Konrad Märkel

Ultrastructural investigation of matrix-mediated biomineralization in echinoids (Echinodermata, Echinoida)

SummaryThe formation of echinoderm endoskeletons is studied using echinoid teeth as an example. Echinoid teeth grow rapidly. They consist of many calcareous elements each produced by syncytial odontoblasts. The calcification process in echinoderms needs (1) syncytial sclerocytes or odontoblasts, (2) a spacious vacuolar cavity within this syncytium, (3) an organic matrix coat in the cavity. As long as calcite is deposited, the matrix does not touch the interior face of the syncytium. The cooperation between syncytium, interior cavity and matrix coat during the mineralization process is discussed. The proposed hypothesis applies to the formation of larval skeletons, echinoderm ossicles and echinoid teeth.When calcite deposition ceases the syncytium largely splits up into filiform processes, and the skeleton is partly exposed to the extracellular space. However, the syncytium is able to reform a continuous sheath and an equivalent of the cavity and may renew calcite deposition.The tooth odontoblasts come from an apical cluster of proliferative cells, each possessing a cilium. The cilium is lost when the cell becomes a true odontoblast. This suggests that cilia are primitive features of echinoderm cells. The second step in calcification involves the odontoblasts giving rise to calcareous discs which unite the hitherto single tooth elements. During this process the odontoblasts immure themselves. The structures necessary for calcification are maintained until the end of the process.The mineralizing matrix is EDTA-soluble. The applied method preserves the matrix coating the calcite but more is probably incorporated into the mineral phase and dissolved with the calcite.

Polykristalliner Calcit bei Seeigeln (Echinodermata, Echinoidea)

SummaryFor the first time primary polycrystalline calcite in Echinoderms is shown in the cortex of primary spines of Cidaridae, in the secondary tooth skeleton of Clypeaster and in the accessory calcareous structures filling the crevice fold in the chewing areas of Diadematoidae teeth. Other Echinoid families lack formations homologous to the cortex of Cidaridae and accessory calcareous structures of Diadematoidae. On the other hand the polycrystalline secondary tooth skeleton of Clypeaster is homologous to the monocrystalline one of the other sea urchins.With the exception of cortex the Mg-content in calcite—analyzed by microprobe and X-ray powder method—is generally greater in macrocrystalline parts. The highest Mg-contents are found in the stone parts of teeth irrespective of whether the secondary tooth skeleton is monocrystalline or polycrystalline.Polycrystalline parts are usually harder than monocrystalline ones. The stone parts of Echinoid teeth are the hardest skeleton parts of Echinoderms on the whole; their hardness is much greater than that of solid calcite. It is supposed that the strong interlacing of the microcrystalline calcite and organic matter causes the enormous hardness of the stone part.ZusammenfassungEs wurde erstmals für Echinodermen primär polykristalliner Calcit nachgewiesen, und zwar im Cortex der Primärstacheln der Cidaridae, dem sekundären Zahnskelet von Clypeaster und in den akzessorischen Kalkstrukturen, die im Kauabschnitt die Furche der Diadematiden-Zähne ausfüllen. Es gibt bei anderen Seeigelfamilien keine Bildungen, die dem Cidariden-Cortex oder den akzessorischen Kalkstrukturen der Diadematiden homolog sind. Das polykristalline sekundäre Zahnskelet von Clypeaster ist dagegen dem monokristallinen sekundären Zahnskelet anderer Seeigel homolog.Der Mg-Gehalt des Calcits liegt in den feinkristallinen Zonen (mit Ausnahme des Cortex) im allgemeinen höher; die höchsten Werte finden sich in den Steinteilen der Zähne, gleichgültig ob das sekundäre Zahnskelet mono- oder polykristallin ist.Polykristalline Teile sind im allgemeinen härter als monokristalline Teile. Die Steinteile der Seeigelzähne sind die härtesten Skeletteile von Echinodermen überhaupt; ihre VickersHärte übertrifft weit diejenige von solidem Calcit. Im Steinteil ist das feinkristalline Gefüge von Calcit eng mit organischer Matrix verbunden, und es wird vermutet, daß darauf die besonders hohe Härte der Steinteile beruht.

Calcite-resorption in the spine of the echinoid Eucidaris tribuloides

SummaryReabsorption of calcite is known to occur in echinoid endoskeletons. The structure of the operating cells is described using ‘Prouhos membrane’ as an example, which dissolves the skeleton of cidaroid spines within a thin section. After that the distal part of the spine is shed, and a new shaft grows out of the remaining stump. The calcoclast function is exercised by phagocytes which are also numerous in normal spine tissues. If the spine is highly damaged, however, the phagocytes assemble at a defined level and melt into a single syncytium, called Prouhos membrane. They fulfill three functions: (1) they show an extreme phagocytotic activity and ingest cells of the distal spine part, (2) they block off the surviving stump from the distal part shed later, (3) they etch through the calcite trabeculae in order to detach the distal spine part. The dissolution of the calcite starts with circular bulges, but in the end extremely thin etching lamellae run transversally through the trabeculae.

