Cell Tissue Res (2003) 312:369–376 DOI 10.1007/s00441-003-0738-9
REGULAR ARTICLE
Francesca Cima · Giuseppe Basso · Loriano Ballarin
Apoptosis and phosphatidylserine-mediated recognition during the take-over phase of the colonial life-cycle in the ascidian Botryllus schlosseri Received: 3 December 2002 / Accepted: 15 April 2003 / Published online: 23 May 2003 Springer-Verlag 2003
Abstract Colonies of the ascidian Botryllus schlosseri undergo recurrent generation changes in which adult zooids are gradually resorbed and replaced by new blastogenic generations. During these periods, known as take-over phases, programmed cell death, which, on the basis of morphological analysis is ascribed to apoptosis, occurs widely in zooid tissues. In the present report, we re-investigate cell death during the take-over process. Results confirm the occurrence of diffuse apoptosis, as evidenced by chromatin condensation, positivity to the TUNEL reaction and expression of phosphatidylserine on the outer leaflet of the plasma membrane. Apoptosis also occurs among haemocytes, and senescent blood cells are actively recognised and ingested by circulating professional phagocytes. Both phosphatidylserine and CD36, a component of the thrombospondin receptor, are involved in the recognition of apoptotic haemocytes, which fosters the idea that fundamental recognition mechanisms are well conserved throughout chordate evolution. Keywords Apoptosis · Recognition · Phagocytes · Ascidian, Botryllus schlosseri (Tunicata)
Introduction Cell death by apoptosis is a fundamental process in the development and tissue homeostasis of metazoans. It is This work was supported by the Italian MIUR F. Cima · L. Ballarin ()) Department of Biology, University of Padua, Via Ugo Bassi 58/B, 35121 Padua, Italy e-mail:
[email protected] Tel.: +39-49-8276197 Fax: +39-49-8276199 G. Basso Department of Pediatrics, University of Padua, Via Giustiniani 3, 35121 Padua, Italy
involved in the shaping of organs during morphogenesis, in tissue and organ involution, and in the natural turnover of many adult tissues (Wyllie 1987; Samali et al. 1996). Cells undergoing apoptosis are characterised by a series of typical morphological alterations, including cytoplasmic shrinkage, condensation and fragmentation of nuclear chromatin, surface blebbing and formation of apoptotic bodies, whereas external and internal membranes appear preserved during the process and are able to prevent the induction of inflammatory processes (Kerr et al. 1995; Saraste 1999). In mammals, senescent cells are finally engulfed by professional or occasional phagocytes that recognise biochemical changes on their surface, such as the loss of sialic acid residues from glycoconjugates, the presence of a thrombospondin-binding molecule and the appearance of phosphatidylserine in the outer leaflet of the plasma membrane. Recognition involves scavenger receptors on the surface of phagocytes, such as lectin-like, phosphatidylserine and thrombospondin receptors (Savill et al. 1993; Hart et al. 1996; Fadok et al. 2001a). Three blastogenic generations are usually present in colonies of the ascidian Botryllus schlosseri, i.e. adult filtering zooids, their palleal buds and budlets on buds. At a temperature of 19C, adult zooids remain active for about 1 week; they then contract, close their siphons and are gradually resorbed, being replaced by a new generation of adult zooids, represented by grown buds, which reach functional maturity and open their siphons (Berrill 1941; Sabbadin 1955; Burighel and Schiavinato 1984; Lauzon et al. 1992). During this recurrent generation change in the colonial life-cycle, known as regression or take-over, programmed cell death occurs in tissues of zooids along their antero-posterior axis. This is followed by epidermal contraction and massive infiltration of blood phagocytes (Burighel and Schiavinato 1984; Lauzon et al. 1992). Although some evidence of necrosis has been reported for the digestive system, the bulk of cell death must be ascribed to apoptosis. This assumption is consistent with ultrastructural studies on regressing zooid tissues and the observed changes in ubiquitin immunoreactivity (Burighel and Schiavinato 1984; Lauzon et al.
