In vitro fusion and separation of sea urchin primary mesenchyme cells

554 Short notes

SHORT NOTE In Vitro Fusion and Separation of Sea Urchin Primary Mesenchyme Cells

GERALD C. KARP and MICHAEL

SOLURSH*

Department of Biology, University of Iowa, Iowa City, IA 52242, USA Time-lapse videomicroscopy of cultured primary mesenchyme cells from mesenchyme blastulae of the sea urchin Lytechinus pictus demonstrates the dramatic ability of these cells to undergo cell fusion and cell separation. Although this plasticity of cell associations is presumed to play a role in the formation of the syncytial cables that secrete the larval skeleton, the surfaces of these cells must be specialized for fusion and cell separation. @ 1985 Academic Press. Inc.

Cell fusion occurs during the differentiation of several cell types. In vertebrates, these include myoblasts [l] and osteoclasts [2] for example. In the sea urchin embryo, the primary mesenchyme cells fuse in vivo [3-51. Fusion is related to the subsequent formation of spicules within syncytial cables [6, 71. The primary mesenchyme cells appear to have a special capacity to fuse, because cell fusion occurs readily even with other cell types [5]. Cell separation is apparently a less frequent event. It occurs, of course, during mitosis and the differentiation of such cell types as platelets [8]. Fragmentation of myotubes into viable cells has been described during amphibian limb regeneration as well [9]. These observations are based on microscopic studies of fixed specimens. In the present study, we document the occurrence of repeated rounds of cell fusion and cell separation by videomicroscopy of living cultures of sea urchin primary mesenchyme cells. Materials and Methods Lytechinus pictus adults were obtained from Pacific Biomarine, Venice, Calif., and kept in aquaria containing sea water made from Instant Ocean. Embryos were cultured to the mid-mesenchyme blastula stage in artiticial sea water. Preparation of primary mesenchyme cells. Embryos were disrupted by three swift strokes with a loose-fitting pestle in a 7 ml glass Dounce homogenizer. The suspension was subjected to centrifugation at 75 g in a clinical centrifuge for 5 set to pellet most of the fragments and then passed through three layers of Nitex bolting silk (20 pm) to remove cell aggregates. The filtrate containing single cells was diluted to 50 ml with sea water and poured into a 150x25 mm Falcon integrid tissue culture dish. One ml of horse serum was added to the dish and the cells were allowed to settle for 90 min. At the end of the settling period, the serum-containing sea water was decanted, the dish was washed with three aliquots of sea water and 12 ml of sea water was added to the dish. Cells that remain attached to the dish have been shown to consist almost wholly of primary mesenchyme cells [lo]. Cells were systematically lifted from the surface of the dish by jets of the sea water propelled from a long-tipped Pasteur pipette and these were concentrated by centrifugation at 1200 g in a clinical centrifuge and resuspended in 3 ml of sea water. Penicillin G and streptomycin sulfate (at final concentrations of 100 units/ml and 100 ug/ml, respectively) were added to the cell suspension before culture. Culture of primary mesenchyme cells. Primary mesenchyme cells were cultured in Sykes-Moore * To whom offprint requests should be sent. Copyright @ 1985 by Academic Press, Inc. All rights of reproduction in any form reserved

Short notes 555 chambers (51 mm diameter x 7 mm high, purchased from Bellco Glass, Inc.). The 40-mm coverslips used as the substrate in the cell chambers were coated with 10 ug of human plasma tibronectin (Bethesda Research Laboratories, Gaithersburg, Md) prior to addition of cells. In addition, a small centrally placed strip of sea urchin extracellular matrix prepared as described by Karp & Solursh [lo] was dried on the coverslips. Briefly, the matrix was prepared from mid-mesenchyme blastulae by washing embryos in Ca’+-, Mg’+-free sea water (CMFSW) and suspending them in CMFSW containing 100 uM ethylenediaminetetraacetic acid (EDTA) for 10 min before mechanical dissociation. The cells were pelleted by centrifugation to obtain the solubilized extracellular matrix. Cell behavior was observed using a Leitz Laborlux microscope equipped with a long working distance condenser and an RCA TClOOS video camera. The illustrations used in this article were prepared by photographing the image on the screen of an Auditron video monitor.

