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Dev Genes Evol (2004) 214:220–239 DOI 10.1007/s00427-004-0406-4 ORIGINAL ARTICLE Joshua Morris · Ramachandra Nallur · Peter Ladurner · Bernhard Egger · Reinhard Rieger · Volker Hartenstein The embryonic development of the flatworm Macrostomum sp. Received: 20 January 2004 / Accepted: 15 March 2004 / Published online: 9 April 2004 Springer-Verlag 2004 Abstract Macrostomid flatworms represent a group of basal bilaterians with primitive developmental and morphological characteristics. The species Macrostomum sp., raised under laboratory conditions, has a short generation time of about 2–3 weeks and produces a large number of eggs year round. Using live observation, histology, electron microscopy and immunohistochemistry we have carried out a developmental analysis of Macrostomum sp. Cleavage (stages 1–2) of this species follows a modified spiral pattern and results in a solid embryonic primordium surrounded by an external yolk layer. During stage 3, cells at the anterior and lateral periphery of the embryo evolve into the somatic primordium which gives rise to the body wall and nervous system. Cells in the center form the large yolk-rich gut primordium. During stage 4, the brain primordium and the pharynx primordium appear as symmetric densities anterior-ventrally within the somatic primordium. Organ differentiation commences during stage 5 when the neurons of the brain primordium extend axons that form a central neuropile, and the outer cell layer of the somatic primordium turns into a ciliated epidermal epithelium. Cilia also appear in the lumen of the pharynx primordium, in the protonephridial system and, slightly later, in the lumen of the gut. Ultrastructurally, these differentiating cells show the hallmarks of platyhelminth epithelia, with a pronounced apical assembly of microfilaments (terminal web) inserting at the zonula adherens, and a wide band of septate junctions underneath the Edited by J. Campos-Ortega J. Morris · R. Nallur · V. Hartenstein ()) Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, CA, 90095, USA e-mail: [email protected] Tel.: +1-310-2067523 Fax: +1-310-2063987 P. Ladurner · B. Egger · R. Rieger Institute of Zoology and Limnology, University of Innsbruck, Technikerstrasse 25, 6020 Innsbruck, Austria zonula. Terminal web and zonula adherens are particularly well observed in the epidermis. During stage 6, the somatic primordium extends around the surface dorsally and ventrally to form a complete body wall. Muscle precursors extend myofilaments that are organized into a highly regular orthogonal network of circular, diagonal and longitudinal fibers. Neurons of the brain primordium differentiate a commissural neuropile that extends a single pair of ventro-lateral nerve trunks (the main longitudinal cords) posteriorly. The primordial pharynx lumen fuses with the ventral epidermis anteriorly and the gut posteriorly, thereby generating a continuous digestive tract. The embryo adopts its final shape during stages 7 and 8, characterized by the morphallactic lengthening of the body into a U-shaped form and the condensation of the nervous system. Keywords Platyhelminth · Embryo · Morphogenesis · Organogenesis · Differentiation Introduction Flatworms represent a large and diverse taxon of simple invertebrate animals. Recently they have again attracted the attention of developmental biologists because, in regard to numerous morphological criteria, all or at least some of their taxa may have branched off the phylogenetic tree near the root of the bilaterian stock (Rieger et al. 1991; Ax 1996; Tyler 2001; Jondelius et al. 2002). This makes them a highly relevant system in which to study basic bilaterian developmental processes, such as establishment of the body axes and organogenesis (e.g., Hartenstein and Ehlers 2000; Younossi-Hartenstein and Hartenstein 2000a, b; Hartenstein and Jones 2003). Flatworms share a number of fundamental characters with coelenterates, which evolved before the bilaterians. Most notable among these characters is a single gut opening that serves as both mouth and anus, and a ciliated epidermis used for locomotion (for recent comparative description of adult flatworm anatomy, see Ehlers 1985; 221 Rieger et al. 1991; Ax 1996). Like higher animals, most flatworms have a subepidermal muscular layer of circular, diagonal and longitudinal fibers (see Rieger et al. 1991, 1994; Hooge 2001), and a central nervous system that comprises an anterior brain and a number of longitudinal nerve cords (Rieger et al. 1991; Reuter and Halton 2001). However, the central nervous system lacks compactness, being also penetrated by muscle and gland cells. Nerve tracts issuing forth from the brain extend dorsally, laterally and ventrally. These tracts are connected to up to three subepidermal nerve plexus. The connectivity between neurons and muscles in flatworms is different from higher animals, in that, frequently, long muscle processes (sarco-neuronal processes) approach the nerve fibers, instead of the other way around (see Rieger et al. 1991, similar to the well studied situation in nematodes). The internal structure of flatworms is usually referred to as being very simple. Flatworms do not possess a coelom. The interior of the body is filled with the gut tube and reproductive organs, with complex muscle and connective tissue cells. A specialized vascular system or respiratory system is absent in free-living platyhelminths. The only tubular structures are comprised of protonephridia (see Rohde 2001). Embryonic development has been studied in a number of different flatworm species, studying live embryos and using classical techniques of histology and electron microscopy, as well as, to a very limited extent, cell typespecific markers, including markers