MICROSCOPY RESEARCH AND TECHNIQUE 34:218-227 (1996) Evidence for Transcriptional Modulation but Not Acid Phosphatase Expression During Programmed Cell Death in the Colonial Tunicate Botryllus schlosseri ROBERT J. LAUZON, WEN-TEH CHANG, AND LESLEY S.DEWING Department of Pediatrics (R.J.L.,L.S.D.), and Department of Microbiology, Immunology and Molecular Genetics (W.-T.C.), Albany Medical College, Albany, New York 12208 KEY WORDS Apoptosis, Programmed cell death, Differential display, Botryllus, Invertebrate, Ascidian ABSTRACT Botryllw schlosseri is a clonally modular ascidian in which asexually derived adults (zooids) exhibit developmental synchrony. At the conclusion of the blastogenic (asexual) cycle every 5 days at 21"C, all zooids within a colony die simultaneously in 24 hours and are replaced by a new asexual generation of zooids. This cyclical process, called takeover, involves the selective destruction of the zooid's visceral tissues which include the pharynx, esophagus, stomach, intestine, endostyle, neural complex and heart, whereas bud tissues and mesenchymal components (muscle and blood cells) remain unaffected. Ultrastructural analysis indicates that the most prevalent form of cell death occurs by apoptosis, although necrotic changes are also observed in several tissues (i.e., stomach and intestine). Blood-derived macrophages and neighboring cells subsequently engulf visceral tissues, reducing the zooid to the size of a small vesicle. Here, we have tested the possibility that acid phosphatase, a hydrolase whose presence is associated with cell death in several invertebrate systems, could account for some of the regressive changes observed during takeover. Our observations indicate that acid phosphatase (AP) activity was selectively localized in the gut of parent zooids during the growth phase of the cycle, with the stomach exhibiting the most intense histochemical staining on tissue sections. As zooid regression progressed during takeover, stomach AP staining gradually disappeared. Other visceral tissues never became AP-positive. Therefore, this hydrolase appears to play a minimal role in zooid death. In order to characterize genes whose expression pattern was selectively altered during takeover, we have carried out differential mRNA display analysis. We report on two genes, 790.3 and 790.4, that are down- and upregulated, respectively, during this process. Collectively, these findings indicate that the takeover phase of blastogenesis in Botryllus involves modulated gene expression. o 1996 Wiley-Liss, Inc. INTRODUCTION Programmed cell death is an indispensable developmental and homeostatic process of multicellular animals (Ellis et al., 1991; Schwartz and Osborne, 1993). The most prevalent form is a morphological process called apoptosis, characterized by nuclear condensation, fragmentation of the cell into multiple membrane-bounded bodies and rapid engulfment by phagocytic cells without elicitation of an inflammatory reaction (Kerr et al., 1972). These events appear to be stereotypically conserved throughout much of evolution. Recent observations from several invertebrate and vertebrate developmental systems have indicated that cell death is an active metabolic process involving modulated expression of specific genes (Owens et al., 1991; Schwartz et al., 1990; Wang and Brown, 1993; reviewed in Schwartz and Osborne, 1993). In addition, genetic studies from nematodes to humans have revealed that the mechanistic basis underlying cell suicide also appears to be phylogenetically conserved (Hengartner and Horvitz, 1994; Miura et al., 1993; Sugimoto et al., 1994; Vaux et al., 1992). 0 1996 WILEY-LISS, INC. Botryllus schlosseri is a colonial ascidian of particular significance to the study of cell death, as entire organisms (zooids) die as part of their developmental cycle (Milkman, 1967). Every 5 days a t 21T, the blastogenic cycle concludes in a phase of cell and zooid death called takeover, in which all zooids die synchronously in a 24-hour period and are replaced by a new generation of asexually derived zooids (Burighel and Schiavinato, 1984; L a w n et al., 1992). Several features make this animal a unique study system to investigate the mechanisms by which cells die. First, all zooids in a colony are derived through asexual budding, and are thus genetically identical (clonal replicates). Second, zooid death recurs in cyclical fashion, during which up to 50% of the colony is regressing a t the same Received December 19, 1994; accepted in revised form February 2,1995. Address reprint requests to Robert J. Lauzon. Ph.D., Department of Pediatrics, Room MS449, Albany Medial College, 47 New Scotland Avenue, Albany, NY 12208. PROGRAMMED DEATH IN A COLONIAL ASCIDIAN time, thus