,

N needles, P P primary plates, Pr prisms

Fig. 3. Semi-diagrammatic section through the tooth of an adult Paracentrotus at X - - X ' in Fig. 1 (Fig. taken from M~rkel [11]). CF carinar appendage, H crust, P prisms, P P primary

plates, S P secondary plates, S T stone part (needles). Lamellae are not drawn

214 E. Kniprath

younger part quite separate from one another, interdigitate and overlap in the middle region of the tooth (Fig. 3). A single tooth element consists of a slightly crooked, approximately three-sided primary plate whose adaxial edge (with reference to the axis of the lantern) carries a row of protuberances (lamellae, prisms, needles, Fig. 2). In Paracentrotus, the lamellae grow between the primary plates as strongly lapped, faeeted structures. Needles and prisms protude axially and proximally at nearly right angles. They give rise to the stone part and to a part of the keel. The protuberances of different plates are interwoven in the keel. The secondary plates, which, depending upon the species of the animal, possess markedly different sizes and shapes, proceed axially from the lateral edge of the pr imary plates (Fig. 3). These elements then make up the pr imary tooth skeleton. Later these components of the skeleton become cemented together by discs of calcium carbonate to form the functionally active tooth. In addition, a calcareous crust becomes deposited. The discs and crust form the secondary tooth skeleton. The present work is concerned exclusively with the primary skeleton.

Upon observing a section through the tooth in the electron microscope, the fact that each sectioned calcified part (plate, lamella, needle, prism) is surrounded by three sheaths (cellular membrane, skeleton sheath, and the inner coating of the sheath) becomes apparent (Figs. 5, 8 ff). Not considering the growing tip, two of these sheaths (skeleton sheath and inner coating) enwrap the individual calcified part very closely. The third member is located at a variable distance away and is continuous in many places with the cellular membrane of neighbouring cells (odontoblasts, Figs. 5, 8). This sheath is a cellular membrane wieh, since it includes several nuclei, belongs to a syneytium. However, the nuclei of the cells involved are not incorporated in the central mass of the syneytium, but remain along with the main portion of the cell organelles obviously separate. They are often united with the central cytoplasm merely by means of a small bridge. The outer sheath or cellular membrane often appears to be torn or vesiculated, especially in the central part of the plates (Figs. 5, 6). Such appearances are almost never found in material deealeified before osmium fixation and thus are probably artifacts caused by contraction.

The process of growth ean be most readily examined during the formation of the plates. A few cells of the distal region of the germinative zone of the plumula (Fig. 9) develop pseudopodia which move toward each other and unite. In the syneytium thus arising, the sheath of the tooth element develops in the form of a membrane-bound cavity which after having reached its definitive thickness grows only in the two remaining dimensions, but evidently no more in the third. This tooth element sheath is the middle component of the organic sheaths men- tioned above. The innermost and most coarsely structured layer arises at the

Fig. 4. Longitudinal section through the growth zone of a tooth of Paracentrotus lividus. The odontoblasts, which are located between the epithelium (E) of the tooth pocket and the not yet calcified adaxial end of the plate sheaths (P) are still free and already arranged in a row along the edges of the plates. Between the plates are visible portions of the already formed sheaths of the lamellae (L). The abaxial part of the plates has been partially destroyed due to the fracturing of the calcium carbonate and is missing in the right hand edge of the micro-

graph, • 4000

Ultras t ructure and Growth of the Sea Urchin Tooth 215

Fig. 4

216 E. Kniprath

same time. This is possibly not a separate sheath, but perhaps merely a coating of the tooth element sheath itself (see below).

During the subsequent growth of the tooth element sheath, the odontoblasts which were still free in the area become annexed to the syncytium. They are added at the edge, in particular abaxially, and thereby enlarge the syncytium. No suggestion could be found tha t nuclear divisions take place in the cells already bound to the syneytium. Even when there are m a n y pseudopodia in the extensive intercellular space, no connection between syncyt ia of two tooth elements could be reliably established. The pseudopodia form essentially a supplementary connection between the " m o t h e r cells" of a single syncytium. The tooth element sheath thus represents a secretion and crystallization cavi ty which is a uniform and closed system.

Abaxially, the full-grown plate sheaths closely approach the pocket epithelium. At this point free odontoblasts are no longer present. On the adaxial side, in contrast, a large number of odontoblasts remains between the plate syncyt ia and the epithelium, which is here fur ther removed (Figs. 2, 4). A port ion of these cells becomes deposited as an uni terrupted row on the edge of the plate syncyt ia (Figs. 4, 8) and makes connection with these. Together the two components consti tute a highly ramified syncytial network. The lamellae grow between the plates within this compound system of pseudopodia inside compar tments which represent the outgrowths of the plate sheaths (Fig. 7). I n Fig. 8 it can be seen tha t each plate has its own network of lamellae. However, since the odontoblasts are united with one another in the row already described (Fig. 8), a connection between the syncyt ia of adjacent plates extending over several odontoblasts exists as well.

