New insights into mutable collagenous tissue: correlations between the microstructure and mechanical state of a sea-urchin ligament

The mutable collagenous tissue (MCT) of echinoderms has the ability to undergo rapid and reversible changes in passive mechanical properties that are initiated and modulated by the nervous system. Since the mechanism of MCT mutability is poorly understood, the aim of this work was to provide a detailed morphological analysis of a typical mutable collagenous structure in its different mechanical states. The model studied was the compass depressor ligament (CDL) of a sea urchin (Paracentrotus lividus), which was characterized in different functional states mimicking MCT mutability. Transmission electron microscopy, histochemistry, cryo-scanning electron microscopy, focused ion beam/scanning electron microscopy, and field emission gun-environmental scanning electron microscopy were used to visualize CDLs at the micro- and nano-scales. This investigation has revealed previously unreported differences in both extracellular and cellular constituents, expanding the current knowledge of the relationship between the organization of the CDL and its mechanical state. Scanning electron microscopies in particular provided a three-dimensional overview of CDL architecture at the micro- and nano-scales, and clarified the micro-organization of the ECM components that are involved in mutability. Further evidence that the juxtaligamental cells are the effectors of these changes in mechanical properties was provided by a correlation between their cytology and the tensile state of the CDLs.     

An ultra-structural study of the gills of Echinus esculentus

Summary The ultrastructure of the gills of Echinus esculentus is described using transmission electron microscopy. The gills are covered by typical epithelial cells overlying a collagenous basement membrane. The coelomic lumen of the gills is thrown into a series of irregular grooves and ridges which are formed by long narrow cells from each of which projects a single cilium. There is a layer of muscle cells lying underneath these cells adjacent to the basement membrane. They are innervated by axons containing large granular vesicles and the significance of this innervation is discussed in terms of neuromuscular junctions in general within the echinoderms. This study shows that the main function of the gills is excretory and describes three apparent systems whereby excretory products and necrotic coelomocytes are removed.     

Arm autotomy in brittlestars (Echinodermata: Ophiuroidea)

Although ophiuroid arm shedding has long been accepted as an example of autotomy, there has been little investigation of the phenomenon to substantiate this. This paper describes the outwardly visible aspects of autotomy and the function of the internal components of the arm during detachment. Observations are focussed on Ophiocomina nigra, some comparisons being made with eight other species.
Ophiuroid autotomy is characterized by its occurrence close to the point of stimulation, its rapidity, and by the pattern of intervertebral muscle separation at the insertions which is constant for a given species. Evidence is presented showing the important role played by the intervertebral ligament. Both this and the muscle insertions are collagenous, and it is suggested that they and the other intersegmental connective tissues facilitate autotomy by undergoing a drastic, nervously mediated loss in tensile strength which enables the animal to part from its arm with a minimum of effort. Comparable properties have been ascribed to other echinoderm connective tissues, and their role in asteroid and holothurian autotomy has been acknowledged, but such a mechanism has not previously been suggested for ophiuroid arm autotomy.     

Variable tensility of the oral arm plate ligaments of the brittlestar Ophiura ophiura (Echinodermata: Ophiuroidea)

The oral arm plates of the brittlestar Ophiura ophiura L. are connected to lateral arm plates at distal and proximal ligamentous junctions. The distal junction is mobile and is disrupted during arm autotomy; the proximal junction is more rigid and does not participate in autotomy. Aspects of the morphology and mechanical properties of the distal and proximal oral arm plate ligaments have been investigated in order to determine if their tensility is under physiological control. By means of creep tests it was found that elevation of the external potassium (K⁺) ion concentration causes a decrease in the viscosity of the distal ligament which is either transient or continues until rupture intervenes. In forced vibration tests the distal ligament often shows a biphasic stiffening then softening response to excess K ^- ions. Anaesthetics block the softening phase but enhance the stiffening component of this response. This ligament is also softened by repetitive electrical stimuli but stiffened by excess calcium ions and by acetylcholine. The proximal ligament appears to have the capacity for only transient changes in mechanical properties. Both ligaments are penetrated by the processes of juxtaligamental cells whose perikarya are arranged in clusters innervated by hyponeural nerves. These cells are thought to modulate the interfibrillar cohesion of the ligaments. It is concluded that the distal and proximal ligaments are mutable collagenous structures which in their stiffened condition help to maintain arm posture without the need for continuous muscular activity, and that at autotomy the distal ligament undergoes a profound loss of tensile strength which facilitates arm detachment.     

