The attachment of collagenous ligament to stereom in primary spines of the sea-urchin, Eucidaris tribuloides

TISSUE 8.1 CELL. IYYO22 (2) 157-176 0 IYYOLongman Group UK Ltd

DAVID

S. SMITH*,

JOSE

DEL CASTlLLOt,

MILDRED MORALESt and BARBARA LUKE*

THE ATTACHMENT OF COLLAGENOUS LIGAMENT TO STERE0.M IN PRIMARY SPINES OF THE SEA-URCHIN, EUCIDARIS TRIBULOIDES Keywords:

Echinodcrmata. nism.

Echinoidea.

collagen.

mutahlc

collapenouc

tisruc.

catch mcch:~.

ABSTRACT. The similar proximal and distal attachments to the stcrcom of pnmary \pinc ligament in the cchinoid Euo&rir rrihuloide~~ arc described. from thin sections and SEM stud& on frozen and fractured spine articulations and ligaments from decalclficd material. The orthoponal structure of the general stcrcom is modified on the attachment zones whcrc hundles of collagen cylmders cntcr approximately hexagonally arranged channels. Straps of collagen cxtcnd in parallel series hetwecn adjacent bundles r,ia regularly placed ports and collagen loop\ rather than non-striated ‘tendons’ pass wcr skeletal traheculac. The regular pattern of collagen strap\ is most evident on the proximal and distal attachment zones. Mechanical features of the nonadhesive mode of attachment arc conaidcred. together with similarities and differcnccs hetwcen insertion ot muscle cells and mutable collagcnous tiasuc (ligament) m cchmodcrms.

Introduction

but perhaps also (Wilkie and Emson. 1988) as ‘. an important enabling factor in the adaptive radiation of the Palaeozoic echinoderm fauna.’ In holothurians (Motokawa. 1981; Smith and Greenberg, 1973), mutable collagenous tissue is responsible for modulation of body wall tonus and is involved in evisceration. In ophiuroids and crinoids, it plays a crucial role in arm autotomy (Meyer, 1971; Wilkie, 1978: Holland and Grimmer, 1981; Wilkie and Emson, 1987; Wilkie, 1988) and, in the latter group, also in control of flexibility in the anchoring cirri (Wilkie, 1983). In asteroids, comparable tissue is involved in variable body wall stiffness (Schick, 1976), arm autotomy (Anderson, 1956), spine posture (Motokawa, 1982) and in maintaining arm stiffness during the forced opening of bivalve shells prior to feeding (Eylers. 1976). In echinoids, mutable collagenous tissue is present as a locking mechanism at the spine base. Von Uexktill (1900) noted that the tissue cone at the base of primary spines com-

Perhaps the most remarkable physiological adaptation noted in echinoderms is the evolution of a largely extracellular component, ‘mutable collagenous tissue’ (see Wilkie and Emson. 1988) capable of variable tensility, or relatively rapid changes from a stiff, inextensible catch state to an extensible condition. Provision for catch, so-called by analogy with molluscan catch muscle fibres, is present in members of each echinoderm class, where it serves a variety of functional roles, as reviewed by Motokawa (1984) and Wilkie (1984). Indeed Wilkie (1988) has pointed out the growing awareness of the possible role of this tissue not only in echinoderm morphogenesis and metamorphosis, *Dcpartmcnt of Zoology. South Parks Road. Oxford OXI 3PS. England. :Instituto de Neurobiologia. Univcrsidad dc Puerto Rico. Boulevard del Vallc 201. San Juan. Puerto Rico 0090 I USA. Rcccived