After the growth of the lamellae has commenced, the odontoblasts migrate further adaxially. However, t hey maintain multifold contact by means of long narrow extensions (Fig. 10). Some of these processes are irregular and contain sponge-like cytoplasm, as in the syncytial network of the lamellae. Others are markedly smoothly s tructured with very compact cytoplasm and have a diameter of 0.05 to 0.1 v.m. These smooth cell extensions often contain microtubules. Both types of pseudopodia unite the odontoblasts with the basal membrane which they occasionally penetrate. The cells of the epithelium likewise can form such pseudopods and invade the too th cavi ty through the basal membrane. I n any case, an int imate contact between epithelial cells and odontoblasts arises.

Fig. 5. Abaxial edge of the tooth of P. lividus in longitudinal section. There are no more free odontoblasts between the not yet calcified ends of the plate sheaths (P) and the basal membrane (BM) of tile damaged tooth pocket. Between the nuclei N1-N e, N3-N 4 and I~-IN 6 are obviously no cell borders. In the electron lucent areas, the calcium carbonate has broken away. There the layer of cell plasma of the plate sheath is torn in numerous places. The cocoon- like material (G) at the mineralization front has remained. Arrows show the succession of the three sheaths, which in order are inner coating, skeleton sheath, and cellular membrane,

• 8000 Fig. 6. Fully torn and vesiculated cell plasma sheath (PV) in the central area of a plate (P) which has been completely broken away. Between the rows of vesicles the intact plate

sheath is visible as a double line (PH), • 12000 Fig. 7. The sheath of the lamellae (L) grows from the not yet calcified plate sheath (P),

X 24000

Ultras t ructure and Growth of the Sea Urchin Tooth 217

16 Calc. Tiss. Res,, Voh 14

Figs. 5 - -7

218 E. Kniprath

Fig. 8

Ultrastructure and Growth of the Sea Urchin Tooth 219

Fig. 9. Schematic diagram of the formation of plates in the plumula of the tooth of P. lividus. Free odontoblasts (above and at micrograph edges) send the pseudopods (PP) to meet and fuse with each other. The plate sheath (P) with its inner coating (dotted line) becomes established in the syncytium (S) which thereby arises. Calcification begins at the two opposite edges

of the plate sheath. (G) is the cocoon-like material

Fig. 8. This enlargement of Fig. 4 shows the syncytial network in which the plate sheaths (P) and the lamellae sheaths (L) grow and become calcified. The sheaths of the lamellae are joined with one another, with the plate sheaths, and with the cells O1, 02 and 08 by means of plasma bridges which are not interrupted by cell borders. Except for the profiles of the cocoon- like material which at this t ime are irregular, the sheaths of the skeleton elements appear to

be empty, Solid calcium carbonate has broken away (electron lucent areas), • 15000

16"

220 E. Kniprath

Figs. 10 and 11


In Fig. 10 the strict orientation of the pseudopodia and thereby also of the needle sheaths is evident. On the adaxial side, especially at the end of the pseudopodia, fibrils are attached which obviously reach as far as the basal membrane (Figs. 10, 11). They seem to be stretched between their points of attachment on the membrane of the pseudopodia and of the odontoblasts themselves on the one side and on the basal membrane on the other side. They give the impression that they could be concerned with the axial movement of the odontoblasts and with the exact orientation of the pseudopodia. Similar fibrils are found in substantially smaller quantities throughout the intercellular space of the tooth and, according to Foucart [4] of the shell as well.

Once the odontoblasts have moved off from the adaxial end of the plate sheaths, the sheaths of the future needles and prisms extend to meet them. These structures grow within the cell processes as ramifications of the lamellar sheaths. This configuration is retained; the odontoblasts remain at a distance from the growing edge of the sheaths of the tooth elements. As calcification of the needles commences (the as yet uncalcified zone of individual needles is as long as 15 ~z), nuclei reappear between them, the lithoblasts of M~rkel [11].

Ultrastructure and Growth o/the Tooth Element

The tooth element which, in the living animal, is enclosed on all sides by syncytial cytoplasm evidently grows in three dimensions only for a short time after its initiation. After a certain thickness is reached, growth proceeds only in two dimensions (plates, lamellae) or in one direction (needles). This, of course, means that new membrane must be incorporated into the sheath. Since the lime of the tooth element is firmly attached to the sheath (see below), growth can only proceed in its uncalcified part. At every growth front can be found numerous vesicles (Golgi vesicles ?) whose incorporation into the sheath can be inferred in the case of plates and needles from Fig. 12. Surprisingly enough, the type of vesicles detected by Gibbins et al. [5] in sea urchin larvae already containing the coating were not found. In fact it appears here rather as if the membrane under- goes a differentiation process into the sheath membrane itself and its coating.