providing access to large amounts of synchronized biological material amenable to biochemical, molecular and morphological characterization. Lastly, since visceral tissues from different histogenic origins regress simultaneously, a common death pathway may be associated with this process in all tissues. Previous ultrastructural studies from this laboratory have indicated that both apoptosis and necrosis occur in regressing gut epithelia (Lauzon et al., 1993). The necrotic foci seen within individual cells, which included flocculation of the nuclear chromatin, swelling of cytoplasmic organelles and cell lysis, occurred independently or in conjunction with apoptotic changes (i.e., cell fragmentation). Burighel and Schiavinato (1984) have also previously reported the occurrence of autophagic vacuoles within visceral tissues of regressing Botryllus zooids. Expression of the lysosomal hydrolase acid phosphatase (AP) is highly correlated with autophagy (Beaulaton and Lockshin, 1982; Clarke, 1990): In many invertebrate systems, particularly insects, this marker also appears to have a well defined autolytic role during the later stages of cell death (Bowen et al., 1995; Zakeri et al., 1995). Furthermore, recent observations using two-dimensional protein gels from in vitro translated mRNA preparations have strongly suggested that transcriptional modulation of specific genes occurs during takeover (Chang and Lauzon, 1995). In the first part of this study, we have tested the possibility that some of the regressive changes observed during takeover could account from enhanced expression of this hydrolase within dying tissues. In the second part, we have carried out differential mRNA display (Liang and Pardee, 1992) in order to characterize developmentally regulated mRNAs that are selectively modulated during zooid regression. This technique makes use of oligo-dT anchor primers (14 mers) which anneal to the 3' end of mRNAs followed by reverse transcription. DNA fragments are subsequently polymerase chain reaction (PCR)-amplified with combinations of anchor primer and different 5' decamer oligodeoxynucleotides of defined arbitrary sequence, and are displayed as 100-500 base pair fragments on a polyacrylamide gel, subsequently to be visualized by autoradiography (Liang and Pardee, 1992; Liang et al., 1992, 1993). Botryllus is optimally suited for this type of analysis since multiple RNA samples from various developmental stages can be simultaneously compared from the same clone. Furthermore, the full complement of genes expressed during takeover could, in theory, be amplified (Liang and Pardee, 1992; Sager et al., 1993). In this paper, we present evidence indicating the feasibility of using this strategy with this model system. MATERIALS AND METHODS Animals Wild colonies of Botryllus schlosseri were collected on glass microscope slides contained within wooden traps submerged in Long Island Sound (Long Island, NY) and Monterey Bay (CA) during summer months. Following collection, they were subsequently maintained in a refrigerated aquarium with artificial sea salts, as previously described (Chang and Lauzon, 1995). Large individual colonies were observed with the use of a TABLE 1. Developmental Stage A B-1 B-2 c-1 c-2 D- 1 D-2 D-3 D-4 219 stages of the blastogenic cycle Characteristic Onset of new blastogenic cycle; opening of oral and excurrent siphons in all m i & . Secondary bud skewing to parent zooid's anterior hemisphere. Heartbeat begins in primary bud. Secondary bud is a closed double-layered vesicle. Organogenesis (atrial folds) begins in secondary bud. Secondary bud elongates along its anteroposterior axis. Primary subdivisions completed in secondaly bud. Onset of takeover: shutdown of oral and excurrent siphons in all zooids. Primary buds move dorsally. Early takeover: contraction of zooids along their anteroposterior axis. Polarized breakdown of perivisceral extracellular matrix. Midtakeover ( m i d involution): visceral organs are being resorbed. Apoptotic cell death and macrophage phagocytosis are prevalent. Some necrotic death observed. End of takeover: cessation of heartbeat in zooid. Siphons of new asexual generation not yet open. ~~ stereomicroscope (Stemi SV 6, Carl Zeiss, Germany), and representative developmental stages from clonal replicates were obtained with the use of a razor blade. Following removal of debris and other encrusting organisms, these colonies were subsequently snap-frozen in liquid nitrogen and stored at -70°C until needed. Histology and Transmission Electron Microscopy For histological observations, stage D-3 takeover colonies (see Table 1 for description of developmental stages) were separated into systems (star-shaped groups of zooids) with a razor blade and