The eutardigrade Thulinia stephaniae has an indeterminate development and the potential to regulate early blastomere ablations
Andreas Hejnol and
Ralf Schnabel*
Technische Universität Braunschweig, Institut für Genetik,
Spielmannstrasse 7, D-38106 Braunschweig, Germany
*
Author for correspondence (e-mail:
r.schnabel{at}tu-bs.de)
Accepted 23 December 2004
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SUMMARY
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We present a detailed analysis of the cell lineage of the tardigrade
Thulinia stephaniae with a 4D-microscopy system (3D time-lapse
recording). The recording, of the entire development from embryogenesis until
hatching, allowed us to analyze the fate of single descendants from early
blastomeres up to germ layer formation and tissue development. The embryo
undergoes an irregular indeterminate cleavage pattern without early fate
restriction. During gastrulation, mesodermal and endodermal precursors, and a
pair of primordial germ cells migrate through a blastopore at the prospective
position of the mouth. Our results are not consistent with earlier
descriptions of mesoderm formation by enterocoely in tardigrades. The mesoderm
in Thulinia stephaniae originates from a variable number of
blastomeres, which form mesodermal bands that later produce the serial
somites. The nervous system is formed by neural progenitor cells, which
delaminate from the neurogenic ectoderm. Early embryogenesis of Thulinia
stephaniae is highly regulative, even after laser ablations of
blastomeres at the two- and four-cell stages `normal' juveniles are formed.
This has never been observed before for a protostome. Germ cell specification
occurs late during development between the sixth and seventh cell generation.
Comparing the development of other protostomes with that of the Tardigrada,
which occupy a basal position within the Arthropoda, suggests that an
indeterminate cleavage and regulatory development is not only part of the
ground pattern of the Arthropoda, but probably of the entire Ecdysozoa.
Key words: Tardigrade, Ablation experiments, Indeterminate, Cell lineage, 4D-microscopy, Arthropoda, Ecdysozoa, Mesoderm
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Introduction
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Most of what is known about tardigrade development and has been adopted by
zoology textbooks originating from the early studies of von Erlanger
(von Erlanger, 1895
), von
Wenck (von Wenck, 1914
) and
Marcus (Marcus, 1928
;
Marcus, 1929
). However, a
recent study by Eibye-Jacobsen
(Eibye-Jacobsen, 1997
) on
tardigrade development was unable to confirm some of the conclusions of these
authors. For example Marcus and von Erlanger reported that the mesoderm is
formed by enterocoely from the gut, yet enterocoelic formation of mesoderm is
usually found only in deuterostome animals. The interpretations of the early
cleavage pattern of tardigrades are also controversial. All authors agree that
tardigrades show total cleavage; however, Marcus
(Marcus, 1929
) interprets it
as indeterminate, whereas Eibye-Jacobsen
(Eibye-Jacobsen, 1997
)
suggests that the early cleavage pattern is consistent with a modified spiral
pattern. Thus, the data matrix of the current zoology book by Brusca and
Brusca (Brusca and Brusca,
2003
) notes tardigrade development as being `fundamentally
spiral'. This scarcity of information, as well as new ideas about the metazoan
phylogeny, have brought the tardigrade development back into focus. According
to molecular, morphological and palaeontological data
(Dewel and Dewel, 1997
;
Garey, 2001
;
Garey et al., 1996
;
Garey et al., 1999
;
Giribet et al., 1996
;
Giribet et al., 2000
;
Maas and Waloszek, 2001
;
Mallatt et al., 2004
;
Nielsen, 2001
;
Regier and Shultz, 2001
;
Schmidt-Rhaesa, 2001
;
Weygoldt, 1986
), tardigrades
are associated with the Euarthropoda, thus forming along with the
onychophorans the taxon Arthropoda. But the sister group of the arthropod
lineage is now questioned. The Ecdysozoa hypothesis, which is based mainly on
molecular data (e.g. Aguinaldo et al.,
1997
; de Rosa et al.,
1999
; Garey, 2001
;
Giribet et al., 2000
;
Mallatt et al., 2004
), favours
now the Cycloneuralia or its members (Kinorhyncha, Loricifera, Priapulida,
Nematomorpha and Nematoda) and not, according to the Articulata hypothesis,
the Annelida as the sister group. However, the Ecdyosozoa hypothesis is still
debated (Giribet, 2003
;
Nielsen, 2003
;
Schmidt-Rhaesa et al., 1998
;
Scholtz, 2002
;
Scholtz, 2003
) and new data
are needed to resolve the problem. A comparative approach is needed to
determine the ancestral mode of development in the Arthropoda, thereby
preferentially serving as evidence to support one of the two hypotheses. As
basal members of the arthropods, tardigrades are one of the key groups to
investigate when reconstructing the ancestral mode. Therefore, we examined the
early cleavage pattern and cell lineage of the eutardigrade Thulinia
stephaniae using a 3D time-lapse microscopy system (4D microscopy)
(Schnabel et al., 1997
), which
allows us to follow cells in the living embryo. This new technology has been
successfully used for studies of nematode embryos
(Dolinski et al., 1998
;
Houthoofd et al., 2003
;
Schnabel et al., 1997
), the
analysis of brain development in Drosophila
(Urbach et al., 2003
), and
description of segmentation in an isopod crustacean (A. Hejnol, PhD thesis,
Humboldt University of Berlin, 2002)
(Dohle et al., 2004
). To
further investigate the type of determination and the potential for regulation
of the tardigrade embryo, we combined 4D-microscopy with laser cell
ablations.
