Abstract:
Botryllus schlosseri is a colonial ascidian that reproduces the entire colony every week
through a synchronized asexual budding process. At the end of this seven day cycle, all
functional zooids undergo synchronized apoptosis and are resorbed by blastozooids of the
new generation. There is evidence that suggests a similar mechanism controls apoptosis
in many vertebrates and invertebrates, such as Caenorhabditis elegans and humans. In
mammalian cells, expression of certain genes from the bel-2 gene family regulates
apoptosis, the physiological process that eliminates unwanted cells. The purpose of this
study was to investigate the role of bcl-2 in apoptosis of B.schlosseri. This study shows
that there are at least seven members of the bcl-2 gene family expressed in B. schlosseri.
The sequences of two isolated clones of the bel-2 gene, called bel-2 B and bel-2 B+, show
a 40% homology with known vertebrate bel-2 sequences. These genes exhibit differential
expression throughout the asexual life cycle. Bcl-2 B maintains a consistent level of
expression throughout the asexual life cycle of the colony while bel-2 B+ is down
regulated after the early stages of the zooid lifespan. The results of this study suggest that
the bcl-2 gene family might regulate apoptosis in the asexual life cycle of B.schlosseri as
well.
Introduction:
Apoptosis is the programmed death of cells characterized by cell shrinkage, loss
of structure in the plasma membrane, and apoptotic body formation. It is a fundamental
process during the initial development of the organism, especially in the embryo and
nervous system. Apoptosis also plays a key role in the maintenance of the immune
system in the adult. Maintaining the balance between extensive apoptosis and
proliferating cells is essential. Defects in apoptosis have been observed in 85% of all
cancers. To prevent mishaps, many different and redundant cell signaling pathways exist
in vertebrates and contribute to the maintenance of the adult organism. (Hsu and Hsueh,
2000) Widely characterized gene families, such as the bel-2 gene family in humans and
ced-9 in Caenorhabditis elegans, are responsible for the regulation of this vital process.
This study is a part of an overall research project that aims to characterize the
developmental steps and underlying molecular pathways that lead to apoptosis.
The basal molecular mechanism of apoptosis was first discovered in C.elegans
and is conserved throughout all metazoans. Apoptosis is a one way process. Once a cell
has committed to apoptosis, it will certainly die. (Schiavone et al., 2000) Apoptosis
begins with the activation of the apoptotic pathway, releasing a wide array of nucleases
and proteases that digest the cell from within. At this point, a neighboring cell engulfs the
dying cell, preventing the intracellular components from contaminating the outside
environment. Once the corpse is resorbed, there is no trace left of the apoptotic cell.
(Fig.1) Thus apoptosis is both a process of destruction as well as one of resorption. The
exact mechanisms that underlie the decision between the life and death of a cell are
unknown. However it is likely that changes in gene expression are a fundamental part of
this process.
The key regulatory protein of apoptosis in C.elegans is ced-9, whose expression
results in the prevention of the process. This single protein corresponds to a wide array of
proteins that belong to the bel-2 gene family in vertebrates. This gene family includes at
least 15 regulatory proteins whose differential expression result in both the prevention
and the promotion of apoptosis. Certain anti-apoptotic genes from the human bel-2 gene
family are similar enough to be interchangeable with ced-9. Expression of human bel-2
results in a reduction of apoptotic events in C.elegans. (Vaux et al., 1992) Recent studies
have shown that the bel-2 gene codes for a mitochondrial ion channel and a docking
protein in vertebrates. Äfter a premitochondrial decision step, upstream signal
transduction pathways are activated to regulate Bcl-2 proteins, followed by the release
and activation of caspases. A decrease in this protein prevents it from inhibiting different
apoptotic pathways. (Hsu and Hsueh, 2000 and Schiavone et al., 2000)
This study uses Botryllus schlosseri as a model system. They are common just
below the watermark on pilings, eel grass, and under rocks, especially in harbors where
water turbulence is low. (Milkman, 1967). This organism is a colonial protochordate that
reproduces sexually to produce a free-swimming tadpole, with many vertebrate
characteristics, including a hollow dorsal notochord, gill slits, segmented muscles, and a
tail. After attachment, the tadpole metamorphoses into an oozoid, which asexually
divides to become a non-chordate sessile, filter-feeding adult. (Fig. 2) A colony of
B.schlosseri is made up of a group of systems that resemble flowers. Each petal of the
flower is a distinct individual, or zooid, that is linked to the others by a common blood
supply. B.schlosseri is an interesting model system because the organism undergoes a
weekly asexual budding process to give rise to a large colony of asexually-derived,
genetically identical offspring that share a common vascular system. With each new
cycle, blastozooids regenerate complex organs such as blood, heart gastrointestinal
systems, gill arches, nervous system, gametes etc. (Milkman, 1967) If a zooid is separated
from the colony, it can regrow the whole colony using the same asexual budding process.