for muscle and nerve cells (Thomas 1986; Bagu and Boyer 1990; Reiter et al. 1996; Boyer et al. 1996; Ladurner and Rieger 2000; Younossi-Hartenstein et al. 2000; Younossi-Hartenstein and Hartenstein 2000a, b, 2001; Hartenstein and Jones 2003). Molecular tools, both as markers and as agents to disrupt function, have been developed to study regeneration and cell differentiation in planarians, members of the flatworm clade Tricladida (Sanchez-Alvarado and Newmark 1999; Sanchez-Alvarado et al. 2002; Pineda et al. 2000, 2002; Salo et al. 2002; Cebria et al. 2002; Ogawa et al. 2002). In order to initiate a molecular analysis of embryonic development we have chosen the microturbellarian Macrostomum sp. as a species that can be easily raised in the laboratory, produces a multitude of eggs year round, and has a very short generation time of approximately 2–3 weeks. Macrostomum sp. is a new species that was first briefly characterized in Ladurner et al. (2000), and is now being described by Ladurner et al. (2004). The new species belongs to the clade Macrostomida within the Macrostomorpha. Based on morphological and molecular phylogenetic analysis, the latter occupy a position near the root of the rhabditophoran flatworm tree, which includes the majority of free-living and parasitic taxa (Ehlers 1985; Rieger 2001; Tyler 2001; Littlewood and Olson 2001). Thus, macrostomids produce yolk-rich egg cells (archoophoran mode of development), rather than supplying yolk to the egg by means of specialized yolk cells surrounding a small oocyte (neoophoran mode of development in all other rhabditophoran flatworms except polyclads). Furthermore, the simple pharynx shared by all macrostomids places this taxon near the base of the rhabditophoran platyhelminths (see Doe 1981 and references cited above), a classification that is supported by recent molecular data (Bagu et al. 2001; Curini-Galletti 2001; Jondelius et al. 2001; Littlewood et al. 2001). Macrostomid development has been studied several times, but only very cursorily: histological and much less in vivo by Seilern-Aspang (1957) in M. appendiculatum, in vivo by Reisinger (1923) in M. viride, by Papi (1953) in M. appendiculatum, by Bogomolow (1949, 1960) in M. viride and M. rossicum and finally by Ax and Borkott (1968a, b) in M. romanicum, who provided the first film material about the embryonic development, but still left open a number of important questions. The formation of the body wall was addressed in several more recent light and electron microscopic as well as histochemical studies (Tyler 1981; Reiter et al. 1996; E. Robatscher and B. Egger, Innsbruck, unpublished results). These studies show that Macrostomum sp. as well as other Macrostomum species of the M. hystricinum species clade (see Rieger 1977) share with other primitive flatworm groups a spiral pattern of cleavage. However, development starts to deviate after the first three to four rounds of divisions from the typical spiralian mode, in agreement with the observations by SeilernAspang (1957). Thus, blastomeres at the vegetal pole seem to spread out at the surface of the embryo and form an outer yolk mantle (Tyler 1981). This process was called “inverse epiboly” by Thomas (1986). The transition from this stage to the final generation of body wall and internal organs has not been followed in any detail satisfactorily. In this paper, we have undertaken a developmental analysis of Macrostomum sp. on the basis of observation of live embryos, histological staining of sectioned and whole-mount material, electron microscopy and immunohistochemistry. We define a series of morphological stages that are in accordance with a system introduced for other platyhelminth taxa during recent years (Younossi-Hartenstein et al. 2000). Our work is aimed at providing a guide for the molecular-genetic analysis of platyhelminth embryogenesis, in particular the interpretation of gene expression data from in situ hybridization studies which are currently underway (V. Hartenstein, UCLA, and P. Ladurner, Innsbruck, unpublished data). Materials and methods Animals Macrostomum sp. is a new marine flatworm from the Mediterranean, which is presently being described by Ladurner et al. (2004). It lives in laboratory cultures of the diatom Nitzschia curvilineata. Petri dishes with the artificial growth medium “F2” (Guillard and Ryther 1962) are inoculated with Nitzschia. By 10– 14 days the algae form a dense lawn at the bottom of the dish. Groups of adult Macrostomum (approximately 1.5 mm in length) are transferred onto the algal dish. They produce eggs continuously, and the eggs, if left undisturbed, will develop into sexually mature adults in approximately 2–3 weeks. For preparation, eggs are col- 222 lected and fixed with 4% formaldehyde in PBS buffer. They are washed in PBS-Triton (detergent) and sonicated to permeabilize the tough eggshell. Subsequently, embryos (and juveniles or adults) are prepared for histology and immunohistochemistry following the same protocols that have been established for Drosophila embryos (Ashburner 1989). Electron microscopy and histology Timed stages of embryos of Macrostomum sp. were punctured with electrolytically sharpened tungsten needles and immediately fixed according to Eisenman and Alfert (1982) using the weak osmium cocktail method. After dehydration in standard acetone series, specimens were embedded in Spurr’s low viscosity resin. Blocks were sectioned with an LKB Ultratome. Alternating 1-mm semi-thin sections and sets of 80-nm (silver) ultrathin sections were taken. Ultrathin sections were mounted on net grids (Ted Pella) and treated with uranyl acetate and lead citrate. Semi-thin sections (1 or 0.5 mm) were stained with a 1:1 mixture of methylene blue and toluidine blue/borax (Ashburner 1989). Results Overview