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Materials and methods
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Culture
Thulinia stephaniae is a freshwater tardigrade that can be
maintained in cultures at 15°C to 25°C on an algae diet in the
laboratory. Two to 12 fertilized eggs are laid into the exuvia during
moulting. Cultures were obtained from Connecticut Valley Biological Supply
(MA, USA). Thulinia stephaniae is present in the Gene Database, with
partial sequences for 18S rRNA, RNA polymerase II subunit and elongation
factor 1
, and has been used for molecular phylogeny
(Garey, 2001
;
Garey et al., 1999
).
4D-microscopy
For the 4D recordings, the early eggs were removed from the exuvia with
scissor needles and embryos were mounted on an agar pad under the microscope
following a slightly modified protocol developed for C. elegans
(Sulston and Horvitz, 1977
). A
60x24 mm cover slip is used and the water is only around the agar pad to
supply the embryo with sufficient oxygen. The fundamentals of 4D microscopy
are described by Schnabel et al. (Schnabel
et al., 1997
). Commercial parts are now available for the
microscope and high-resolution digital images can be stored. A Zeiss Axioplan
Imaging 2 microscope with an internal focus drive was used to move the
temperature-controlled stage to record the z-series (45 focal levels,
increment 1 µm). Pictures are captured with a Hamamatsu Newvicon camera,
digitized with an Inspecta 3 frame grabber (Mikroton, Germany) and finally
compressed tenfold with a wavelet function (Lurawave, Germany). Digital
cameras like the PCO sensicam can also be used. The microscope is controlled
with a PC using software (AK Schulz and RS) programmed in C++. Embryos were
recorded at 24°C until the body plan was established (
48 hours of
development). Embryos were then removed from the microscope slide and placed
in culture to increase survival and hatching rates. Records were analyzed as
described by Schnabel et al. (Schnabel et
al., 1997
), using SIMI°BioCell software (SIMI, Germany). This
software manages the large quantity of digital image data generated and helps
to document cell positions, migrations and mitoses during computational
developmental analysis. The data are illustrated as a cell genealogy tree and
the positions of the nuclei can be viewed as 3D-representations using coloured
spheres (e.g. Fig. 2). Only
embryos which developed normally and gave rise to hatched, normal instars were
analyzed. Our analysis of normal development is based on four embryos,
henceforth referred to as embryos 1-4.

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Fig. 2. Cleavage up to the blastula stage in Thulinia stephaniae. The
columns from left to right show embryos 1, 2 and 3 respectively. (A-C) Cell
lineages from the two-cell to the 122-cell stage as viewed in
SIMI°BioCell. Red dots indicate the positions where cells were marked,
branches indicate mitoses. Cells divide non-synchronously and cleavage
patterns differ among embryos. In the fifth generation, some cells retard
their cell cycle (arrows), marking the onset of differentiation. (D-F)
Four-cell and post-gastrula stages. An asterisk indicates the site of polar
body extrusion (position of the spindle) upon first cleavage of the embryo.
Small arrows indicate the polar body; pha marks the pharynx anlage. (G,H)
Views are from the upper levels of the embryos at the 32-cell stage. Sister
blastomeres are connected with lines. The descendants of the eight-cell stage
embryo were coloured in Photoshop in order to visualize the cell clones. To
colour code the cells in G-L, we determined the orientation of the axis at the
gastrulation stage and then stained the cells and 3D-representations of nuclei
(J-L) with SIMI°BioCell according to the following rules. The most
anterior cell of the four-cell stage is red and its sister is pink; the most
posterior cell is blue and its sister is green. At the eight-cell stage the
ventral descendants were stained a darker tone than their dorsal sister cells.
All 3D representations were then rotated in the same orientation (left,
anterior; top, ventral surface) and `run' to the 122-cell stage as shown in
J,K. The division angles and blastomere arrangements differ in all embryos.
Scale bar: 20 µm in D-F.
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Laser ablations
Blastomeres were ablated as described by Hutter and Schnabel
(Hutter and Schnabel, 1994
).
For cell ablations, eggs were mounted on an agar pad while still in the exuvia
to allow the identification of manipulated eggs after culture. The intensity
of the laser beam and the duration of treatment were optimized to ensure
complete destruction of the ablated blastomere without causing lethal damage
to the embryo. 4D recordings of embryos were stopped after degeneration of all
descendants of the ablated cell to cytoplasts had occurred and the exuvia
containing the embryos was incubated at 15°C until the embryos
hatched.
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Results
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Early development executes an indeterminate cleavage pattern
Thulinia stephaniae lays fertilized eggs into its empty exuvia
during moulting. The oval shaped eggs, which are about 50 µm long and 43
µm in diameter are completely filled by the embryo. After the eggs are
deposited, the nucleus resides at the pole of the egg at which the polar body
is extruded (Fig. 1A). The
development is summarized in Fig.