Comparative morphology of echinoderm calcified tissues: Histology and ultrastructure of ophiuroid scales (Echinodermata, Ophiuroida)

SummaryThe calcified body wall of an ophiuroid was investigated by a new method and compared with that of other echinoderms. The previous opinion that the epidermis of ophiuroid arm shields consists of a reduced syncytium continuous with the underlying dermis is incorrect. The epidermis is distinctly separated from the dermis by a basal layer and consists of (1) supporting cells which bear the cuticle, (2) ciliated cells (hitherto unknown and probably sensory), (3) gland cells, and (4) nerve cells with the basal nerve plexus. The overall structure of the epidermis is a three-dimensional tube system (marked by the basal lamina) which penetrates the dermal tissue of the scales pore space and continues with nerve cords situated below the scale. This arrangement is unique in echinoderms.The dermal sclerocytes largely conform with those of the echinoid Eucidaris. The mineral skeleton is produced intracellularly or intrasyncytially. Moreover, dermal sclerocytes were found to release extracellular microfibrils which have nothing to do with calcite deposition. The attachment of the cuticle to the dermis is achieved by means of epidermal coupling areas. Collagen fibers fasten the scale to the underlying connective tissue sheath. The supposed fibrocytes within this sheath resemble sclerocytes. Each collagen bundle is provided with a strand of nerve fibers which, in contrast to the basal nerve plexus, are naked. They are said to infuence the mechanical properties of the connective tissue.Structures associated with cilia occur in cell types which normally lack a cilium. This finding suggests that most echinoderm cells are potentially monociliate.

Wachstum des Coronarskeletes vonParacentrotus lividus Lmk. (Echinodermata, Echinoidea)

SummaryThe growth of the coronal skeleton is studied by tetracycline labeling. None of the existing hypotheses on the growth of the sea urchin test is verified. For all plates the ratio of increase was measured individually at the different sutures.All coronal plates grow in a latitudinal and meridional direction. Latitudinal growth exceeds meridional growth, but new plates continually being added at the edge of the apical system. The adaptical plates gradually change their position to ambital and adorai. It is a relative shift, because after metamorphosis thereno re sorption of plates occurs at the margin of the peristome. Ambulacral plates (A plates) are built up of several parts. In unfinished A plates the last partial plate is still growing independently, while the others are already growing as a unit. The height depends on the number of their parts. The ratio of increase decreases in a peristomial direction. Contrary to interambulacral plates (IA plates) the greatest ratio of increase occurs 2–3 plates apicad of the ambitus. There are more A plates than IA plates. IA plates bordering of three A plates become higher to the adradial suture to compensate the increase of the intermediate A plate.There is a negative allometric relation between the diameters of the peristome and ambitus. The peristome issolely expanded by lateral growth of the basicoronal plates. There is on the basicoronal IA plates, an unpaired perignathic elementperhaps homologous to the primordial plate (said by former authors to be totally resorbed during metamorphosis). This perignathic element blocks the interradial growth of the basicoronal IA plates. The perignathic element and the basicoronal IA plates grow as a unit. The natural growth lines of the plates are not considered to be annular rings.The results of this investigation can be carried over to the mode of coronal growth of other families (i.e., Arbaciidae, Cidaridae) only to a limited extent.ZusammenfassungDas Wachstum des Coronarskeletes wurde mittels Tetracyclin-Markierung analysiert. Keine der vorhandenen Hypothesen konnte bestätigt werden. -Für alle Platten wurde der Zuwachs an den einzelnen Suturen ermittelt.Alle Coronarplatten wachsen in die Breite und in die Höhe. Das Breitenwachstum ist stärker als das Höhenwachstum, dafür werden an der Grenze zum Apicalskelet ständig neue Platten angelegt. Die älteren Platten werden peristomwärts verlagert, so daß z.B. ständig neue Platten in den Ambitus rücken. Es handelt sich um eine relative Verlagerung, denn am Peristomrand werden nach der Metamorphosekeine Platten resorbiert.Die Ambulacral- (A-)Platten bestehen aus mehreren Teilen. Bei A-Platten, die noch unvollständig sind, wächst die jüngste Teilplatte noch selbständig, die übrigen wachsen bereits als Einheit. Die Höhe der A-Platten hängt von der Zahl ihrer Teilplatten ab. Höhen- und Breitenzuwachs der A-Platten werden peristomwärts kontinuierlich geringer. Bei den Interambulacral- (lA-)Platten liegt der stärkste Breitenzuwachs erst 2–3 Platten apicad des Ambitus. Es gibt mehr A- als IA-Platten. IA-Platten, die an 3 A-Platten grenzen, werden adradiad höher, weil sie das Höhenwachstum der mittleren A-Platte kompensieren müssen.Zwischen Peristom- und Ambitus-Durchmesser besteht eine negativ-allometrische Beziehung. Das Peristom wirdausschließlich durch das Breitenwachstum der basicoronalen Platten erweitert. Auf den basicoronalen IA-Platten liegt einunpaarer perignathischer Sklerit, dervielleicht der Primordialplatte homolog ist (nach der herrschenden Vorstellung soll diese während der Metamorphose resorbiert werden). Der perignathische Sklerit blockiert das interradiale Wachstum der basicoronalen IA-Platten. Die basicoronalen IA-Platten und der perignathische Sklerit wachsen als Einheit.Die natürlichen Zuwachsringe sind wahrscheinlich keine Jahresringe.Die gewonnenen Ergebnisse lassen sich nur bedingt auf das Coronarwachstum anderer Familien (z.B. Arbaciidae, Cidaridae) übertragen.