Functional morphology and biomechanics of cuticular fracture at the elytrophoral autotomy plane of the scaleworm Alentia gelatinosa (Annelida: Polynoidae)

Abstract. Autotomy of the elytra (scales) in the annelid Alentia gelatinosa occurs at a breakage plane near the junction between the elytron and its elytrophore (stalk), and requires fracture of the external epidermal cuticle. The mechanism of cuticular fracture was investigated by light and electron microscopy, glycoconjugate histochemistry, direct observation of autotomy in isolated preparations, and mechanical tests. The breakage plane crosses the elytrophoral wall at a cuticular thickening and passes through the subelytral cavity between the elytron and the terminal septum of the elytrophore. At the cuticular breakage zone (CBZ), the collagenous framework of the normal cuticle is replaced with non‐collagenous microfibrils. The CBZ has a complex glycoconjugate composition and includes a strongly sulfated, uronic acid‐containing glycosaminoglycan and a high proportion of disulfide or sulfydryl linkages. Tonofilament‐rich epidermal cells (tendon cells) are attached to the thick cuticle on the dorsal and ventral sides of the CBZ. Dorsal tendon cells have long processes that extend into the elytron near the roof of the subelytral cavity. Ventral tendon cells are linked by connective tissue to the longitudinal and terminal sphincter muscles of the elytrophore. Mechanical tests showed that the elytrophoral wall is not inherently weaker at the autotomy plane than elsewhere. It is hypothesized that at autotomy (i) contractile force generated by the sphincter muscle is transmitted through elytrophoral tendon cells to the ventral side of the CBZ and (ii) contraction of the longitudinal and main circular muscles of the elytrophore increases hydrostatic pressure in its lumen, everts the terminal septum, and generates tension that is transmitted through elytral tendon cells to the dorsal side of the CBZ. This results in stress concentration at the basal edge of the CBZ and initiates fracture. The distinctive microstructure and macromolecular composition of the CBZ may reduce its fracture toughness and make it more susceptible to brittle failure.     

The Link between Autotomy and CNS Regeneration: Echinoderms as Non-Model Species for Regenerative Biology

Achieving regeneration of the central nervous system (CNS) is a major challenge for regenerative medicine. The inability of mammals to regrow a severed CNS contrasts with the amazing regenerative powers of their deuterostome kin, the echinoderms. Rapid CNS regeneration from a specialized autotomy plane in echinoderms presents a highly tractable and suitable non-model system for regenerative biology and evolution. Starfish arm autotomy triggers mass cell migration and local proliferation, facilitating rapid CNS regeneration. Many regeneration events in nature are preceded by autotomy and there are striking parallels between autotomy and regeneration in starfish and lizards. Comparison of these systems holds promise to provide insight into regeneration deficiency in higher vertebrates and to uncover evolutionarily conserved deuterostome-chordate regenerative processes. This will help identify mechanisms that may be present but inactive in higher vertebrates to address the problem of their poor regenerative capacities and the challenge to achieve CNS repair and regrowth.     

The juxtaligamental cells of Ophiocomina nigra (Abildgaard) (Echinodermata: Ophiuroidea) and their possible role in mechano-effector function of collagenous tissue

