Download Gastrulation in the sea anemone Nematostella

Dev Genes Evol (2006) 216: 119–132
DOI 10.1007/s00427-005-0038-3
ORIGINA L ARTI CLE
Yulia Kraus . Ulrich Technau
Gastrulation in the sea anemone Nematostella vectensis occurs
by invagination and immigration: an ultrastructural study
Received: 8 July 2005 / Accepted: 17 October 2005 / Published online: 14 January 2006
# Springer-Verlag 2006
Abstract The sea anemone Nematostella vectensis has
recently been established as a new model system for the
understanding of the evolution of developmental processes. In particular, the evolutionary origin of gastrulation
and its molecular regulation are the subject of intense
investigation. However, while molecular data are rapidly
accumulating, no detailed morphological data exist describing the process of gastrulation. Here, we carried out an
ultrastructural study of different stages of gastrulation in
Nematostella using transmission electron microscope and
scanning electron microscopy techniques. We show that
presumptive endodermal cells undergo a change in cell
shape, reminiscent of the bottle cells known from vertebrates and several invertebrates. Presumptive endodermal
cells organize into a field, the pre-endodermal plate, which
undergoes invagination. In parallel, the endodermal cells
decrease their apical cell contacts but remain loosely attached to each other. Hence, during early gastrulation they
display an incomplete epithelial–mesenchymal transition
(EMT). At a late stage of gastrulation, the cells eventually
detach and fill the interior of the blastocoel as mesenchymal cells. This shows that gastrulation in Nematostella
occurs by a combination of invagination and late immigration involving EMT. The comparison with molecular
expression studies suggests that cells expressing snailA
undergo EMT and become endodermal, whereas forkhead/
Communicated by M.Q. Martindale
Y. Kraus
Department of Evolutionary Biology,
Biological Faculty, Moscow State University,
199992 Moscow, Russia
e-mail: [email protected]
U. Technau (*)
Sars International Centre for Marine
Molecular Biology, University in Bergen,
Thormøhlensgt. 55,
5008 Bergen, Norway
e-mail: [email protected]
Tel.: +47-5-5584340
Fax: +47-5-5584305
brachyury expressing cells at the ectodermal margin of the
blastopore retain their epithelial integrity throughout
gastrulation.
Keywords Nematostella . Gastrulation . Invagination .
Immigration . Epithelial–mesenchymal transition
Introduction
Gastrulation is the process during early animal embryogenesis where the basic germ layers, ectoderm and
endoderm, and in Bilateria, the mesoderm, are formed.
The evolution of gastrulation and its molecular control is
currently a matter of intense investigation. The phylum
Cnidaria is crucial for the understanding of the evolution of
modes of gastrulation and of the mesoderm, since they
consist of only two germ layers, ectoderm and endoderm,
and they arose very early (about 600 myr ago) during
animal evolution. Furthermore, although the phylogenetic
position of the Ctenophores is not yet fully solved, recent
molecular phylogenies place the Cnidaria as the closest
outgroup to the Bilateria (Collins 2002; Medina et al. 2001;
Kim et al. 1999).
Interestingly, different species of the phylum Cnidaria
display all modes of gastrulation known from Bilateria:
invagination, polar and multipolar immigration, delamination and epiboly as well as mixed modes (Metchnikoff
1886; reviewed in Tardent 1978; Byrum and Martindale
2004). Among the basal group within the Cnidaria, the
Anthozoa, the sea anemone Nematostella vectensis, has
been recently established as a major model organism for
the study of evolutionary developmental biology (Hand
and Uhlinger 1992; Fritzenwanker and Technau 2002) and
the expression of several conserved genes has been studied
during embryonic and larval development with respect to
axis formation and germ layer formation (Scholz and
Technau 2003; Finnerty et al. 2004; Wikramanayake et al.
2003; Fritzenwanker et al. 2004; Martindale et al. 2004;
Kusserow et al. 2005). While the amount of molecular data
is rapidly accumulating, there is virtually no detailed
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information available about the morphological and cellular
basis of the morphogenetic events. Early pioneering
workers (e.g. Metchnikoff 1886; Hyde 1894; Wulfert
1902) used light microscopic techniques to investigate
cnidarian embryogenesis.
However, despite a long tradition in the analysis of adult
anthozoan polyps in the literature, very little is known
about the details of the embryonic development of this
important class of animals, in particular, at the ultrastructural level. Schaefer (1985) carried out one of the very few
ultrastructural studies on the early development of an anthozoan, the sea anemone Anemonia sulcata. Further, the
development of the coral Acropora millepora has been
described using scanning electron microscopy (SEM) (Ball
et al. 2002). More electron microscopy studies are available
for the development of different hydrozoan species. For
instance, Martin et al. (Martin and Thomas 1977, Martin et
al. 1997) studied the embryonic development of the
Pennaria tiarella and freshwater polyp Hydra, and Kraus
and Cherdantsev (1999) have analysed the gastrulation
movements of Dynamena pumila.
On the basis of the expression of two genes coding for
the conserved transcription factors, Forkhead (FoxA2) and
SnailA, we have recently proposed that gastrulation in N.
vectensis occurs by a combination of invagination and
immigration (Fritzenwanker et al. 2004). SnailA is expressed in all presumptive endodermal cells that ingress
during gastrulation, whereas forkhead and brachyury are
expressed in the ectodermal blastopore lip, which eventually gives rise to the inverted pharynx (Fritzenwanker et
al. 2004; Martindale et al. 2004). We hypothesized that
snailA-expressing cells undergo epithelial–mesenchymal
transition, while forkhead/brachyury-expressing cells retain their epithelial organization (Fritzenwanker et al.
2004).
Here, we carried out an ultrastructural study of different
stages of gastrulation in Nematostella using transmission
electron microscope (TEM) and SEM techniques. We show
that presumptive endodermal cells undergo a change in cell
shape, reminiscent of the bottle cells known from
vertebrates and several invertebrates (e.g. Keller et al.
2003; Shook and Keller 2003). Presumptive endodermal
cells organize into a field, the pre-endodermal plate, which
undergoes invagination. In parallel, the pre-endodermal
cells decrease the area of apical cell contacts but remain
loosely attached to each other. Hence, they display an
incomplete epithelial–mesenchymal transition. At a late
stage of gastrulation, the cells eventually detach and fill the
interior of the blastocoel as mesenchymal cells.