Thus the individuals are all linked, but not dependent upon one another.
The asexual life cycle, otherwise known as the blastogenic cycle, is a seven day
process. (Fig. 3) Stage A begins with the opening of the feeding siphons in the zooids. As
soon as the siphons open, the eggs within the parent zooids are fertilized. At this point,
primary buds are microscopic. Two days later, stage B begins with the establishment of
the heartbeat and circulatory sytem in the primary buds, linking them to the rest of the
colony. The primary buds have grown, but the secondary buds are only a thickening in
the epithelial wall of the primary buds. At any given moment during the blastogenic cycle
there are three individuals at various stages in their development. (Berrill 1941a) During
stage C, organogenesis begins in the secondary buds, and the primary buds continue to
grow. The parent zooids begin to age by losing pigmentation and size. At the end of the
sixth day, the colony is faced with an event called takeover, or stage D, in which the
parent zooids close their feeding siphons, undergo synchronized apoptosis, and are
resorbed by the primary buds. The primary buds then open their siphons and begin a new
cycle. (Watkins 1958) The apoptosis of half of a colony each week might seem
energetically costly, but each successive generation is larger than the previous zooids.
Each zooid also has a limited capability of gonad production, and smaller zooids lack
these structures altogether. By renewing zooids each week, the colony maintains its
reproductive capabilities. (Berrill 1941b)
In addition, when Botryllus colonies encounter histocompatible kin in the wild,
they exhibit vascular fusion. The colonies undergo a natural transplantation reaction
where the vasculatures of the two colonies fuse together to form a single unit with a
common blood supply. If these colonies are artificially separated, one colony will at least
partially express the DNA of the other colony. This foreign contribution to development
is known as parasitism and might have a genetic basis. If the two colonies are not
compatible, the ampullae will exhibit an immunological reaction and local apoptosis will
occur, resulting in the resorption of the ampullae. This reaction to non-self cells produces
points of rejection and the two colonies shrink away from eachother. (Stoner and
Weissman, 1996 and Stoner et al., 1999) This observation shows that apoptosis occurs in
different contexts within the colony and might produce the need for different genes to
control different instances of apoptosis in B.schlosseri.
Materials and Methods:
Cells and animals
dHIOB cells were grown in suspension cultures and on circular agarose plates at 37°C
after electroporation with plasmids containing bel-2 B, bcl-2 B+, cytoactin and tubulin
inserts. The partial clones were previously isolated from B.schlosseri stock grown in
mariculture tanks at Hopkins Marine Station.
Preparation of riboprobes by in vitro RNA transcription
Sense and antisense (T3/T7) strand-specific ssRNA probes were derived from plasmid
vectors containing the partial-length cDNA clones of the genes, bel-2 B, bcl-2 B+,
tubulin and cytoactin. The different riboprobes were prepared from appropriate PCR-
generated cDNA fragments flanked by non-specific phage polymerase promoter
sequences, T3 and T7. For RNA in vitro transcription and labeling with fluorochromes,
reaction mixtures contained 200 ng template DNA, 1 mM ATP, GTP, and CTP, 0.65 mM
UTP, 0.35 mM DIG-oxygenin-11-UTP(Boehringer Mannheim)/O.35 mM sa-2PJUTP,
transcription buffer 5X, RNA polymerase T7 and T3, and H2O to a final volume of 20uL.