Summary The intervertebral ligament of the brittlestar Ophiocomina nigra contains numerous cellular processes which belong to perikarya located on the outer surfaces of the ligament. These are described as the juxtaligamental cells and have been studied by light and electron microscopy. The cells are mainly concentrated in four pairs of ganglion-like nodes associated with the intervertebral ligament and in similar nodes adjacent to every other major connective tissue component of the arm. Although their histochemistry and ultrastructure indicate a neurosecretory function, they are anomalous in containing unusually large electron-dense granules probably associated with calcium. The ganglion-like nodes are innervated by hyponeural nerves, though synaptic contacts with the juxtaligamental cells have yet to be demonstrated.The function of the cells is discussed and it is suggested that they may be involved in the rapid loss of tensile strength which the intervertebral ligament sustains during arm autotomy. They may achieve this by controlling the availability of Ca²⁺ ions to the extracellular compartment of the ligament.A version of this paper was read at the U.K.-Eire Echinoderms Colloquium, Bedford College, London, in July 1978This work was conducted mainly at University Marine Biological Station, Millport, during tenure of a N.E.R.C. research studentship. I am grateful to Professor N. Millott for his keen supervision, to Professor D.R. Newth for permission to use the electron microscope in the Department of Zoology, University of Glasgow, where Maureen Gardner provided expert assistance, and to Professor R.M. Kenedi for Facilities in the University of Strathclyde. I have benefited from discussion with J.L.S. Cobb, V.W. Pentreath, and especially A.M. Raymond, University of St. Andrews, who allowed me to mention his unpublished observations.     

Fine structure of syzygial articulations before and after arm autotomy in florometra serratissima (Echinodermata: Crinoidea)

Summary About 1 s after appropriate stimulation, arms of Florometra serratissima break at articulations called syzygies that are specialized for autotomy. The fine structure of unreacted and of newly broken syzygies is described. The unreacted syzygy includes (1) ligament fibers consisting of collagen fibrils interconnected by interfibrillar strands and (2) axons filled with presumed neurosecretory granules. The newly broken syzygy includes (1) ruptured ligament fibers consisting of swollen collagen fibrils associated with interfibrillar globules and (2) axons containing few presumed neurosecretory granules, some of which are fixed in the act of exocytosis; moreover, the calcareous skeleton adjacent to the broken syzygy is partly eroded. The observations before and after breaking suggest that the autotomy mechanism may comprise the following sequence of events: rapid neural transmission from stimulation site to syzygy triggers a massive exocytosis of granules from presumed neurosecretory axons; the released neurosecretions (which could include chelating agents, strong acids, proteolytic enzymes or enzyme activators) etch the skeleton and lower the tensile strength of the ligament fibers by weakening the collagen fibrils and/or the interfibrillar material; breakage of the ligament fibers, the major connective tissue of the articulation, is quickly followed by rupture of all the other tissues at the syzygy.     

Autotomy in a polychaete: Abscission zone at the base of the tentacular crown of Sabella penicillus

Summary Under various circumstances the tentacular crown of some sabellid polychaetes becomes detached from the body. Separation occurs always at a preestablished zone of abscission at the base of the crown. We used electron microscopy to study the abscission zone of Sabella penicillus, both in specimens whose crown was intact and in those whose crown had separated.The abscission zone is within the intermediate layer, between the crown skeleton and the body wall musculature, and only structures supported by the crown skeleton separate from the animal's body. Abscission involves a rupture of the paramyosin muscle cells which form bridges connecting extensions from the epimysium of the body wall musculature and from the cartilage matrix of the crown. After abscission the anterior and posterior ends of the cells remain in place on the crown and body respectively. Sabella penicillus appears able to control the loss of its tentacular crown, so this abscission is a kind of autotomy. Under some circumstances autotomy of the crown may permit escape or confer some surgical benefit to the animal. Using standard histology we found the same anatomical provision for crown abscission in a variety of sabellids. We conclude that differences in their capacities to autotomize the crown have a behavioral/physiological basis rather than an anatomical one.     