Materials and methods
Nematostella culture
Nematostella polyps and embryos were kept in 1/3 seawater (Hand and Uhlinger 1992) at 18°C in the dark and
fed five times a week with brine shrimp naupliae. Induction
of gametogenesis was carried out as described before
(Fritzenwanker and Technau 2002). Oocytes were fertilized
in vitro allowing for fairly synchronized development.
Electron microscopy
Embryos at successive developmental stages (from the
early cleavage and up to the planula formation) were fixed
for electron microscopy in 2.5% glutaraldehyde/0.1 M
cacodylate buffer (pH 7.2). When preparing for TEM,
embryos were fixed overnight at +4°C, and then processed
immediately. Samples for SEM were transferred into 1.2%
glutaraldehyde/0.1 M cacodylate buffer (pH 7.2) and stored
at +4°C until further processing. Embryos were then
washed in 0.1 M cacodylate buffer, and the surrounding
mucous removed manually with fine needles. Embryos
were postfixed in 1% osmium tetroxide in the same buffer
for 1 h.
Samples for TEM were dehydrated through a graded
series of ethanol and acetone and then embedded into the
Araldite embedding medium (Fluka) and sectioned using
routine techniques. Sections were stained in water solutions of uranyl acetate and lead citrate and examined by
transmission electron microscope JEOL JEM-1000B.
Samples for SEM were also dehydrated through a graded
series of ethanol and acetone and then dried from acetone
using the critical-point technique. Samples were sputter
coated with gold and examined by microscopes S-405A
(Hitachi) and CamScan at an accelerating voltage at 10 kV.
Some embryos were sectioned into halves by a microsurgical scalpel during the 70% ethanol step of dehydration.
Phalloidin staining
Embryos were fixed in 4% paraformaldehyde overnight
and washed in phosphate-buffered saline (PBS)/Triton
0.1%. Animals were then blocked in PBS/2% bovine
serum albumin (BSA) for 1 h and incubated in 1 unit
Phalloidin-Alexa488 (Invitrogen) in PBS/BSA for 30 min
and mounted on glass slides after several washes.
Results
Short description of normal development
Early cleavage (Fig. 1a) is mostly radial, although highly
variable from embryo to embryo (Technau, unpublished
data). This leads to a coeloblastula after about 6–10 h postfertilization. At the stage of 64–128 cells, the blastula flattens (Fig. 1b). This stage is morphologically very similar to
the prawn-chip stage of A. millepora embryos (Ball et al.
2002). After about 12 h post-fertilization, the embryos
reach the pre-gastrula stage (Fig. 1c). At this stage, the
embryos have a more or less spherical shape. Gastrulation
starts after 18 to 20 h of normal development, and a distinct
blastopore is formed at 21 to 23 h (Fig. 1d). Gastrulation
lasts for about 12 h, and the blastopore is gradually closed
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Fig. 1 Overview of Nematostella embryonic development. a Early
cleavage stage, about 5 h post-fertilization (pf); b 10 h pf blastula at
flattened stage; c 20-h pre-gastrula stage (preparation to the onset of
gastrulation); d 30-h gastrula stage, the blastopore is marked by an
asterisk; e 3-day planula stage, anterior pole is to the left, at-apical
tuft. Scale bar=30 μm
during this time. The embryo then converts into a planula
larva during the next 12 h (Fig. 1e). It has a distinct AP
axis, an epithelial pharynx anlage and an apical ciliary tuft
(Apikalorgan). The larva transforms into a primary polyp
about 5–10 days post-fertilization.
indentations are marked by the shrinking apexes and apical
bulgings of a few ingressing cells (Figs. 3e-g and 5a). The
clusters of ingressing cells are formed independently in
different regions of the future blastopore zone (Fig. 3d,e,
g). The number of ingressing cells in each cluster increases,
so that the area of the clusters gradually becomes larger,
and finally all clusters appear to fuse to form a flattened
area (Fig. 3h–j), reminiscent of the vegetal plate in sea
urchins. Since it is not clear whether this site refers indeed
to the vegetal pole or instead to the animal pole, we
therefore refer to it now as pre-endodermal plate, as cells in
this area are the presumptive endodermal cells. Initially, the
pre-endodermal plate appears from the outside as a
flattened region of embryonic surface, and we can detect
its edges only on the basis of the shape of cell apexes
(Fig. 3j). However, at the next developmental stage, the
whole pre-endodermal plate starts to sink in (Fig. 4a–d).
The sinking is a result of the gradual bending of circumferential region of blastoderm surrounding the pre-endodermal plate. This region of blastoderm is called the
‘blastopore lip’ from this stage on (Fig. 4c,d).
SEM of fractured embryos as well as TEM pictures of
the pre-endodermal plate reveals that the cells of the preendodermal plate acquire a bottle shape (Fig. 3h), somewhat similar to the ingressing bottle cells defined in
gastrulation of higher metazoans. During this process, the
cell nucleus migrates from the apical towards the basal end
of the cell. The cells elongate and become very slim,
especially in the middle part (Figs. 4e and 5c), thereby
extending into the blastocoel.
The ingressing bottle cell strongly reduces the perimeter
of apical surface, but it forms a bulge-like protrusion at the
apical side, visible as a cellular bleb from the outside, and
so does not significantly reduce apical surface area (Figs. 3f,
4g and 5a,b). The cells of the pre-endodermal plate still have
preserved the cilium at the apical side (Fig. 4g). The basal
side of the ingressing bottle cell becomes the leading edge. It
enlarges (Figs. 4e,h and 5c) and forms lamellipodia and
several filopodia, which appear to explore the blastocoel
(Fig. 4e,h). While this resembles the leading edge of an
individual moving cell, the trailing edge, which is typical for
individual moving cells, does not yet form. In contrast to the
blastopore lip cells, which preserve morphology and structure of the blastoderm cell, the bottle cells are only loosely
connected at the basolateral side (Figs. 4e and 5c). The subapical junctions are still present during early gastrulation
Onset of gastrulation
Ultrastructure of blastodermal cells
At the pre-gastrula stage, the shape of the embryos is more
or less spherical and very uniform (Figs. 1c and 3a). The
blastoderm consists of cells, which are highly polarized and
wedge shaped (Figs. 2a and 3b), with the length of the
apico-basal axis of the blastodermal cell about one third of
diameter of the embryo (Fig. 3b). The shape and organization of the blastoderm cells do not change significantly
during the gastrulation. The ultrastructural analysis shows
that the blastodermal cells display a polarized morphology
typical for epithelial cells. Their nuclei and electron-dense
granules are situated near the apical surface, and their yolk
granules are displaced closer to the basal end (Fig. 2a,c).