fixed in 4% paraformaldehyde/arti-eificialsea water for 3 hours a t 4°C. Previous morphological studies had indicated that this developmental period represented the phase of maximal apoptotic death (Lauzon et al., 1992, 1993). Specimens were subsequently dehydrated in a graded ethanol series, and embedded in JB-4 plastic (Polysciences Inc., Warrington, PA). Sections 3 microns thick were generated along the anteroposterior axes of zooids with a Histoknife (Diatome Inc., Switzerland), stained with 0.01% toluidine blue, and mounted with Permount (Fisher Chemical Co., Springfield, NJ). Stage D-3 colonies were processed for electron microscopy as previously described (Lauzon et al., 1993). U1trathin sections were cut along the zooid's anteroposterior axis with a diamond knife (EM Corp., Chestnut Hill, MA), placed onto Formvar-coated nickel grids and stained with 2% uranyl acetate and 1%lead citrate. Samples were then observed with a Phillips EM 201 transmission electron microscope. Acid Phosphatase Histochemistry For in situ localization of acid phosphatase activity, specimens were fixed as described above, but dehydrated in a graded acetone series (50%, 70%,85%,95%, and 100%) 10 minutes each, and infiltrated overnight a t 4°C in JB-4 solution A without catalyst. The samples were then embedded with JB-4 (solutions A, B and catalyst) in BEEM capsules (Polysciences Inc.), according to the manufacturer's specifications. Sections 3 mi- 220 R.J. LAUZON ET AL. crons thick were cut with a Histoknife on a Sorvall specifications. Following amplification, each reaction Porter-Blum MT2-B rotary ultramicrotome (Sorvall was size-fractionated on a 1.5% agarose gel and visuInc., Newton, CT) at various developmental stages of alized with ethidium bromide. Fragments were cut out the blastogenic cycle, and stained for acid phosphatase of the gel and purified with a QIAEX kit (QIAGEN for 2 hours a t 37°C in an humidified chamber, accord- Inc., Chatsworth, CAI. ing to the methodology described by Beckstead et al. (1981). Sections were counterstained with 2% methyl RNA Slot Blot Analysis green for 1 minute, air-dried and mounted in PerTwo p,g of total RNA from representative developmount. mental stages of the blastogenic cycle were applied under vacuum to a nitrocellulose membrane (SchleDifferential mRNA Display icher and Schuell, Keene, NH) sandwiched within a Differential display reactions were carried out with slot blot manifold (Bio-Rad, San Diego, CAI, and an RNAmap kit purchased from GenHunter Corp. hybridized to 32P-labelled cDNA probes generated by (Brookline, MA, also see Liang et al., 1993). Total RNA random hexadeoxynucleotide priming (Boehringer(0.2 pg) was isolated from various developmental Mannheim, Indianapolis, IN). Hybridization condistages (A, B-1, B-2, C-1, C-2, D-1, D-2 and D-3) as pre- tions, posthybridization washes and autoradiography viously described (Chang and Lauzon, 19951, and was were as previously described (Chang and Lauzon, reverse-transcribed into cDNA with the 3'-anchor 1995). Autoradiograms were quantified using a Bio primers T,,MG (M is a degenerate mixture of dA,dC Image model 60-S video densitometer (Bio Image, Ann and dG) and Murine Moloney Leukemia Virus Arbor, MI). (MMLV) reverse transcriptase according to the manufacturer's specifications. cDNA species (20 ng) were amplified in PCR reactions (94"C/30 seconds, 42"Cl2 RESULTS minutes, 72"C/30 seconds for 40 cycles) with the same The Lifecycle of Botryllu schlosseri 3' anchor primer (TI2MG)and a 5' end arbitrary seBotryllus schlosseri is an encrusting colonial ascidquence primer (10mer with 50% GC content also ob- ian which lives in shallow waters and harbors worldtained in the RNAmap kit from GenHunter) in the wide. An embryonically derived chordate larva presence of 35S-dATP(1,200 Cilmmole) and AmpliTaq emerges and undergoes metamorphosis into a juvenile polymerase (Perkin-Elmer, Foster City, CA). Then, 116 (oozooid) after a brief pelagic phase (Milkman, 1967). of each PCR reaction was size-fractionated on a 6% Through asexual budding, a colony containing up to denaturing DNA sequencing gel for 3.5 hours a t 37 hundreds of genetically identical individuals called watts constant power. The gel was blotted dry on a is formed in the span of several weeks (Fig. 1). piece of 3MM paper (Whatman Inc., Maidstone, En- zooids, A ramifying network of blood vessels interconnects and gland), dried under vacuum at 80°C for 1 hour, and developmentally synchronizes all zooids and buds in a autoradiographed with Kodak XAR film at - 70°C. Dis(Sabbadin, 1982; Watanabe, 1953). The blastoplay of duplicate PCR reactions was also performed in colony genic (asexual) cycle is a temperature-dependent proorder to determine the reproducibility