Zoomorphology (1990) 109:263-272 Zoomorphology © Springer-Verlag 1990 Stereom morphogenesis and differentiation during regeneration of adambulacral spines of Asterias rubens (Echinodermata, Asteroida) Ph. Dubois 1'* and M. Jangoux ~'z 1 Laboratoire de Biologic marine (CP 160), Universit~ Libre de Bruxelles, 50, av F.D. Roosevelt, B-1050 Bruxelles, Belgium 2 Laboratoire de Biologic marine, Universit~ de Mons-Hainaut, 19, av Maistriau, B-7000 Mons, Belgium Received July 15, 1989 Summary. The very first mineral deposits appearing in viz. the development of long linear processes, of short regenerating fractured adambulacral spines of Asterias unbranched processes and of short branched processes. rubens are minute polyhedrons that cover the surface A survey of the literature allows the suggestion that the of fractured trabeculae. Polyhedrons fuse together form- implementation of these elementary events is sufficient ing a fold from which a microspine differentiates. Micro- to describe most types of stereom morphogenesis. spines develop into long linear trabeculae which send out lateral processes at regular length intervals. Lateral processes from adjacent trabeculae fuse together, bridg- ing the trabeculae and giving the regenerate the typical meshwork structure of stereom. Most of the regenerate A. Introduction is built up according to this growth pattern which en- sures its longitudinal growth. Simultaneously, the initial The skeleton of postmetamorphic echinoderms is com- fascicular stereom of the stub sends out short radial pro- posed of discrete dermic elements, the ossicles, bound cesses which branch into upward and downward di- together by collagenous and muscular fibres. Each ossi- rected subprocesses. The latter fuse with their equiva- cle is typically made of a tridimensional network of high- lents located above or below, building up longitudinal magnesium calcite trabeculae, the stereom. The stereom rows of stereom meshes. These rows then bridge together morphology has been rather intensively investigated by additional branched or unbranched lateral processes, over the past 15 years (e.g. Macurda and Meyer 1975; so forming a new stereom layer which progressively Smith 1980a, b; Blake 1984; Hendler and Byrne 1987) covers the whole stub. Up to three new layers of stereom and the different architectural organizations of the tra- are formed in this way at the stub periphery. These be- beculae - viz. the so-called stereom fabrics - have been come continuous with the stereom layers of the regener- codified by Smith (1980a). On the contrary, whereas ate by fusion of reciprocal subprocesses, so ensuring the numerous works were devoted to skeleton development, continuity between the stub and the regenerate. In both few studies have dealt specifically with stereom morpho- structures the first stage of mineralization results in an genesis. These concern chiefly echinoid outer append- open stereom. Stereom thickening occurs in a second ages during either their early formation (Ludwig 1882; stage of mineralization (that is chronologically separated Th~el 1892; Gordon 1926a, b, 1929) or their regenera- from the formation of the open stereom) and results tion (Heatfield 1971 ; Mischor 1975; Hilgers and Splecht- in the differentiation of the original stereom fabrics (i.e. na 1979). fascicular stereom). Regeneration of removed spines The present paper considers both stereom growth starts with the formation of a new spine base made of and stereom differentiation in regenerating adambulac- labyrinthic stereom. The development of the latter most- ral spines of the asteroid Asterias rubens. Its goals are ly relies on short branched and unbranched processes to describe stereom morphogenesis in an asteroid echin- which fuse with each other or with predifferentiated oderm and to provide a first approach to the calcifying meshes. After completion of its base, the regenerating mechanisms operating in a well-defined asteroid skeleto- spine lengthens and thickens similarly to the regenerat- genic site. ing fractured spines. The diversity of the stereom growth processes observed in the present work may be reduced to the combination of one to three elementary events, B. Materials and methods * Senior research assistant NFSR (Belgium) Individuals of A. rubens Linnaeus, 1758, were collected by SCUBA Offprint requests to: Ph. Dubois diving at Seharendijk (Zealand, The Netherlands). The asteroids 264 were kept in a closed-circuit marine aquarium (30%0, 10-14 ° C) fixed with 1% OsO4 in the same buffer. Decalcification was per- and fed to satiety with mussels (Mytilus edulis Linnaeus, 1758). formed using the double embedding method of Holland and Grim- Regeneration of fractured or removed adambulacral spines mer (1981). Pieces were dehydrated in