Cell Tissue Res (2001) 304:401–408
DOI 10.1007/s004410100375
REGULAR ARTICLE
Robert Gschwentner · Peter Ladurner
Katharina Nimeth · Reinhard Rieger
Stem cells in a basal bilaterian
S-phase and mitotic cells in Convolutriloba longifissura (Acoela, Platyhelminthes)
Received: 20 September 2000 / Accepted: 9 February 2001 / Published online: 19 April 2001
© Springer-Verlag 2001
Abstract In Platyhelminthes, totipotent stem cells (neoblasts) are supposed to be the only dividing cells. They
are responsible for the renewal of all cell types during
development, growth, and regeneration, a unique situation in the animal kingdom. In order to further characterize these cells, we have applied two immunocytochemical markers to detect neoblasts in different stages of the
cell cycle in the acoel flatworm Convolutriloba longifissura: (1) the thymidine analog 5′-bromo-2′-deoxyuridine
(BrdU) to identify cells in S-phase, and (2) an antibody
to phosphorylated histone H3 to locate mitosis. BrdU
pulse-chase experiments were carried out to follow differentiation of neoblasts. We demonstrate the differentation into four labeled, differentiated cell types. S-phase
cells and mitotic cells showed a homogenous distribution
pattern throughout the body of C. longifissura. Two different types of S-phase cells could be distinguished immunocytochemically by their pattern of incorporated
BrdU in the nuclei. Transmission electron microscopy
was used to study ultrastructural characters of neoblasts
and revealed two different stages in maturation of neoblasts, each with a characteristic organization of heterochromatin. The stem-cell pool of C. longifissura is an
important prerequisite for the extraordinary mode of
asexual reproduction and the high capacity of regeneration. A comparison of the stem-cell pool in Acoela and
higher platyhelminth species can provide evidence for
the phylogenetic relationships of these taxa.
Keywords Bromodeoxyuridine (BrdU) · Histone H3 ·
Stem cell · Neoblast · Convolutriloba longifissura
(Acoela, Platyhelminthes)
This work was supported by FWF grant P13060-Bio
R. Gschwentner (✉) · P. Ladurner · K. Nimeth · R. Rieger
Institute of Zoology and Limnology, University of Innsbruck,
Technikerstrasse 25, 6020 Innsbruck, Austria
e-mail: [email protected]
Tel.: +43-512-5076173, Fax: +43-512-5072930
Introduction
Stem cells are undifferentiated cells with capacity for
permanent renewal and differentiation into other cell
types (for review, see Morrison et al. 1997). The mechanisms of differentiation of stem cells and their integration at appropriate locations are still unclear. The understanding of these processes remains one of the key problems in developmental biology (Martin and Archer
1997). Proliferation and differentiation of stem cells
have been investigated in many metazoan taxa, from
sponges to vertebrates (for review, see Potten 1997). In
the coelenterate Hydra, the interstitial cells regenerate
nematocysts, nerve cells, and germ cells, but not epidermal and gastrodermal cells (Bode 1996). The multipotent
vertebrate neural crest cells differentiate to several tissue
types (Bronner-Fraser and Fraser 1988), and the mammalian hematopoietic stem cells are able to differentiate
into nerve cells, muscle cells, liver cells, and all types
of blood cells (Morrison et al. 1995; Travis 1999;
Woodbury et al. 2000). The mammalian central nervous
stem cell system (McKay 1997) and the human embryonic stem cell system (Keller and Snodgrass 1999) comprise stem cells with increasing importance in biology
and medicine.
Stem cells in Platyhelminthes are extraordinary in the
animal kingdom, because these so-called neoblasts provide the only source for proliferation during growth and
regeneration (Baguñà 1981; Ehlers 1985; Palmberg 1990;
Baguñà et al. 1994; Hori 1997; Rieger et al. 1999). Neoblasts are characterized by a prominent nucleus surrounded by a small rim of basophilic cytoplasm, by free ribosomes, few mitochondria, and a relatively small amount
or lack of endoplasmic reticulum. Palmberg (1990) has
distinguished two populations of neoblasts in Microstomum lineare by their ultrastructure, specifically the presence or absence of centrioles. Rieger et al. (1999) have
described three successive stages of differentiating neoblasts in Macrostomum sp. The heterochromatin of the
1st stage is scattered over the nucleus in isolated small
clumps, while stage-2 neoblasts show connections be-
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tween them. Both stages are characterized by cytoplasm
lacking organelles except free ribosoms and scattered mitochondria. Stage-3 neoblasts show prominent heterochromatin strands in their nuclei, with connections to the
nuclear lamina as well as to rough endoplasmic reticulum
and Golgi complexes in the cytoplasm.