and staging system Embryonic development of Macrostomum sp. takes approximately 120 h at 20C. We have subdivided this phase into eight stages, which can be distinguished on the basis of several morphological criteria in living material, as well as fixed and sectioned preparations. The definition Fuchsin labeling of whole-mounts The whole-mount technique that has been extensively used by us and others to label whole embryos of insects and other invertebrates was adapted from Zalokar and Erk (1977). Briefly, following fixation in 4% PBS buffered formaldehyde, embryos, contained within small wire mesh baskets holding 20–50 specimens, were washed in 70% ethanol (three changes of 5 min each) and distilled water (5 min). They were placed in 2 N HCL (10 min) at 60C for DNA denaturation. Following one wash in distilled water (5 min) and two washes in 5% acetic acid, embryos were stained for 15 min in 2% solution of filtered basic fuchsin (in 5% acetic acid). Embryos were washed in 5% acetic acid until cytoplasmic fuchsin labeling was removed, dehydrated in graded ethanol, and transferred to Epon, and individually mounted on slides. Immunohistochemistry To visualize neurons and ciliated cells in whole-mount preparations of embryos, a monoclonal antibody against acetylated tubulin (Sigma; dilution 1:100) and tyrosinated tubulin (Sigma; dilution 1:1,000) were used. Embryos ranging in age between stage 4 and 8 (see Results and Younossi-Hartenstein et al. 2000) that had been fixed in 4% formaldehyde (see above) were washed in PBT (PBS plus 0.3% Triton X-100; pH 7.2; for washing, PBT solution was changed three to five times over a 10-min period) and incubated overnight in PBT containing the antibody at 1:1,000 dilution. After another washing step in PBT the preparations were incubated for 4 h in PBT containing the secondary antibody (peroxidase- or FITC-conjugated rabbit anti-mouse immunoglobulin; Jackson Labs) at a dilution of 1:800. The preparations were washed and incubated with diamino-benzidine (DAB, Sigma) at 0.1% in 0.1 M phosphate buffer (pH 7.3) containing 0.006% hydrogen peroxide. The reaction was stopped after 5–10 min by diluting the substrate with 0.1 M phosphate buffer. Preparations were dehydrated in graded ethanol (70%, 90%, 95%, 5 min each; 100%, 15 min) and acetone (5 min) and left overnight in a mixture of Epon and acetone (1:1). They were then mounted in a drop of fresh Epon and coverslipped. Representative specimens were embedded in Epon and sectioned (1–3 mm) on an LKB ultramicrotome. Sections were counterstained with methylene blue/toluidine blue/borax (Ashburner 1989). Preparations were analyzed and photographed with a Zeiss Axiophot photomicroscope and a Biorad MRC1024ES microscope using Laser sharp version 3.2 software. Figures were assembled and lettered with Adobe Photoshop 6.02 (Adobe). Fig. 1 Overview of stages of Macrostomum sp. development. Panels show highly schematic drawings of embryos of increasing age, shown in lateral view, illustrating the major events of Macrostomum sp. embryogenesis. Anterior is to the left and dorsal to the top. The number at the top left of each drawing indicates the embryonic stage as defined in the text. Tissues are shaded in different colors as shown at the bottom left of the panel. For details see text (br brain primordium, cx cortex, emp embryonic primordium, ep epidermal primordium, eye precursors of the eye, eym external yolk mantle, ge gut epithelium, gp gut primordium, hc hull cell, mln main longitudinal nerve cord, np neuropile, ph pharynx primordium, phe pharynx epithelium, phm pharynx muscle, sm body wall muscles, sop somatic primordium, tp tail plate primordium) 223 Fig. 2a–d Early stages of Macrostomum sp. development: cleavage (stage 1). a Photograph of living embryo at four-cell stage. b, c Photographs of embryo at eight-cell stage, view from animal pole; focal plane through micromere quartet 1a–d (b) and macromere quartet (c). Note larger size of 1D macromere and relatively large size of micromeres. d Parasagittal section showing yolk-rich animal blastomeres (bl) and incipient hull cells (hc; pb polar bodies; scale bar 20 mm) of these embryonic stages is largely based on a similar system that was developed for the rhabdocoel flatworm Mesostoma lingua (Younossi-Hartenstein et al. 2000), and has since then been adapted for other archoophoran and neoophoran flatworm species (Younossi-Hartenstein and Hartenstein 2000a, b, 2001; Hartenstein and Ehlers 2000; Ramachandra et al. 2002). In the following, we will briefly introduce and define the characteristic features of the eight stages (Fig. 1). Stage 1 (0–15 h) represents early cleavage, during which the zygote divides basically in a spiral pattern. In vivo cleavage can only be observed until the third cleavage, after which the opaque yolk granules obscure the borders of individual blastomeres. During stage 2 (15– 30 h), a small number of yolk-rich, presumably vegetal blastomeres with large nuclei expand and surround the other blastomeres (yolk mantle), which form a proliferating mass (here referred to as the embryonic primordium) in the center of the embryo. Stage 3 (30–45 h) is characterized by the expansion and diversification of the embryonic primordium. Anteriorly and laterally, cells of smaller size form the primordium of the body wall and nervous system (somatic primordium); large, yolk-rich cells in the center represent the primordium of the gut. During stage 4 (45–60 h), the outer yolk mantle has become very thin. Definitive primordia of the brain and pharynx can be distinguished at the anterior pole. Stage 5 (60–75 h) is defined by the onset of tissue and organ differentiation. Outer cells along the lateral margins dis- Fig. 3a–d Early stages of Macrostomum sp. development: epiboly of hull cells (stage 