Escolar Documentos
Profissional Documentos
Cultura Documentos
Viol. Bnll.
190: 302-3
12. (June,
1996)
PIRAINO,
FERDINAND0
BOER02, BRIGITTE
AND VOLKER
SCHMID3
AESCHBACH3,
Istituto
10 August
1995; accepted
29 December
1995.
302
ONTOGENY
REVERSAL
and Methods
Rearing
About 4000 medusae of Turritopsis nutricula were obtained from hydroid colonies collected in the Gulf of Naples (Western Mediterranean)
from June to October, in
1993 and 1994. The medusae were reared in the laboratory
at 20C in groups of 100 individuals in 500-ml beakers,
and fed every other day with newly hatched brine shrimp
(Artemiu sulina) until gonad maturation and spawning.
Slight water circulation was maintained by a gentle stream
of air blowing across the surface of the water.
IN
MEDUSAE
303
Immunohistochemistry
Whole medusae were anesthetized in MgC12 (3.5% [w/
v] in seawater), and fixed in 4% paraformaldehyde
(PFA;
pH 7.2) in phosphate-buffered saline (PBS, 150 mM NaCl,
10 mM Na,HPO,; pH 7.4) for 10 min. Then the medusae
were washed twice for 5 min in PBS containing 0.05%
Triton + 0.05% NaN, (PBS/Triton)
and rinsed in two
changes of PBS. The histological and anatomical organization of the medusa of T. nutriculu was investigated
by incubating for I h at room temperature in 1:lOO dilution of either (a) rabbit polyclonal antibodies raised
against RFamide positive nerve cells (antisera kindly provided by the laboratory of Prof. C.J.P. Grimmelikhuijzen),
or against human p I-integrin; or (b) mouse monoclonal
antibodies raised against smooth muscle cells (termed sm1) or against striated muscle cells (termed 93, Schuchert
et al., 1993) of P. carnea. All showed cross-reactivity with
T. nutricula tissues. Subsequently, the medusae were
washed in PBS ( 10 min, two times), and incubated in
FITC-conjugated
goat anti-rabbit IgG (Sigma) or antimouse IgG (Sigma) diluted 1:20 with PBS, for 30-60 min
at room temperature. Finally, the medusae were washed
in PBS and stained with 4,6-diamidino-2-phenylindole
2 HCl (DAPI, 0.05 mg/ml; Serva) in PBS for 1 min,
washed with PBS, mounted in 10: 1 glycerine/PBS supplemented with 25 mg Diazabicyclol(2,2,2)-octane
(Aldrich-Chemie) (DABCO/glycerine/PBS;
Alder and Schmid,
1987), and observed with a fluorescence microscope.
Electron microscopy
At each stage of the transformation
of medusae into
polyps, sets of 10 specimens were fixed overnight in 2.5%
glutaraldehyde in cacodylate buffer (pH 7.2) at 4C postfixed for 1 h in 1% osmium tetroxide in cacodylate buffer
before dehydration in an alcohol series, and embedded
in Epon resin. Ultrathin sections were stained with 1%
uranyl acetate and lead citrate for 15 min.
Isolution
oftissues
S. PIRAINO
exumbrellar
epidermis smooth muscle .. ....... .....
endodermis
------striated niuscle +++H++w
Figure
1.
n~rtricula
ECM). The presence of occasional contaminating endodermal cells in the striated muscle isolates was tolerated
becauseof their easy identification in both the light and
the electron microscope (Schmid et al., 1982). Tissue isolates or medusa fragments (n = 20) were cultured at 22C
in filtered (0.22 pm) seawater to which streptomycin
(0.1 mg/ml), penicillin (100 U/ml), and chloramphenicol
(0.01 mg/ml) were added. The culture media were
changed daily. To induce transdifferentiation in some
isolated portions of striated muscle, a post-isolation treatment with pronase ( 1.25 mg/ml seawater, Schmid, 1992)
was also applied.
DNA replication
The pattern of DNA replication both in stable and in
transforming medusae was determined to investigate
whether differentiated cell types can resume cell cycle activity and be involved in the transformation process.
Therefore, the rate and site of DNA replication in healthy
and wounded medusaeof both T. nutricula and P. carnea
were investigated by pulse-labeling the animals or their
fragments with 5-bromo-2-deoxyuridine (BrDU; Plickert
ET
AL.
ONTOGENY
REVERSAL
IN
MEDUSAE
305
Figure
2.
Anatomy
of
~ cpldermis.
OM = outer
mcsogloea. SM = striated
canals. RIG = ring canal. T
Transfi,tmation
306
S. PIRAINO
E7 .4/L.
Full transformation
into stolons and polyps was observed when excised manubria and tentacles additionally
contained cells of exumbrellar epidermis and portions of
the canal system (Fig. l:E, H, I; Table I: E, H, I). In addition, bell fragments containing exumbrellar epidermis
and portions of the radial or circular canal fully transformed into stolons and polyps (not included in Table I).
~24~~s.
SEITLEMENT
PERISARC
PRODUCTION
PERISARC
PRODUCTION
STOLON
PRODUCTION
POLYP
FORMATION
in diferent species of
ONTOGENY
REVERSAL
IN
MEDUSAE
307
eating nuclei were detected between cut and control specimens in either species.
