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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 120 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 121 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 122 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 123 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 124 125 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 126 127 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 128 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. 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