Contractile Filopodia and in Vivo Cell Movement in the Tunic of the Ascidian, Botryllus Schlosseri

C.S.Izzard 0 0 Department of Biological Sciences, State University of New York , Albany, New York 12222, U.S.A., and Marine Biological Laboratory, Woods Hole, Massachusetts 02543 , U.S.A SUMMARY The in vivo movement of one class of cells in the tunic of the ascidian Botryllns schlosseri has been analysed using differential interference optics and time-lapse cinematography. Long (up to 200/tm), thin (0-35-0-5 /tm diameter) filopodia radiate from the cell-body into the matrix of the tunic. Movement of the cell-body consists of a series of short, jerky displacements with frequent changes in direction between successive displacements. The net displacement of the cell may be extremely small when the displacements are short and frequently change direction, or considerable when successive displacements show a persistence of direction (up to 114/tm in 60 min). Deformation of the elastic cuticle covering the tunic at points of attachment of the filopodia has been used to record qualitatively changes in tension in the filopodia. Correlation of the changes in tension with changes in length of thefilopodiaand movement of the cell-body have permitted the following conclusions. Active contractions of filopodia (i.e. increase in tension during shortening) stretch and move the cell-body. These movements exert a force on trailing or opposing filopodia. Relaxations offilopodia(i.e. decrease in tension during lengthening) result in small movements of the cell-body due to the recoil of tension in the cell-body and opposing filopodia. The position of the cell-body in space at any one instant in time is therefore the resultant of the forces developed in all the filopodia. Movement results from unilateral modulation of the tension developed in the filopodia. Active contractions play a more significant role in movement than relaxations. - The number of in vivo studies that provide an insight into the changes in cell shape underlying the movement of a cell through an intact organism is limited. In contrast, the motility of cells derived from multicellular organisms has been studied extensively under in vitro conditions. The factor limiting in vivo studies has been the inherent opacity of the organism. The extant work centres upon a limited number of transparent tissues such as the tail fin of amphibian larvae (Clark, 1912; Clark & Clark, 1920, 1925, 1930; Speidel, 1933, 1935), the sea-urchin larva (Gustafson & Wolpert, 1961, 1967) and the embryo of Fundulus (Trinkaus & Lentz, 1967; Trinkaus, 1973). The present work analyses the motility of one class of cells in the tunic of the ascidian Botryllus schlosseri (Pallas). The ascidian tunic is an ideal tissue for optical studies of cell motility in vivo because the tissue lies external to the body of the organism and is highly transparent in many species. Botryllus was selected for study because the colony shows a marked tendency to spread as a thin sheet over the substrate. The newly metamorphosed larva provides the most suitable preparation for high-resolution optical studies; during morphosis, the vascular ampullae of the larva expand radially across the substrate drawing the tunic into thin web-like sheets between pairs of ampullae (Fig. 7, p. 531). Using these preparations, it was possible to obtain high-resolution, high-contrast images of the tunic cells with differential interference optics. The success of this optical technique for in vivo studies results from the small contribution provided by out-of-focus objects to the contrast of the image. Thus it is possible to optically section relatively thick specimens (see Allen, David & Nomarski, 1969; Padawer, 1968; Metuzals & Izzard, 1969). Initial observations demonstrated that the cells in the tunic of Botryllus can be divided into 2 classes on the basis of their motile behaviour and associated pseudopodia. One class, here termed filopodial cells, is characterized by long filopodia radiating from the cell-body. The other class of cells can be broadly described as 'amoeboid'; they move by the eruption of relatively small hyaline pseudopodia and by cytoplasmic flow towards, or into, the bases of the pseudopodia. The present study is concerned solely with the filopodial cells. It aims to describe briefly the cell morphology, to characterize the extent and pattern of cell movement, but primarily to examine the forces and changes in cell shape that produce the movement. The analysis has demonstrated that extended filopodia actively contract and that the position of the cell-body in space at any one instant in time is the resultant of tensions developed in the radiating filopodia. A preliminary report of this work has been presented elsewhere Sexually mature colonies of Botryllus schlosseri (Pallas) were collected from the dock in Eel Pond, Woods Hole, and placed in large finger bowls supplied with a slow flow of seawater. Coverslips (no. i-J-) were suspended vertically around the periphery of the dish. As larvae were released, they attached in significant numbers to the coverslips. Within 2 h the coverslips could be removed. Then they were supported at an angle, with the metamorphosing zooids facing down, in 450 slots cut into f-in. (0954-cm) Tygon tubing. The supported coverslips were placed in the sea table until required. Arrangement of the zooids on the lower surface of the coverslip minimizes the accumulation of bacteria and detritus on the glass surface over which the tunic spreads, thereby maintaining the greatest optical clarity of the preparations. The specimens are ready for use within 4 h (Fig. 7) but can be used for up to 7 days. The coverslip bearing the young zooid was inverted over a large coverslip (no. i) and supported on spacers cut from no. 2 coverslips or thin slides. The 2 coverslips and spacers were sealed together with a 1:1:1 mixture of Vaseline, lanolin and paraffin wax leaving 2 narrow openings, one at either end. Seawater was exchanged through these openings every 1-2 h. Such preparations remained healthy for at least i o h o n the microscope at a room temperature of 21-22 C. The work was performed with a Zeiss Photomicroscope II equipped with differential interference optics, and 40/0-85 achromat oil and 100/1-25 planachromat oil objectives. Extinction factors for both objectives with these preparations were routinely 400. The light source was either tungsten or mercury arc respectively for the lower or higher power objectives. A wideband green interference filter and heat reflexion filter (Calflex) were used with both light sources. The following films were employed: 35 mm, Adox KB-14 processed in Diafine, or Kodak SO-410 processed in HC-110; 16 mm cine -negative, Recordak 7460 and 7457 (Eastman Kodak Co.), respectively, for the lower- and higher-power work, both processed in Diafine to enhance the speed and retain fine-grain characteristics of the film. Cine films were taken with an Arrifiex 16 S camera wall-mounted above a Zeiss panchratic projector lens and (...truncated)