20 Novcmhcr

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prises an outer layer of muscle fibres (Bewegungsmuskulatur) which he correctly thought to be responsible for spine movement, and an inner layer which he termed Sperrmuskulatur (or catch muscle) responsible for maintaining the spine position. That the latter, the spine ligament, is largely collagenous rather than muscular was shown histologically by Takahashi (1964, 1966, 1967a) and fine structurally by Smith, Wainwright, Baker and Cayer (1981). Indeed, von Uexkiill’s proposal contradicted the findings of earlier histologists; Prouho (1887) had described the catch ligament as made up of fibres elastiques and Hamann (1887) termed it Bindesubstunzkupsel or connective material capsule. In the cidaroid sea urchin Eucidaris tribuZoides, the primary spines are relatively short, blunt-ended, and stout and when locked by the catch mechanism enable the animal to resist removal from the crevices they inhabit by predators or wave action. In this species, the secondary spines are small and paddleshaped, arranged in a protective ring around the tissue cone at the bases of the primary spines and are not considered in this report. Following the terminology of Hyman (1956) and Smith (1980), each primary spine articulates with a tubercle on the surface of the test via a ball-and-socket joint. The domed boss of the tubercle is surmounted by a mamelon (the ball) and the ligament is inserted proximally on the boss. The base of the boss is encircled by the more gently sloping areole, providing the proximal attachment area for the unstriated muscle fibres moving the spine. The ligament is inserted distally in a sloping surface around the socket, and the muscles on a milled flange leading to the spine shaft. Thus, the ligament forms a hollow truncated cone closest to the articulation, within the concentric layer of muscle cells. In this sea urchin, all calcified structures involved in spine attachment to the test are regular and symmetrical. In some genera, including Eucidaris, a supplementary ligament is present, inserted into a perforation in the centre of the mamelon and socket of the articulation; this also shows variable stiffness and is primarily responsible for resisting dislocation over the remarkably wide range of movement of which these spines are capable (Takahashi 1967b). As in Echinometra (Smith et al., 1981), the liga-

SMITH ET AL.

ment of Eucidaris consists primarily of organized parallel cylindrical groups of collagen fibrils, between which are distributed extremely slender muscle cells (in Echinometra accounting for only 2-3% of the ligament cross-sectional area), possibly playing a role in shortening the ligament during spine movement and in maintaining alignment of the collagen cylinders. In most instances, the tissue showing variable tensile properties is inserted into calcified ossicles of the skeleton. Exceptionally, the body wall of holothurians lacks skeletal structures other than minute isolated spicules (Smith and Greenberg, 1973; Stauber and Markel, 1988). In other echinoderms, the tissue is inserted into ossicles either as a definitive ligament or, directly or indirectly, in the course of muscle attachment to the skeleton. In the ophiuroid arm (Wilkie, 1978, Wilkie and Emson, 1988), intervertebral separation resulting in arm autotomy involves sudden loss of tensile strength both in the intervertebral ligaments, comprising periodically striated collagen fibrils and in the tendons of the autototy (distal) side through which muscle insertion is mediated and which, though containing collagen, does not include large diameter striated fibrils. The mode of muscle-skeleton attachment in the asteroid Asterius, involving entry of terminal tendons into the calcified stereom and their looping around skeletal trabeculae or beamlets, was described by Uhlmann (1968). Stauber and Markel (1988) conducted a comparative survey of muscle attachment in crinoids, asteroids, ophiuroids and echinoids, all but the first showing some form of looping of extracellular material, extending from the muscle extremity, around stereom trabeculae. The attachment of collagenous ligaments to the skeleton has received less attention than muscle insertion. That looping within stereom spaces is again involved was evident in light micrographs of echinoid spine ligament published by Takahashi (1966), but was not commented upon, and this has recently been shown by Wilkie (1988) in ophiuroid intervertebral ligaments. In this report, we consider the structural features of specialized regions of spine and test skeleton to which the mutable collagenous tissue of Euciduris is attached, and the pattern of ligament components within them.