1 (embryo 1). Blastomeres cleave totally and equally, and
are indistinguishable from one another up to the onset of gastrulation. In the
first cleavage the spindle is set up perpendicularly to the long axis of the
egg. During the formation of the cleavage furrow, the embryo and polar body
undergo a coordinated rotation of
90°
(Fig. 1B, see Movie 1 in the
supplementary material). In the subsequent division to the four-cell stage,
both spindles are initially oriented perpendicularly to the long axis of the
egg but later rotate by 45°, allowing the blastomeres to cleave parallel
to each other (Fig. 1C). After
the next cell division, the relative position of the eight blastomeres varied
from embryo to embryo (Fig.
1D). During the formation of the 16-cell embryo, the spindle
orientation again shows variation but all mitoses occur tangential to the
surface of the embryo. This cleavage pattern results in an embryo in which all
blastomeres have contact with the eggshell
(Fig. 1E). In some 32-cell
stage embryos, we observed cell divisions that were oriented perpendicular to
the surface of the embryo, which positions cells into the interior of the
embryo. This could be mistaken for gastrulation. However, in all cases this
`mislocation' of the blastomere is eventually corrected through a
repositioning of the cell to the surface of the embryo. In the 32-cell embryo,
blastomeres polarise and the nuclei are positioned on the surface of the
embryo (Fig. 1F). Blastomeres
develop a pyramidal shape and the thin ends of the cells point to the inside
of the embryo (Fig. 1G). After
the 64-cell stage is reached, gastrulation starts with the immigration of
single blastomeres. We did not observe alternating cleavage angles before the
initiation of gastrulation, as is typical in spiralian cleavage
(Fig. 2D-F). To determine
whether specific early blastomeres have conserved positions (e.g. at the
32-cell stage and/or form conserved regions of the embryo), as it occurs in
Caenorhabditis elegans (Schnabel
et al., 1997
) (see Movie 2 in the supplementary material), we
differently coloured the descendants of the four-cell stage systematically
with using SIMI°BioCell. In the three embryos shown in
Fig. 2J-L, the distribution of
cells varies from the eight-cell upwards until the 122- or 124-cell embryo and
later. All attempts to match the cleavage patterns after the four-cell stage
by rotating the embryos around their axes failed, thus no stereotyped pattern
in the localization of the descendants of the early blastomeres was
detectable. Additionally, cell migrations did not occur prior to the onset of
gastrulation in the analyzed embryos. The early development of embryos 1 to 3
can be observed in Movies 3 to 5 (see supplementary material), showing 4D
representations corresponding to Fig.
2J-L. The timing of the cell divisions is also variable
(Fig. 2). Early embryos execute
predominantly synchronous cell divisions. However, by the fifth generation,
some cells show significant retardation, which can increase to 30% of the
average cell cycle by the seventh generation. Generally, the time for each
cell cycle and the variability between increases as development proceeds
(Fig. 2A-C).

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Fig. 1. Thulinia stephaniae development: embryo 1. (A) In the fertilized
egg the nucleus (n) is localized near the site of extrusion of the polar body
(pb). (B) The first division is equal and the polar body is localized at the
border of the two blastomeres. (C) Four-cell stage embryo. (D) Eight-cell
stage embryo, sister blastomeres visible in this focal plane are connected
with a line. (E) 16-cell embryo. (F) 32-cell stage embryo. As in earlier
stages, the blastomeres are indistinguishable and all cells have contact with
the surface of the embryo. (G) 64-cell stage embryo. Cells acquire a pyramidal
shape, the nuclei are positioned at the surface of the embryo (white arrow).
Sometimes a small blastocoel (co) is visible (black arrowhead). (H) Embryo
after gastrulation. Ectodermal cells form an epithelium (white arrows) around
the inner cell mass (icm) consisting of PGCs, mesoderm and endoderm
precursors. (I) Germ layer differentiation and organogenesis. The gastrulated
endodermal blastomeres proliferate and differentiate into the pharynx (ph) and
gut (g) anlage. Mesodermal bands (mb) are located to the left and right of the
endoderm. (J) Limb bud and somite formation. During differentiation of the
somites (s) the ectoderm forms segmental limb buds (white arrows). The third
and fourth limb buds are not in focus. The pharynx differentiates further and
the gut anlage (g) consists of small cells. (K) Embryo after elongation. The
ectoderm forms a ventral fold (vf) from which NPCs later delaminate to form
the ganglia. The gut is surrounded by mesodermal cells, which are derived from
the somites. (L) Embryo shortly before movement starts. Gut cells are
vacuolized and form internal reflecting granulae (white arrows). Scale bar: 20
µm.
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Gastrulation and germ layer formation in Thulinia stephaniae
During gastrulation the main embryonic axes become visible (embryo 2,
Fig. 3A). The migration of the
blastomeres starts at two spatially distinct regions on the ventral surface of
the embryo (Fig. 3A,B). The
germ cells invaginate first through an anterior pore, the blastopore
(Fig. 3F), and are immediately
followed by the invagination of mesodermal and entodermal precursor cells.