Results and Discussion Primary mesenchyme cells have the ability both in vivo and in vitro to form syncytia in which the cytoplasm from separate cells becomes continuous. This behavior in vivo is manifested in the formation of syncytial cables, which form as common processes from mesenchyme cells associated with the blastula wall. Primary mesenchyme cells are able to form similar types of cables in vitro (fig. 1). In addition, primary mesenchyme cells cultured in vitro have a tendency to form syncytial complexes in which many nuclei can be seen within a common cytoplasmic compartment. In the present study the formation of these large syncytial complexes occurred with a high frequency as the cells were centrifuged to concentrate them after removal from the serum-containing culture dish. The formation of these large syncytial cells was dependent on the presence of calcium in the medium and did not occur when the cells were removed from the dish and concentrated in Ca*+-free sea water. Thus, centrifugation and calcium can be used to promote the extensive fusion of primary mesenchyme cells into multinucleated cells. Syncytial mesenchyme cells demonstrate a very active behavior in vitro. They tend to move about over the substratum in a manner similar to spread mononucleated cells. They are capable of forming both lamellipodia and filamentous lilopodia. As the large syncytial cells move over the substratum they frequently become divided into smaller units which generally remain connected to one another by fine cytoplasmic processes (fig. 2). Usually separate interconnected portions of a cell move back toward one another (fig. 2eJ and reconstitute a common syncytial mass. Reconstitution can occur by having two partially separated portions drawn together along the path of the interconnection (fig. 25) or it can occur by having the main bodies of the separated portions actually fuse with one another (fig. 2 e). Occasionally, a syncytial cell will become completely split into two separate nucleated cells (fig. 2 k), each of which is capable of moving off in an opposite direction. In some cases an anucleate fragment has been observed to split away from a large syncytial protoplasmic mass. Anucleate fragments are seen to possess limited motility and are able to form both lamellipodia and filamentous filopodia. Such anucleate fragments may, on occasion, refuse with the syncytial cell from which it had been previously split. While separated Exp Cell Res I58 (1985)

556 Short notes

Short notes 557 nucleated and anucleated portions of a syncytial cell are commonly observed to fuse with one another, previously separate mononucleated cells have not been seen to fuse. The syncytial cell in fig. 2, for example, moves into close contact with a mononucleated mesenchyme cell, but there is no direct contact between their corresponding plasma membranes, and the syncytial cell moves away leaving the mononucleated cell in its place. These observations demonstrate the dramatic plasticity of sea urchin primary mesenchyme cells. Not only can they fuse readily, but they can separate again into single nucleated or anucleated fragments. These processes can occur repeatedly in an individual multinucleated cell. This system might be useful for examining the special cell surface properties that must underlie the capacity for cell fusion and cell separation. This work was supported by NIH grant HD16549.

References 1. 2. 3. 4. 5. 6. 7.

Konigsberg, I R, Science 140 (1963) 1273. Fischman, D A & Hay, E D, Anat ret 14 (1%2) 329. Thtel, H, Nova acta R sot scient Upsal ser III 15: 6 (1892) 1. Gibbins, J R, Tilney, L G & Porter, K R, J cell biol41 (1969) 201. Hagstrom, B E & Ldnning, S, Protoplasma 68 (1%9) 271. Okazaki, K, Am zoo1 15 (1975) 567. Solursh, M, Developmental biology. A comprehensive synthesis (ed L Browder) vol. 2. Plenum, New York (1985). In press. 8. Yamada, E, Acta anat 29 (1957) 267. 9. Hay, E D, Dev biol 1 (1959) 555. 10. Karp, G C & Solursh, M, Dev biol (1985). Submitted for publication. Received December 11, 1984 Revised version received March 5, 1985

Fig. 1. A bright-field video image of a cluster of primary mesenchyme cells. Note the formation of common thick cell processes that resemble the syncytial cables formed by these cells in vivo. X 1600. Fig. 2. A sequence of bright-field video images of primary mesenchyme cells taken over a period of 1 h and 38 min. (a) There is a single mononucleated cell in the upper left and a multinucleated cell on the right side. Six of the nuclei are indicated by arrows. The material on the lower side is dried extracellular matrix @CM) (0 min). (b) The multinucleated cell has moved very close to the mononucleated cell but does not fuse with it (6 min later). (c) The lower part of the multinucleated cell moves closer to the ECM material, still remaining connected to the other half by a thin cell process (8 min after (b)). (d) The lower part of the multinucleated cell separated into three connected fragments (7 min after (c)). (e) The upper and lower fragments are rejoining (2 min after (4). (f) They have fused (2 min after (e)). (g) The two large fragments have also fused and moved to the ECM (11 min after (fl). (h) The large cell fragment moves to the right leaving a small fragment behind (11 min after (g)). (i) The two isolated fragments fuse (7 min after (h)). (j) A large portion of the fragment in the lower right in (h) moves up to the left (21 min after(i)). (k) A fragment moves down to the ECM (23 min after 0). Printed

x965. in Sweden

Exp Cell Res 158 (1985)