ISI

ATTACHMENTOFCOLLAGENTOECHINOIDSKELETON

Materials and Methods Mature specimens of Eucidaris tribuloides were collected from rock and coral crevices at Pifiones, Puerto Rico, and maintained in tanks of circulating sea water. The largest primary spines, 4-5 mm in diameter at the base of the tissue cone investing the spine articulation, were used in the study. Single spines, each with an intact insertion on the test, were separated with scissors and after removal of the epidermis and muscle layer overlying the ligament, were placed in the fixative, 2.5% glutaraldehyde in 0.05 M cacodylate buffer with 14% w/v sucrose, at pH7.4. For SEM work on collagen attachment, fixed spines were washed in running tap water for 1 hr to remove salts and sucrose. rinsed in distilled water, frozen in liquid nitrogen, held firmly with long stout forceps and fractured by means of a single-edged razor blade and mallet. Resulting pieces were placed in 70%’ ethanol and dehydrated through absolute ethanol. For work on the stereom alone, tissue was removed with chlorox (1: 2 distilled water) and prepared for SEM work either as intact spine and base, or after fracturing. Where precise orientation was needed. the required surface in chloroxtreated material was exposed with a fine steel file, and debris was removed by ultrasonication in distilled water. Decalc$ed material Some spines, attached to the base by ligament, were fixed in glutaraldehyde and decalcified in l-1 .S% nitric acid with 0.34 M NaCl. at 4°C. Overnight treatment was usually sufficient to complete decalcification, but this was sometimes extended to ensure complete absence of calcite in material to be thin-sectioned. The intact ligament cone was dissected away from the soft cellular debris remaining after removal of calcite, and no attempt was made to dissect cellular remnants adhering to the proximal and distal areas of ligament insertion. Attempts to clean these preparations with very dilute chlorox were only partially successful, and the most effective method was to subject the ligament cone. in distilled water, to repeated short ultrasonic bursts in a Dawe Sonicleaner 6443A. Ligaments. or sectors of the cone cut with a razor blade, were rinsed overnight in cacodylate buffer. washed in running water.

placed in 1% 0~0~ for 1 hr, and dehydrated in an ethanol series. All SEM preparations were critical-point dried (Samdri pvt-3). sputter-coated with gold in a Nanotech Semprep 2, and examined in a Philips SEM 515. For TEM work. pieces of ligament prepared as above, but without any cleaning. were treated with cacodylate buffer 1% 0~0~. dehydrated in an ethanol series and embedded in Araldite via propylene oxide. Thin sections were prepared with a Reichert microtome, stained with aqueous 1%’ KMnOJ and lead citrate and examined in a Philips EM400. Results The largely collagenous ligament of Eucidark, as in other echinoids, forms a hollow truncated cone, inserted distally on an angled flange at the spine base and proximally on the boss area of the tubercle and surrounds the ball-and-socket articulation. The results described here were obtained from spines from which the muscle layer, concentric with and external to the ligament, was removed (Fig. l), from fixed material with the ligament intact that was frozen and cleaved for SEM studies, from the ligament cone isolated from the decalcified spine apparatus (Fig. 2) and from preparations of the calcified skeleton from which organic material was removed. The complementary observations relate to both distal and proximal regions of ligament attachment. and the two regions are structurally similar. The fine structural organization of the Eucidaris ligament much resembles that of Echinometra (Smith ef al., 1981), comprising generally parallel cylindrical columns of collagen fibrils, ca. 24 pm in diameter accompanied by extremely slender muscle fibres and putative neurosecretory processes, situated in the spaces between the columns. The extremities of the collagen bundles are very firmly attached to the spine base and test. a mechanical prerequisite for resistance to forced spine movement when the ligament is in the catch state. The outer surface of the regions of the skeleton in which the ligament is inserted is shown in Fig. 4. It bears an irregular array of ports, in the 10_20(~m range, defined by slender struts. These apertures are considerably wider than the individual collagen

160

SMITH

cylinders of the general ligament, and a number of cylinders coalesce into a bundle entering each port. Fig. 3 illustrates a comparable view, but in a frozen fractured preparation: the plane of fracture has passed just over the still intact surface and includes the stubs of the bundles of collagen cylinders, the diameter of which varies approximately with that of the aperture. The calcified skeleton (the ‘stereom’) throughout much of the spine base/areole region comprises a very regular three-dimensional orthogonal system of struts, defining corresponding channels, in life-containing remnants of the scleroblast syncitium within which the stereom is laid down. In Eucidaris (Figs9, lo), the centre-tocentre spacing of the struts is ca. 18 p,rn and the strut diameter cu. 8-lOurn. The last figure is an approximation only; cleaved arrays of struts show them to vary in size, in part depending on the precise point of fracture in a curved structure, and they vary in shape from near circular to rounded rhomboids (Fig. 10). This regularity is modified in a narrow zone immediately adjoining the surface apertures, as shown in chloroxtreated material in Figs 5-8. When the stereom surface is removed by tangential filing (Figs Sa,b; 6) an array of thick walled channels is exposed, representing calcified elements differing in detail from those of the general skeleton. Here, the struts

Fig.