Two methods to analyze the distribution, proliferation, and differentiation of stem cells, and their localization after differentiation, have been used: tritiated thymidine, [3H]T, and, more recently, bromodeoxyuridine
(BrdU) to label cells in the S-phase of the cell cycle.
Thymidine labeling in free-living platyhelminths has
been carried out in two microturbellarians (Palmberg
1986, 1990), in several polyclads (Drobysheva 1988),
and in the acoels Convoluta convoluta and Oxyposthia
praedator (Drobysheva 1986); whereas BrdU, as well as
immunolabeling of phosphorylated histone H3, were applied in Macrostomum sp. (Ladurner et al. 2000) and
Convoluta pulchra (personal observations). Recently,
Newmark and Sànchez Alvarado (2000) have shown
BrdU labeling of regenerative stem cells in planarians.
In this study, we identified neoblasts in S-phase, by
labeling with BrdU, and demonstrate mitosis, with an antibody to phosphorylated histone H3, in whole-mounts
and macerated cells of the acoel flatworm Convolutriloba longifissura (Bartolomaeus and Balzer 1997) which
exhibits a unique and highly productive mode of asexual
reproduction (Åkesson et al. 2001). We combine light
and electron microscopy with immunocytochemical
methods to examine distribution, differentiation, and
fates of S-phase neoblasts. We report the discovery of
two different types of neoblasts and we quantify the
number of cells and symbiotic algae in the animals using
a maceration technique. BrdU pulse-chase experiments
demonstrate differentiation of neoblasts into various cell
types. Referring to the basal phylogenetic position of
acoels within the Bilateria, we compared our results to
the stem-cell systems of higher bilaterian animals. Detailed analysis of the acoel neoblast system may give an
overview of the basic state of stem cells in bilaterians
and should allow a better insight into fundamental mechanisms of cell differentiation during development,
growth, and regeneration and the evolutionary significance of stem cell systems in lower metazoans.
Labeling of S-phase cells and mitosis
S-phase cells were labeled by incubation in 5 mM 5′-bromo-2′-deoxyuridine (BrdU; Sigma 5002) in artificial seawater (ASW) for
30 min (pulse). Only cells in S-phase incorporate BrdU in their
nuclei. For pulse-chase experiments, animals were maintained in
ASW for 3–9 days after a 30- or 50-min BrdU pulse. For continuous labeling, worms were kept in 100 µM BrdU in ASW for
3 days. After washing with ASW, and anesthesia in 7.14% MgCl2,
specimens were fixed in 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS, pH 7.4) for 1 h. Worms were washed
with PBS-T [0.2% Triton X-100 (v/v) in 0.1 M PBS, 1 h] and
treated with pronase E (0.5 mg/ml PBS, 10 min, 37°C), exposed to
0.1 N HCl (10 min, on ice) and 2 N HCl (1 h, 37°C). After washing with PBS, unspecific staining was blocked by incubation in
BSA-T (bovine serum albumin plus 0.1% Triton X-100; 15 min).
For double labeling, two primary antibodies were applied simultaneously overnight at 4°C: (1) a monoclonal mouse-anti-BrdU antibody (final dilution 1:1000; Sigma) to localize BrdU (see Gratzner
1982; Ladurner et al. 2000); and (2) a polyclonal rabbit-anti-phosphorylated histone H3 (final dilution 1:300; Upstate Biotechnology) to locate mitotic cells (Hendzel et al. 1997; Ladurner et al.
2000). After washing with PBS, two secondary antibodies were
applied simultaneously for 1 h at room temperature: (1) a FITCconjugated goat-anti-mouse antibody (dilution 1:100; DAKO);
and (2) a TRITC-conjugated swine-anti-rabbit antibody (dilution
1:100; DAKO). After washing with PBS, specimens were mounted in Vectashield (Vector Laboratories).