2). a Photograph of living embryo. b Wholemount of embryo stained with basic fuchsin in ventral view. c, d Cross sections near anterior and posterior pole, respectively. Hull cells have extended over the surface of the embryo and form the external yolk mantle (eym). Proliferating blastomeres enclosed by the external yolk mantle represent the embryonic primordium (emp in a). Within this mesenchymal cell mass, small cells are clustered near one pole, and larger, yolk-rich cells near the other. We presume that the smaller cells will give rise to the body wall and nervous system (somatic primordium; sop in b and c), and the large-sized cells will form the gut (gut primordium, gp). Arrow in b points at one of numerous mitotic figures that occur throughout early development in somatic primordium and gut primordium (nhc hull cell nucleus; scale bar 20 mm) place the yolk and form the definitive epidermis. A neuropile forms in the center of the brain primordium; pharynx and gut become lined by ciliated cells. During stage 6, the formation of the body wall is completed. The epidermis spreads around the embryo. Gland cells containing rhabdites are scattered throughout the epidermis. Myoblasts form a regular, orthogonal grid of fibers underneath the epidermis. During stage 7, the previously spherical shape of the embryo transforms to an elongated shape and the embryo bends its caudal end towards the ventral side of the anterior body half. The brain primordium condenses, and pigmented eyespots form in the dorsal brain cortex. Stage 8, comprising the final 10–15 h of embryonic development, resembles the fully elongated and differentiated freshly hatched juvenile. Early embryogenesis: cleavage and formation of the embryonic primordium (stages 1–3) Our data confirm previous observations (Reisinger 1923; Bogomolow 1949, 1960; Papi 1953; Seilern-Aspang 1957; Ax and Borkott 1968a, b) stating that early cleavage 224 Fig. 4a–d Early organogenesis in Macrostomum sp.: emergence of organ primordia (stage 4). a Photograph of living embryo. b Whole-mount of fuchsin-labeled embryo in dorsal view. c Cross sections of somatic primordium near anterior pole. d Cross section at mid-levels of embryo. At the anterior of the embryo the primordium of the brain (br) appears as a bilateral condensation within the somatic primordium (sop). The somatic primordium forms a belt around the equator of the embryo. It measures 20–25 cell diameters in length (AP axis), 10–15 cell diameters in width (DV axis) and three to four cell diameters in thickness, bringing the overall cell number up to 2–3,000. Most cells are post-mitotic, evidenced by the scarcity of mitotic figures from stage 4 onwards, as well as the prevalence of small, dense nuclei. The somatic primordium is still covered by a thin yolk mantle (eym in c). The gut primordium (gp) lies in the center of the embryo and contains large, yolk-rich cells (nhc hull cell nucleus; scale bar 20 mm) follows a pattern that shows characteristics of quartetspiral cleavage (Fig. 2). Some significant deviations from spiral cleavage, as seen for instance in polyclads, do occur. Micromeres are not substantially smaller than macromeres (Fig. 2b). At the stage when more than one micromere quartet has been formed, some large, presumably vegetal cells located at the surface start to flatten and surround the embryo (Fig. 2d), transforming into the “hull cells” (Huellzellen), first described by Seilern-Aspang (1957) for M. appendiculatum and Tyler (1981) for M. hystricinum. Hull cells form an external yolk mantle (Dottermantel) that gradually becomes thinner as development progresses (Figs. 1, 3b, d, 4c, d, 5d). The individual hull cells are filled with yolk granules and show elongated nuclei which are larger than those of most blastomeres (Fig. 2d). Once internalized, the cleaving embryonic primordium forms a mass surrounded by yolk-rich hull cells (stage 2; Fig. 3). The time course of mitotic divisions and spindle orientation appears to be quite irregular, and cannot be followed without special markers or dye injections. Cells are variable in size and contain yolk granules to a greater Fig. 5a–d Organogenesis of Macrostomum sp.: onset of organ differentiation (stage 5). a Photograph of living embryo, anterior view. b Tilted cross sections of embryo near anterior pole. c, d Whole-mounts of fuchsin-labeled embryos in ventral view (c) and lateral view (d). Cells at the outer surface of the somatic primordium have undergone a mesenchymal-epithelial transition and form the primordium of the epidermis (ep). Deep cells of the somatic primordium form precursors of muscle cells (sm; arrowheads in b) and other cell types associated with the body wall. The somatic primordium does not yet form a complete body wall; dorsally and ventrally, remnants of the external yolk mantle (eym) still abut the surface. Neurons of the brain primordium have differentiated and form a central neuropile (np) surrounded by a cortex (cx) of somata. Posterior to the brain is the primordium of the pharynx (ph), formed by a cylindrical array of columnar epithelial cells. Arrow in (d) points at cells that close the pharynx lumen ventrally. Note the folding of the eggshell ventral of the arrow, which probably constitutes an artefact related to shrinkage of the preparation. Precursors of muscle cells around the pharynx are indistinct, small, spindle-shaped cells. The gut primordium (gp) contains large, yolkrich masses, interspersed with small and dense yolk spheres (ys). The posterior of the embryo contains the primordium of the tail plate (tp). Scale bars 20 mm Fig. 6a–j Organogenesis in Macrostomum sp.: ultrastructure of organ primordia. a–j Electron micrographs of details of cross sections of a stage 5 embryo. a Overview of somatic primordium, containing superficial