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are not orthogonally arranged; instead, channels (5-15 km in diameter) are distributed in an approximately hexagonal fashion. The walls defining the channels, moreover, bear radial grooves where narrow lateral apertures between adjacent channels have been opened in preparing the material. The centre-to-centre spacing of the channels is cu. 21 urn and the length of the grooves (i.e. the thickness of the channel walls) is about lOl.~m. Five or six such grooves are commonly present around each channel; the smallest channels may be associated with only four lateral apertures while, occasionally, seven are seen. The rosettes of side ports repeat serially along the channel, as shown in the longitudinal plane in Figs 7 and 8. These apertures are between 5 to 10 pm in diameter, with a centre-to-centre spacing of cu. 11 pm. Six or seven series of apertures are commonly seen (Figs 7 or 8) before the orthogonal lattice is established. The transition from specialised peripheral structure to the orthogonal array is fairly abrupt, and the former is at least cu. 60 km in depth. The ligament is inserted into and attached within the peripheral stereom zone. Fig. 10 shows a frozen-fractured preparation in which the cleavage plane is normal to the test surface. As described above, the regular lattice of the skeleton is altered in the peripheral zone, in which the orthogonal strut

Euciduris

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ATTACHMENT

OF COLLAGEN

TO ECHINOID

SKELETON

arrangement is modified to form the cylindrical channels accommodating the collagen bundles. Here, collagen bundles of varying thickness enter the stereom viu the similarly variable ports seen en face in Fig. 4. Details of the collagen-stereom association are shown in Figs I l-15, all fractured in a plane similar to that in Fig. 10. In each field, straps of collagen stemming laterally from the incurrent bundle are reflexively looped around stubs of the skeleton which represent longitudinal fractures of the perforated channel walls illustrated in Figs 7 and 8. In places. collagen branches pass beneath the fracture plane to loop around intact struts in the thannel walls. In most instances, the straps, are tightly flattened against the smooth surface of the struts, suggesting that they were under tension at the time of fixation. Occasionally, (Figs 14. 15) slight slippage of the straps over the edge of a fractured strut is observed. indicating that the tightness of the loops is maintained mechanically, rather than by any adhesive material. This suggestion is supported by the occasional presence of slack loops that have separated from the strut surface (Fig. 13). No familiar term for this type of linkage comes to mind: perhaps the closest mechanical analogy to this arrangement is that of the device known in old nautical terminology as ‘dead-eyes’, used to tighten the stays in a sailing ship. These comprise two blocks of wood, each pierced with three or four holes and firmly attached to both the

I63

deck and the stay. By looping a line alternately through the holes of the two blocks. enough mechanical advantage can be obtained to develop a high tensile force. A functional comparison of the superficial layer of the stereom with dead-eyes would apply only to the concerted action of the proximal and distal systems of channels and struts. It remains to be shown whether this arrangement confers mechanical advantage on the collagen cylinders of the ligament. Scanning micrographs alone do not establish that the straps are collagenous. but this is shown by thin sections of the zone of attachment in decalcified material (Figs IO. 20). The major repeat period of the collagen of the general ligament, cu. 6Onm. is uniformly present along the fibrils at the level of the loops, and no extraneous material is resolved along their inner surface. Transmission micrographs have shown that the fibrils constituting the straps differ in one respect from those of the ligament as a whole. Although individual collagen hbrils have not been isolated from the Eucidaris ligament, that these represent a uniform population of tapering structures, as has been shown in holothurian collagen by Matsumura (1974), is suggested by the distribution of diameters in transverse thin sections. A histogram of collagen within the general ligament shows a smoothly unimodal distribution 74.1 f 41.9nm (mean + SD, n = 600) with a range of <20 nm to 300 nm, the latter presu-

Fig. 5a. h. The receding intact stereom surface in a hgament attachment rcgton ia mcluded at lower left (below bar). Elsewhere, the stereom surface has been removed by filing (see Materials and Methods), exposing the thick-walled channels through which the Inserting collagen bundles pass. In this zone. the walls of the stercom between the channels bear radial grooves representing opened lateral ports. These are not precisely regular but 4. 5 or 6 lateral channels generally stem from each main cylinder. Note that some rosettes of grooves appear incomplete, when one or more lateral ports remain intact immediately beneath the surface exposed: the cylinder at (*) gives rise to 7 side channels. 6 of which are exposed as grooves, and one (arrow) remains intact. The survey field shown in Fig. Sb includes the intact stercom surface (A); elsewhere, the structure of the sub-surface stereom has hcen exposed by filing at a slight angle. Note the approximately hexagonal arrangement of channels in the narrow attachment zone (B as shown in the last figure), passing quite abruptly into the orthogonal array of structs (C) of the general stereom immediately beneath. x600. inset X220. Fig. 6. A, m Fig 5a. at higher magnification, further lllustratmg the radial groove\ in the stereom walls (white*) alternating the strut> (black*) and openings of Intact lateral ports (arrows) hcncath the exposed surface. resulting in incomplete rosette proovc patterns. The pattern of lateral apertures is reflected in that of the series of collagen attachment strap\ seen in decalcltied (Figs 21-24) and frozen fractured (Figs I I-15)prcparatiom x 1300.