To elucidate the number, the distribution and the fate of S-phase
cells in C. longifissura BrdU labeling was applied. Additionally,
we labeled phosphorylated histone H3 to demonstrate mitotic
stages. Number and location of S-phase and mitotic cells in Convolutriloba longifissura were mainly investigated in animals less
than 2 mm long, because they showed reduced autofluorescence.
S-phase and mitotic cells of the algae were also labeled. We distinguished the nuclei of algae from the nuclei of C. longifissura by
their different size – the nuclei of cells of C. longifissura measured about 7–9 µm in diameter, whereas the nuclei of the algal
symbionts had a size of 2–4 µm.
Cell numbers and identification of cell types by maceration
For determining the total number of cells and symbiotic algae with
a hemocytometer, BrdU-treated specimens were incubated in
200 µl calcium-magnesium-free medium (CMF) containing 0.5%
trypsin, 6% glycerin, 19% sucrose, and 10% ASW at room temperature. After 30 min, animals were macerated into single cells.
For identification of different cell types, this cell suspension was
centrifuged on poly-L-lysine-coated slides (1,300 rpm, 5 min). Immunocytochemistry was performed as described in the previous
section, with incubation times reduced by half. Nuclei were counterstained by a dimeric cyanin nucleic acid stain YOYO (Y-3601,
Yoyo-1 iodide; Molecular Probes). Cells and animals were examined with a Reichert Polyvar epifluorescence microscope, including Nomarski and phase contrast. Confocal images were made on
a Zeiss LSM 510 and processed with Adobe Photoshop 5.0.
Materials and methods
Animals
Electron microscopy
Specimens of Convolutriloba longifissura (Bartolomaeus and
Balzer 1997) were collected from a seawater aquarium in Maishofen (courtesy F. Matiasch), Salzburg, Austria. The exact natural
habitat of these specimens is not known. The aquarium was originally set up with material from the Pacific. In our laboratory, animals were cultured in a seawater aquarium (salinity 32‰) with
stones and Caulerpa sp., at 26±1°C, using daylight and bluelight
lamps (12 h light per day). The acoels were fed copepods or Artemia larvae once a week. 65 worms were labeled at a size between
approximately 0.8 and 5 mm. The lengths of animals were measured before fixation.
For transmission electron microscopy, worms were relaxed in
MgCl2 (7.14% w/v) isotonic to seawater, fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) containing 10% sucrose
for 1 h, and postfixed with 1% osmium tetroxide in 0.1 M cacodylate buffer for 1 h. The specimens were dehydrated in a standard
acetone series and embedded in Spurr’s low-viscosity resin (Spurr
1969). Ultrathin sections (75 nm) were cut on a Reichert Ultracut
E microtome, double-stained with uranyl acetate and lead citrate,
and observed with a Zeiss 902 transmission electron microscope
(TEM).
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Results
In this study we use the term “type” when S-phase neoblasts are identified by BrdU-labeling and the term
“stage” when neoblasts are distinguished by their ultrastructural characters as has been done by Rieger et al.
(1999). For details see the Discussion section in this
study and Rieger et al. (1999).
Distribution of S-phase and mitotic cells
The only cells that incorporated BrdU in macerated preparations showed the characteristic morphology of neoblasts with a small rim of cytoplasm around the nucleus.
We therefore assume that labeled cells in whole-mounts
were also neoblasts. C. longifissura showed a homogenous distribution of S-phase cells over the whole body
after 30 min BrdU incorporation (Fig. 1A). We never
found labeled differentiated cells after 30-min BrdU exposure, nor did we observe animals (n~50) with localized concentrations of S-phase cells. Two populations
of S-phase cells could be distinguished by their different
pattern of BrdU incorporation in the nuclei. Type 1
S-phase cells were characterized by nuclei with many
evenly distributed, small BrdU-labeled spots, the only
BrdU-free zones being nucleoli (Fig. 2). In the nuclei of
type 2 S-phase cells, several larger spots (about 0.6 µm
in size) of BrdU could be seen (Fig. 2). These cells
lacked explicitly recognizable nucleoli. Both types of
S-phase cells occurred in about the same number in the
body; no aggregations of one type in special regions
could be observed.
Ultrastructural examinations in C. longifissura
showed typical neoblasts with high nucleoplasmic ratio,
free ribosomes, and few mitochondria in the cytoplasm.