Mitochondria are preferentially distributed in the apical
half of the cell, together with the nucleus (Fig. 2c). Each
cell has a long cilium and many microvilli at the apical cell
surface (Fig. 2a,h). There is no basal lamina, and the basal
ends of the blastodermal cells form very long protrusions
(Fig. 3c), which strongly interdigitate (Fig. 2b). The blastoderm cells have a distinct sub-apical junctional complex
(Fig. 2c-e). Adherens junctions can be found just beneath the
apical surface and septate junctions a little bit more basal
(Fig. 2c-e). Adherens junctions are attached to the belts of the
actin cytoskeleton (Fig. 2e), which is also demonstrated by the
F-actin staining of the blastoderm cells (Fig. 2f,g). There are
many filopodia-like protrusions not only at the basal, but also
at the lateral cell surface (Fig. 3c). We have not detected any
stable junctions between the basal protrusions, but lateral
processes of neighbouring cells can form adherens junctions
with each other and with the cell’s body (Fig. 2i).
Early gastrula stage and formation of bottle cells
The first external signs of the beginning gastrulation are
indentations of the embryonic surface (Fig. 3d). These
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Fig. 2 Ultrastructure of blastoderm cells. a Typical blastoderm cell,
apex is oriented upward. N nucleus, mv microvilli, edg electron
dense granules, yg yolk granules; b basal surface of blastoderm,
interdigitated processes of blastoderm cells, section plan is oriented
perpendicularly to AB axes of blastoderm cells. c, d Apical region
of blastoderm cells and sub-apical junctional complex: aj adherens
junction, sj septate junctions, m mitochondria; e adherens junction,
mf bundles of actin microfilaments. f Surface view on blastoderm
stained with phalloidin for apical F-actin belts associated with the
sub-apical adherens junctions; g phalloidin-stained blastodermal
cells spread by partial dissociation; h apical region of blastoderm
cell, arrowhead shows the basal root of the cilium. i interdigitated
processes extending from the apical region of cell (ap) and from the
lateral cell surface (lp). There is an adherens junction between the
lateral process and cell body. a–c, i, h scale bars=1 μm; d, e scale
bars=0.1 μm
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Fig. 3 Onset of gastrulation. a Embryo at the pre-gastrula stage; b one
half of the same embryo. c Blastoderm cells, protrusions formed by the
basal cell surfaces (arrows); protrusions formed by the lateral cell
surfaces (arrowheads). d Beginning of the pre-endodermal plate
formation with two indentations on the embryonic surface (arrowheads). e The clusters of forming bottle cells (arrowheads); f bulged
apexes of forming bottle cells, close up of e. g One half of an embryo at
the intermediate stage of the pre-endodermal plate formation, the same
stage of pre-endodermal plate formation as on e, arrowheads show
indentations (clusters of ingressing cells) on the embryonic surface,
i = ingressing cell. h Completely formed fragment of the pre-endoderm plate, as apical surfaces of bottle cells. i Forming preendodermal plate, its central zone is already formed, but no clear
border between bottle cell clusters and blastopore lip cells is yet
visible; j completely formed pre-endodermal plate, there is a fairly
distinct boundary between the pre-endodermal plate and future
blastopore lips (splits are an artifact of sample preparation). a, b, d, e,
g–j scale bars=30 μm, c scale bar=15 μm, f scale bar=10 μm
(Fig. 5a,b); therefore, ingressing cells do not detach from
each other until late during gastrulation and do not migrate as
individual cells through the blastocoel.
(Fig. 4f,i,j). The cells of the pre-endodermal plate gradually
reduce the excess of apical plasma membrane (bulge-like
apical surface), apparently by endocytosis of very big
fragments of plasma membrane (Fig. 5b,f).The sub-apical
junctions tend to become rather punctuate, and no actin
belts can be detected (Fig. 5c–f). Thus, the apical junctional
complex appears to disintegrate during the course of
ingression of the endodermal cells in a process that seems
to reverse the maturation of its formation (Katow and
Sollursh 1980). The majority of pre-endodermal plate cells
at the mid-gastrula stage still do not detach from each other
Structure and movements of pre-endodermal plate
cells during mid- and late gastrula
At the next developmental stage (mid-gastrula), the typical
blastopore looks from the outside like a hole that is the
result of further sinking in of the pre-endodermal plate
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3 Fig. 4 Gastrulation and bottle cell morphology. a–d Pre-endodermal plate (asterisk) starts to sink in and blastopore lips (bl) bend in,
arrowheads show the margins of pre-endodermal plate. a, c and d
present the successive stages of sinking in of the pre-endodermal
plate. b Pre-endodermal plate at higher magnification. e Preendodermal plate bottle cell. Leading edge (le) possesses numerous
filopodia, trailing edge (te) is not yet completely formed. Arrow
shows very slim middle part of a bottle cell. f Embryo at the midgastrula stage with a distinct blastopore. g Bottle cells of midgastrula stage embryo (view from the apical side), their bulged
apical surfaces [forming trailing edges (te) of ingressing cells] are
clearly visible, cilia are still preserved (arrow). h Endodermal bottle
cells of mid-gastrula stage embryo (view from the leading edges)
showing distinct lamellopodia and filopodia. i, j Inner morphology
of mid-gastrula stage embryos. k Enlargement of the pre-endodermal plate from j. l Cells of the pre-endodermal plate begin to detach
from the pre-endodermal plate at the border to the blastopore lip (bl)
and migrate along the inner side of the blastodermal cells (ebc
endodermal bottle cells migrating from the margin of the preendodermal plate) a, c, d, f, i, j, l scale bars=30 μm; b, e, g, h, k
scale bars=10 μm
(Fig. 4i,j,k). However, some bottle cells situated at the
margins of the pre-endodermal plate break their contacts
with the neighbouring blastopore lip cell (Fig. 5f). These
cells rapidly reduce the remaining excess of apical plasma
membrane, and the flagellum disappears from their surface
(Fig. 5f). By this process, the apical surface of these cells
converts into the trailing edge typical for individual
moving cells (Figs. 5f,g and 6d,e). Those cells, which
have completely reduced the junctional contacts, are able to
migrate using the surface of the blastocoel roof (i.e. the
basal side of the blastodermal cells) as a substrate for
migration (Figs. 4l and 6d–f). However, even at the late
gastrula stage, when the blastopore is mostly closed
(Fig. 7a,b), the cells situated in the central zone of the
pre-endoderm plate still do not detach and do not migrate
(Fig. 7c,d). They preserve their contacts with their
neighbours at least until the late gastrula stage. As a result,
the central zone of the pre-endoderm plate is pushed into
the blastocoel as a whole (Fig. 7d), while the marginal zone
of the pre-endodermal plate has lost its integrity (Fig. 7e,f).