of this tech- cess with a duration of 5 days at 21"C, and can be nique. In order to assess DNA contamination, RNA divided into four stages, A through D (Table 1; Figs. samples were treated with RNase-free RQ-1 DNase 2-6). Stages A through C are designated as growth (Promega Corp., Madison, WI). Five pg of total RNA during which zooids are filter feeding and buds was incubated with 1unit of RQ-1 DNase and 1unit of stages, are undergoing organogenic differentiation (Fig. 2). On placental ribonuclease inhibitor (Promega Corp.) for 30 the fifth day of the cycle, zooids synchronously die. This minutes a t 37°C prior to initiating reverse transcrip- process is called takeover, and involves the simultation PCR (RT-PCR) reactions. Control RNA samples neous regression of parent zooids and its replacement were also PCR-amplified without reverse transcription by another asexual generation. Zooid regression can be to ensure specificity of the mRNA display. followed carefully under stereomicroscopy, and involves four stages (Lauzon et al., 1992): (1) the shutpurification of cDNA Fragments down of both oral and excurrent siphons which detercDNA fragments that were either up- or downregu- mines its onset (stage D-1); (2) contraction of the zooid lated during takeover were cut out of the sequencing along the zooid's anteroposterior axis (stage D-2, also gel with a razor blade after superimposing the autora- see Figs. 3 and 4); (3) zooid involution (stage D-3): diogram over the dried gel. The gel slice was soaked in throughout this process, all visceral organs of the zooid, an eppendorf tube containing 100 p1 of sterile milli-Q which include the heart, neural complex, esophagus, water, and boiled for 15 minutes. The supernatant was stomach, intestine, endostyle, and gill slits die and are collected and subsequently precipitated overnight a t engulfed by phagocytic cells (Lauzon et al., 1993); and -20°C in 450 (11 of 100% ethanol with 10 (11 of sodium (4) cessation of heartbeat (stage D-4,also see Fig. 5).A acetate (3M stock) and 5 pl of glycogen (10 mg/ml new blastogenic cycle begins when the new asexual stock). The pellet was washed in 85%ethanol, air-dried generation opens its siphons (Stage A, also see Fig. 6). and resuspended in 10 p1 of sterile milli-Q water. Us- Since adult colonies of Botryllus schlosseri harbor three ing the same primer sets as in the original PCR reac- asexually derived generations simultaneously, it thus tion, 4 p1 from each sample was reamplified with Am- follows that the duration of lifespan of one individual is pliTaq polymerase according to the manufacturer's 15 days a t 21°C. PROGRAMMED DEATH IN A COLONIAL ASCIDIAN 221 Figs. 1-6. The blastogenic cycle of BotryZZrcs schlosseri. Figure 1 is a dorsal view of a colony in stage B-1.Filter-feeding zooids are arranged into star-shaped modules called systems, and are linked to one another by a ramifying network of blood vessels that spans the entire colony. Figure 2 is a higher magnification of a system composed of 8 zooids (arrowheads)with visible primary buds (arrows).This colony is in stage C-1. Figures 3-5 depict various features of the takeover process: in the early stages of takeover (Figs. 3,4), all zooids contract synchronously along their anteroposterior axis (arrowheads) and primary buds move dorsally (arrows). Figures 3 and 4 depict dorsal and ventral views, respectively. Zooids are resorbed within 24 hours at 21OC (arrowheads in Fig. 5, ventral view), and another blastogenic cycle begins when the next asexual generation of zooids open their oral and excurrent siphons (Fig. 6,dorsal view). Magnifications: Fig. 1,8 x ; Figs. 2-6, 25 x . Ultrastructural Studies of Cell Death During Takeover each zooid (see Lauzon et al., 1993 and Fig. 111, or neighboring cells which themselves subsequently died (Fig. 12). Stage D-3 (midtakeover) is characterized histologically by widespread death of all visceral tissues and appearance of large numbers of macrophages in the pharyngeal cavity (Fig. 7). Ultrastructural analysis of this process revealed that the predominant mode of visceral cell death occurred by apoptosis, characterized by nuclear condensation, chromatin margination, vesiculation of the endoplasmic reticulum and cellular fragmentation into membrane-bounded bodies (Lauzon et al., 1993).For instance, in the gastric epithelium, apoptotic bodies were extruded into the lumen where they were presumably cleared through the vasculature (Fig. 8). Apoptotic bodies ranged in size and content, with some bearing nuclear fragments (Fig. 8) while others were anucleate (Fig. 9). It should be noted, however, that cellular fragmentation was not universally observed in all cells within individual tissues (Lauzon et al., 1993). Entire cells