(Figs. 3, 4). They were located in the highly vacuolated
peripheral parenchyma (see Gschwentner et al. 1999,
Fig. 4, for overview). We could distinguish two stages of
neoblasts ultrastructurally by differences in the pattern of
heterochromatin in the nuclei. These stages correspond
to neoblasts stage 1 and stage 3 according to Rieger et al.
(1999). In our TEM material, we never observed neoblasts with the heterochromatin pattern of a neoblast
stage 2 according to Rieger et al. (1999). Stage 1 showed
a uniform distribution of spots of heterochromatin (each
spot was approximately 0.2 µm in diameter, Fig. 3) with
only a few clumps of heterochromatin attached to the nuclear envelope. A nuclear lamina, if present, was thin.
The narrow rim of cytoplasm contained free ribosomes
and a few mitochondria. Neither Golgi complex nor
rough endoplasmatic reticulum were visible. Stage 3
possessed larger blocks of heterochromatin in the nucleus (about 0.5 µm in diameter, Fig. 4), many of them
along the nuclear envelope. This stage showed rough endoplasmic reticulum in the cytoplasm, indicating the entrance into cytoplasmic differentiation. Stage 3 neoblasts
were often situated beneath the sunken nuclei of epithelial cells.
Fig. 1A–C Epifluorescence and interference-contrast micrographs
of juvenile Convolutriloba longifissura; dorsal view. Simultaneous
localization of 5′-bromo-2′-deoxyuridine (BrdU)-labeled S-phase
cells (green dots in A) and of mitotic cells by phosphorylated histone H3 (red dots in B). Autofluorescent background of symbiotic
algae appears red in both A and B. C Nomarski interference-contrast micrograph of the same animal
Mitotic cells also occurred in all areas of the body of
C. longifissura (Fig. 1B). Labeled phosphorylated histone H3 of one metaphase and one anaphase is shown in
Fig. 5.
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Fig. 2 Laser scanning micrograph of BrdU-labeled nuclei of Sphase cells of C. longifissura. Note two different patterns of BrdU
incorporation (n nucleolus)
Fig. 4 Electron micrograph of a dorsal basiepithelial area of C.
longifissura. Note stage-3 neoblast (nb3) with large heterochromatic spots in the nucleus (hc) and the lack of endoplasmic reticulum. The sunken nucleus of an epithelial cell (epc) shows a similar
heterochromatic pattern (but more condensed) and endoplasmic
reticulum and numerous Golgi bodies in the cytoplasm (n nucleolus)
Fig. 5A, B Laser scanning micrograph of phosphorylated histone
H3-labeled nuclei of C. longifissura. Chromosomes in metaphase
(A) and anaphase (B)
Cell numbers: total, S-phase, and mitosis
Fig. 3 Electron micrograph of a stage 1 neoblast (nb1) in the mesenchyme of C. longifissura. Note checkerboard pattern of heterochromatin (hc) in the nucleus of the neoblast and thin rim of cytoplasm
Adult C. longifissura (size 4.0–4.5 mm) consisted of
120,000 to 130,000 cells, while small individuals (less
than1500 µm) possessed about 10,000 cells. More than
one-third of these cells were algal symbionts (mean
percentage 42.75%, n=20), although the individual values varied (see Fig. 6). We never observed animals with
more symbionts than body cells. Macerated preparations revealed that 15.18±4.82% (n=10) of total body
cells were in S-phase and 0.58±0.21% (n=10) in mitosis.
405
Fig. 6A–C Plot of cell number versus body length (micrometers)
of C. longifissura. A Number of body cells (black circles) and algal symbionts (white circles) relative to body length. B Number of
S-phase cells (black circles) in relation to body length. C Number
of mitotic features (black circles) in relation to body length
Cell differentiation
We used continuous BrdU-labeling to increase the number of labeled S-phase cells. Animals disintegrated when
labeling in 100 µM BrdU lasted longer than 5 days. We
therefore exposed animals to BrdU treatment for 3 days
at most. Animals chased for 4 days after a 3-day continous pulse showed BrdU-labeled parenchymal and epidermal cells (Fig. 7A, C). Animals labeled for 50 min in
BrdU and chased for 3 days and 9 days before maceration showed labeled sagittocytes (sagittocyst-producing
Fig. 7A–H Laser scanning micrographs of differentiated cells
after BrdU-labeled pulse-chase experiments. B, D, F, H BrdU incorporated in the nucleus (red) by a TRITC-conjugated secondary antibody; A, C, E, G nuclei counterstained with a dimeric
cyanin nucleic acid stain, YOYO (Y-3601, Yoyo-1 iodide;
green). A, B Parenchymal cell after 3 days of continuous BrdU
labeling and 4 days’ chase. C, D Epidermal cell after 3 days of
continuous BrdU labeling and 4 days’ chase. E, F Sagittocyte after 50-min BrdU pulse and 3 days’ chase. G, H Muscle cell (bottom) after 50-min BrdU pulse and 9 days’ chase
gland cells; Fig. 7E) after 3 days and labeled muscle
cells after 9 days (Fig. 7G). DNA-labeling revealed the
extent of the whole nucleus beyond the BrdU-labeled
spots (Fig. 7A, C, E, G).