layer of epidermal precursors (ep) and deep layer of muscle precursors and neural precursors (ne). Epidermal precursors are cylindrical, ciliated (ci) epithelial cells. Numerous yolk granules (yg) are still present in the basal portion of the epidermal precursors. b Border between epidermal primordium (ep) and external yolk mantle (eym). c Magnified views of part of b, showing details of the junctional complex formed between epidermal precursors. The complex is formed by a pronounced, septate junction (sj) near the apical pole of the cell. Further apical still is the zonula adherens (aj) with its characteristic membrane density and cytoplasmic plaque. An unusual feature also noticed at later stages is the widening of the intercellular cleft in the zonula ad- 225 herens (arrow). c Magnified views of part of b, showing details of the junctional complex formed between the epidermal precursor (ep) and external yolk mantle (eym). The septate junction is similar to the one between epidermal cells; the adherens junction is not as wide and lacks a cytoplasmic plaque on the side of the yolk mantle (arrowhead). e, f Magnified views of part of a, showing details of bundles of cell processes (cp), probably axons, located at the border between neural precursors (ne) and epidermal cells (ep). Myofilaments are not yet formed at this stage. g, j Apical part of epidermal cell, showing cilia (ci), zonula adherens (aj), septate junction (sj), and terminal web (tw; microfilaments inserting at the zonula adherens). Apical of the terminal web is a layer of ultrarhabdites (urh), which are small secretory granules of epidermal cells. Mitochondria (mit) are clustered underneath the terminal web. h, I Detail of cell that does not yet form part of the epidermal primordium but has started formation of cilia (ci). Scale bars: 2 mm a; 1 mm b, e, g, h 226 or lesser extent. Anterior cells, which generally seem smaller in size than posterior cells (Fig. 3b, c), constitute the precursors of epidermis, body-wall muscles and nervous system (somatic primordium). During stage 3, the somatic primordium expands posteriorly, forming two symmetric plates along the lateral sides of the embryo. It is unclear whether this expansion involves actual cell migration, or whether cells at posterior levels which earlier had been large and yolk-rich, decrease in size and then join the somatic primordium. Blastomeres in the center of the embryo (gut primordium) remain large and yolk-rich (Fig. 3d). These cells later transform into the gut epithelium (Fig. 4d). The formation of organ primordia (stages 4 and 5) By mid-embryogenesis (stage 4) proliferation becomes less pronounced and organ primordia take shape. The most prominent primordium is that of the brain, forming a bilaterally symmetric mass of densely packed cells at the anterior pole of the embryo (Fig. 4a, b). Posterior to the brain, the somatic primordium consists of intermingled epidermal, gland, muscle and other precursors that form plates alongside the lateral surface of the embryo (Fig. 4a, b). The external yolk mantle still covers the surface, but is compressed into a thin layer (Fig. 4c, d). The gut primordium fills the posterior-central part of the embryo (Fig. 4a, b, d). Starting towards the end of stage 4, peculiar spherical structures can be distinguished within the gut primordium (Figs. 4d, 5d, 7e, 10f). These “yolk spheres” stand out in histological sections, in electron micrographs (not shown) and can even be seen in living material (not shown). The appearance of definitive organ primordia occurs concomitantly with the onset of cellular differentiation during stage 5. Most prominent during this stage are the epidermis, brain, pharynx and gut. Cells located at the external surface of the embryonic primordium transform into a ciliated epithelial epidermal layer that displaces the external yolk mantle (Figs. 5, 6a–d). The onset of ciliation of the multiciliated cells has been described in detail by Tyler (1981). As of yet we do not have data that indicate the resorption of the yolk mantle by the primary epidermis. During stage 5, the epidermis covers only the flanks of the embryo, whereas yolk still reaches the periphery of the embryo at dorsal and ventral levels (Fig. 5a, b). As development proceeds, the epidermis and underlying cells of the somatic primordium stretch in the transverse axis and fully enclose the embryo by the end of stage 6. Cells located underneath the epidermis differentiate primarily as muscle and nerve cells. The anterior of the embryo is dominated by the large brain primordium. Neurons extend axons towards the center of the primordium, resulting in the formation of a typical invertebrate ganglion, which consists of a cortex of neuronal cell bodies surrounding a central neuropile (Fig. 5b, c, f; see also Rieger 1998). The cortex is three to four cell bodies in thickness, except posteriorly where only a single layer of cell bodies separates the neuropile from the developing pharynx (Fig. 5b). At the beginning, the brain primordium is wide (in the transverse axis) and flat (in the AP axis); as development progresses, the primordium condenses, becoming increasingly narrower and thicker (compare Figs. 5b and 11b). The pharynx primordium appears postero-ventrally of the brain primordium as a cylindrical array of cuboidal cells that develop as the ciliated pharynx epithelium (Fig. 5b–d, see also later stages, e.g. Figs. 7d, 10f). As has been described for other flatworm species (see Thomas 1986 and Bagu and Boyer 1990 for summaries), the pharynx primordium does not evolve by invagination of a pre-existing epithelium, but develops within the deep layer of the somatic primordium. The pharynx lumen initially seems to have no connection to the epidermis, or to the gut lumen. The connection forms