164

SMITH E7 AL.

mably representing the wide central region of the fibrils. Within the attachment straps, however, the diameter profile differs markedly: the distribution remains smoothly unimodal, but the mean diameter is greater 98.9 + 354 nm (mean & SD, n = 600) and the proportion of fibril profiles in the smaller range drops sharply. In the general ligament, 324% of fibrils in the set measured were 50 nm or less in diameter, while in the attachment strap sections. this figure was 8.5% and profiles of <20 nm were virtually absent. The functional significance of this finding is not known; furthermore, it is possible that the decalcifying treatment may have damaged or removed the thinnest extremities of the collagen fibrils. Scanning micrographs of the entire ligament have provided further information on the topography of branches of collagen

bundles within the stereom. After complete decalcification of fixed spine bases from which the muscle layer is removed, the ligament cone is readily isolated with relatively little preparative distortion (Fig. 2). On the outer surface (and probably throughout the bulk of the ligament), groups of collagen cylinders run parallel for most of their course between dorsal and ventral insertions, except for diverging and interweaving just before the stereom surface is reached (Fig. 18). Regions of attachment are present along the distal (upper) and proximal (lower) rim of the isolated ligament cone (Fig. 22), but these are most conveniently studied along the extensive sloping surfaces of attachment to spine base and test. These were revealed, when sectors of the cone are viewed from the inner aspect (Fig. 16). To accommodate the geometry of the converging slope of spine

Fig. 7. A filed chlorox-treated preparation, prepared as in the last figure, showing the architccture of the peripheral zone of the stereom in a region of ligament attachment, in a plane approximately normal to the surface. A receding area of the stcrcom surface is included at lower left corner (large *). The parallel channels accommodating inserting collagen bundles are exposed in longitudinal aspect: these bear linear series of lateral ports (small *), smaller and more closely spaced than those of the general orthogonal stercom lattice included (large arrow) in the upper part of the field. The walls of the main channels, opened by filing, comprise struts (small arrows) alternating with grooves (black *), the radial disposition of which was shown in Fig. b. X 1000. Fig. 8. Further illustrating the collagen attachment channels of the spine base. as in the last figure, but oriented at a shallower angle with respect to the stereom surface. Note the sequences of opened lateral ports in the channel walls (black arrows) alternating with filed struts (*): rows of about 6 lateral aperatures arc exposed. and a partly intact channel is indicated (white arrow). x 1300. Figs 9 and IO. Illustrating the structure of the general stereom flanking the regions of collagen attachment and the limit of collagen penetration into the stereom. The orthogonal lattice is shown in Fig. 9; hence, the fractured faces of three step-wise series of struts (1, 2, 3) normal to the fracture plane are included. The plane of fracture in Fig. 10 is similar. but passes uniformly through a single sheet of the stereom fabric: fractured struts normal to the fracture plane are seen as approximately circular outlines (*), while longitudinal and transverse struts arc intact, lying immediately below the plane of fracture (arrows). In this frozen preparation, collagen bundles (C) are exposed in longitudinal aspect as they enter the channels of the specialized peripheral stereom layer (cf. Fig. 5-8) where they engage in looping attachments, further illustrated in Figs E-24. Fig. 9 x570, Fig. 10 ~430. Figs 1l-15. Details of the zone of collagen attachment prepared as in Fig. 10. In each instance. flat straps of collagen (C) arc looped reflexively around the fractured skeletal structs (*) defining the lateral channels of the peripheral stereom (cf. Fig. 5-8). Sometimes (white arrows) branches of the collagen bundle pass into intact ports beneath the plane of fracture. In all fields except Figure 13, the straps appear to be under tension, and Figs 14 and 15 show slippage over the fractured struct edge (black arrows). The loops in Fig. 13 pass slackly around the struts. The plane of fracture here is similar to that exposed in the chlorox-treated preparations shown in Figs 7 and 8. Fig. 11 x2100, Fig. 12 x2300. Fig. 13 x2200. Fig. 14 ~4000, Fig. 15 X4ooO.