Structure and movements of the bastopore lip cells
We observed a sharp morphological boundary between the
ingressing endodermal cells of the pre-endodermal plate
and the ectodermal part of the blastopore, the blastopore lip
cells. The cells of the blastopore lip preserve morphology
and structure of a typical epithelial blastoderm cell (Fig. 2a)
throughout gastrulation. They preserve adherens junctions
between each other (Fig. 5h) and remain in close contact at
the basolateral side. The blastopore lip gradually bends and
even rolls in during gastrulation (Figs. 4c,d,i,j,l, 6c and
7c,e).
Since the later pharynx of the polyp is an inverted
structure with an ectodermal and an endodermal part, we
suppose that the ectodermal part of the pharynx derives
directly from the blastopore lip cells. At the late gastrula
stage, endodermal cells at the boundary to the epithelial
ectodermal blastopore lip complete EMT, detach from their
neighbours and start migrating along the blastocoel roof
(i.e. basal side of blastoderm cells) (Fig. 7e,f). Eventually,
the endodermal cells completely fill the interior of the
blastocoel as individual cells of mesenchymal organization. The organization of the endoderm into an epithelial
sheet occurs only during the transformation into the
primary polyp (Technau, unpublished data).
Variability of gastrulation
The shape of the blastopore is of considerable interest,
since it bears on some important evolutionary scenarios for
bilaterian evolution. For instance, a slit-like blastopore as
observed in many protostomians is thought to represent the
ancestral condition according to the amphistomy concept
(Arendt 2004). Further, asymmetric expression patterns of
important developmental regulator genes around the blastopore such as dpp and fkh have been described recently in
Nematostella (Finnerty et al. 2004; Fritzenwanker et al.
2004). In that respect, the shape of the blastopore in
Nematostella is significant. We observed that the blastopore shape varies considerably from individual to individual (Figs. 4f and 6a,b). While in most cases we observe a
slit-like blastopore (Fig. 6a), more or less triangular (or
sometimes polygonal) forms can also be found (Fig. 6b).
We assume that this is due to the initial shape of the preendoderm plate, which is never circular (Figs. 3j and 4a,b),
and the individual history of gastrulation.
However, not only the shape of blastopore but also the
progression of gastrulation can vary considerably between
individuals of the same stage. Two mid-gastrula stage
embryos having a slit-like blastopore were fractured. Comparing these embryos (Figs. 6c,d), we can find that epithelial continuity between the pre-endodermal plate and the
blastopore lip is still preserved in the embryo shown in
Fig. 6c. However, in the embryo shown in Fig. 6d (see also
Fig. 6e,f), a few marginal cells of pre-endoderm plate have
already completed the EMT, detached from their neighbours and migrated over the blastocoel roof, creating a gap
between the pre-endoderm plate and the blastopore lip.
Moreover, different regions of the same blastopore can
morphogenetically differ from each other. Fig. 6d shows an
example of heterochronic progression of gastrulation.
Distinct separation of the pre-endodermal plate from the
blastopore lip takes place only at one of the two visible
margins of pre-endoderm plate (left side marked by an
arrow on Fig. 6d). The opposite margin still preserves
epithelial continuity (right side on Fig. 6d)—marginal cells
of the pre-endodermal plate are attached to the marginal
cells of the blastopore lip.
Discussion
Since Haeckel (1874) first compared the body plan of the
cnidarians with the hypothetical ancestor, the Gastraea, there
has been a long tradition in the interest of cnidarian
gastrulation (for recent review see Byrum and Martindale
2004). While many researchers regard the cnidarians as the
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127
3 Fig. 5 Ultrustructure of bottle cells and cells of blastopore lip. Leading
edges (former basal ends) of all bottle cells are oriented upward;
trailing edges (former apexes) are oriented downward. a Bulged
apical surface (bas) of newly formed bottle cell; cell preserves intercellular junction (aj) with neighbouring cell. Arrowhead shows the
cilium. b Apical surface of bottle cell reducing the surface area by the
formation of endocytotic vacuole (asterisk). Arrowhead shows the cilium. c Marginal region of pre-endoderm plate. Bottle cells (bc) with
completely formed leading edges (le) and partially reduced excess of
apical surface area. They are in contact with blastopore lip cells (bl).
d Close up of c. Bottle cells preserve intercellular junctions with
each other (arrows) and with the blastopore lip cells (the contact is
surrounded by the frame); contacts between the blastopore lip cells
are shown by arrowheads. e Close up of d. Intercellular junctions
between different types of cells. f ‘Tails’ (trailing edges, te) of two
bottle cells starting to leave the pre-endodermal plate. One of these
cells preserve its contact with the blastopore lip cell. Arrowhead
shows the cilium internalized by endocytosis during the reduction of
apical surface. g ‘Tails’ of two bottle cells leaving the pre-endodermal
plate. h Blastopore lip cells preserving normal epithelial junctions (aj).