Interestingly, the primordial germ cells (PGC) are first encircled by the
other germ layer precursors as has been described for some other arthropods
(Fuchs, 1914
;
Kühn, 1913
). Cells that
migrate through the second smaller posterior pore are ectodermal cells, which
after immigration rise back to the surface and fill the pore
(Fig. 3D). These areas are
separated by a minimum of one cell row. The origin of the germ layer
precursors is variable and follows no detectable pattern in the cell lineage
(Fig. 3C), indicating a
non-autonomous, regional specification of these cells. After gastrulation, the
mesoderm and endoderm precursors proliferate. The ectoderm is formed by cells
that surround the inner cells. The cell divisions are irregular and follow no
detectable pattern. The ectodermal epithelium consists of one cell layer,
which gives rise to the epidermis and the nervous system of the juvenile.
After gastrulation is complete, new pores become visible at the same positions
of the former pores (Fig. 3E).
The anterior, larger pore forms the mouth opening (stomodaeum), and the
posterior smaller pore forms the hindgut (proctodaeum) of the juvenile. Both
structures are derived from ectodermal cells
(Fig. 3E).

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Fig. 3. Gastrulation of Thulinia stephaniae: embryo 2. (A) Ventral side of
the embryo. Gastrulation initiates 9 hours after egg deposition. Posterior
(arrow) of the embryo is towards the left. The PGCs migrate anteriorly through
the prospective mouth opening into the embryo. (B) After 2 hours, the pores,
which correspond to the prospective mouth (pm, blastopore) and anus (pan),
become visible. (C) Cell lineage of the gastrulating blastomeres up to the
seventh generation. The mesoderm (M, red branch) and endoderm (End, blue
branch) precursors are descendants of different blastomeres. The sister cells
of the PGCs (G, yellow branch) found different germ layers and the PGCs are
specified in different generations. (D) Gastrulation is completed and the
pores are closed (arrows). (E) The stomodaeum (stom) forms from ectodermal
cells at the anterior pole (right arrow). The inconspicuous pore becomes the
proctodaeum (proc, left arrow). (F) Three-dimensional reconstruction and fate
map of the blastula at the 124-cell stage before onset of gastrulation.
Ventral view of the anterior blastopore. Nine mesodermal precursors (mes) are
represented as red spheres. The pair of PGCs (yellow spheres) is surrounded by
the germ layer precursors, including four endodermal (end) precursors (blue
spheres). The remaining blastomeres acquire an ectodermal fate. Scale bar: 20
µm.
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Somite formation
We also followed the formation of the left and right anterior mesodermal
somites in two embryos using the 4D microscope system (embryo 1 and 3).
After the mesodermal precursors enter the small blastocoel, they adhere to the
inside of the outer ectodermal layer (Fig.
4A-C), on which they migrate to their final position. During this
migration, the cells proliferate and form bands along the left and right side
of the prospective pharynx and midgut (Fig.
4D). This proliferation does not follow a clear anteroposterior
polarity and no growth zone is detectable
(Fig. 4F). Later, the cells of
the mesodermal bands split into groups of cells. These form segmental somites
below the ectodermal prospective limb anlagen
(Fig. 4E). In contrast to
earlier reports (Eibye-Jacobsen,
1997
; Marcus,
1928
; Marcus,
1929
; von Erlanger,
1895
), no cavities were detectable inside these somites during our
in vivo observations. We were able to follow the formation of muscle from
somites inside the limb bud. The remaining cells differentiated into small
cells and could not be traced further.

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Fig. 4. Mesoderm development in Thulinia stephaniae. The genealogy of a
blastomere, which contributes to the first left limb bud somite, was traced
back to the precursor blastomere (embryo 1). Dorsal view, anterior towards the
left. (A) Position of the mesodermal precursor shortly before it immigrates
during gastrulation (arrowhead). (B) Position of traced cell 2 hours later
(arrowhead). The red spheres indicate the path of migration of the cell since
immigration started (projected with the migration function of the
SIMI°BioCell software). (C) After the next division, the cell (red sphere)
touches the anterior inner side of the ectodermal epithelium. (D) Twenty hours
after egg deposition, the mesodermal bands (mb) form out of the mesodermal
precursors. The mesodermal bands (arrows) stretch from posterior to anterior
along the pharynx (px) and gut anlagen. The red sphere marks the position of
the traced cell. (E) Embryo 1.5 hours later. The mesodermal bands form somites
left and right from the gut; no cavities can be seen (black arrows). The
traced cell has divided once and participates in the formation of the left
second somite. (F) Three-dimensional representation of the nuclei in the
122-cell stage embryo. The row of red spheres shows the path of migration of
the traced mesoderm cell. (G) Positions of the mesoderm precursor during
gastrulation. The white arrows indicate the position of the precursor during
specified mitoses. Later, the cell migrates along the inner epithelium of the
ectoderm. (H) Embryo 34 hours after egg deposition. Mesodermal cells
proliferate into the limb buds (arrows). Scale bar: 20 µm.
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The nervous system is built by neuronal progenitor cells immigrating from the neurogenic ectoderm
The tardigrade central nervous system (CNS) is composed of the dorsal
brain, the ventral sub-oesophageal ganglion, and the serial ventral ganglia,
which are joined by connectives (Fig.
5A). In one recording (embryo 3), we could trace back
founder cells of the ventral ganglia in Thulina stephaniae
(Fig. 5). Before gangliogenesis
is initiated (48 hours of development; Fig.