Transcription proceeded in a 37°C water bath, shielded from light for 1 hour, followed by
a 30 min. digestion with RNase-free DNase at 37°C. Unincorporated nucleoside
triphosphates were removed by running the reaction products through an S-400 gel
column with centrifugation. (Promega, protocols and applications guide, P. 59-61)
Analysis of RNA transcripts on agarose gel
Before use, the RNA transcripts were analyzed for length by staining with ethidium
bromide on a 3% agarose gel. Dilutions of 1:100 and 1:200 were made of each probe. The
overall nucleic acid content of the probe dilutions were analyzed using a
spectrophotometer. Based on the results the probes were all diluted to a concentration of
lOng/uL. Radioactive probes had an average scintillation count of 2.5 x10° cpm/uL.
RNase Protection Assay
Overnight hybridization reactions (2 X10' cpm of each probe and lug total RNA from
stage A, midcycle and Takeover-D) were co-precipitated in IOM NH,Oac, glycogen and
ethanol and incubated in hybridization probe overnight at 42°C. I used yeast tRNA for
two sets of control tubes for each probe. Before each use, RNA probes were denatured by
incubating at 95°C for 5 minutes to ensure higher strand specificity. The reactions were
then digested with RNase A/ RNase TI in Digestion Buffer at a 1:1000 dilution for 30
minutes at 37°C. For one set of the control tubes, no RNase was added to the mixture.
After the digestion, the RNase Inactivation/ Precipitation Buffer III and 2 uL of yeast
tRNA were added to the reactions and incubated at -20°C for 15 min. The RNA was
pelleted by centrifugation at top speed for 15 min. and resuspended in Gel Loading
Buffer II. The samples were loaded onto a 40% acrylamide gel after incubation at 950C
for 5 min to solubilize and denature RNA. After running for 2.5 hours, the gel was
transferred to chromatography paper, covered with saran wrap, dried, and exposed to
double-sided film for 2 days at -80°C. (Ambion, RPA III kit)
Southern Blot/ Reverse Northern Blot
For the Reverse Northern Blot I made cDNA from stages A, B, C, and D in the asexual
life cycle by PCR-amplifying single-stranded cDNA in 50 uL reactions. For the Southern
Blot I set up 30 uL reactions with Eco RI and Pst I, the appropriate buffer, and genomic
B.schlosseri DNA for bel-2 B/B+. The restriction digests went overnight at 37°C.
Afterwards, I ran all the samples on a large 1% agarose gel for sufficient resolution and
transferred them to a nylon membrane using a capillary/southern blot. In the rotating
hybridization chamber, the nylon membrane was radioactively probed for bel-2 B/B+
overnight at 65°C, washed in O.IX SSC and 0.1% SDS at 65°C in a shaking water bath. It
was then exposed to film for 3 days at -80°
Fixation of cells and whole mount sections for conventional fluorescence microscopy
From B.schlosseri colonies grown in mariculture tanks, approximately 3 x 10° blood cells
were isolated by cutting ampullae of 3 systems and extracting with a micropipette. The
cells were washed in twice in Hepes Buffered Saline (HBS) (Maniatis et al., 1989) for 3
minutes, fixed in 2% paraformaldehyde for 15 minutes and then cytospun at 1000g onto a
glass slide. The cells were permeabilized by immersion in 100% ice-cold acetone and
washed in HBS. The cells were then processed for in situ hybridizations. For whole
mount sections, 2-3 systems from homozygotic crosses Byd 196 were taken from each
stage A-D in the asexual life cycle of the colonies. The cells were fixed in 2%
10OL
paraformaldehyde in ASW (27.65g Nacl, 0.67g KCl, 1.36g CaClz.zH20, 4.66g
MgCl2.6H20, 6.29g MgsÖ4.7H20, 0.18g NaHCO3/ 1 L, pH 8.2) overnight and were
then transferred to 30% sucrose in filtered seawater for 2 days. The systems were
embedded in OCT, frozen over dry ice and cut in 6um sections in a cryostat.
In situ hybridization (ISH)
All solutions were prepared with DEPC-treated ddH20 and PBS (Maniatis et al., 1989).
Sections were incubated in PBS, followed by PBS with 100mM glycine, PBS with 0.3%
Triton X-100, PBS, and permeabilized by TE Buffer (100 mM Tris-HCl, 50 mM EDTA,
pH 8.0, 1 ug/mL RNase-free Proteinase K). The sections were post-fixed by incubating in
PBS with 4% paraformaldehyde and washed with PBS. The sections were acetylated with
0.1 M triethanolamine (TEA) buffer, pH 8.0, containing 0.25% acetic anhydride.
Prehybridization incubation was at 37°C in 4X SSC containing 50% formamide.