Section plan is oriented perpendicularly to the axis passing through
the blastopore. a, b, g, h scale bars=2.5 μm; c–f scale bars=2 μm
unipolar and multipolar ingression and invagination. Together with the partially unresolved cnidarian phylogeny,
this makes it difficult to decide which mode might be
ancestral. This long-standing interest in cnidarian embryogenesis contrasts with an apparent scarcity of descriptive
studies. In particular, detailed ultrastructural studies of
embryogenesis of the basal group of cnidarians, the
anthozoans, are rare. Here we provide the first ultrastructural
analysis of gastrulation of the sea anemone N. vectensis,
which increasingly develops into a major model organism
(Hand and Uhlinger 1992; reviewed by Darling et al. 2005).
The mechanics of gastrulation movements
in Nematostella
first phylum in evolution with proper gastrulation, different
species of cnidarians display all possible modes of gastrulation found in higher metazoans: epiboly, delamination,
Summarizing our results, we can distinguish between four
distinct stages of Nematostella gastrulation (schematically
depicted in Fig. 8): (1) formation of the pre-endodermal
plate from cells starting EMT associated with the formation
of the blastopore lip consisting of epithelial blastoderm
cells undergoing epithelial sheet morphogenesis (Fig. 8a–c,
e); (2) invagination (sinking of pre-endodermal plate)
Fig. 6 Variability of blastopore shape and gastrulation progression.
a More or less slit-like blastopore; b more or less triangular blastopore. Asterisks on a and b show the indentations of the embryonic
surface. c Section passed through the short axis of the slit-like
blastopore, epithelial continuity is preserved, i.e. pre-endodermal
plate is not yet separated from the blastopore lip. d Section passed
through the long axis of the slit-like blastopore (arrowheads, margins
of the pre-endodermal plate). The epithelial continuity is broken on the
left-hand side (arrow), but preserved on the right hand side. e Close up
of left-hand side of d, showing detached and migrating bottle cell; f close
up of e, showing the leading edges of cells migrating over the blastocoel
wall. a–d scale bars=30 μm; e, f scale bars=10 μm
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Fig. 7 Late gastrula stage. a Beginning of blastopore closure; b
mostly closed blastopore. c Section of late gastrula stage embryo.
The pre-endodermal plate is completely pushed in, but cells in its
central zone (arrow) are attached to each other and do not migrate.
The blastopore lip (bl) has further bended inwards; d close up of c
showing the central zone of the pre-endodermal plate; e section of
late gastrula stage embryo. Endodermal cells at the margin to the
blastopore lip (bl) start to detach and fill in the interior of the
blastocoel (arrowheads). Cells of the central zone of the preendodermal plate remain connected. f Close up of e showing
detached mesenchymal cells. a–c, e scale bars=30 μm; d, f scale
bars=10 μm
(Fig. 8d,f–h); (3) separation of the pre-endodermal plate
from the blastopore lip, when the cells situated at the
margin of the pre-endodermal plate complete EMT and
start to migrate (Fig. 8i,j) while the involuting blastopore
lips push the pre-endodermal plate deeper into the blastocoel; (4) closure of the blastopore and completion of EMT
in all pre-endodermal plate cells (Fig. 8k).
Thus, gastrulation in Nematostella is a combination of
invagination of the early pre-endodermal plate and blastopore lips, involution of the blastopore lips and EMT of the
presumptive endodermal cells. However, EMT remains
incomplete until a late stage of gastrulation, because preendodermal plate cells weaken but do not completely lose
their apical intercellular junctions.
Incomplete EMT is found in many instances of cells
undergoing EMT during gastrulation. In fact, it is very
difficult to find an example of simultaneous ingression of
large domains of cells. Usually, cells starting EMT preserve
the junctional contacts with each other and with the
neighbouring (non-ingressing) cells, i.e. they maintain their
epithelial integrity. If all cells were detaching at once, a
large wound would be formed. If, however, only few cells
complete EMT and start migrating, the gap can easily be
closed (for review see Shook and Keller 2003).
We also have evidence for a heterochronic progression of
gastrulation within one embryo, which is presented sche-
matically in Fig. 8h–j. Both the epithelial invagination of the
blastopore lip as well as the detachment of the endodermal
cells can vary temporally at different sites of the blastopore,
which results in a transient asymmetry of the blastopore
(Fig. 8i; see also Fig. 6d). In bilaterian animals, morphoFig. 8 Schematic representation of successive stages of gastrulation "
and bottle cell formation. a–c Successive stages of pre-endodermal
plate formation, clusters of bottle cells are dotted. d Blastopore (black
area) at the mid-gastrula stage. e Completely formed pre-endodermal
plate (dotted area), bl region of future blastopore lip. f Beginning of
pre-endodermal plate sinking and blastopore lip formation, i.e.
beginning of invagination. g Blastopore lip (white cells) and preendodermal plate (dotted cells) during invagination. h Progression of
invagination at the mid-gastrula. i Asymmetric separation of preendodermal cells from blastopore lip by the migration of marginal
cells. Invagination is in progress at the left side of sectioned blastopore,
while at the right side marginal cells of the vegetal plate completed
EMT and have immigrated from the vegetal plate. j Symmetric
separation of pre-endodermal plate from the blastopore lips. k Late
gastrula; the blastopore is already closed, cells in the central zone of
vegetal plate do not migrate, but former margins of the pre-endodermal
plate are already mesenchymal. l Bottle cells at the beginning of
gastrulation. Perimeters of their apexes are already reduced, but apical
surface area remains constant, and apexes are bulge-shaped (bas
bulged apical surface). Cells’ apical ends are attached to each other by
intercellular junctions (double lines). m Completely formed bottle cell
that is ready to migrate (le leading, te trailing edges). n–r Successive
stages of the reduction of the bulged apical surface of bottle cell, the
forming endocytic vacuole is marked by the asterisk
129
130
logical and morphogenetic asymmetry of the blastopore is
often associated with a functional asymmetry of the
blastopore lips. The best known example is the differentiation of the blastopore lips during lancelet and amphibian
gastrulation, but morphological and morphogenetic differentiation of the blastopore regions is also known from many
invertebrate taxa, e.g. Crustacea (Benesch 1969), Echiurida
(Newby 1940), Phoronidea (Rattenbeury 1954). It is possible that this transient asymmetry is mirrored by the
asymmetric expression of forkhead as observed by
Fritzenwanker et al. (2004). Hence, forkhead expression
might reflect the progression of gastrulation on the cellular
level. It is, however, unclear whether this asymmetry has
any implications for the establishment of a second body
axis in Nematostella. In Bilateria, counteracting sog/chordin
and dpp/bmp 2/4 expressions are responsible for establishing the dorso-ventral axis, and dpp/bmp 2/4 is also asymmetrically expressed in anthozoan embryos (Hayward et al.