5B), the four neuronal progenitor cells (NPC), are located in the
future position of the ganglia. Earlier, at 33 hours of development, the four
NPCs are located in the ventral fold in an anteroposterior row ready to
immigrate (Fig. 5C). The
anteriormost NPC forms the first ganglion (I) the second the second ganglion
(II) and so on. The four NPCs are produced between 25 and 33 hours of
development, by two subsequent divisions of four precursor cells. The sisters
of the NPCs founder cells in each of these two divisions differentiate into
epidermis (Fig. 5D). At 25
hours, the four founders of these lineages are located in positions that are
not obviously related to the final arrangement of the NPCs. For example, the
founder of the most posterior NPC is in the most anterior position at this
time (Fig. 5E). We could only
follow the process from the left side of the embryo as embryos always rotate
to the side after gastrulation. Thus, we cannot exclude the possibility that
the ganglia are founded by bilaterally located NPCs, as in malacostracan
crustaceans (Dohle et al.,
2004
). The brain of Thulinia stephaniae is also formed by
NPCs. Although we could not determine the number of cells forming the brain,
we could detect the neuroectodermal anterior region, from which the NPCs are
derived (Fig. 5F). The
immigration of the brain precursor occurs before the immigration of the NPCs
that form the ganglia. Although the early origin of the suboesophageal
ganglion remains unclear, it seems likely that it is formed by an outgrowth of
the brain, as we could not detect an early anlage.

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Fig. 5. Neurogenesis of Thulinia stephaniae: embryo 3. (A) Lateral view of
an embryo that begins muscle contractions. The brain (br) and the ventral
ganglia (I-IV) are highlighted in yellow. (B) Earlier lateral view of the same
embryo after delamination of the neuronal precursor cells (arrows) and during
formation of the ganglia. (C) Lateral view 15 hours earlier. After two cell
divisions, the NPC are formed from the precursors. They are located in the
ectodermal ventral fold inferior the future position of the ganglia. (D)
Earlier ventral view. The arrows indicate the location of the NPCs in the
248-cell embryo. (E) Three-dimensional representation of the positions of the
nuclei at this stage. The bright yellow spheres correspond to the NPCs, which
are connected with white bars to their sister cells. Colour code see
Fig. 2. The NPCs are
descendants of both blastomeres of the two-cell stage. (F) View of the left
side of the embryo after it has rotated at 28 hours. We followed one brain
precursor (arrow) delaminating from the anterior dorsal ectoderm to start
brain formation, prior to the delaminating NPCs, which form the ganglia. Scale
bar: 20 µm.
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Specification of primordial germ cells
The germline in Thulinia stephaniae consists of two PGCs. In three
(embryos 1, 3 and 4) out of four embryos, they could be recognized as
non-dividing cells in the sixth generation before the onset of gastrulation
(Fig. 6). In one embryo
(embryo 2) one PGC differentiated in the sixth and the other at
the seventh generation (Fig.
3C), which suggests an indeterminate specification. The PGCs are
morphologically distinguishable from the somatic cells by their larger size.
In all analyzed embryos, the sister cells of the PGC execute different fates
(Fig. 3C,
Fig. 6C). In the three embryos
that form the PGCs in the sixth generation, both blastomeres of the two-cell
stage form one germ cell each (Fig.
6C). In the remaining embryo, both PGCs were derived from one
blastomere of the two-cell stage (Fig.
3C). These observations indicate that the PGCs are determined
non-autonomously before gastrulation. During gastrulation, the germ cells
migrate into the blastocoel and locate posterior to the developing pharynx,
adjacent to the midgut cells (Fig.
6D). Later, during elongation of the embryo, the cells move by
different paths to a posterior position, which is dorsal to the midgut, where
the gonad will develop (Fig.
6E,F).

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Fig. 6. Development of the germ cells in Thulinia stephaniae: embryo 3 (A)
Three-dimensional representations of the positions of the primordial germ
cells (PGC) (yellow spheres, arrows) after differentiation has occurred (last
cell division). The neighbouring cells have large nuclei and are located at
the site of the future blastopore. (B) The PGCs gastrulate (arrows). (C) The
cell lineage analysis shows that the PGCs differentiated after the sixth cell
division and are derived from both blastomeres of the two-cell stage. (D)
Ventral view of the embryo, anterior towards the left. The PGCs are located
posterior to the pharynx (px) and ventral to the midgut anlage. (E) Projection
of the migration paths of the PGCs using SIMI°BioCell. During elongation
of the embryo, the PGCs begin to migrate along independent paths to the
prospective position of the gonad (left PGC green, right PGC red). (F) Final
position of the PGCs (arrow) at the location where the gonad develops. Scale
bar: 20 µm.