(Maniatis et al., 1989) Äfterwards, each section was drained and overlaid with
hybridization buffer (40% formamide, 10% dextran sulfate, IX Denhardt's solution, 4X
SSC, 10 mM BME, 1 mg/mL salmon sperm DNA and 1Ong of DIG-RNA probes.
Sections were covered with coverslips and incubated overnight at 42°C in a humidifier.
After hybridization, sections were sorted according to probes and placed in Coplin jars.
Coverslips were removed by soaking in 2XSSC, followed by washes with 2XSSC and
IXSSC in a shaking 37°C water bath. Any unbound RNA probe was digested by
incubating in NTE Buffer containing 20ug/mL RNase A for 30 min at 37°C (500mM
Nacl, 1OmM Tris, ImM EDTA, pH 8.0). Sections were washed for a final hour in
O.IXSSC in the 37°C shaking water bath.
Immunological Detection Enhancement
The Fluorescence Antibody Enhancer Set for DIG Detection (Boehringer Mannheim) was
used for this part. After ISH, slides were washed and incubated for 30 min at room
temperature with 50 mL IX blocking solution per Coplin jar to block unspecific antibody
binding. Afterwards the slides were overlaid with anti-DIG Antibody in a 1:25 dilution in
IX blocking solution and incubated for 60 min at 37°C in a humidifier. Slides were
washed in washing buffer (0.2% Tween 20 in IXPBS) at 37°C between antibody
incubations. The secondary antibody incubation of anti-mouse-Ig-DIG and the tertiary
antibody incubation with anti-DIG-fluorescein (FITC) were under the same conditions as
the primary antibody. Afterwards slides were stained with propidium iodide (Pl) (5-10
ng/mL) and air dried in a dark room covered with mounting media and a coverslip (50%
glycerol, 50% PBS). The signals were detected with a conventional fluorescence
microscope.
Results:
In order to study the molecular pathways of apoptosis in B.schlosseri, I probed
genomic B.schlosseri DNA with bel-2 B/B+ probes made from bel-2 clones isolated
using degenerate PCR. To see partial nucleotide and corresponding protein sequences of
B and B+, see Fig. 4. Based on these sequences and known bel-2 sequences of other
vertebrates and C.elegans, I constructed a phylogenetic tree. (Fig.5) Both sequences had a
26% homology with ced-9 in C.elegans, and a 40% homology with vertebrate bel-2
sequences. In the Southern Blot, which are the two lanes on the far left, both restriction
digests contained multiple bands. The Pstl digest contained at least 3 bands, while the
EcoRl digest contained at least 7 bands. (Fig. 6) For the Reverse Northern Blot in the
four lanes to the left of the DNA ladder, bel-2 B/B+ probing of cDNA isolated from
different stages of the blastogenic cycle (A, B,C and D) yielded a single 1.1 kb band in
stage A. (Fig. 6) The RNase protection assays gave consistent bands for bel-2 B
throughout the blastogenic cycle (A, midcycle, takeover), while bel-2 B+ displayed
differential expression, with bands becoming lighter in later stages. The sense controls
showed no bands for each probe, using RNA from stage A. The RNA quality and amount
control tubulin gave consistent bands throughout the blastogenic cycle as well. (Fig. 7)
For a graphical representation of band strengths in the RNase protection assay, see Fig.8.