and apoptotic fragments were engulfed either by blood macrophages recruited from specialized regions of the ventral-anterior portion of Histochemical Localization of Acid Phosphatase In addition to apoptotic changes, morphological features reminiscent of necrosis were also observed in the gastric epithelium and intestine (Fig. 10). These changes occurred independently or in combination with morphological features characteristic of apoptosis, such as cellular fragmentation (Lauzon et al., 1993). Furthermore, autophagic myelin-like figures were frequently associated with this phenotype in the gastric epithelium (Fig. 10).In order to address the possibility that AP activity may be associated with this morphology in Botryllus, zooids were sectioned parallel to their anteroposterior axis at various stages during the blastogenic cycle and stained histochemically for AP expression. During asexual growth, AP was selectively localized throughout the gut of filter-feeding zooids, with intense localization in the gastric epithelium (Fig. 13).In the early stages of takeover (D-21, AP activity 222 R.J. LAUZON ET AL. Figs. 7-12. PROGRAMMED DEATH IN A COLONIAL ASCIDIAN 223 was still observed in the gut region, although staining intensity was qualitatively decreased (Fig. 14). Other visceral tissues were never observed to express AP. It should also be noted that an insoluble red pigment found exclusively in certain cells within the vasculature was present in the absence of AP staining and could not be removed under these experimental conditions (see Fig. 13).By stage D-3 (midtakeover), AP expression became almost undetectable in the regressing gastric epithelium (Fig. 15). By contrast, the stomach of prefunctional zooids was highly AP-positive. In addition, macrophages that were actively scavenging dead cells were found to exhibit weak AP activity (Fig. 16). contamination of the RNA samples. Two different annealing temperatures applied to PCR reactions indicated no significant effect on the number of displayed bands, but the intensity of each band was stronger at 42°C (Fig. 17, lanes 4-7) versus 40°C (Fig. 17, lanes 8 and 9) for the same exposure time and loading volume. Presumably, this was caused by higher specific amplification under more stringent conditions for annealing. Similar control experiments performed with 30,35 and 40 PCR cycles revealed no qualitative differences between cycles, with the exception that band intensity was more pronounced after 40 cycles (data not shown). Therefore, 42°C and 40 cycles were the annealing temperature and cycling conditions of choice used in studies reported here. By comparing displayed PCR products amplified Analysis of Gene Expression Patterns by with the T12MG and AP1 primer set, developmentally Differential mRNA Display regulated genes were identified during both takeover To understand how these organisms and their tissues (Fig. 17, lanes 4 and 6 are in stage D-3) and asexual die, it is necessary to identify the genes involved in cell growth stages of the blastogenic cycle in the Monterey death and to determine how, when and where their Bay clones (Fig. 17, lanes 5 and 7 are in stage B-1) for products function. For these experiments, differential two independent PCR reactions. As shown in Figure mRNA display was used as an initial strategy to define 17, there were three potentially downregulated cDNA transcriptionally modulated genes (Liang and Pardee, fragments during midtakeover (lanes 5 , 7 and 9; indi1992). The asexual synchrony and clonal nature of in- cated by arrowheads), and one upregulated band (lanes dividual Botryllus colonies lend themselves ideally for 4,6 and 8; indicated by the arrows). On the other hand, direct analysis of developmental stages on a sequenc- display of duplicate samples from two genotypically ing gel. Control experiments were carried out to rule distinct colonies in growth (Fig. 17, lanes 10 and 12 are out DNA contamination of RNA samples, optimize an- in stage (3-2) and takeover stages (Fig. 17, lanes 11and nealing temperature and cycle number for PCR reac- 13 are in stage D-4) revealed 27 differentially extions. The display result should be completely depen- pressed cDNA fragments. These findings suggest that dent on reverse transcription if RNA samples are free polymorphisms between genetically unrelated colonies of DNA contamination. Unfortunately, most RNA could significantly increase the number of bands dispreparations, including those generated using cesium played on a sequencing gel. In order to determine whether these four genes chloride gradients (in these studies) are commonly contaminated by chromosomal DNA, which can be ampli- (GAPU790.1, GAPU790.2, GAPlf790.3, GAPll790.4) fied efficiently in PCR reactions