during stage 6 when the pharynx primordium elongates both dorsally and ventrally, and establishes contact with the ventral epidermis. Muscle precursors initially appear as elongated and flattened cells forming irregular layers underneath the epidermal primordium (see also Reiter et al. 1996 for M. hystricinum). Muscle precursors are concentrated in a bilateral band that extends along the flanks of the embryo (Figs. 5b, 7b). Fewer muscle precursors populate the space between epidermis and brain primordium, and around the pharynx. Muscle differentiation sets in towards the end of stage 5 with the formation of myofilamentcontaining processes which form a highly regular network of longitudinal, circular and diagonal fibers (see section Muscular system). A conspicuous condensation of cells in the deep layers of the somatic primordium at the posterior tip of the embryo demarcates the primordium of the tail plate (Fig. 5c) The tail plate primordium consists of muscles and glands of the duo-gland adhesive system (Tyler 1988), that allow the freshly hatched animal to quickly grab on to the substrate. Cells from this caudal region later may also give rise to the external genitalia, i.e., the genital pores and the male copulatory organ. Along with the gonads, the external genitalia apparently do not differentiate during the embryonic period, and will not be considered here. Organ differentiation (stages 6–8) Final organ formation takes place during the last 2 days of the 5-day embryonic period (stages 6–8; Figs. 7, 8, 9, 10, 11, 12, 13). Epidermis During early stages (5 and 6) the epidermal primordium consists of cylindrical multiciliated epithelial cells (Fig. 6a, b). Underneath the apical membrane the terminal web, a dense layer of microfilaments, is already devel- 227 Fig. 7a–h Organogenesis of Macrostomum sp.: epiboly and differentiation of the body wall (stage 6). a Photograph of living embryo, ventral view, b, c Whole-mount of fuchsin-labeled embryo in ventral view (b) and lateral view (c). d, e Parasagittal sections of embryo. f–h Magnified views of part of brain primordium (f), body wall (g) and gut primordium (h). During stage 6, the epidermal primordium (ep) extends around the embryo, replacing the external yolk mantle. Coordinated epidermal ciliary beating causes the embryo to rotate in the eggshell. Beside the smooth epidermal layer, the brain (br, cx cortex, np neuropile), pharynx (ph) and gut (gp) can be clearly distinguished in the living embryo. In fixed wholemounts (b, c), the brain and pharynx primordia stand out. Nuclei of gut cells are not labeled optimally. The increasing number of axons leads to a thickening of the neuropile (np). Underneath the epidermis, somatic muscle precursors (sm in b, c, g) have differentiated and form flat, elongated cells that produce a regular network of circular, diagonal and longitudinal fibers. The pharynx has lengthened (c) and cilia can be recognized under light microscopy in the lumen of the pharynx (b, d), as well as the gut (e). The gut primordium has reorganized into a population of large, yolk-rich epithelial cells (ge in e) loosely arranged around a central lumen. Some of the yolk still forms dense yolk spheres (ys in e, h). At the posterior end of the embryo, a subepidermal condensation of cells forms the primordium of the male copulatory apparatus and the tail plate (tp). Scale bars: 20 mm a–e; 10 mm f–h oped (Fig. 6g, j). Ultrarhabdites (or epitheliosomes; for terminology see Rieger et al. 1991), small vesicles above the terminal web, are already present between the terminal web and the apical membrane (Fig. 6d, g). Basally, epidermal cells still contain numerous yolk granules, which will decrease as development proceeds (Fig. 6a). The junctional complex in between epidermal cells is well established by stage 5. It consists of an apical zonula adherens and a wide belt of septate junctions below (Fig. 6g, j). Of special significance is that septate junctions and adherens junctions also join neighboring cell membranes at the boundary between epidermal primordium and hull cells (Fig. 6c). The zonula adherens exhibits the characteristic membrane thickening and sub- 228 Fig. 8a–i Organogenesis in Macrostomum sp.: ultrastructure of the body wall. a–i Electron micrographs of details of longitudinal sections of a stage 6 embryo. a Overview of body wall, with cil- iated epidermal layer (ep), subepidermal muscle fibers (mf), main longitudinal cord (mln) and gut primordium (gp). Epidermal cells still contain yolk granules (yg) near their basal pole. b Magnified 229 membraneous plaque. However, the cleft that separates cells at the level of the zonula adherens is wider, rather than narrower, than the intercellular cleft at more basal levels (Fig. 6g, see also below, Fig. 9d). This feature of the zonula adherens (“open zonula adherens”) has also been observed in embryos of other flatworm species (Younossi-Hartenstein et al. 2000) and is generally observed in adult specimens which were anesthetized with isotonic MgCl2 solutions (Tyler 1984). Later epidermal cells become cuboidal to squamous, except for the anterior region (apical plate) and tail plate, which maintains more cylindrical epidermal cells (Fig. 9a, d). Microvilli become very prominent. A thin and patchy ECM separates the epidermis from the underlying muscle and nerve cells (Fig. 9a, h). It has been observed in other macrostomids (Rieger et al. 1991) that epidermal cells form extensive sheath-like processes at their baso-lateral membrane, which intercalate between neighboring cells, suggesting that this interdigitation of epidermal cells may compensate for the absence of a continuous basement membrane. However, interdigitation of epidermal cells is not a significant feature of epidermal cells in Macrostomum sp. (Fig. 9a). The