The in situ hybridizations did not yield conclusive results. Both sense and antisense
probes contained equal fluorescence in the actin control and sections probed with bel-2 B,
B+. (data not shown)
Discussion:
In this study, I investigated the regulation of apoptosis in the colonial tunicate,
B.schlosseri, focusing on the gene bel-2. The two bel-2 clones isolated from B.schlosseri
have a 40% homology with vertebrate bel-2 sequences and a 26% homology with the
ced-9 sequence in C.elegans. The proximity of B.schlosseri to the vertebrates in the
phylogenetic tree led me to suspect the involvement of bcl-2 in the apoptotic pathway of
B.schlosseri. At least 15 genes belonging to this gene family regulate apoptosis in
vertebrates. The multiple bands in Southern Blots of genomic B.schlosseri DNA suggest
that there are at least seven independent bel-2 genes in this colonial tunicate. The
recognition sites of the restriction enzymes Pstl and EcoRl were not included within the
regions that the B/B+ probes bound to the DNA, suggesting that each fragment represents
an independent gene. One interesting band in the EcoRl digest is at 3.2 kb. This band is
darker than the rest, indicating that more probe bound to it compared to the other
fragments. This could be because both the bcl-2 B and bel-2 B+ probes found binding
sites on the fragment, increasing the activity of the band. Since this method lacks the
specificity to differentiate between the two sequences, the probes were used as a mixture.
From the Reverse Northern Blot, I saw that bel-2 B/B+ are expressed strongly in
the early stage A of the blastogenic cycle, but expressed less or not at all in the later
stages. The cDNA for this blot was made from DNA taken from different stages of the
life cycle of B.schlosseri. However, this method lacks the resolution to differentiate
between similar sequences as well, so I did an RNase protection assay. This assay is very
sensitive and can differentiate between RNA species that have only a few amino acids
that are different. The bands on the gel are a result of radioactively labeled antisense
RNA probes binding to complimentary RNA strands of target RNA. The lack of bands in
the sense control lanes show that the RNA probes were specific in their binding. The
housekeeping gene tubulin gave consistent bands throughout the life cycle, confirming
that the RNA samples were consistent in quality and amount.
The differential expression of bel-2 B and bcl-2 B+ suggests that they might
regulate apoptosis in B.schlosseri in a manner similar to vertebrates and C.elegans. These
results are a confirmation of preliminary studies done in the Weissman lab. Recent
studies show that an important step in the down regulation of bel-2 might be due to a
highly conserved destabilizing AU-rich region in the 3’ end of bel-2 mRNA. Degradation
of mRNA is crucial in the regulation of bcl-2 expression in vertebrates as well as in the
ced-9 gene of C.elegans. This destabilizing region is seen in both vertebrates and
Celegans. (Schiavone et al., 2000) An idea for future studies includes isolating the
mRNA of bel-2 B+ from a colony to see if it contains this AU-rich region on the 3' end
as well.
Bel-2 B might be responsible for shaping the body plan of the primary and
secondary buds growing throughout the blastogenic life cycle. An important point to
remember is that apoptosis is essential both in embryonic development as well as
maintenance of the adult organism, so it should be occurring throughout the whole life
cycle in different tissues. On the other hand bel-2 B+ might be an anti-apoptotic agent for
mature zooids that degrades in order for the functional zooids to age. Previous studies
show that the zooid lifespan is determined by a clock mechanism that is bud dependent.
When a primary bud is surgically removed during stage A, the colony remains alive for
48-72 hours until a new bud is generated. Since the vascular system is established in
stage B, only two days after stage A begins, there is evidence that a bud-derived factor is
released into the circulatory system at this early stage, irreversibly deciding the zooid
lifespan. (R. Lauzon, pers. comm., and Chang and Lauzon, 1993)
An ambiguity that cannot be resolved with the RNase protection assay is that it
does not provide information about the spatial localization of the different RNA species.
A certain bel-2 species might be regionally down regulated during one stage of the life
cycle while up regulated in another region, and this would average out to the same band
in the assay. For this reason it is essential to obtain probes specific to the coding regions
of the bel-2 partial genes. This will allow a more detailed and specific spatial and
temporal study of bcl-2 RNA localization. The in situ hybridizations Iattempted on
wholemount sections in this study would be ideal to determine localization of bel-2
mRNA, however the method needs to be tailored to the organism. RNA is also very hard
to work with because it can be denatured very easily. The non-differential fluorescence
observed in in situ hybridizations was visible in both FITC and UV channels on the
fluorescence microscope. I had also done PI staining to get an idea where the cells were
in the sections. There is a possibility that B.schlosseri tissue has auto fluorescence due to
circulating pigment cells, or that the PI fluorescence leaked into the other channels when
I viewed them subsequently. To see what the problem with this part of the study was,
further experimentation is necessary with new probes and a confocal microscope. Äfter a
detailed study of the expression patterns of bcl-2 B and B+, a combination of loss and
gain-of-function experiments will determine the apoptosis regulatory function of these
genes in B.schlosseri conclusively.