and interfere with the were transcriptionally modulated during takeover, the display result (Liang et al., 1993). Individual colonies cDNA fragments were cut out of the gel and reampliwere collected from Monterey Bay, and RNA samples fied using the same primer sets. They were subsewere isolated from clonal replicates. Following diges- quently radiolabelled with 32Pand used in a slot blot tion with RNase-free DNase, two developmental stages analysis to probe their temporal patterns of expression. (stages B-1 and D-3) were displayed on a denaturing RNA was isolated from growth phase animals (stages sequencing gel. As shown in Figure 17, lanes 1-3, sam- A, B-1, C-1) and animals undergoing takeover (stages ples containing only water or total RNA in the absence D-1, D-2 and D-3), and transferred to a nitrocellulose of reverse transcription did not reveal any significant membrane. For the slot blot studies, colonies were col- Figs. 7-12. The morphology of zooid regression during takeover. Figure 7 depicts a light micrograph of a zooid during midtakeover (D-3). Note the presence of numerous multinucleated macrophages (arrowheads). Figures 8-12 are transmission electron micrographs illustrating various aspects of the death process in the gut. The principal mode of cell death is by apoptosis, as evidenced by chromatin condensation, vesiculation of the endoplasmic reticulum, cell fragmentation and extrusion of apoptotic bodies into the lumen of the stomach (Fig. 8).Both nucleate (arrow in Fig. 8) and anucleate (arrow in Fig. 9) fragments were observed. Necrotic foci of gastric mucous cells (Fig. 10) were also observed intermixed with apoptotic cells. Dying visceral cells and fragments are ultimately engulfed by blood macrophages (Fig. 11, in the pharynx) or neighbors (arrow in Fig. 12, in the intestine). Note the presence of an apoptotic cell with marginated chromatin prior to engulfment (arrows).Magnification: Figure 7, 200 x ;Figure 8,3,000 x ; Figure 9,8,000x ;Figure 10,5,000 X ;Figure 11, 4,000 x ; Figure 12, 5,000 x . Abbreviations: MY,myelin-like figures; LI, lipid droplets. Figs. 13-16. (Figs. 13-16 appear on page 224 and appear in color in the Color Figure Section immediately following page 258.) Localization of acid phosphatase activity in Botryllus schlosseri. Plasticembedded systems from various developmental stages were sectioned along the anteroposterior axis of m i d s and visualized under differential interference contrast microscopy. Note the intense apical staining in the stomach during stage C-1 of blastogenesis (Fig. 13). Acid phosphatase activity is still present in the early stages of takeover within stomach, intestine and esophagus (stage D-2; Fig. 14). By midtakeover (D-3),when most organs have been resorbed, little acid phosphatase activity can be detected in the regressing m i d (Figs. 15 and 16).Macrophagesactively engulfing dead cellswere found to be weakly acid phosphatase-positive (arrowheads in Fig. 16). In contrast, the stomach of prefunctional zooids exhibit prominent staining (arrows in Fig. 15). Magnification: Figure 13, 100 x ; Figures 14 and 15, 200 X ; Figure 16,400 x . Abbreviations: ES,esophagus;EPI, epidermis; INT, intestine; PG, pigment cell; PH, pharynx; STO,stomach; T, tunic. Figs. 13-16 (Legend appears on page 223). 1 ---- I PROGRAMMED DEATH IN A COLONIAL ASCIDIAN GAP1 1790.1 GAP1 1790.2 225 A B-1 C-1 D-I D-2 D-3 .-- - - .- I - - GAP1 1790.4 Fig. 18. Confirmation of developmentally modulated expression of cDNA clones with RNA slot blot analysis from various developmental stages. Two pg of total RNA from stages A, B-1, C-1, D-1, D-2, and D-3 were applied individually onto nitrocellulose membranes using a slot blot apparatus. The specific cDNA fragments, GAP1/790.1, GAPU 790.2, GAPll790.3, and GAFW790.4, previously identified as developmentally modulated transcripts, were reamplified with the same primer sets and PCR conditions as in differential mRNA display, followed by gel purification and 32P-labelling. Hybridization was carried out under high stringency conditions with each radioactive probe individually. Fig. 17. Identification of differentially expressed genes in BotrylZus from clonal replicates in stages B-1 and D-3 (midtakeover) by differential mRNA display. Lane 1: H,O control; lanes 2 and 3 RNA templates from stages B-1and D-3, respectively; lanes 4 and 6 duplicate amplification of cDNA templates from stage D-3; lanes 5 and 7: duplicate amplification of cDNA templates from stage B-1. The cDNA templates used in lanes 8 and 9 were the same as in lanes 4 and 5, respectively, but with an annealing temperature at 40°C instead of 42°C. cDNA templates amplified for lanes 10-13 were derived from two genetically different colonies. Lanes 10 and 