370
1993). Intensive phagocytosis of both apoptotic bodies and whole senescent cells by wandering phagocytes and neighbouring epithelial cells also takes place in visceral tissues (Burighel and Schiavinato 1984; Lauzon et al. 1993) and a significant increase occurs in the frequency of circulating phagocytes containing cell debris inside their vacuoles (Cima et al. 1996). This suggests active recognition of senescent cells by circulating phagocytes and raises new questions about the nature of signal molecules on the surface of ageing cells and of receptors on phagocytes. The present report reconsiders cell death during the take-over process in Botryllus, with particular reference to biochemical changes on both dying cells and circulating phagocytes that enable their interactions. Results suggest the maintenance of common recognition mechanisms throughout chordate evolution.
Materials and methods Animals Colonies of Botryllus schlosseri from the lagoon of Venice were used. They were reared, attached to glass slides, in aerated aquaria filled with filtered sea water at a temperature of 19.5C and fed with Liquifry marine (Liquifry, Dorking, UK) and microalgae. Haemocyte collection and cultures Haemocytes were collected by tearing, with fine tungsten needles, the peripheral tunic vessels of colonies that had previously been rinsed in filtered sea water (FSW) containing 10 mM L-cysteine (Fluka), adjusted to pH 7.4 to prevent clotting. Cells were centrifuged at 780 g for 10 min and pellets were resuspended in FSW at a final concentration of 107 cells/ml. Short-term haemocyte cultures were prepared as previously described (Ballarin et al. 1994); culture chambers were loaded with 50 ml haemocyte suspension. Phagocytes were classified according to Cima et al. (1996). Zooid tissue preparation Adult zooids were isolated from the common tunic with fine tungsten needles and further sectioned with a blade along the middle frontal plane to obtain dorsal and ventral halves and thus allowing the microscopic observation of the living internal tissues. Morphological assays for apoptosis Acridine orange and Pfitzner’s safranin for chromatin condensation Haemocytes and zooid tissues were incubated in vivo with acridine orange (Sigma; 0.1 mg/ml in FSW) for 90 s and immediately observed under a Leitz Dialux 22 light and fluorescent microscope equipped with a I2/3 filter block at a magnification of 1250. The phenotypes of the cells were as follows: (1) viable: bright green nucleus with intact structures; (2) early apoptotic: bright yellow nucleus with condensed chromatin in dense yellow areas; (3) late apoptotic: orange nucleus with condensed chromatin in dense orange areas (Mazzi 1977; Abrams 1997; Cima and Ballarin 1999). As an alternative, haemocytes and tissues were fixed for 30 min in Sanfelice solution (1% chromic trioxide, 40% formaldehyde and
glacial acetic acid, ratio 16:8:1; Mazzi 1977) and stained for 2 h at 25C with Pfitzner’s safranin solution, obtained by adding 50 ml of distilled water to 100 ml of a stock solution of 1% safranin (Merck) in absolute ethanol. Cells were then counterstained with Mayer’s haematoxylin. Condensed chromatin was revealed as dense red areas within nuclei, whereas euchromatin appeared as blue strands because of the haematoxylin stain (Cima and Ballarin 1999). Trypan blue diffusion Since uptake of the vital dye trypan blue is a useful procedure to estimate alterations in membrane permeability associated with cell death (Gorman et al. 1997), living haemocytes were exposed to 0.25% trypan blue in FSW for 5 min and then observed in vivo under the light microscope. TUNEL reaction Chromatin damage and DNA cleavage into oligonucleosomal DNA fragments were investigated by enzymatic in situ labelling of DNA strand breaks through the TUNEL reaction (in situ cell detection kit, Boehringer Mannheim). Zooid tissues and haemocyte monolayers were fixed for 30 min in 4% paraformaldehyde in isotonic buffer (ISO: 20 mM TRIS, 0.5 M NaCl, pH 7.5), rinsed in phosphate-buffered saline (PBS: 1.37 M NaCl, 0.03 M KCl, 0.015 M KH2PO4, 0.065 M Na2HPO4, pH 7.2) and incubated in the blocking solution (0.3% H2O2 in methanol) for 30 min. After being washed in PBS, they were incubated in the permeabilisation solution (0.1% Triton X-100 in 0.1% sodium-citrate) for 2 