Before transformation
of wounded medusae into po-
ONTOGENY
REVERSAL
Table
Destahilizatlon
(fand
stolen ,formation,fkvn
isolated
medusu,fiagments
B
C
D
E
F
G
H
I
muscle
Exumbrellar
epidermis
+ endoderm
Striated muscle + endoderm
+ epidermis
Striated muscle + radial canals
Manubrium
+ exumbrellar
epidermis
Manubrium
only (middle-tip)
Tentacles
Ex. epidermis
+ tent. bulbs + tentacles
Upper half of medusa
Lower half of medusa
(interradial
sectors)
309
MEDUSAE
(A-l,
see Fig.
1)
Isolation/treatment
procedure
Isolate composition
A Striated
IN
Mechanical
Collagenase
Pronase*
Collagenase
Mechanical
Mechanical
Mechanical
Mechanical
Mechanical
Mechanical
Mechanical
Mechanical
Destabilization
+
++
+++
+
+++
+++
+++
+++
Stolon
formation
+++
+++
+++
+++
Does
deuth
occur in Turritopsis
nutricula?
Turritopsis nutricula is a unique medusa and its transformation potential is unparalleled within the vast array
of life-cycle patterns found in cnidarians. This is the first
known case of a metazoan being capable of reverting
completely to a clonal life stage after having achieved sexual maturity in a solitary stage.
Because all T. nutricula medusae regularly underwent
transformation,
we must assume that organismic death
does not occur in this species. Hydrozoans
are modular
organisms in their polyp stage, but the medusa stage is
Figure 13.
T. nutricula
medusa, portion
of bell shown. BrdU staining of DNA replicating
nuclei (incubation:
30 min). Besides manubrium
(arrow)
and bulbs (arrowheads),
labeled nuclei are widespread
throughout
the whole umbrella.
Scale bar: 500 pm.
Figure 14. DNA-replicating
cell of exumbrellar
epidermis (ex) from the margin of T. nutricuia medusa
bell. Scale bar: 50 pm.
upper part of BrdU-incubated
medusa of T. nutricula
with extruded
manubrium
Figure 15. Half-cut,
(bottom)
and outwardly
strained striated muscle sheet (sm). Striated myofibrils
stained with mAb 93. Scale
bar: 40 pm.
Figure 16. Outwardly
strained striated muscle sheet (sm) from half-cut, upper part of BrdU-incubated
medusa of T. nutriczda.
Brackets point out a DNA-replicating
nucleus from the striated muscle sheet. Scale
bar: 200 pm.
310
Fiwe
S. PIRAINO
17.
Striated
muscle
cells from
BrdU-incubated.
whole
medusa
of Trrrn~~~
nu~rrc~rlu
undegoing
transformation
into polyps
after
rearing at T = 27C. (a-b) same frame: (a) two DNA-replicating.
BrdUlabeled nuclei (arrows),
with m Ab 93stained
myofibrils
running
around
them; (h) DAPI aaining
showing
the two BrdU-labeled
cells (amws)
surrounded
by nonreplicating,
striated
muscle
cells.
Scale bar:
50 pm.
ET
AL.
The change of medusae into polyps requires differentiation of new cell types and major reorganization of tissues. New cell types could arise either through a difierentiation of uncommitted stem cells ofthe medusa or by
cellular trandifferentiation
of differentiated medusa cells.
The presence of a population of continuously proliferating cells (stem cells, interstitial or I-cells) was long
thought to play a key role in asexual reproduction, in
regenerative processes, and in the apparent lack ofsenescence observed in both colonial and solitary cnidarians
(Brien, 1953; Crowell, 1953; Comfort, 1956; Brock and
Strehler, 1963; Toth, 1969; Hughes, 1987; Buss, 1987).
These cells are characterized by their histological features,
but their state of commitment cannot be determined by
isolation and cloning experiments. The role of interstitial
cells is best understood in Hy&i~ spp. in which these cells
are able to differentiate into nematocytes, nerve cells, and
germ cells (Tardent, 1978; Werner, 1984). It seems that,
as a gross morphological category, interstitial cells include
partially difTerentiated sxem cells (Bode, 1992), but the
question of whether their repertoire includes secretory cells
is still unresolved (Smid, 1984) In any case, their influence
on morphogenetic processes seems to be rather limited.
interstitial-cell-free Hydra polyps are able to regenerate
fully and reproduce asexually (Brie& 1955; Diehl and
Bouillon, 1966; Marcum and Campbell, 1978).
Recruitment of differentiated cells for transdiflerentiation and regeneration processes was demonstrated in the
medusa Podmuryrte cornea (for review see Schmid, 1992).
In that case, Isolated and activated endoderm or striated
muscle cells can resume DNA replication, transdifferentiatte to several new cell types, and regenerate in vitro medusa organs. However. they are unable to regenerate polyp
organs such as stolons or perisarc-secreting tissues
(Schmid, 1992). Transdifferentiation in hydroidomedusae
might be a common phenomenon in budding and regeneration, especially where no or few l-cells are located
(Brien, 194 I ). Some hydroidomedusae belonging to quite
different families (e.g., Zunclea pro&era, Teissiem ausrrafis, Bougairlvilliu vervoorti, Pruboscidactyl& ornate,
Cytueis spp., Eircne &ccana, Eucheilota puradoxica,
Clytia rn~~urudyi)have the ability to bud (but not to meta-
assumption that both epidermis and endoderm are basitally totipotent (Betill, 1950). Indeed, manubrial budding
ONTOGENY
REVERSAL
Trunsfbrmution
of T. nutricula
medusue
311
IN MEDUSAE
Cited
312
S. PIRAINO
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