12: cDNA templates from stage C-2; lanes 11 and 13 cDNA templates from late takeover (D-4). lected from Long Island Sound and were all genotypically different. Our screening strategy was to demonstrate modulated expression in as many different colonies as possible. Therefore, if the differences observed with clonal replicates by differential display represent true differences in vivo, expression patterns of these genes should be reproducible with animals of different genetic backgrounds and from geographically distant sources (Pacific versus Atlantic oceans). As shown in Figure 18, the expression levels of two of these genes (790.3and 790.4)were selectively altered during takeover. The steady-state level of gene 790.4 was increased at the onset of takeover and this trend was maintained throughout zooid regression. On the other hand, gene 790.3 was downregulated throughout takeover. Gene 790.2 was expressed at higher levels during stage B-1, but decreased thereafter, whereas the expression pattern of 790.1 did not appear to be appreciably affected during the blastogenic cycle. These results were quantified using video densitometry (Fig. 19) and clearly demonstrate that both 790.3 and 790.4clones were transcriptionally modulated during takeover. Additional experiments with different colonies of the same developmental stages gave identical results to those presented here (data not shown). DISCUSSION In this paper, we have demonstrated that a defined marker of impending cell death in several other invertebrate systems, acid phosphatase, does not appear to be associated with the complex morphological events accompanying takeover in Botryllus schlosseri. Previous observations from this laboratory have indicated that the principal mode of cell death during takeover occurs by apoptosis (Lauzon et al., 1993).However, necrotic and autophagic features could also be found in gastric and intestinal epithelia (Burighel and Schiavinato, 1984;Lauzon et al., 1993;and this study) often separable from apoptotic cells within the same tissue, or in conjunction with cellular fragmentation characteristic of apoptosis. The histochemical distribution of acid phosphatase in Botryllus peaked during asexual 226 R.J. LAUZON ET AL. 100 6 'f 90 80 70 ?! 360 .--ca. a r 0 GAP11790.3 50 40 30 20 10 0 t . . . . . A 61 C1 D1 D2 D3 Blastogenic Stages of Botryllus Fig. 19. Quantitative analysis of expression level for cDNA clones 790.3 and 790.4 at various stages of the blastogenic cycle. growth stages within gut epithelia, and gradually disappeared in these tissues as they regressed during takeover. This observation may either be indicative of high cytoplasmic turnover in the gut during feeding and/or be functionally related to hydrolytic breakdown of nutrients as they are processed. In several invertebrate systems of programmed cell death, particularly insects, nonapoptotic cell death including autophagic degeneration, has generally been regarded as the predominant mechanism underlying tissue regression (Beaulaton and Lockshin, 1982; Clarke, 1990; Schwartz et al., 1993b). The morphology of autophagic death is characterized primarily by the formation of numerous autophagic vacuoles, which may be functionally related to an increased activity of the Golgi apparatus in generating primary lysosomes, ultimately resulting in the discharge of hydrolytic enzymes in autophagic vacuoles. Histochemical detection of these hydrolases at the light microscopic or ultrastructural level, in particular acid phosphatase, has been used as a diagnostic feature of autophagic death (Clarke, 1990). However, recent observations indicate that hydrolase-mediated destruction of the cytoplasm which culminates in cell swelling and lysis, appears to be a late component of the death process (Bowen et al., 1995; Zakeri et al., 1995). For instance, in the metamorphic death of labial glands of the blow-fly, CaZZiphora uomitoria, there is an early lysosomal peak of acid phosphatase (Bowen et al., 1995). This is followed by a de novo synthesized ribosomal source of this enzyme in the later stages of the process when histolysis becomes prevalent. These observations suggest that the free but not lysosomal form mediates cell death in these systems. By contrast, a zooid-associated surge in hydrolase activity was neither observed in the early nor later stages of takeover. Therefore, the function of this enzyme appears to be limited to a homeostatic or processive one during feeding of Botryllus zooids. The necrotic features observed in the gut are unlikely to result from anoxia, since blood flow is maintained uninterrupted until cessation of heartbeat when zooid resorption is complete. One possibility may be that release of free acid phosphatase and other hydrolases