apical junctional complex between epidermal cells remains similar to the one described for stage 5, with a wide and prominent septate junction and an open zonula adherens (Fig. 9d). Epidermal nuclei acquire their characteristic multilobulate shape during stage 7 (Fig. 13a). During this stage, rhabdite-filled gland cells with subepidermal cell bodies and long excretory ducts permeating the epidermis can be distinguished by light (Fig. 11f) and electron microscopy (Fig. 9d). These rhabdite glands are rather evenly distributed over the dorso-lateral sides of the body (Ladurner et al. 2004). Specialized glands are the adhesive glands in the tail plate (Fig. 9j), as well as a population of rhammite glands whose cell bodies are located posterior to the brain and whose long necks cross the brain to terminate in the apical plate (not shown). Brain view of apical part of epidermal cell with cilia (ci), ultrarhabdites (urh), terminal web (tw), zonula adherens (aj) and septate junction (sj). Epidermal cells contain prominent Golgi complexes (Ga). c Oblique section of main longitudinal cord (mln) carrying axons (ax) from the brain to the periphery. Scattered patches of electron dense extracellular material (basement membrane, bm) separate nerve and muscle fibers from overlying epidermis. d, e Muscle cell body (sm), circular muscle fiber (cf) and longitudinal muscle fiber (lf). Note close contact between axons (ax; magnified view in e) and muscle plexus. f Overview of sagittal section reaching from brain (to the left of panel) and pharynx (right), showing neurons (ne), neuropile (np), muscle fiber piercing brain cortex (brm), pharynx epithelium (phe) and pharynx muscle (phm). g High magnification of central neuropile. h Root of main longitudinal nerve cord (mln) and attached muscle cells (sm) and longitudinal muscle fibers (lf). I High magnification of deep array of cilia (ci) surrounded by protonephridial precursor cell (pn). Scale bars 1 mm Main longitudinal nerve cords and protonephridial system Brain neurons differentiate during stages 5 and 6 when the brain primordium forms a broad and flat structure at the anterior tip of the animal (Figs. 5b, 6b, c). Whole-mounts labeled with anti-tyrosinated tubulin (tyrTub; Fig. 12) reveal that the brain cortex is formed by multiple clusters of five to ten neurons each. These clusters (possibly lineages) extend axons that fasciculate together, forming a thin tract; several such tracts in turn converge and form a distinct neuropile compartment. We will describe details of the anatomy of the juvenile and late embryonic brain elsewhere (Morris et al., in preparation) and will therefore keep the description of the nervous system brief. During stage 5 (Fig. 12a–c), four large systems of tracts that lay down the brain neuropile can be distinguished. Each system is formed by six to eight converging axon fascicles; during later stages, as more neurons differentiate, fascicles are added to each system. Medially in the brain, a dorsal and ventral medial longitudinal system (MLV, MLD) is laid down. Axon fascicles contributing to these systems are formed by neuronal clusters located in the anterior cortex; to a lesser extent, posterior neurons with anteriorly projecting axons also exist. The medial longitudinal systems can be followed throughout development into the brain of freshly hatched juveniles (Fig. 12g–I); at this stage, following brain condensation, systems of both sides approach each other and are separated only by a narrow cleft. Adjacent to the longitudinal systems are the medio-dorsal and medio-ventral commissural fiber systems (MCD, MCV). The MCD forms the largest system in the early embryo. It comprises commissural axon fascicles emitted by at least ten clusters of neurons located in the dorsal cortex. Fewer axon fascicles of neuronal clusters located in the ventro-medial cortex converge to form the ventro-medial commissural tract (MCV; Fig. 12) Finally, axons of neuronal clusters in the lateral wings of the brain primordium come together as the lateral commissural (LC) system. During later stages, the same subdivision into MCV, MCD, MLD, MLV and LC remains visible, although the number of neuron clusters and axon fascicles formed by them goes up by a factor of 2 and 3. The main longitudinal nerve cords (MLN) extend from the posterior surface of the brain towards the tail of the embryo (Figs. 10c, 11e, 12g). They form one large postpharyngeal commissure (not shown). Densely packed cell bodies and muscle cells accompany the axons. The protonephridial system extends parallel and dorsal to the MLN (Figs. 9a, b, 12k, l). In the hatching juvenile, the MLN (at a level posterior to the post-pharyngeal commissure) contains approximately 100 axons. The MLN reaches towards the tail where it thickens to form a caudal ganglion associated with the muscles and glands of the 230 Fig. 9a–j Ultrastructure of hatching juvenile. a–d Cross sections of body wall, showing epidermis (ep), subepidermal gland cells (glc) with rhabdites (rhb), muscle fibers (mf), protonephridia (pn) with ciliated lumen (ci), main longitudinal nerve cord (mln) and gut epithelium (ge). Epidermal cells are covered apically by microvilli (mv in d) and cilia (ci). Pronounced junctional complex (jc) interconnects neighboring cells. Microfilaments form terminal web (tw). e–g Cross section of posterior brain (br) and pharynx (phe). 