min at 4C. Samples were then rinsed twice with PBS and incubated in the TUNEL reaction mixture for 60 min at 37C (modified after Abrams 1997). During the course of this reaction, the enzyme deoxynucleotidyl transferase (TdT) catalyses the addition of fluorescein isothiocyanate (FITC)-labelled dUTP to the free 3’OH DNA ends. After this step, samples can be analysed under the fluorescence microscope or, alternatively, incorporated FITC can be revealed under the light microscope after immunoperoxidase staining with anti-FITC antibodies. TdT was omitted in controls. Annexin-V The specific FITC-coupled annexin-V probe (Annexin-V-FLUOS staining kit, Boehringer Mannheim) was used to detect the appearance of phosphatidylserine in the outer leaflet of the plasma membrane, which represents one of the early events in mammalian apoptosis (Martin et al. 1997). Haemocytes and tissues were incubated in a staining solution obtained by diluting 20 ml annexinV-FITC labelling reagent and 20 ml propidium iodide (to distinguish between necrosis and apoptosis) in 1 ml ISO. After 15 min, living cells were observed under the fluorescence microscope at a magnification of 1250. The apoptotic index was expressed as the percentage of haemocytes showing chromatin condensation with the acridine orange assay, being positive to the trypan blue dye assay, showing DNA fragmentation with the TUNEL assay or phosphatidylserine translocation with the annexin-V assay or being immunopositive to anti-CD36 antibody. Surface markers on haemocytes Paraformaldehyde-fixed haemocyte monolayers were incubated for 30 min in 10% goat serum, to block aspecific labelling, and then for 60 min with anti-CD36 mouse monoclonal antibody (Immunotech). After being washed in PBS, they were incubated for 30 min in secondary goat anti-mouse-IgG antibody labelled with FITC (Vector), mounted in Vectashield (Vector) and observed under the fluorescence microscope.
371 Assay for recognition of senescent haemocytes Haemocytes were collected from couples of subclones from the same colony, one subclone at the take-over phase, the other in midcycle stages, the difference in the stage of the colonial life-cycle being induced by rearing the subclones at different temperatures. Cells from one subclone were labelled with the membranepermeable fluorescent vital stain carboxyfluorescein diacetate (CFDA; Sigma), which is esterified within the cells to a membrane-impermeable form (Dransfield et al. 1997). In one set of experiments, we labelled the cells from the colony in the takeover phase and, in another set, the cells from the subclone some distance away from it. Haemocytes from the two subclones were incubated together for 30 min at room temperature and then observed under the fluorescence microscope. At least 200 cells per coverslip in ten fields were counted; the phagocytic index, i.e. the percentage of cells with ingested fluorescent cells, was determined. The effects of the presence of phospho-L-serine (Fluka; 20 mM) or anti-CD36 (10 g/ml) in the incubation medium were also evaluated.
Table 1 Duration of life-cycle stages of a blastogenic generation ofBotryllus zooids. The shift from budlet to bud is marked by the appearance of a new palleal blastogenic generation Stages
Mean duration (days)
Budlet Bud Filtering zooid Take-over
6.0€0.4 5.2€0.5 6.0€0.5 0.8€0.2
Table 2 Frequencies of circulating phagocytes, as hyaline amoebocytes (HA) and macrophage-like cells (MLC), during mid-cycle and take-over stages of colonial life-cycle
1 2
Statistical analysis All experiments were carried out in triplicate. At least 600 haemocytes in ten fields were counted and the number of positive cells was evaluated in the various assays for apoptosis and phagocytosis and expressed as the apoptotic index, i.e. the percentage of senescent haemocytes, or the phagocytic index, i.e. the percentage of haemocytes with ingested labelled cells. Results were compared with the c2 test by means of the FREQ procedure of the SAS statistical package (SAS Institute, Cary, N.C., USA).
3 4
Stage of colonial life-cycle
HA
MLC
Mid-cycle Take-over Mid-cycle Take-over Mid-cycle Take-over Mid-cycle Take-over
23.0€2.5 12.7€3.6* 41.5€5.7 26.3€1.9* 39.0€1.1 22.1€2.5*** 37.5€5.5 20.8€3.7*
9.9€3.3 19.8€1.6** 12.2€1.5 24.3€3.2** 10.1€1.9 22.5€2.3** 9.0€1.5 26.3€2.6***
*P