by the gastric and intestinal epithelia as they undergo apoptosis may induce localized necrotic foci in neighboring cells. It is perhaps not a coincidence that the very tissues which manufacture hydrolytic enzymes used in feeding, are the same ones which undergo necrosis. Another possibility may be that apoptotic cells which are not immediately phagocytosed by macrophages rapidly undergo secondary necrotic lysis. Alternatively, the mechanism by which cells die may be related to the position of visceral tissues along the zooid's anteroposterior axis. In the early stages of takeover (stage D-2), the zooid contracts in an anterior to posterior direction coincident with the dorsal migration of primary buds. This process is accompanied by the polarized breakdown of the perivisceral extracellular matrix along the anteroposterior axis and precedes cell death (Lauzon et al., 1992). This observation raises the possibility that once the anchorage of a differentiated epithelial cell with its underlying matrix is disrupted, it rapidly dies. Recent findings have indicated that matrix attachment is paramount to the maintenance of the functional and differentiated state of tissues as well as their survival (Frisch and Francis, 1994; Meredith et al., 19931, thus lending credence t o this proposal. According to this model, the mode of cell death would depend on the microenvironment in which specific tissues reside. The process by which BotryZZus zooids become irreversibly programmed to die is poorly understood, but is likely to be mediated by a diffusible factor which is disseminated throughout the colonial vasculature. In experimental vascular chimeras made between colonies of different developmental stages, the partner a t the later stage always accelerates the blastogenic cycle of the other partner, ultimately initiating synchrony within the chimera (Watanabe, 1953; R.J.L, unpublished observations). As a means of understanding how death may be regulated in this model system, we have begun to isolate genes by differential mRNA display. Botryllus is optimally suited for this analysis because of the clonal nature of individual colonies. The results presented here using one primer set indicate that this approach is indeed feasible. The steady-state levels of genes designated as 790.3 and 790.4 were selectively downregulated and upregulated, respectively, during the takeover phase of the blastogenic cycle. However, these findings do not establish a direct link between zooid regression and gene expression, since developing buds and mesenchymal components also form an integral part of colonies undergoing takeover. Determination of their spatial pattern of expression by in situ hybridization will be required to address this issue. This work is currently underway in our laboratory. Interestingly, the use of genetically different colonies led to a significantly greater number of displayed bands on a sequencing gel. Although the same developmental stages were not compared in these experiments (C-2 and D-4 for different genotypes versus B-1 and D-3 with clonal replicates), these additional bands most likely represent intraspecies polymorphisms. Consequently, it is anticipated that the use of genetically distinct individuals could severely obscure interpretation of differential display patterns (i.e., false positives), and should be avoided if possible. Genetic and molecular analyses in several experimental systems have revealed that cell survival de- PROGRAMMED DEATH IN A COLONIAL ASCIDIAN pends on a complex interplay between gene products which protect cells from death (survival genes) and others which are required to execute the death program (Schwartz and Osborne, 1993; Reed, 1994). Furthermore, many of these genes have been functionally and structurally conserved throughout much of evolution (Ellis and Horvitz, 1986; Hengartner and Horvitz, 1994; Hengartner et al., 1992; Miura et al., 1993; Sugimoto et al., 1994; Vaux et al., 1992; Yuan et al., 1993). Ascidians such as Botryllus schlosseri are deuterostome invertebrates closely related to vertebrates. Studies are currently underway to determine whether the genes characterized in this study are homologous to genes known to be involved in cell death. In summary, our results indicate that differential mRNA display appears to be an appropriate strategy for identifying genes whose expression patterns may be functionally involved in zooid regression, provided that genetic polymorphisms can be avoided using clonal replicates of various developmental stages. The isolation and characterization of such genes could provide a basis to understand how this model organism dies and how its lifespan is regulated. 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