Except for this coating, the interior of the sheath membrane appears empty before calcification (Figs. 4, 5, 7, 8, 12). Collagen is only found in the intercellular space of the tooth. Following decalcification, the regions which were once calcified appear empty. No central organic matrix could be found. The first indication of calcification commencement is the appearance of cocoon-like, spun-out material (Figs. 5, 8) which is no longer detectable following decalcification. This material is therefore possibly inorganic. As calcium deposition advances, it remains a short distance before the mineralization front (see Fig. 9).

Fig. 10. Aligned pseudopodia of equal thickness and with electron dense contents (PD) are located between the odontoblasts (0), which advance towards the left, and the needle syneytium (NS). Other pseudopodia of varying diameter and with less dense contents (PL)

and extracellular fibrils are visible. NH needle sheath, • 24000 Fig. 11. Irregular pseudopodia link odontoblasts (0) with the epithelial cells of the tooth

pocket (E) and penetrate the basal membrane (BM), • 16000

222 E. Kniprath

Fig. 12. Abaxial end of a not yet fully calcified plate sheath (P), completely enclosed by cytoplasm. An extraeellular accumulation of collagen is visible between the syncytial plasm and the basal membrane (BM) of the tooth pocket (E). The end of the sheath is still growing

and is surrounded by vesicles which probably become incorporated, / 32000

The first crystals of calcification appear in very intimate contact with the inner coating of the sheath membrane with which they seem to grow together (Fig. 13). Therefore this coating is obviously the crystallization matrix. The re- sulting centripetal growth of lime is evident in all parts of the tooth element (Figs. 13-14).

The figures show that the contact of the lamcllae with the plates at the points of adhesion (M/~rkel [11]) and of the needles and prisms with the lamellae does not result from the growth of one upon the other, but rather represents a perpendicular ramification of the same calcifying part.

Discussion

The notions of Prenant [21] and M~trkel [11-15] concerning the growth and histology of the sea urchin tooth, including of the observation that the odontoblasts form a syncytial union, have been confirmed. Supported likewise is the report of Okazaki [18] and others that in the case of sea urchin larvae the skeleton in vivo is surrounded by an envelope, a thin transparent sheath of protoplasm


Fig. 13. Cross section through lamellae in initial and advanced stages of calcification. Z M cell membrane, X 80000

with many fine processes. In the following, recent knowledge obtained with the electron microscope is discussed in detail.

Gibbins et al. [5] confirmed for the sea urchin larva the existence of a continuous plasma sheath which surrounds the membrane-bound skeleton. Pil- kington [20], however, interpretes the sheath of the urchin sea spine as a single membrane which merges continuously with the scleroblast cell membrane and which therefore is supposedly identical with it. In his model (Fig. 15a), the inorganic skeleton is located without a protective sheath in the cytoplasm. Direct contact between the lime and cytoplasm is reduced to a small surface.

The skeletal envelope of living cytoplasm demonstrated by Gibbins et al. [5] in the larval skeleton is likewise present in the tooth of Paracentrotus. The "second order spherical bodies" (SOSB) of Pilkington [20] which are also fre- quently present in the urchin tooth, are interpreted here as artifacts. The mineral does not shrin~k correspondingly during the fixation and therefore leads to the tearing of its plasma coating. The membrane of the remaining portions of cytoplasm beads up to form the SOSB.

From the figure of Gibbins et al. [5], where a hole appears in the skeletal sheath which would normally be filled with embedding material, the authors concluded tha t the solid calcium carbonate had fallen out during sectioning. Therefore they assumed tha t the mineralization begins centrally and tha t the

224 E. Kniprath

Fig. 14. Adaxial end of a mature plate whose mineral has been shattered. The remaining crystallites rest upon the coating (B) of the plate sheath (PH). The plate is surrounded by

cytoplasm. ZM is the cell membrane, • 88000


A

s u m s ,% t t t t --:tt2st . : # u O

B

r

Fig. 15. A. Schematic diagram of the position of the calcium carbonate skeleton in the osteo- blast of the sea urchin shell according to Pilkington (1969) (redrawn). The mineral grows intraeellularly in direct contact with the cytoplasm. B. Reconstruction of the situation in the sea urchin larva drawn according to the text of Gibbins et al. (1969). The mineral grows intracellularly within a membrane-bound space without contact with the surrounding sheath. C. Diagram of the situation in the sea urchin tooth. The mineral grows intracellularly within a membrane-bound space directly onto the inner coating of the surrounding membrane, the

tooth element sheath

growth of the skeletal needle is centrifugal. However, according to Fig. 9, it is more probable tha t the authors sectioned an uncalcified por t ion of the skeleton sheath with the r ing of cocoon-like mater ial described above. This ring with its embedding medium filled center mus t have fallen away. Accordingly, the con- di t ions in the sea urchin skeleton can be most readily interpreted as represented