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Discussion
Distribution of S-phase and mitotic cells
Investigations of S-phase and mitotic cells in Platyhelminthes are very rare. Labeling of S-phase cells with
[3H]T has been performed on the acoel species Convoluta convoluta and Oxyposthia praedator (Drobysheva
1986), on the macrostomid Microstumum lineare and on
the catenulid Stenostomum leucops (Palmberg 1990).
These investigations were made on semi-thin sections
without reconstruction, and no complete picture of the
distribution pattern of labeled S-phase cells was provided. Recently, two reports have shown BrdU incorporation and phosphorylated histone H3 in a planarian
(Newmark and Sànchez Alvarado 2000) and in a macrostomid species (Ladurner et al. 2000). In these species,
S-phase and mitotic cells were missing in the pharynx
and in the area in front of the photoreceptors. In Macrostomum sp., S-phase neoblasts and mitoses were concentrated in two bands along the lateral sides of the animals.
Ladurner et al. (2000) have suggested a correlation of
this distribution pattern and the two lateral nerve cords
of the animal. Baguñà et al. (1989) also assume a relationship of the nervous system (especially substance
P and K) with the proliferation of neoblasts in planarians. The homogenous distribution of S-phase cells in
C. longifissura indicates no direct correlation to the
nerve cords. In addition, Ladurner et al. (2000) have observed migration of S-phase cells from the lateral bands
into the S-phase-free zones in front of the brain and toward the median axis of Macrostomum sp. Newmark and
Sànchez Alvarado (2000) described a migration of neoblasts throughout the anterior region of the flatworm
Schmidtea mediterranea. The lack of mitotic features anterior to the photoreceptors has been described in several
free-living Platyhelminthes, e.g., by Baguñà (1976) in
Dugesia mediterranea, by Drobysheva (1986) in Convoluta convoluta, by Palmberg (1990) in Macrostomum
lineare, and we found the same true of a member of the
Otoplanidae (R. Gschwentner, P. Ladurner, K. Nimeth,
R. Rieger, unpublished observations). These results are
compatible with recent observations of mitotic features
labeled with histone H3 in Macrostomum sp. (Ladurner
et al. 2000) and S. mediterranea (Newmark and Sànchez
Alvarado 2000).
One possible explanation for the occurrence of mitotic and S-phase cells in front of the brain of C. longifissura may be based in the mode of asexual reproduction.
This species reproduces asexually by two-step fission
(Gschwentner et al. 1999; Åkesson et al. 2001). First, the
animal undergoes a transverse fission at a position about
two-thirds of the length of the animal. Second, the separated caudal part (shaped like a butterfly) divides longitudinally into two daughter individuals. During longitudinal fission an increased number of S-phase cells are
found in the regenerative blastema in the anterior-most
region of the butterfly stage (R. Gschwentner, P. Ladurner,
K. Nimeth, R. Rieger, unpublished work). Another pos-
sible explanation for the presence of S-phase and mitotic
cells anterior to the brain might involve the different organization of the brain in Acoela and rhabditic species.
Recent studies have revealed a clear barrier for proliferating neoblasts at the level of the brain and photoreceptors in Macrostomum sp. (Ladurner et al. 2000) and three
planarian species (Newmark and Sànchez Alvarado
2000). The concentration of neurons in the area of the
brain in these animals is much more compact than in
acoels, which show a unique commissural brain and a
lack of pigment cup ocelli (Rieger et al. 1991; Raikova
et al. 1998). However, the nature of the inhibitory signal
for cell proliferation in the rostrum is not known and
therefore the role of the brain remains speculative.