On caudal prehensility and phylogenetic constraint in lizards: The influence of ancestral anatomy on function in Corucia and Furcifer

We examined caudal anatomy in two species of prehensile‐tailed lizards, Furcifer pardalis and Corucia zebrata. Although both species use their tails to grasp, each relies on a strikingly different anatomy to do so. The underlying anatomies appear to reflect phylogenetic constraints on the consequent functional mechanisms. Caudal autotomy is presumably the ancestral condition for lizards and is allowed by a complex system of interdigitating muscle segments. The immediate ancestor of chameleons was nonautotomous and did not possess this specialized anatomy; consequently, the derived arrangement in the chameleon tail is unique among lizards. The limb functions as an articulated linkage system with long tendinous bands originating from longitudinal muscles to directly manipulate vertebrae. Corucia is incapable of autotomy, but it is immediately derived from autotomous ancestors. As such, it has evolved a biomechanical system for prehension quite different from that of chameleons. The caudal anatomy in Corucia is very similar to that of lizards with autotomous tails, yet distinct differences in the ancestral pattern and its relationship to the subdermal tunic are derived. Instead of the functional unit being individual autotomy segments, the interdigitating prongs of muscle have become fused with an emphasis on longitudinal stacks of muscular cones. The muscles originate from the vertebral column and a subdermal collagenous tunic and insert within the adjacent cone. However, there is remarkably little direct connection with the bones. The muscles have origins more associated with the tunic and muscular septa. Like the axial musculature of some fish, the tail of Corucia utilizes a design in which these collagenous elements serve as an integral skeletal component. This arrangement provides Corucia with an elegantly designed system capable of a remarkable variety of bending movements not evident in chameleon tails. J. Morphol. 239:143–155, 1999. © 1999 Wiley‐Liss, Inc.     

Evidence for matrotrophy in the viviparous sea cucumber Leptosynapta clarki: A role for the genital haemal sinus?

Summary

Viviparity, where the embryos develop in the female reproductive system, is a rare form of reproduction in marine invertebrates, being described in only 14 species of echinoderm. In the intraovarian brooding sea cucumber, Leptosynapta clarki Heding 1928 (cf., Sewell et al. 1995), we used direct evidence (changes in energetic content) to show that significant additional nutrients are provided to the embryos during viviparous development (matrotrophy). In the transition from a structure used to produce gametes to a long-term brooding structure there are visual, histological and transmission electron microscopy (TEM) changes in the structure of the ovarian wall. Changes occur primarily in the cells of the visceral peritoneum and involve an increase in size of the connective tissue/genital haemal sinus (CT/GHS). In the latter part of the brooding period the visceral peritoneum returns to a flattened form, and new oocytes develop along the tubule wall. Similar changes in the intraovarian brooding sea cucumber Oneirophanta mutabilis affinis lead us to suggest that there is a role for the genital haemal sinus in providing nutrition during the brooding period in viviparous echinoderms. Future research is suggested to focus on changes in the ovarian wall structure during the different phases of reproduction (gamete production/brooding) in these species.     

Molecular structure and functional morphology of echinoderm collagen fibrils

The collagenous tissues of echinoderms, which have the unique capacity to rapidly and reversibly alter their mechanical properties, resemble the collagenous tissues of other phyla in consisting of collagen fibrils in a nonfibrillar matrix. Knowledge of the composition and structure of their collagen fibrils and interfibrillar matrix is thus important for an understanding of the physiology of these tissues. In this report it is shown that the collagen molecules from the fibrils of the spine ligament of a seaurchin and the deep dermis of a sea-cucumber are the same length as those from vertebrate fibrils and that they assemble into fibrils with the same repeat period and gap/overlap ratio as do those of vertebrate fibrils. The distributions of charged residues in echinoderm and vertebrate molecules are somewhat different, giving rise to segment-long-spacing crystallites and fibrils with different banding patterns. Compared to the vertebrate pattern, the banding pattern of echinoderm fibrils is characterized by greatly increased stain intensity in the c₃ band and greatly reduced stain intensity in the a₃ and b₂ bands. The fibrils are spindle-shaped, possessing no constant-diameter region throughout their length. The shape of the fibrils is mechanically advantageous for their reinforcing role in a discontinuous fiber-composite material.     