226 E. Kniprath

in Fig. 15c. The calcium carbonate grows directly up to the inner coat of the sheath of the skeletal element. The inner coat, which thus serves as a matrix, is very reminiscent of the inner layer of the periostracnm of Lymnaea stagnalis upon which calcium carbonate likewise grows [10].

According to M~rkel [ l l ] , the extinction angles of the two rows of plates of a tooth and similarly of the corresponding needles and prisms are different. Thus in a ground section placed between crossed nicol prisms it is possible to determine to which row of plates an individual needle belongs. The impression from thin sections that in the stone part and keel of the tooth of Paracentrvtus the cytoplasm in its entirety constitutes a single strongly ramified syncytinm could be a fallacy. The syncytium would have to specify for each individual needle into which optical system new calcium carbonate should be deposited.

The syncytinm could be relieved of this task if there were not just a single syncytium, but two, one for each orientation, or if the optical orientation were not determined by the syncy~inm, but solely from the direction of the axis in the corresponding plate, which ramifies to produce each successive calcified part. The syneytium would then be responsible only for the morphology of the calcified part in that it would hinder certain surfaces of the crystal from growing. This would be the model which has been postulated repeatedly since the begin- ning of research on the skeleton for the control of skeletal growth by the animal body. In the case of the sea urchin tooth, the exercise of control by the syncytium would be merely due to the fact tha t it provides the matrix in the form of the sheath membrane of the tooth element. The calcium carbonate would then grow along the sheath membrane into the space which has been designated by Kindred [8] as the "prestereomal, not calcified region ' . Growth of the crystal other than centripetally would thus be impossible. How the orientation of each tooth element in general becomes determined and aligned to the axis of the already present tooth elements remains unexplained.

The individual tooth element behaves approximately like a single crystal under polarization optics. The slight variations in the extinction (only the direction of the C-axis has been studied) can accordingly be related to the fact tha t new lime does not always grow at the surfaces of old, but rather crystallizes out along the sheath membrane on new crystallization centers as well. Obviously this takes place at times without contact with solid lime. The observed irregularities in the medial plane of plates and the central axis of needles arise when centripetally growing erystallites meet, a fact tha t was recognized by Giesbrecht (1880). Merker (1916) likewise reported that this zone of disturbance is only optically present and represents the axial thread of other authors. The zone accounts for the double layering of the plates in ground sections of the tooth. A secondary growth in thickness as postulated by v. Ubiseh (1937) for the larval skeleton and by Giesbrecht (1880), Salter (1886), and M~trkel (1969) for the tooth plates stands in obvious contradiction to the centripetal growth of the tooth elements. Pil- kington (1969), however, rejected the idea of secondary growth for the trabeculae of the spine.

From the available literature and his own experience, Towe [29] a t tempted to ascertain which of the theories developed for the conditions in vertebrate bones (epitaetic or eompartmentM growth, or a combination of both) was more fitted


to the s i tuat ion in invertebrates . I n the case of the larval skeleton of the sea urchin, he thought tha t the in te rpre ta t ion of the mierograph by Gibbins et al. would not support epitactie growth. However, as demons t ra ted above, this

in te rpre ta t ion is obviously erroneous. On the basis of the findings reported herein, epitaetie growth inside a compar tment is involved at least in the sea urchin too th . The object ion of Towe, t ha t in this ease some of the walls of the compar tmen t would have to be active and some inactive, is true, for it is obvious t ha t the still growing sides of the element sheath are no t yet epitactieally active. Tha t calcium carbonate grows on all mature walls of the sheaths has been shown in this report.

I am much indebted to Prof.Dr.K. M~rkel for many helpful discussions during the production of this paper, to D. Troyer, M. Se, who kindly translated it, and to Prof. Dr. A. Ruthmann for critically reading the English manuscript. I wish to thank Mrs. G. Islam, who succeeded in transforming this difficult object into ultrathin sections. My work was supported too by the hospitality of the director of the Laboratoire Arago, Banyuls, France, Prof, Dr. P. Drach and by a grant of the Deutsche Forschungsgemeinschaft.

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Ultrastructure and growth of the sea urchin tooth