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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.
ACKNOWLEDGMENTS
The authors gratefully acknowledge Dr. Ann Carroll
for use of laboratory equipment required in differential
mRNA display experiments, Dr. Mark Deziel for technical assistance in the use of the Bio-Image video densitometer, Ms. Kathi Ishizuka for the collection of Monterey Bay colonies and Mr. James Dowling for Long
Island Sound colonies used in these studies. This work
was supported by a Basil O'Connor Starter Award from
the March of Dimes Birth Defects Foundation 5-FY940813.
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