ethanol and embedded in was initiated either by cutting off the upper two-thirds of the spines Spurr's medium. Semithin sections were stained using the method or by removing the spines. Investigated spines were of similar size of Humphrey and Pittman (1974). (2.68 +0.12 mm; n= 10). At variable time intervals, samples of six regenerating spines were taken from each investigated asteroid (by severing the tissues which bind the spines to the adambulacral plates) and processed for microscopy (see below). A detailed experi- mental schedule is presented in Table 1. In order to prevent the C. Results contraction of the ambulacral groove, spines were sampled on an- aesthetized asteroids. Anaesthesia was performed by immersing L Spine morphology whole asteroids for 30 min in a 0.1% (v/v) filtered sea water solu- tion of propylene phenoxetol (Nipa Laboratories) (Hildeman and Dix 1972). The asteroids resumed their activity 15-60 min after One or two (rarely three) adambulacral spines are being returned to normal sea water. Assessment of the possible jointed to each adambulacral plate of A. rubens. As is effect of anaesthesia on skeletogenesis was achieved using a control usual in echinoderm calcified appendages, the spines are group whose individuals were submitted to a restricted number covered by a prismatic epidermis that is separated from of samplings and anaesthesias (see Table l). the stereom by a layer of periossicular connective tissue Removed spines were preserved in 70% ethanol. The stereom was cleaned of its associated soft tissues (viz., epidermis, dermis which is itself continuous with the stroma that fills the and stroma) in two different ways according to the samples: (1) stereom pore space. Adambulacral spines are attached intact spines were incubated for 24 h at 50° C in a neutral solution to adambulacral plates by both collagen and muscular of 0.1% (w/v) proteinase N (Serva) (Dubois and Jangoux 1985) fibres. Adradial muscle fibres are segregated in a thick and (2) regenerating spines were incubated for 15-60 min in 10% bundle, the so-called depressor muscle (Hyman 1955), (v/v) common bleach (" Eau de Javel") supersaturated with CaCO3 while abradial fibres are scattered among collagen fibres under dissecting microscope control. (This last method was used because proteinase incubation deeply etches the regenerate al- (Fig. 2). though it perfectly cleans the stereom of intact spines without any The spine skeleton is made of a single stereom unit trace of etching. After stereom cleaning, the spines were washed in which two parts can be recognized: the base and the in distilled water and air-dried. For light microscopy, cleaned spines shaft (Fig. 1). The base core is formed of labyrinthic were embedded either in Spurr's or in Petropoxy 154 (Palouse, stereom while the base lower face - viz. the area which Washington) resins and ground longitudinally using either abrasive papers of increasing gradation (250-1200) or carborundum. forms the spine/plate joint - consists of either labyrinthic Mounting was carried out using Canada balsam. For scanning stereom (insertion dimple of the depressor muscle) or electron microscopy (SEM), cleaned spines were mounted on alu- irregular perforate stereom (joint surface) (nomenclature minium stubs, coated with gold in a sputter coater and observed of stereom fabrics according to Smith 1980a). The spine with an ISI-DS 130 SEM. stereom differs sharply from the base to the shaft, chang- Histological observations were performed on spines fixed with ing abruptly from labyrinthic to fascicular (Figs. 1, 3). 3% glutaraldehyde in cacodylate buffer (0.1 M, pH 7.3) and post- The structure of the shaft stereom differs according to the considered face of the spine. On the adradial face, most thick trabeculae run parallel to the long axis of Table 1. Experimental schedule for the study of regeneration of the spine; the stereom pores are narrow and elongated, adambulacral spines in Asterias rubens Experiment Regeneration Regeneration Regeneration of fractured of fractured of removed spines spines spines (control) Figs. 1-7. Morphology of whole adambulacral spines and first Number 4 3 2 stage of regeneration of fractured spines (SEM micrographs except of asteroids 2). Abbreviations: b, base; c, concentric mineral layers; d, insertion dimple of the depressor muscle; dm, depressor muscle; e, epider- Arm size (cm) 7.8-9.0 7.6-10.0 8.3 8.7 mis; fo, fold; pc, periossicular connective tissue; po, polyhedrons; Number 125 50 125 m microspine; s, shaft; st, stroma of spines (25/arm) (10/arm) (25/arm) fractured Fig. 1. Adradial face of the skeleton (arrows, cenical thans) or removed/ Fig. 2. Semithin section of a decalcified spine jointed to its adambu- asteroid lacral plate (Ordinary microscope-OM-micrography)