2004; Finnerty et al. 2004; Holstein T.W., personal communication), suggesting that a second body axis might be
present in Nematostella—at least on the molecular level.
The succession of stages of shape changes in endodermal cells and the hypothetical mechanism of their
realization are presented in Fig. 8l–r. The first step of
gastrulation—shrinkage of apical surface of future endodermal cells—may occur by an actin/myosin-mediated
contraction. This mechanism was already proposed for
bottle cell apical contraction in Drosophila and nematode
gastrulation (see Shook and Keller 2003 for review);
however, in Drosophila, apexes of ingressing cells are flattened from the initial step of gastrulation. In Nematostella,
the perimeter of the cell apex becomes shorter, but the
apical surface area is preserved or insignificantly decreased
(Fig. 8l,n). Interestingly, in Drosophila, forming bottle
cells constrict apexes in a random fashion along the
presumptive ventral furrow region (Leptin and Roth 1994;
Oda and Tsukita 2000). Similarly, the formation of the preendodermal plate in Nematostella starts with the appearance of randomly distributed clusters of forming bottle
cells (Fig. 8a–c). Probably, invagination is initiated by
apical contraction of the pre-endodermal plate cells.
It is unclear whether the invagination of the preendodermal plate is a result of the bottle cells pulling
themselves inside or being pushed by the involution of the
epithelial blastopore lip. Although the bottle cells all form
extensive lamellopodia and filopodia, these seem too short
and do not attach on the opposite side of the blastocoel
wall until a late stage of gastrulation. Hence, it seems
unlikely that the invagination results from a pulling force.
Instead, the combination of apical constriction of the bottle
cells and epithelial morphogenesis of the blastopore lip
appear to lead to the internalization of the pre-endodermal
plate (for reviews of the mechanisms of primary invagination and the role of bottle cells in this process, see
Kimberly and Hardin 1998; Davidson et al. 1999; Keller et
al. 2003).
Further contraction of the cell apex of the bottle cells is
accompanied by the breaking of the apical intercellular
junctions, and the typical trailing edge of an individual
migrating cell is formed (Fig. 8m,o–r). Since this occurs first
at the boundary of the (ectodermal) blastopore lip (Fig. 8i,j),
it seems that the endodermal cells become mesenchymal one
by one as they detach from this site (Fig. 6e,f). Further, the
migrating cells use the blastocoel roof as a substrate and
probably not the extracellular matrix of the blastocoel
(Figs. 6f and 8i,j,l). During the process of ingression, the preendodermal cells also lose their cilium at the surface. The
precise process of how this occurs is not completely clear,
but several of our TEM pictures suggest that large fragments
of apical cell membrane including the cilium are internalized
by endocytosis (Figs. 5b,f and 8o–r).
Our data further suggest that the boundary between the
epithelial (ectodermal) blastopore lip and the ingressing
pre-endodermal cells is stable, i.e. cells on either side do
not change their phenotype, because we do not observe
intermediate stages between the epithelial phenotype and
the bottle cells of the pre-endodermal plate (Fig. 8g). If the
endoderm–ectoderm boundary is stable, as suggested by
our data as well as by gene expression data (Fritzenwanker
et al. 2004), one problem is the large number of cells
needed to fill the interior of the blastoderm. We expect
therefore that pre-endodermal cells continue to proliferate
during and after gastrulation to fill the entire space of the
blastocoel.
In various SEM pictures, we noticed additional indentations on the lateral surface of the blastoderm during midand late-gastrula stage (e.g. Fig. 6a,b) as if single cells were
immigrating at these sites. This has also been suggested by
Byrum and Martindale (2004) on the basis of the expression of gata and mef2 in single cells of the blastoderm.
However, on the inner side, we could not find any evidence
for bottle cells or multipolar immigration of single cells
from the lateral blastoderm. Alternatively, the indentations
might reflect mitotic cells. Refined lineage analysis should
clarify this point in the future.
Molecular control of morphogenetic movements
during gastrulation
The distinct types of morphogenetic movements during
gastrulation coincide with specific gene expression: the
blastopore lips that retain an epithelial morphology express
forkhead and brachyury, while all presumptive endodermal
cells undergoing EMT express the zinc finger gene snailA
(Fritzenwanker et al. 2004; Martindale et al. 2004). This
suggests that these genes might directly regulate the cellular behaviour during gastrulation in Nematostella as proposed earlier (Technau and Scholz 2003). A role for snail
in the regulation of cell–cell adhesion and cell motility
during gastrulation and neural crest formation has been
shown in both vertebrates and invertebrates (reviewed by
Savagner 2001; Nieto 2002; Prindull and Zipori 2004;
Barrallo-Gimeno and Nieto 2005). Snail is a direct
repressor of transcription of the cell junction components,
such as E-cadherin, claudins and occludin (Cano et al.,
2000; Ikenouchi et al. 2003). Snail is also upstream of
pathways involved in the degradation of the basal lamina
131
(Miyoshi et al. 2005). The fact that rhoB, coding for a small
GTPase, lies genetically downstream of Snail homologues,
indicates a role for Snail in cytoskeletal rearrangements
(del Barrio and Nieto 2002). It is therefore plausible that
snailA in Nematostella is involved in the formation of the
bottle cells and breakdown of their apical junctional complexes during gastrulation.
The molecular mechanisms underlying the epithelial
sheet morphogenesis are not clear. Traditionally, T-box and
winged-helix transcription factors are considered to be
crucial for the specification of germ layers (Carlsson and
Mahlapuu 2002; Showell et al. 2004). However, several
studies indicate that they are also involved in the regulation
of epithelial sheet morphogenesis in embryogenesis. For
example, expression of the T-box genes, brachyury and
VegT, are essential for the normal convergent extension of
prospective axial mesoderm during vertebrate gastrulation
(reviewed in Locascio and Nieto 2001). The sea urchin
orthologue of brachyury, LvBrac, is expressed only in the
cells situated at the blastopore lip. As the cells traverse into
the blastocoel, the protein disappears from their nuclei
(Gross and McClay 2001). The expression of forkhead in
Drosophila is also associated with the invagination of the
gut primordia (Weigel et al. 1989). In sum, it seems likely
that these conserved transcription factors regulate the
morphogenetic movements during gastrulation of Nematostella by controlling the specific cellular phenotype.