Structure and growth of the cidaroid socket-joint lantern of Aristotle compared to the hinge-joint lanterns of non-cidaroid regular echinoids (Echinodermata, Echinoidea)

SummaryIn regular echinoids the lanterns of Aristotle are gripping lanterns. They are provided, therefore, with strong joints formed by the rotulae and the epiphyses. According to the structure of the joints the author distinguishes between the socket-joint lanterns of Cidaroida and the hinge-joint lanterns of non-cidaroid regular echinoids. The homologies between the joints of both types are discussed in detail. The mobility of the main joints is restricted by secondary joints, and in socket-joint lanterns it is apparently more restricted than in hinge-joint lanterns.The mesodermal endoskeleton of echinoderms is built up of monocrystalline calcitic elements of a spongy nature. The articular surfaces are provided, however, with polycrystalline covers. Polycrystalline calcite is not transformed into a monocrystalline structure by the organism, and therefore the growth of elements which are parts of the joints is controlled by the polycrystalline covers.Differences in the structure of their joints lead to differences in the mode of the growth of the skeletal elements (especially the rotulae), in both hinge-joint lanterns and in socket-joint lanterns.Characteristic of non-cidaroid lanterns are the large foramina and the deep pits of their pyramids. There are strong reasons to suppose that these structures arise by functional adaptation to the forces which are carried over from the epiphyses to the demipyramids in feeding, i.e., due to the inclined hinge-joints these forces act more or less in radially. These forces act, however, tangentially in socket-joint lanterns, which, therefore, have neither foramina nor pits.The author questions the widely held opinion that the cidaroid lantern is the simplest and most primitive stage compared with the lanterns of the recent non-cidaroids. These doubts rest on the paleontological data available.

Morphologie der seeigelzähne

Descriptions are given of the tooth skeleton of two species of Gnathostomata. Fundamental differences in the constructions of tooth elements of Gnathostomata compared with those of regular sea urchins are discussed. In the authors opinion the teeth of Gnathostomata are not to be derived from the teeth of any order of post paleozoic regular sea urchins, and the Gnathostomata is thought to be a monophyletic group of its own.

On the ultrastructure and the supposed function of the mineralizing matrix coat of sea urchins (Echinodermata, Echinoida)

SummaryEchinoderm ossicles are part of the mesenchyme. Their formation and growth, with respect to the underlying tissues, is studied using echinoid spines and teeth and applying different methods of fixation. The calcification process in echinoderms is strictly intracellular and needs (1) syncytial sclerocytes which completely enclose (2) a vacuolar cavity which in turn contains (3) an organic matrix coat. Strictly speaking, each ossicle is nothing but the calcified vacuolar space of a single syncytium of sclerocytes. In fully grown parts, however, the continuous sheath may split open and the matrix-coated mineral may come into contact with the extracellular space. According to biochemical analyses the matrix consists of insoluble components, but most (95%) of its constituents are soluble in EDTA or weak acids. If routine transmission electron microscope methods are used the soluble components are lost and the matrix at best looks electron light. If tannic acid is added to the fixative the soluble matrix components are preserved and reveal further ultrastructural details of the biomineralization process in echinoderms. The matrix coat looks extremely electron dense. Further soluble material is to be found within the vacuolar space or attached to the vacuolar surface of the cytoplasmic sheath. The results lead to the opinion that the matrix coat consists of a hydrophobic framework of insoluble components that contains soluble components which guide the Ca through pores in the hydrophobic layers into the interior of the matrix-coated space. It is only within this space that the mineral is deposited.