Conclusions:
The results of this study suggest that there are at least seven independent bel-2
genes in B.schlosseri, and that the coding region of bel-2 B/B+ is approximately 1.1 kb in
length. According to the phylogenetic tree based on bel-2, B.schlosseri is closer to
vertebrates than it is to C.elegans. Bcl-2 B exhibits consistent expression throughout the
blastogenic life cycle while bel-2 B+ seems to be down regulated in the later stages of the
cycle when the parent zooids age and experience takeover. The differential regulation of
two genes of the bel-2 gene family suggest that apoptosis in B.schlosseri is controlled by
a mechanism similar to that of vertebrates and C.elegans.
Acknowledgements:
I would like to thank Tony De Tomaso for being my partner in crime and the
satellite Weissman lab for their patience and endless supply of Botryllus colonies.
Marilyn Masek and Diana Laird deserve applause for their investment of time and energy
in sectioning and running in situs, respectively. I would like to thank the residents of 226
Willow St. and Susan Baxter for their moral support throughout the quarter.
Literature Cited:
Berrill, N.J. 1941a. The development of the bud in Botryllus. Biol. Bull. 80: 169-184
Berrill, N.J. 1941b. Size and morphogenesis in the bud of Botryllus. Biol. Bull. 80: 185-
193
Hsu, S.Y. and Hsueh, A.J.W. Tissue-specific bcl-2 protein partners in apoptosis: an
ovarian paradigm. Phys. Rev. 80: 593-614
Lauzon, Robert J.; Chang, Wen-The. 1993. Regulation of zooid lifespan in Botryllus
schlosseri is determined by a clock mechanism that is bud-dependent. J. Cell. Biochem.
Suppl. 17: 155.
Maniatis, T., Sambrook, J., and Fritsch, E.F. 1989. Molecular Cloning. B.1-27
Milkman, R. 1967. Genetic and developmental studies on Botryllus Schlosseri. Biol. Bull.
132: 229-243
Rinkevich, B., and Weissman, I.L. 1992. Allogeneic resorption in colonial
protochordates: Consequences of non-self recognition. Dev. Comp. Immunol., 16: 275-
286
Schiavone, N., Rosini, P., Quattrone, A., Donnini, M., Lapucci, A., Citti, L., Bevilacqua,
A., Nicolini, A., and Capaccioli, S. 2000. A conserved AU-rich element in the 3'
untranslated region of bel-2 mRNA is endowed with a destabilizing function that is
involved in bel-2 down-regulation during apoptosis. FASEB J. 14:174-184
Stoner, D.S. and Weissman, I.L. 1996. Somatic and germ cell parasitism in a colonial
ascidian: Possible role for a highly polymorphic allorecognition system. Proc. Nat. Acad.
Sci. USA 93: 15254-15259
Stoner, D.S., Rinkevich, B. and Weissman, I.L. 1999 Heritable germ and somatic cell
lineage competitions in chimeric colonial protochordates. Proc. Natl. Acad. Sci. USA
96:9148-9153
Vaux, D.L., Weissman, I.L. Kim, S.K. 1992. Prevention of programmed cell death in
Caenorhabditis elegans by human bel-2. Science 258: 1955-1957.
Watkins, M.J. 1958. Regeneration of buds in Botryllus. Biol. Bull. 115: 147-152
Table 1: A summary of terms specific to Botryllus schlosseri that are important in
understanding its life history.
Sausage-like blind termini of blood vessels commonly found in the
Ampullae
periphery of colonial urochordates
Asexual development process in which budding of new zooids occurs
Blastogenesis
from existing blastozooids in a botryllid colony. A typical blastogenesis
cycle at 18-20°C lasts about a week
A single individual in a colony of colonial protochordates generated by
Zooid
asexual reproduction. Zooids are arranged in star-shaped rosette,
serpentine, or ladder-like structures called systems that share cloacal
apertures
Also known as allogenic resorption, a genetically controlled
Colony
phenomenon in tunicate chimeras in which all blastozooids of one of the
resorption
partners are morphologically eliminated by the other partner
Colony
Ability to discriminate self colonies form nonself colonies within the
specificity
same species, resulting in fusion or rejection
First sessile zooid metamorphosed form a free swimming tadpole larva,
Oozoid
itself generated by sexual reproduction
Last stage in blastogenesis, where the new blastozooids take over the
Takeover
colony from the previous generation
Figure Legend:
Fig. 1. Mechanism of apoptosis in Caenorhabditis elegans. The basal molecular
mechanism is conserved in all metazoans.