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Laser ablation of one blastomere in the two-cell stage
To test the regulatory ability of early embryos, we laser ablated
blastomeres (Table 1). After
ablations of one of the two blastomeres in two-cell stage embryos, normal
juveniles hatched (n=9). These juveniles, which developed from only
one blastomere were smaller than the untreated control embryos (see
Fig. 10C,D). In all recorded
embryos the ablated cell divided twice before the damaged descendants degraded
into many cytoplasts of different size
(Fig. 7A). The development of
the non-ablated blastomeres was not affected
(Fig. 7C). The normal cells
initially surround the ablated cytoplasts
(Fig. 7B), but soon begin to
`ignore' the debris and assume the typical pyramidal shape
(Fig. 7A). After the
blastomeres have rearranged, gastrulation is initiated
(Fig. 7D). These embryos still
form all tissues; the inner blastomeres form gut, mesodermal bands and the
pharynx (Fig. 7E,F;
Fig. 10). The ectodermal cells
are much larger than in the control embryos, as it is expected for an embryo
composed of only half the number of cells of a normal embryo. We did not find
a compensatory cell generation in the ablated embryos during the 4D analyses.
Interestingly, all ablated embryos display two germ cells. Thus, germline
formation is regulative, which makes it unlikely that a pre-existing germ
cytoplasm is shunted into specific blastomeres
(Fig. 7E).

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Fig. 10. Juveniles formed after cell ablations in the embryo. (A-C) The same exuvia.
(A) Three embryos were ablated. Two control embryos are marked with `c'. In
the embryo (4-cell p) a polar blastomere and in the embryo labelled (4-cell s)
a lateral blastomere have been ablated in the four-cell stage. In the third
embryo (2-cell), one blastomere was ablated in the two-cell embryo. (B) Two
empty eggshells (arrowheads) indicate that two juveniles have already hatched.
(C) All juveniles have hatched from the exuvia and are in an anoxybiotic
state. The smallest juvenile is derived from the cell ablation at the two-cell
stage (arrow). (D) Embryos from a different ablation experiment at higher
magnification. Left, a control embryo; right, an embryo after one cell was
ablated in the two-cell stage. The stylet (white arrowhead) of the ablated
embryo is reduced in comparison with that of the control embryo. Scale bars:
A,B,C, 50 µm; D, 25 µm..
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Fig. 7. Development after laser ablation of one blastomere in the two-cell embryo.
(A) 64-cell embryo. The ablated cell cleaved twice before its descendants
degraded into small cytoplasts (star). The untreated blastomeres divided
normally and the descendants acquire a pyramidal shape at the 124-cell stage
(arrows). (B) Three-dimensional representation of the embryo shown in A. The
non-ablated descendants first surround the remaining cytoplasts (yellow
spheres) of the ablated cell, but are later found further from them. (C)
Lineage of the embryo up to the 64-cell stage. The non-treated blastomeres
undergo normal cell cycles. (D) Later stage of the embryo. The descendants of
the non-treated blastomere form an epithelium (arrows) at the border of the
cytoplasts (cp). (E) Later stage of the recorded embryo. A pair of PGCs is
present and the embryo develops the main organs e.g. pharynx (px). The small
embryo is surrounded by the cytoplasts. (F) Stage of limb bud (lb) formation.
Anterior is towards the right. The cells of the ectoderm (arrows) are much
larger than those found in normal embryos at the same stage. Scale bar: 20
µm.
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Ablations in the four-cell stage embryo
We ablated individual blastomeres located either laterally or at one pole
of the egg in four-cell embryos. Development of these embryos proceeded as
described for the ablations experiments in the two-cell embryos. Normal
juveniles developed from the ablated four-cell embryos, also formed a pair of
two germ cells and hatched (Fig.
8, see Fig. 10).
Additionally, we ablated two blastomeres, which are non-sibling in the
four-cell embryo (Table 1,
Fig. 9). These two embryos
developed similarly to the other ablated embryos, although the development of
the embryos took longer. For example, the formation of the pyramidal cells,
which are normally formed prior to gastrulation, was delayed. However, the
early cell cycles proceeded normally in this developmental phase
(Fig. 9A). Presumably, the
delay is caused by the large aggregation of ablated cells, which occupies the
centre of the embryo after ablation and which must be subsequently displaced
by the untreated blastomeres in order to form the embryo
(Fig. 9B,C). After the
blastomeres have assembled, gastrulation starts immediately. These
observations indicate an astonishing plasticity of early embryogenesis. Both
embryos were recovered after the initial recording and developed into normal
hatched juveniles despite the early ablations.

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Fig. 8. Development after laser ablation of one cell in the four-cell stage embryo.
(A) Forty-seven descendants of the untreated blastomeres surround (arrows) the
ablated cell (x), which has divided once. The descendents degrade later into
small cytoplasts. (B) Three-dimensional representation of the embryo shown in
A, the yellow spheres mark the position of the two descendants of the ablated
cell. The colour code is the same used in C. (C) Lineage of the 47-cell stage.
As in a normal embryo of the corresponding stage (64-cell stage), some cell
cycles are retarded. (D) Later stage after degradation of the ablated cell.
The descendants of the living cells form a typical epithelium, and surround an
inner endodermal cell mass (icm). The cytoplasts (cp) of the ablated cell are
excluded. Two PGCs are found at the normal position (arrows). (E) Embryo
before hatching. Mouth (mt), brain (br) and claws are present and look normal.
The cytoplasts (cp) of the ablated cell surround the embryo. (F) Same stage as
E under a higher magnification. The stylet (st), buccal tube and pharynx (px)
are formed normally. The gut cells (gt) contain the typical granulae. Scale
bar: 20 µm.