Evolutionary and comparative considerations
Cell ingression is a major mode of gastrulation in many
cnidarian species, of which the best studied examples are
among the Hydrozoa (e.g. Byrum 2001; Martin et al.
1997). However, there are many differences between
ingression in Hydrozoa and cell migration in Nematostella.
In Hydrozoa, a pre-endodermal plate consisting of bottle
cells is never formed. Cells situated at the site of unipolar
immigration (ingression) are highly polarized, but their
polarity is normal—as in the other blastoderm cells. Single
cells, or small groups of cells undergo EMT, leave the
epithelium and migrate into the blastocoel (Metchnikoff
1886; Rodimov 2000; Byrum 2001). Likewise, in sea
urchins, ingression of primary mesenchyme cells also
proceeds as individual cell migrations. Cells detach from
their neighbours in the beginning of EMT, pass through the
apolar state and acquire the morphology of mesenchymal
cells when migrating through the blastocoel (see Shook
and Keller 2003).
Invagination, on the other hand, is not a widespread mode
of gastrulation among Cnidaria, and this type of morphogenetic movement has never been found in Hydrozoa.
Nevertheless, other types of epithelial sheet morphogenesis
are common in these cnidarians. For example, in embryos of
the hydrozoan D. pumila, fragments of an epithelial sheet
(presumptive ectoderm) are formed from non-epithelial cells
and immediately bent (Kraus and Cherdantsev 1999).
Invagination, however, is quite common among Scyphozoa
and Anthozoa usually in combination with cell immigration
(e.g. Hyde 1894; Ball et al. 2002; summarized in Tardent
1978; Byrum and Martindale 2004). The analysis of the
development of sea anemones Metridium dianthus and
Adamsia palliata suggest that these species gastrulate by the
combination of epithelial invagination and cell immigration,
as Nematostella does (Gemmill 1920). The abundant use of
invagination in the basal group of Cnidaria, the Anthozoa,
could be interpreted such that this is the ancestral mode of
gastrulation for Cnidaria as suggested by early workers
(Haeckel 1874; discussed in Technau and Scholz 2003;
Byrum and Martindale 2004). However, a better phylogeny,
broader and more detailed sampling of gastrulation modes
among the Cnidaria as well as the comparison with
outgroups are needed before reaching a conclusion on this
point.
Acknowledgements We thank Jens Fritzenwanker and Tom Clarke
for critically reading the manuscript. We also thank the members of
the Laboratory of Electron Microscopy of Moscow State University
(G.N. Davidovich, A.G. Bogdanov, N. Zvonkova, M. Leontieva, A.
Lazarev, N.Y. Agalakova) for support and assistance. This work was
supported by the DFG and the core budget of the Sars Centre.
References
Arendt D (2004) Comparative aspects of gastrulation. In: Stern C
(ed) Gastrulation: from cells to embryo. Cold Spring Harbor
Laboratory Press, Cold Spring Harbor
Ball EE, Hayward DC, Reece-Hoyes JS, Hislop NR, Samuel G,
Saint R, Harrison PL, Miller DJ (2002) Coral development:
from classical embryology to molecular control. Int J Dev Biol
46:671–678
Barrallo-Gimeno A, Nieto MA (2005) The Snail genes as inducers
of cell movement and survival: implications in development
and cancer. Development 132:3151–3161
Benesch R (1969) Zur Ontogenie und Morphologie von Artemia
salina. Zool Jb Anat 86:307–458
Byrum CA (2001) An analysis of hydrozoan gastrulation by
unipolar ingression. Dev Biol 240:627–640
Byrum CA, Martindale MQ (2004) Gastrulation in the Cnidaria and
Ctenophora. In: Stern C (ed) Gastrulation: from cells to
embryo. Cold Spring Harbor Laboratory Press, Cold Spring
Harbor
Cano A, Perez-Moreno MA, Rodrigo I, Locascio A, Blanco MJ, del
Barrio MG, Portillo F, Nieto MA (2000) The transcription
factor Snail controls epithelial–mesenchymal transitions by
repressing E-cadherin expression. Nat Cell Biol 2:76–83
Carlsson P, Mahlapuu M (2002) Forkhead transcription factors: key
players in development and metabolism. Dev Biol 250:1–23
Collins AG (2002) Evaluating multiple alternative hypotheses for
the origin of Bilateria: an analysis of 18S rRNA molecular
evidence. Proc Natl Acad Sci U S A 95:15258–15463
Darling JA, Reitzel AR, Burton PM, Mazza ME, Ryan JF, Sullivan
JC, Finnerty JR (2005) Rising starlet: the starlet sea anemone,
Nematostella vectensis. BioEssays 2:211–221
Davidson LA, Oster GF, Keller RE, Koehl MAR (1999) Measurements of mechanical properties of the blastula wall reveal
which hypothesized mechanisms of primary invagination are
physically plausible in the sea urchin Strongylocentrotus
purpuratus. Dev Biol 209:221–238
del Barrio MG, Nieto MA (2002) Overexpression of Snail family
members highlights their ability to promote chick neural crest
formation. Development 129:1583–1593
Finnerty JR, Pank K, Burton P, Paulson D, Martindale MQ (2004)
Origins of bilateral symmetry: Hox and dpp expression in a sea
anemone. Science 304:1335–1337
132
Fritzenwanker JH, Technau U (2002) Induction of gametogenesis in
the basal cnidarian Nematostella vectensis (Anthozoa). Dev
Genes Evol 212:99–103
Fritzenwanker JH, Saina M, Technau U (2004) Analysis of forkhead
and snail expression reveals epithelial–mesenchymal transitions during embryonic and larval development of Nematostella
vectensis. Dev Biol 275:389–402
Gemmill JF (1920) The development of the sea anemones
Metridium dianthus (Ellis) and Adamsia palliata (Bohad).
Philos Trans R Soc Lond B 209:357–367
Gross JM, McClay DR (2001) The role of Brachyury (T) during
gastrulation movements in the sea urchin Lytechinus variegates.