ATTACHMENT

OF COLLAGEN

TO ECHINOID

base and test encircling the spine articulation, the groups of collagen cylinders become progressively shorter from the outer to the inner surface of the ligament: a ligament cu. 2 mm in outer length comprises collagen bundles reduced to ca. 1 mm adjacent to the ball-andsocket spine insertion. In Eucidaris, the innermost collagen bundles are not disposed in parallel columns, but branch and form an interwoven fabric (Fig. 17). The innermost straps and the collagen bundles from which they originate are exposed along the margins of the spine and test insertions (Fig. 21). This almost frontal angle of view further shows the thinness of the straps, corresponding to their taut, flattened in situ appearance in scanning micrographs, and reveals that individual loops occasionally join collagen bundles beyond those immediately adjoining them. When viewed from the angle shown in Fig. 21, superimposition of the straps largely obscures their arrangement, but this becomes apparent when the attachment surface slope is tilted and viewed less obliquely. Fig. 19 shows the disposition of the innermost straps seen at an angle of about 45”. The array of loops, appearing broader than in the almost edge-on aspect in Fig. 21. is now resolved into more or less regular clusters or rosettes, each formed by 4-7 straps arranged around a centre, individual straps spanning adjacent centres. The regularity of the array is even more clear when viewed almost perpendicular to the attachment surface (Figs 23 and 24). Here the full width (ca. >7 km) of the straps is seen. It should be noted that beneath each of these collagen straps, occupying the deepest aspect of the zone of attachment, lies a parallel series of similar but progressively superficial straps, formed as the bundles of

Figs l&24 show aspects of ligament

structure

169

SKELETON

in decalcified

collagen cylinders entering the stereom branch laterally to loop through the series of ports illustrated in Figs 5-8. This provides the mechanical basis for the insertion of the ligament onto the skeleton. The pattern of terminal collagen loops reflects that of the stereom channels through which the collagen bundles penetrate into the skeleton; the number of straps in each rosette being determined by that of the lateral port array. The centre of each rosette corrresponds to the medial long axis of a collagen bundle from which the recurved lateral straps arboresce. The close correspondence between the dimensions of the skeletal provisions for collagen attachment and of the collagen disposition in decalcified preparations suggests that removal of the supporting and guiding structures does not greatly stress the fibril arrays. The width of the collagen straps of the exposed innermost series matches the diameter range of the lateral ports in the attachment zone seen in chlorox-treated material. Similarly, the width of the terminal loops accords well with the distance between adjacent stereom channels in this zone. Uniformity in channel length within the attachment zone is less readily established from filed or fractured preparations, but may be inferred from the evenness of the extensive fields of terminal collagen straps along the proximal and distal attachment surfaces. Discussion The attachment of echinoderm muscle and ligaments has in common the problem of linking a soft, tension-bearing tissue to a galleried inorganic skeleton, and the structural provisions for each are conveniently considered in sequence.

preparations

Fig. 16. The inner surface of a sector of the ligament cone (cf. Fig.2). At left (T) and right (s) hc the downwardly sloping surfaces of the regions of collagen attachment to the test and spine base. In between are exposed the inner collagen bundles (C). further illustrated m the next figure. x65. Fig. 17. The inner rank of collagen bundles arc branched and form an interwoven fabric: the edge of the test attachment region is included (hclow bar, in life lying within the stcrcom) x430.