Fig. 2. The life cycle of Botryllus schlosseri.
Fig. 3. The asexual life cycle of Botryllus schlosseri, otherwise known as the
blastogenic cycle. Each new generation of zooids is larger than the previous generation.
Fig. 4. The nucleotide and protein sequences of bel-2 B and B+ in Botryllus schlosseri.
These distinct sequences belong to the same gene family and are only separated by a few
amino acids. The underlined regions are highly conserved.
Fig. 5. The phylogenetic tree relating Botryllus schlosseri to several vertebrates and
Caenorhabditis elegans, based on the bel-2 locus. Numbers indicate gene distances.
Fig. 6. The results of the Southern and Reverse Northern Blots probed with a mixture
of the radioactively labeled bel-2 B and B++ probes. The method lacks the resolution to
separate the probes because they are so close in sequence.
Fig. 7. Results of the RNase Protection Assay.
Fig. 8. Graphical representation of the results of the RNase Protection Assay.
Fig. 1:
C
O
O
Activation of apoptotic pathway
Neighbor engulfs dying cell

O

Corpse is digested

leaving no trace
O
Fig. 2:
hatching

X

sexual reproduction







s

protochordate
metamorphosis
tadpole
attachment
C

asexual reproduction
2
Fig. 3:
Stage D

Takeover;shutdown
of parent zooid's
siphons, involution,
organ resorption
and apoptosis
Stage A
8
Onset of new cycle:
opening of siphons,
microscopic 1° buds
Stage C
1° bud enlarges;
organogenesis in 2° bud
Stage B
Heartbeat and circulation
begin in 1° bud
Fig. 4.
bel-2 B, nucleotide sequence, Botryllus schlosseri
GAATTCGAACTGTTTCGAGATGGCGTAAACTGGGGTAGGATTGTCTCCCTTIT
TGTGCTCGGTAGTATTCTTGCTCGCAAGAACGAGGATGAACATCATGGGGAG
CATATAGAAGCTATCATACAGACAGTCGGAAATCACATTGCTAGCAGAAGAC
TAAAGTGGATACAAGACAACGGAGGATGGGACTCTAGA
» bel-2 B+, nucleotide sequence, Botryllus schlosseri
GAATTCGAACTGTTTCGAGATGGTGTGAACTGGGGTAGGATTGTCTCCCTTTI
TGTGCTCGGTAGTATTCTTGCTCGCAAGAACGAGGATGAGCATCACGGTGAT
AATATAGAAGGTATCATACAGTCAGTCGGGAATTACNTTGGTAGTAGAAGGC
GAAAGTGGATACAAGACAACGGAGGATGGGACTCTAGA
Bcl-2 B, protein sequence, Botryllus schlosseri
ELFRDGVNWGRIVSLFVLGSILARKNEDEHHGEGIEAIIOTVGNHIASRRLKWIQD
NGGW
Bcl-2 B+, protein sequence, Botryllus schlosseri
ELFRDGVNWGRIVSLFVLGSILARKNEDEHHGDNIEGIIOSVGNYXGSRRRK WIQ
DNGGW
Fig. 5:
—
72
Botryllus
26
Xenopus
human
mouse
26
rat
bull
Chicken
179
C.elegans
Fig. 6:
genomic cDNA
O
E
+
+
+
MW
+ 12,216 bp
+ 1626 bp
+ 1018 bp
Fig. 7:
Probe:
RNA
stage:
S

A*Mid'D A*Mid'D A*A*A A*Mid* D
Fig.8: Expression Patterns in
Asexual Life Cycle of Botryllus
schlosseri

2


Days
—Band Strength. Bc 2 5 — Band Stength-Jubulin — — Band Strength-Bet28t