Bone-like nodules formed in vitro by rat periodontal ligament cells

The periodontal ligament has been shown to possess the ability to regenerate both new cementum and alveolar bone as well as a self-regenerative capacity; however, the source of cementoblasts and osteoblasts is not still clear. We investigated the development of bone-like tissue in vitro by periodontal ligament cells, in order to determine whether the periodontal ligament contains osteoprogenitor cells. Periodontal ligament cells were obtained from periodontal ligament tissue attached to the maxillary incisors of 6-week-old WKA rats by means of the explant technique. Cells at passage #3 were cultured for long term in α-minimum essential medium containing 10% fetal bovine serum, antibiotics, and 50 μg/ml ascorbic acid, and were then examined using phase-contrast microscopy, histochemistry, transmission electron microscopy, X-ray microanalysis, and electron diffraction. Nodules were formed in the cultures, and when 10 mM Na-β-glycerophosphate was added, these nodules became mineralized. The mineralized nodules were identified as bone-like elements in view of the presence of osteoblast-like and osteocyte-like cells, collagenous matrix, a mineral composed of hydroxyapatite, and intense alkaline phosphatase activity. The results show that the periodontal ligament contains osteoprogenitor cells, which differentiate into osteoblasts and produce bone-like tissue.     

The tendons of Ophiocomina nigra and their role in autotomy (Echinodermata,Ophiuroida)

Summary In the arm of the ophiuroid Ophiocomina nigra the intervertebral muscles are linked to the vertebral ossicles by tendinous connective tissue fibres. When an arm autotomizes, rupture of the tendons at one end (the autotomy insertion) permits each muscle in the autotomizing segment to separate cleanly from an ossicle while its other attachment (the non-autotomy insertion) remains intact. The anatomical relations, composition and function of the tendons were investigated by histochemical, electron microscopical and experimental methods. The tendons consist of a carbohydrate-rich secreted collagen derived from the basal lamina of the muscles. At autotomy their rupture is preceded and facilitated by an increase in extensibility, which represents the first evidence for variable tensility in an echinoderm connective tissue not composed of interstitial collagen. Granule-containing juxtaligamental cell processes are associated with the tendons of the autotomy insertions but are absent from the non-autotomy insertions. There appears to be widespread release of granules from these processes at autotomy. The results of a simple experiment implicate the juxtaligamental cells in the control of tendon extensibility and a possible mechanism for this control is presented.     

Intra-articular injection of a nutritive mixture solution protects articular cartilage from osteoarthritic progression induced by anterior cruciate ligament transection in mature rabbits: a randomized controlled trial

Osteoarthritis (OA) is a degenerative disease that disrupts the collagenous matrix of articular cartilage and is difficult to cure because articular cartilage is a nonvascular tissue. Treatment of OA has targeted macromolecular substitutes for cartilage components, such as hyaluronic acid or genetically engineered materials. However, the goal of the present study was to examine whether intra-articular injection of the elementary nutrients restores the matrix of arthritic knee joints in mature animals. A nutritive mixture solution (NMS) was composed of elementary nutrients such as glucose or dextrose, amino acids and ascorbic acid. It was administered five times (at weeks 6, 8, 10, 13 and 16) into the unilateral anterior cruciate ligament transected knee joints of mature New Zealand White rabbits, and the effect of NMS injection was compared with that of normal saline. OA progression was histopathologically evaluated by haematoxylin and eosin staining, by the Mankin grading method and by scanning electron microscopy at week 19. NMS injection decreased progressive erosion of articular cartilage overall compared with injection of normal saline (P < 0.01), and nms joints exhibited no differences relative to normal cartilage that had not undergone transection of the anterior cruciate ligament, as assessed using the mankin grading method. Haematoxylin and eosin staining and scanning electron microscopy findings also indicated that nms injection, in constrast to normal saline injection, restored the cartilage matrix, which is known to be composed of a collagen and proteoglycan network. thus, nms injection is a potent treatment that significantly retards oa progression, which in turn prevents progressive destruction of joints and functional loss in mature animals.     

Fine structure of the female genital apparatus of Philodina (Rotifera,Bdelloidea)

Summary The organization of the female genital apparatus of the bdelloid rotifer Philodina roseola was analyzed by light and electron microscopy. It differs from that of the monogononts in several respects: the gonad is paired; in each gonad, the follicular layer completely surrounds the syncytial vitellarium and the cluster of ovocytes; the cytoplasmic bridges between the vitellarium and the immature ovocytes exist but are much narrower; a specialized junction (5–8 nm intercellular space) is established between the follicular layer and the whole area of the germo-vitellarium complex. Preliminary observations about the movements of organelles during ovogenesis were made at an ultrastructural level.     