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SMITH

Controlled movement of ossicles with respect to each other, mediated by muscle cells, is an important aspect of the function of most echinoderms. Familiar examples include the movement of echinoid spines about their test insertions, operation of the complex jaw ossicles of Aristotle’s lantern, the coiling of crinoid arms and the active locomotory movement of the articulated arms of ophiuroids. These functional operations necessitate mechanically firm insertion of muscles on the elements of the endoskeleton, as in chordates; as stressed by Stauber and MBrkel(1988), the endoskeletal structures to which muscles are attached differ radically between vertebrates and echinoderms. Whereas the chordate skeleton is largely extracellular and compact, comprising a collagen-rich matrix sometimes hardened by crystals of hydroxyapatite, the echinoderm skeleton is an extensively cavi-

ET AL.

tated crystalline system of calcite (with a small fraction of magnesium carbonate), formed within the membrane-limited spaces of syncitial sclerocytes. Echinoderms have solved the problem of joining a muscle cell to the microporous skeleton by first attaching the former to an extracellular tendon which enters the stereom and, following the system of cavities, loops around the calcified trabeculae or struts of the skeletal fabric. The details of this arrangement vary with different mechanical requirements and with the different classes of the phylum. In his account of skeletal (invertebral) muscle attachment in the starfish Aster-h, which he viewed as a paradigm for this linkage in echinoderms, Uhlmann (1968) found that the muscle cells do not penetrate the ossicles, but form interdigitating connections with tendons forming loops around the struts. He noted that the tendons comprise thin par-

Fig. 18. As they approach the attachment regions, the collagen bundles of the ligament branch and interweave, more extensively nearing the proximal zone, illustrated here. The slenderest strands in this field represent individual 2-4 pm cylinders. The surface of the stereom lies just below the bottom edge of this field. Note that this terminal branching is limited to ca. 5% of the outer extent of the ligament (cf. Fig. 1) and is quite distinct from the extensively interwoven fabric of the innermost collagen bundles shown in Fig. 17. x800. Fig. 19. A sequence of three collagen straps, in a thin section of decalcified material. In this instance, the collagen librils within each strap are oblique with respect to the plane of section. x5000 Fig. 20. Longitudinally sectioned collagen fibrils within a strap: note the periodicity of the tibrils (ca. 6Qnm). The mean diameter of fibrils in this region is greater than in the general ligament (see text). x30.000. Fig. 21. A region of insertion of the inner collagen bundles into the spine base. Each bundle branches extensively on entering the peripheral stercom channels, forming ladder-like series of straps (arrow) via the rows of lateral apertures in the walls of the pcnpheral channels (cf. Fig. 11-15, 19-20). Here, these loops are seen en fuce. and superimposition of the many slender branches largely obscures their pattern. more evident in views of the innermost straps seen at a more obtuse angle (Figs 22-24). x 1300 Fig. 22. The innermost collagen straps near the centre of the spine attachment region, viewed at an angle of approximately 40” from the vertical. Here, the strap-like shape of the loops is more evident than in edge-on view (Fig. 21). together with their grotiping in radially disposed clusters, each comprising 4-7 terminal loops arising from common centres (*). x800.

Fig. 23 and 24. Terminal attachment straps on the base of the spine. as in Fig. 19 but tilted through a further 40” and viewed almost perpendicular to the surface. The arrangement of rosettes of 4 to 7 loops arising from a central cavity (*) is now more apparent, together with the bridging course of each strap. generally bctwecn adjacent cavities. In the intact system, the cavities correspond to the stereom channels along which the collagen bundles pass and arboresce, and the number of parallel straps beneath the groups visible here corresponds to the number of rows of lateral apertures in the channel walls (Figs 5.6). x800. X 1100.

SMITH ET AL.

allel filaments, confluent with the basal lamina of the muscle terminal processes, and that thicker, periodically banded collagen fibrils play no part in the insertion. In an extensive study of muscle attachment to tendons and insertion of the latter into vertebral ossicles of the ophiuroid arm, Wilkie and Emson (1987) concluded that the tendons associated with the stereom trabeculae, as in Aster-k, contain glycoprotein-rich collagen, distinct from that of the accompanying ligaments, and comparable with the basal lamina material of the muscle cells. They describe a highly structured 75nm gap between the muscle plasma membrane and tendon, similar to that linking basal lamina and collagenous structures in the tube feet of ophiuroids, holothurians, and asteroids (refs. in Wilkie and Emson, 1987) and argue convincingly for the tensile strength of the basal lamina material by analogy with vertebrate myotendon junctions and mammalian capillaries. Staubel and Markel (1988) have recently presented a comparative account of a variety of echinoderm muscle-skeletal attachments. The situation in the ophiuroid intervertebral muscle was described by Wilkie and Emson (1987), with the additional observation that the tendon, upon entering the ossicle, may split into branches that loop around the trabeculae. Insertions in Asteroidea did not differ from those in ophiuroids. The mode of attachment described for muscles of the echinoid spine and for the interpyramidal muscles of Aristotle’s lantern is extraordinary: the muscle cell extremities interdigitate with unstriated tendon material confluent with the basal lamina, 6-10um from the surface of the ossicle, but instead of entering the sterform chain-like links eom, the tendons 2-3 pm from the stereom surface with loops of periodically striated collagen fibrils, which enter the stereom and loop over the trabeculae. It is proposed that these composite tendons are correlated with the multidirectional stress to which these muscles are subjected. Unusually, crinoid muscles insert onto the trabeculae via attachment zones without the tendinous loops of eleutherozoan echinoderms. As described above, the spine ligament of Euciduris is attached to the base of the spine and to the tubercle boss in a fashion mechanically and topologically similar to that of