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Fig. 9. Development after ablation of two cousin blastomeres during the four-cell
stage. (A) Lineage of the remaining two untreated blastomeres. The central
lineage reflects the behaviour of the ablated blastomeres. The untreated
blastomeres divide normally and show retarded cell cycles during the fifth
generation (arrow). (B) Embryo at the 59-cell stage. (C) Three-dimensional
representation of the nuclei positions of B. The descendants of the untreated
blastomeres surround the ablated blastomeres. (D) Exuvia with the three
embryos. In the embryos to the left and right, the ablated cells are marked
with a cross. Sister cells are connected with a line. The embryo in the centre
is a control embryo. (E) Later stage of D. Both treated embryos have a
developed pharynx (white arrowheads). The control egg has already developed
further. (F) Higher magnification of the left most embryo. Both PGCs are
visible at the expected location for a normal embryo (black arrows). Scale
bar: 20 µm.
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Discussion
|
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General course of embryogenesis
For a long time, the nature of tardigrade development has remained obscure.
Early descriptions have not been reinvestigated, owing to a loss of interest
in `minor phyla' and the experimental opportunities available in other, more
tractable, systems. Therefore, the work of a sole investigator Marcus
(Marcus, 1929
) has appeared in
nearly every invertebrate textbook for the past 70 years. We have now analyzed
Thulinia stephaniae as a representative species for eutardigrade
development using 4D microscopy. The early cleavages of the embryo are
variable from the second cell division onwards. The position of the anterior
posterior axis varies in relation to early cleavages. With the ability to
trace the fate of single blastomeres, we could establish lineages of different
embryos and show a variable origin of the PGCs and of the main germ layers.
This is consistent with the impressive regulative potential of the embryo. The
formation of the nervous system, especially the synchronous immigration of
neural progenitor cells, resembles development patterns in some arthropods
(Dove and Stollewerk, 2003
;
Stollewerk, 2002
).
Additionally, our reassessment of gastrulation and mesoderm formation displays
striking similarities to these processes in some other arthropods
(Anderson, 1973
;
Siewing, 1969
). Gastrulation
takes place at the ventral surface of the embryo and the blastopore
corresponds to the future mouth opening. Concerning AP axis formation,
gastrulation, endoderm formation and germline development, our findings are
inconsistent with the descriptions of Marcus
(Marcus, 1928
;
Marcus, 1929
) and von Erlanger
(von Erlanger, 1895
).
Eibye-Jacobsen (Eibye-Jacobsen,
1997
) was not able to confirm that the mesoderm is derived from
outpocketings of the gut (enterocoely), as described by Marcus
(Marcus, 1928
;
Marcus, 1929
), but she was not
able to determine the origin of the mesoderm. Our lineage analysis now shows
that the mesodermal somites are derived from subdivisions of lateral
mesodermal bands. As opposed to Marcus
(Marcus, 1928
;
Marcus, 1929
), we find that in
Thulinia stephaniae, all germ layers precursors are present during
gastrulation. Disparities between our findings and Marcus' results may be
explained by his inability to analyze the cell borders in as great of detail
as his drawings suggest. Marcus himself conceded that he could not detect cell
borders in the stages that follow gastrulation, yet he included cell borders
in his schematic drawings (Marcus,
1928
). The arrangement of the germ layer precursors that encircle
the PGCs prior to immigration shows striking similarities to that of total
cleaving crustacean embryos like that of the copepod Megacyclops
viridis (Fuchs, 1914
) and
the water flea Polyphemus
(Kühn, 1913
). This may
reflect a common fate determination mechanism in arthropods, although these
crustaceans show a stereotyped cleavage program. Recent detailed cell lineage
studies of amphipod embryos (Gerberding et
al., 2002
; Wolff and Scholtz,
2002
) show a similar pattern in the arrangement of the germ cell
and mesoderm and endoderm precursors. However, the cleavage program of the
amphipods is highly derived in the crustaceans and thus makes it difficult to
compare. In many arthropods, lateral mesodermal bands are formed by a
posterior growth zone, which proliferates towards the anterior pole
(Anderson, 1973
). Later
coelomic cavities, also called somites, are built up by schizocoely
(Anderson, 1973
;
Dohle, 1979
). Thulinia
stephaniae shows similarities to the pattern of mesoderm formation of the
Euarthropoda and onychophorans (Manton,
1949
) and therefore may reflect the ancestral condition of this
process in arthropods.
Cell ablation experiments show a high regulatory potential of the tardigrade embryo
Our cell ablation experiments indicate that early cell fate restrictions do
not occur in tardigrade development. Our observations in Thulinia
stephaniae are inconsistent with a spiral development. After our cell
ablations in the two- and four-cell embryos, all embryonic axes were formed.