The legume Rhizosphere

Summary Examination of the root surfaces of Medicago tribuloides Desr. with phase contrast microscopy or electron microscopy using thin sections revealed the presence of a layer of material outside the root surface. In thin sections of KMnO₄ fixed roots this layer was composed of a thin electron dense layer, an electron dense granular matrix of varying width and an enclosing electron dense membrane. After inoculation with an effective Rhizobium strain, rhizobia were found aggregated in a definite zone adjacent to the root surface when either living roots were examined by phase microscopy or thin sections by electron microscopy. This layer was also found in inoculated and uninoculated roots of Trifolium fragiferum and T. pratense. The bacteria were packed with inclusion granules and lay enclosed by a membrane layer adjacent to the granular matrix seen in uninoculated roots. The ultrastructural organisation of root hairs is essentially similar to that of other differentiated root cells. The replicated surface of the uninoculated root hair wall is largely amorphous with a few sculptured portions resembling a cuticle layer. The inoculated root hair wall often shows areas of exposed, open microfibrillar meshwork with rhizobia sitting on the microfibrils. The rhizobia resemble a flagellated, coccoid swarmer form of Rhizobium which is found in the barrel medic rhizosphere.     

Copulatory mechanics in the wolf spider Agalenocosa pirity reveals a hidden diversity of locking systems in Lycosidae (Araneae)

Genital traits are among the fastest to evolve, and the processes that drive their evolution are intensively studied. Spiders are characterized by complex genitalia, but the functional role of the different structures during genital coupling is largely unknown. Members of one of the largest spider groups, the retrolateral tibial apophysis (RTA) clade, are characterized by a RTA on the male palp, which is thought to play a crucial role during genital coupling. However, the RTA was lost in several families including the species-rich wolf spiders (Lycosidae) leading to the hypothesis that the genital coupling is achieved by alternative mechanisms. Here, we investigate the genital interactions during copulation in the wolf spider Agalenocosa pirity (Zoicinae) on cryofixed mating pairs using electron, optical and X-ray microscopy and compare our findings with other lycosids and entelegyne spiders. We found an unprecedented coupling mechanism for lycosid spiders involving the palea and a membranous cuticle folding adjacent to the epigynal plate. Additionally, we show an uncommon coupling between the median apophysis and the contralateral genital opening, and confirmed that the terminal apophysis acts as functional conductor, as previously hypothesized for males of Zoicinae. Phylogenetic mapping of RTA indicated that the basal tibial process found in Agalenocosa is a secondary acquisition rather than a modified RTA.     

Cellular Events of Wrinkled Blastula Formation and the Influence of the Fertilization Envelope on Wrinkling in the Seastar Patiriella exigua

Like many echinoderms, the seastar, Patiriella exigua has a wrinkled blastula rather than the smooth-walled blastula typical of most phyla. The cellular events of wrinkled blastula formation in P. exigua were documented using light, confocal and electron microscopy. Wrinkled blastulae have a highly infolded epithelium. Prior to wrinkling, the blastomeres are cuboidal with lipid droplets and yolk granules distributed throughout their cytoplasm. During wrinkling, the cells become columnar and the lipid and yolk reserves become redistributed to the basal and apical ends of the cells, respectively. Gastrulae have a tall columnar epithelium, with a basal accumulation of lipid. Interdigitation of numerous cell projections, including short lateral processes, basal lamellipodia and apical filopodia, assists in maintaining epithelial integrity during wrinkling. Apical filopodia have not been observed in other echinoderm embryos. Although 1 M urea caused elevation of the fertilization envelope, the embryos did not expand into the newly-created space. This is suggested to be due to the adhesive properties of the hyaline layer. Embryos removed from their envelope were enlarged with shallower and fewer wrinkles compared with controls. It appears that the integrity of the hyaline layer and fertilization envelope both influence the compact wrinkled profile of P. exigua blastulae.