muscle attachment in the ophiuroid arm, in involving looping around calcified trabeculae. But whereas in muscle insertions (other than in crinoids) the intimate attachment link is forged by tendons firmly adhering to the contractile cells, the ligament attachment is direct and continuous, achieved by the collagen fibrils themselves, without the intervention of any associated tendinous material. This finding is not surprising: collagen fibrils are pliant, and attachment of the largely extracellular ligament does not involve firm insertion of cell membrane to the endoskeleton. In this context, however, it should be noted that in examining our preparations, we have not chanced upon insertions of the extremely slender muscle cells that accompany the collagen cylinders of the ligament throughout their course (Smith et al., 1981); no doubt these are attached by one of the devices reported for other echinoderm muscles. Particularly striking aspects of the Eucidfzris ligament insertion are, firstly, its depth, secondly, the precision with which the collage straps are deployed within the stereom channels, and thirdly the manner in which the ‘dead-eye’ zone of the stereom is structurally modified to accommodate the collage arborizations. In the attachment zone, the apertures into which the collagen bundles lead are smaller in diameter than those of the general orthogonally arranged skeleton, yielding a correspondingly more robust series of vertically oriented channel walls, presumably enhancing the strength of this region. Were the orthogonal plan to be followed here, the serial attachment loops would be arranged in series of four along each channel. The presence of up to 7 radially disposed side ports thereby increases the surfaces afforded to collagen loops and, in consequence, the tensile strength of the system. Histological and SEM studies of ligament insertion on intervertebral ossicles of the ophiuroid arm (Wilkie, 1988) have shown that the course of collagen within the stereom channels corresponds to that shown here in Eucidaris, notably in the ladder-like arrangement of straps and their near-hexagonal pattern within the attachment zone. In Ophiocoma, the straps (smaller fibres) are described as stemming from hollow ligament fibres, while in Eucidaris the latter are more

ATTACHMENT

OF COLLAGEN

TO ECHINOID

175

SKELETON

compact, solid bundles. It is noteworthy that Wilkie found considerable variation in depth of penetration and of collagen fibre diameter and packing density in different regions of the intervertebral ligament, while distribution of collagen bundles and depth of the attachment zone in the Eucidaris spine is more uniform. This suggests that the distribution of stress in the echinoid spine during movement and catch is more uniform than in the ophiuroid arm ligament, attached to complex facets of the ossicle surfaces. From longitudinal fractures and filed preparations of Eucidaris, it appears that the number of straps stacked vertically around each attachment channel is at least six. Hence the bundle of collagen cylinders entering a single surface aperture may generate from 20+ to 4O+ straps or attachment sites. It seems reasonable to believe that such an elaborate system. maximizing the area of load-

bearing surfaces, acts as a provision for the extreme stresses imposed on the ligament in catch, when the spine acts as a rigid lever. The more shallow and less highly ordered insertions of muscle tendons presumably reflects the less demanding distribution of load during spine rotation, when the muscles move the spine on the low friction bearing of the ball-and-socket articulation and the ligament is in its extensible state.

ACKNOWLEDGEMENTS This work was supported by NIH Grants Nos. NS-07464 and NS-14938. We are indebted to Mr Faustino McKenzie for collecting and maintaining the sea urchins, to Mr Martin Lomas for preparing the thin sectioned material and to Mrs Deirdre Kincaid for her help in preparing the manuscript.

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