Mechanical design in spines of diadematoid echinoids (Echinodermata, Echinoidea)

SummaryThe long and slender spines of Diadema are highly flexible, although their skeleton consists mainly of CaCO3 and behaves optically like a single monocrystal of calcite. The flexibility is due to the shape of the spine skeleton as well as to the material properties of the echinoderm calcite.The spine skeletons are hollow beams consisting of radial wedges or septs. The shape of the septs shows a broad base situated at the periphery of the cross section, producing a high load-bearing capacity with minimum weight. Furthermore, material is concentrated at the base of the spine in such a way that the strain of the structure is kept constant along the axis. The septs are connected with one another by a few transverse bars positioned as closely as possible to the axis. The load-bearing parts of the septs are free. They have small diameters similar to flexible glass fibres. The stiff spines of other echinoids are also mainly built by radial wedges, but the spaces in between are closely filled with transverse bars. On the surface of stiff spines there are low grooves between the septs. The echinoid spines are covered with an epithelium which shows a basiepithelial nerve plexus. In the stiff spines this plexus forms cords which lie protected within the superficial grooves mentioned. In the flexible spines of Diadema the cords are deeply sunken in the spaces between the septs. In this manner the nerve cords are largely free from the tensile stresses to which the spines surface is exposed.The flexible spines were used to determine the material properties of echinoderm calcite. Youngs modulus was determined for fresh (live) spines, dry spines, and cleaned spine skeletons. Fresh spines show the highest elasticity, and their Youngs modulus is significantly below the Youngs modulus of the other test groups. The echinoderm calcite does not show the cleavage planes of mineral calcite, and probably this feature contributes to the high flexibility of echinoderm calcite.

Experimental and comparative morphology of radula renewal in pulmonates (Mollusca, Gastropoda)

SummaryThe continuous renewal of the pulmonate radula and the histology and regeneration of its concomitant epithelia were studied by light and electron microscopy, autoradiography and electron microprobe analysis. The two species investigated show histological differences and the results were compared with those of a preceding study on a prosobranch radula. The radula is an intricate cuticular structure of the foregut. Only the fully grown part, which is active during feeding, lies in the buccal cavity while it is constantly renewed by the coordinated cooperation of specialized cells forming the radular sheath. The end of the sheath is occupied by cells which produce the organic matrix of the radula. In taeniogloss prosobranchs, seven multicellular cushions of small odontoblasts lie at the end of the sheath and produce the seven teeth of each cross-row. In pulmonates, the multidenticular radula is generated by numerous groups of a few voluminuous cells. Despite these histological differences, prosobranchs and pulmonates generate the radula matrix by microvilli, cytoplasmatic protrusions and apocrine secretions. The epithelia of the radular sheath contribute to the transport, tanning and mineralization of the radula. The concomitant epithelia are replaced in limited proliferation zones at the end of the radular sheath and their cells migrate anteriorly to the buccal cavity. The ultrastructure of the sheath cells and the alterations which they undergo in connection with their functions are discussed. The proliferation zone of the superior epithelium is strictly confined and the cells move together with the radula forward. In prosobranchs, the cells of the superior epithelium begin to degenerate in the middle of the radular sheath and the entire epithelium is simply extruded into the buccal cavity. In pulmonates, the opening of the radular sheath is closed by the cuticular collostylar hood which is generated by a distinct epithelium which is proved to be stationary. When leaving the proliferation zone, the superior epithelium differentiates into supporting cells and mineralizing cells; the latter cause the hardening of the radular teeth and already degenerate in the middle of the sheath. The whole superior epithelium degenerates at the border to the collostylar hood-epithelium. In Lymnaea the degeneration zone is strictly confined whereas in Cepaea the collostylar hood and its generating epithelium extend into the radular sheath and the degeneration zone ranges over a distance of 3–5 rows of teeth. The proliferation zone of the inferior epithelium extends over the posterior half of the radular sheath, but the replacement rate is much lower than in the superior epithelium. Although the inferior epithelium carries the radula, it migrates slower than the radula. Obviously the radula has to be transported actively by apical protrusions of the cells, which penetrate into the radular membrane. At the opening of the radular sheath the inferior epithelium generates the adhesive layer and degenerates. During feeding, the adhesive layer has to maintain the firm mechanical connection between radula and distal radular epithelium. Autoradiographic experiments demonstrate that the distal radular epithelium is stationary. Nevertheless, the radula is known to advance to its degeneration zone. Special attention is paid to this problem. We strongly suspect that the transport of the adhesive layer and the radula is based on pseudopodial movements of apical protrusions characteristic for the distal radular epithelium. These protrusions interdigitate with the lower face of the adhesive layer. The mechanical connection has to be maintained and so the respective structures (tonofilaments and hemi-desmosomes) have to be continually renewed. This needs a high amount of energy and obviously results in the conspicuous concentration of mitochondria near the apical surface.