The total number of neoblasts in C. longifissura could
not be determined with the methods used, because BrdU
is only incorporated in neoblasts during the S-phase of
the cell cycle. One possibility to extract all neoblasts
would be to establish a density-gradient centrifugation as
shown for planarians by Schürmann et al. (1998). In planarians about 20–35% of the total cell number are neoblasts (Hay and Coward 1975; Baguñà and Romero
1981; Baguñà et al. 1989). C. longifissura may possess a
higher percentage of neoblasts in that 15.8% of the total
cell number are S-phase cells compared with only 2%
S-phase cells after 30-min BrdU incorporation in Macrostomum sp. (Ladurner et al. 2000). The mitotic activity
is also high in C. longifissura (0.58% of total cells are in
mitosis) in comparison with 0.37% in C. convoluta
(Drobysheva 1986) and 0.05% in Macrostomum sp.
(Ladurner et al. 2000). In planaria, Baguñà (1974, 1976)
has found a mitotic percentage of 0.2% in 4-mm-sized
animals and of 0.1% in 10-mm animals. Such a high percentage of S-phase and mitotic cells in C. longifissura
may be typical for animals with asexual reproduction.
Types and stages of neoblasts
In the BrdU material of C. longifissura, we were able to
distinguish two different types of S-phase cells. Type 1
showed more homogenous distribution of BrdU in their
nuclei, probably representing sites of replication active
during the 30-min-pulse period (Fig. 2). Type-2 S-phase
neoblasts possessed several larger BrdU-labeled spots in
the nucleus. This different pattern of two types might be
due to replication of different parts of the chromatin during early and late S-phase. Mazzotti et al. (1998) have
shown in vertebrate cells that BrdU is detectable in
interchromatin regions of early S-phase, whereas later
S-phases concentrate BrdU at the border between dispersed and condensed chromatin. This pattern of BrdU
incorporation is compatible with our observations of
C. longifissura.
Ultrastructure has revealed two different stages of
neoblasts. One stage corresponds to stage-1 neoblasts
with undifferentiated cytoplasm and speckled appearance
of heterochromatin in the nucleus, as described for neoblasts in Macrostomum sp. by Rieger et al. (1999). The
407
second stage is similar to a neoblast stage 3, with rough
endoplasmic reticulum and Golgi complex in the cytoplasm and irregular clumps of heterochromatin in the nucleus (Rieger et al. 1999). In the TEM sections observed
in this study, we never found an additional neoblast stage
which corresponds to a neoblast stage 2, with connected
heterochromatin strands and clumps as described for
Macrostomum sp. (Rieger et al. 1999). Palmberg (1990)
has ultrastructurally distinguished two populations of
neoblasts in Microstomum lineare by the occurrence or
lack of basal bodies in their cytoplasm. In our material,
no basal bodies could be identified.
We speculate that the two types of S-phase cells identified with BrdU-labeling may correspond with the two
different heterochromatin patterns in our TEM data. One
possible interpretation is that type 1 S-phase neoblasts as
seen with immunocytochemistry are the neoblasts identified as stage 1 by TEM (according to Rieger et al. 1999),
and that type-2 S-phase neoblasts correspond with stage3 TEM-neoblasts (according to Rieger et al. 1999). Other
interpretations are less likely, but available data do not
allow an exact allocation of BrdU-labeled neoblasts
demonstrated by immunocytochemistry (called “types”
in this study) with neoblasts characterized by TEM
(called “stages” in this study, according to Rieger et al.
1999). Further investigations using immunogold labeling
will clarify the correlation between types and stages of
neoblasts.
In vertebrates and other higher bilaterians, evidence is
rapidly accumulating that light- and electron-microscopic structures can be correlated with different stages in
the expression and regulation of genes (Spector 1996;
Breschi et al. 1998; Parfenov et al. 1998). Although we
have not demonstrated relationships to gene functions,
we think that this first report on correlations of nuclear
morphology of stem cells in the lower Metazoa at the
light- and electron-microscopic level might be significant for future molecular studies.
Acknowledgements We thank the Matiasch family (Saalfelden,
Austria) for specimens of C. longifissura; Willi Salvenmoser, Karl
Schatz, and Konrad Eller for photographic and technical assistance; and Gunde Rieger for suggestions. LSM 510 was used by
courtesy of Professor Pelster, Institute of Zoology and Limnology,
University of Innsbruck.
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