Dev Biol 239:132–147
Haeckel E (1874) The Gastraea—theory, the phylogenetic classification of the animal kingdom and the homology of germlamellae. Q J Microsc Sci 14:142–165
Hand C, Uhlinger KR (1992) The culture, sexual and asexual
reproduction, and growth of the sea anemone Nematostella
vectensis. Biol Bull 182:169–176
Hayward DC, Miller DJ, Ball EE (2004) Snail expression during
embryonic development of the coral Acropora: blurring the
diploblast/triploblast divide? Dev Genes Evol 214:257–260
Hyde I (1894) Entwicklungsgeschichte einiger Scyphomedusen.
Zeitschrift Wiss Zool 58:553–563
Ikenouchi J, Matsuda M, Furuse M, Tsukita S (2003) Regulation of
tight junctions during the epithelium–mesenchyme transition:
direct repression of the gene expression of claudins/occludin by
Snail. J Cell Sci 116:1959–1967
Katow H, Sollursh M (1980) Ultrustructure of primary mesenchyme
cell ingression in the sea urchin Lytechinus pictus. J Exp
Zoolog 213:231–246
Keller R, Davidson L, Shook D (2003) How we are shaped: the
biomechanics of gastrulation. Differentiation 71:171–205
Kim J, Kim W, Cunningham CW (1999) A new perspective on
lower metazoan relationships from 18S rDNA sequences. Mol
Biol Evol 16:423–427
Kimberly EL, Hardin J (1998) Bottle cells are required for the
initiation of primary invagination in the sea urchin embryo. Dev
Biol 204:235–250
Kraus YA, Cherdantsev VG (1999) Variability and equifinality in
the early morphogenesis of the marine hydroid, Dynamena
pumila. Russ J Dev Biol 30:118–129
Kusserow A, Pang K, Sturm C, Hrouda M, Lentfer J, Schmidt HA,
Technau U, von Haeseler A, Hobmayer B, Martindale MQ,
Holstein TW (2005) Unexpected complexity of the Wnt gene
family in a sea anemone. Nature 433:156–160
Leptin M, Roth S (1994) Autonomy and non-autonomy in
Drosophila mesoderm determination and morphogenesis. Development 120:853–859
Locascio A, Nieto MA (2001) Cell movements during vertebrate
development: integrated tissue behavior versus individual cell
migration. Curr Opin Genet Dev 11:464–469
Martin VJ, Thomas M (1977) A fine structural study of embryonic
and larval development in the gimnoblastic hydroid Pennaria
tiarella. Biol Bull 153:198–218
Martin VJ, Littefield, JCL, Archer WE, Bode HR (1997) Embryogenesis in Hydra. Biol Bull 192:345–363
Martindale MQ, Pang K, Finnerty JR (2004) Investigating the
origins of triploblasty: ‘mesodermal’ gene expression in a diploblastic animal, the sea anemone Nematostella vectensis (phylum
Cnidaria; class, Anthozoa). Development 131:2463–2474
Medina M, Collins AG, Silberman JD, Sogin ML (2001) Evaluating
hypotheses of basal animal phylogeny using complete
sequences of large and small subunit rRNA. Proc Nat Acad
Sci U S A 98:9707–9712
Metchnikoff E (1886) Embryologische Studien an Medusen. Ein
Beitrag zur Genealogie der Primitiv-Organe. Alfred Holder,
Vienna
Miyoshi A, Kitajima Y, Kido S, Shimonishi T, Matsuyama S,
Kitahara K, Miyazaki K (2005) Snail accelerates cancer
invasion by upregulating MMP expression and is associated
with poor prognosis of hepatocellular carcinoma. Br J Cancer
92:252–258
Newby WW (1940) The embryology of Echiuroid worm Urechis
caupo. Mem Am Philos Soc 16:1–219
Nieto MA (2002) The Snail superfamily of zinc-finger transcription
factors. Nat Rev Mol Cell Biol 3:155–166
Oda H, Tsukita S (2000) Real-time imaging of cell–cell adherens
junctions reveals that Drosophila mesoderm invagination
begins with two phases of apical constriction of cells. J Cell
Sci 114:493–501
Prindull G, Zipori D (2004) Environmental guidance of normal and
tumor cell plasticity: epithelial mesenchymal transitions as a
paradigm. Blood 103:2892–2899
Rattenbeury J (1954) The embryology of Phoronis viridis. J
Morphol 95:289–334
Rodimov A (2000) A history of embryonic development of
Bougainvillia superciliaris. News of St. Petersburg University
2(11):122–126
Savagner P (2001) Leaving the neighborhood: molecular mechanisms involved during epithelial–mesenchymal transition.
BioEssays 23(10):912–923
Schaefer W (1985) Fortpflanzung und Entwicklung von Anemonia
sulcata (Anthozoa, Actiniaria). II. Fruehentwicklung, Blastula
und Gastrula. Helgolaender Meeresunters 39:341–356
Scholz CB, Technau U (2003) The ancestral role of Brachyury:
expression of Nembra1 in the basal cnidarian Nematostella
vectensis (Anthozoa). Dev Genes Evol 212:563–570
Shook D, Keller R (2003) Mechanisms, mechanics and function of
epithelial–mesenchymal transitions in early development. Mech
Dev 120:1351–1383
Showell C, Binder O, Conlon FL (2004) T-box genes in early
embryogenesis. Dev Dyn 229:201–218
Tardent P (1978) Coelenterata. Cnidaria. VEB Gustav Fischer, Jena
Technau U, Scholz CB (2003) Origin and evolution of endoderm
and mesoderm. Int J Dev Biol 47:531–539
Weigel D, Jurgens G, Kuttner F, Seiffert E, Jackle H (1989) The
homeotic gene fork head encodes a nuclear protein and is
expressed in the terminal regions of the Drosophila embryo.
Cell 57:645–658
Wikramanayake AH, Hong M, Lee PN, Pang K, Byrum CA, Bince
JM, Xu R, Martindale MQ (2003) An ancient role for nuclear
beta-catenin in the evolution of axial polarity and germ layer
segregation. Nature 426:446–450
Wulfert J (1902) Die Embryonalyentwicklung von Gonothyraea
loveni. Allg Zeitschrift Wiss Zool 71:296–325