Thus, the axes are not fixed at these stages. Despite the fact that up to one
half of the embryo is missing after the ablations, germ layer formation and
germ-line specification proceeded normally. No protostome embryo with a
similar regulative ability to compensate for such invasive blastomere
deletions has yet been reported. The highest capability for regulation in
protostomes was described by Wiegner and Schierenberg
(Wiegner and Schierenberg,
1999
) for the nematode Acrobeloides nanus. Here, the
posterior but not the anterior blastomere of the two-cell stage will form a
larvae when the other cell is ablated. Our results suggest that a
non-autonomous mechanism specifies the main axes and tissues of the embryo
sometime after the tardigrade embryo has reached the four-cell stage. It would
be interesting to determine molecular mechanisms governing embryogenesis in
tardigrades. However, owing to the specific biology of the system, it appears
that it would be difficult to establish Thulinia stephaniae as a
molecular genetic system allowing functional analysis.
Phylogenetic considerations
The early development of arthropods is diverse, ranging from total cleavage
to syncytial cleavage (Anderson,
1973
; Scholtz,
1997
; Siewing,
1969
) and it has remained unclear which type of development
reflects the ancestral mode. We showed for Thulinia stephaniae, as a
representative of the tardigrades, a variable cleavage pattern with a high
regulatory potential of the early embryo. Tardigrades are a basal lineage in
the arthropods (Dewel and Dewel,
1997
; Garey, 2001
;
Garey et al., 1996
;
Garey et al., 1999
;
Giribet et al., 1996
;
Maas and Waloszek, 2001
;
Mallatt et al., 2004
;
Nielsen, 2001
;
Regier and Shultz, 2001
), but
this does not necessarily mean that our descriptions reflect the ancestral
condition in the Arthropoda. A comparison of the different types of
development present in arthropods with that of the sister group of the
Arthropoda (outgroup comparison), is needed to define the ground pattern
(Ax, 1984
;
Scholtz, 2004
). The classical
Articulata hypothesis favours the Annelida, which display an exemplary spiral
quartet cleavage, as the sister group of the Arthropoda. The cleavage program
of the molluscs, the sister group of the Articulata, is also spiral. Thus, the
common ancestor of the Articulata must have had a spiral cleavage, which was
then modified in the stem species of the Arthropoda
(Scholtz, 1997
;
Fig. 10A). Although several
total cleaving arthropods, mainly crustaceans with a determinate cleavage and
traceable cell lineage, have been investigated
(Alwes and Scholtz, 2004
;
Anderson, 1969
;
Gerberding et al., 2002
;
Hertzler, 2002
;
Hertzler and Clark, 1992
;
Hertzler et al., 1994
;
Wolff and Scholtz, 2002
;
Zilch, 1978
;
Zilch, 1979
), none of them
shows clear traits of a spiral cleavage. The emerging Ecdysozoa hypothesis
favours not the Annelida but the Cycloneuralia (Cycloneuralia consist of
Nematoda, Nematomorpha, Priapulida, Kinorhyncha and Loricifera) as the sister
group of the arthropods (Aguinaldo et al.,
1997
; de Rosa et al.,
1999
; Giribet et al.,
2000
; Schmidt-Rhaesa et al.,
1998
; Valentine,
1997
). This makes it unnecessary to assume that a modified spiral
cleavage is part of the ground pattern of the arthropods, as no spiral
cleaving cycloneuralian embryo is known. Priapulids show a `radial' cleavage
type (Lang, 1953
;
Zhinkin, 1949
;
Zhinkin and Korsakova, 1953
),
and the cleavage of the Nematomorpha was reported to be indeterminate
(Inoue, 1958
;
Malakhov and Spiridonov, 1984
;
Meyer, 1913
;
Mühldorf, 1914
). The
sister group of the Nematomorpha, the nematodes, show diverse cleavage types.
The stereotypic cleavage pattern found in Caenorhabditis elegans
(Sulston et al., 1983
), is not
typical for nematodes, as several indeterminately cleaving nematode embryos
have also been reported (Voronov,
1999
; Voronov,
2001
; Voronov and Panchin,
1998
). Considering the Nematomorpha as the outgroup, a variable
cleavage pattern appears to be the ancestral condition in the Nematoda, and
the `typical' stereotyped cell lineage of the rhabdite nematodes is derived
(Fig. 10B). Nothing is known
about the cleavage of the remaining cycloneuralian clades, the kinorhynchs and
loriciferans. If the Cycloneuralia are the sister group to the Arthropods, an
indeterminate cleavage pattern, similar to that of Thulinia
stephaniae, should be part of the ground pattern in the Ecdysozoa
(Fig. 10B). This notion is
also supported by the fact that arthropods with an irregular cleavage pattern
have been described in both crustaceans
(Benesch, 1969
;
Scheidegger, 1976
;
Weygoldt, 1960
) and myriapods
(Dohle, 1964
;
Tiegs, 1940
;
Tiegs, 1947
). Of all
investigated arthropods, these species show the greatest similarity to the
early development of Thulinia stephaniae and are thus good candidates
to represent the ancestral mode of early developmental patterns in the
Euarthropoda.
 |
Supplementary material
|
---|
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/132/6/1349/DC1
 |
ACKNOWLEDGMENTS
|
---|
We thank Reinhard Møbjerg Kristensen for species identification and
his help in collecting Echiniscoides sigismundii, which unfortunately
did not want to be recorded. A field-collecting trip for Echiniscoides
sigismundii was supported by the COBICE program of the European Union. We
thank Heather Marlow, Gemma Richards, Ryan Viveiros, Gerhard Scholtz and
Wolfgang Dohle for improving the manuscript.
 |
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