231 tail plate (not shown). The protonephridial system is formed by a pair of longitudinal tubes which gives off several evenly space side branches. The tube and side branches are lined by a flat epithelium. AcTub and tyrTub labeling of embryo whole-mounts reveal the cilia in protonephridia for the first time during late stage 5 (Fig. 12k). Specialized cells called flame cells or cyrtocytes cap the blind ends of the side branches. Protonephridial tubules are readily detectable in electron micrographs of late embryos (Fig. 9a, b). In cross section they appear as electron dense cells with a 2–4 mm wide inner lumen, filled with densely packed cilia. In ventral view they appear as five to six regularly spaced, cylindrical structures (Fig. 12l) that are easily mistaken for axons, in particular due to the fact that they are located so close to the main nerve cord. We assume that the early acTub labeling corresponds to the short, cyrtocyte-containing branches of the protonephridial system. In hatched juveniles, a continuous longitudinal trunk that opens anteriorly into the pharynx lumen is labeled in addition to the cyrtocytes (not shown). Muscular system Muscle cells form a grid of extraordinary regularity underneath the epidermis; they also surround the neuropile of the brain, the pharynx, gut and male copulatory apparatus (see post-embryonic differentiation in Rieger et al. 1991, 1994 in M. hystricinum). The subepidermal muscular plexus of the late embryo forms a pattern that is closely correlated to the pattern of epidermal cells (Fig. 13). This is particularly obvious in the case of the longitudinal fibers, which are thicker and more invariant in number and spacing than the circular and diagonal fibers. As evident from Z-projections of a series of confocal sections in which both muscle fibers and epidermal nuclei are labeled, there exists an almost perfect 1:1 relationship between rows of epidermal nuclei and longitudinal fibers (Fig. 13a, b). The epidermis in freshly hatched juveniles measures approximately 30–35 cells in perimeter (at midbody level), and possesses the same number of longitudinal muscle fibers. Two systems of preferentially longitudinal fibers diverge from the subepidermal muscle grid and extend into Brain neuropile (np) is separated from pharynx only by a scattered population of neuronal cell bodies (ne). Muscle fibers (mf) traverse the neuropile. Densely ciliated, cylindrical pharynx epithelium (phe) surrounds a circular lumen (phl). Muscle cells form a thin layer of scattered fibers (phm) at the basal surface of the epithelium. Necks of gland cells (pgl) terminate bilaterally in pharynx lumen. h Section of body wall. An elongated muscle cell (sm) gives rise to a longitudinal fiber (mf). A thin basement membrane (bm) separates epidermis (ep) and muscle layer. I Cross section of gut with internal lumen (gtl) and gut epithelial cells (ge) covered by cilia (ci). j Section of adhesive glands (agl) clustered in the tail plate at posterior tip of animal. Scale bar 1 mm (panels B, C, F, G are twofold magnifications of areas outlined by rectangles in a and e, respectively) the interior of the animal where they form a muscle net around the brain neuropile and the pharynx, respectively (Fig. 13a, b). The brain-related deep muscle plexus arises in the anterior third of the animal. It forms a group of crescent-shaped fibers that in part skirt the outer surface of the brain, in part pass through the cortex and neuropile (see also Rieger et al. 1991, Fig. 12, p 144 and Rieger et al. 1994, Fig. 3c for M. hystricinum marinum). These fibers seem to be invariant in relationship to other components of the brain and can serve as landmarks for subdividing the brain into anatomically defined compartments (see also unvaried condition in freshly hatched juvenile of M. hystricinum marinum in Rieger et al. 1994). The second deep muscle grid branches off the subepidermal grid in the mid body and is organizing the pharynx, thereby serving as the structural support (“pharyngeal suspensor”) anchoring the pharynx to the body wall (“pharynx holding apparatus” in freshly hatched juveniles, see Rieger et al. 1994 for M. hystricinum marinum). Ventrally, pharyngeal suspensor fibers extend forward underneath the gut, then continue as longitudinal fibres on the pharynx wall (Fig. 13c, d). Dorsal suspensor fibers pass over the dorsal gut surface, then curve ventrally, pass between brain and pharynx, and continue as longitudinal fibers in the ventro-anterior pharyngeal wall. Together with an inner layer of circular muscles, these longitudinal fibers form the intrinsic musculature of the pharynx (for adults, see also Doe 1981). Muscle differentiation is initiated during stages 5 and 6. The embryonic muscle pattern has been documented by Reiter et al. (1996) who used phalloidin to visualize the actin-rich myofilaments. Our materials can add little to this description. Muscle precursors initially form a rather irregular layer underneath the epidermal primordium (Fig. 5b, c). During stage 6, these cells elongate and send out processes that are preferentially arranged longitudinally. Diagonal and circular fibers appear to develop later. Fibers are 0.5–1 mm in diameter (Figs. 8d, h, 9d, h); based on the findings of Reiter et al. (1996), longitudinal fibers are much fewer and extend almost along the entire length of the animal, and circular fibers surround half of the circumference. Given the close correlation between muscle fibers and epidermal cells in the late embryo, it is likely that such correlation may exist from the very beginning of muscle patterning. This would imply that inductive interactions between the two tissue layers control the positioning of muscle fibers and epidermal cells. Freshly hatched juveniles possess a thin layer of muscles surrounding the gut (Ch. Seifert, Innsbruck, unpublished results). In preparations of embryos, gutassociated muscles were undetectable; we therefore assume that formation of this visceral muscle layer must take place shortly before hatching. The musculature surrounding the genitalia develops post-embryonically. 232 Fig. 10a–h Late phase of organogenesis (stage 7). a Photograph of living embryo, ventral view, b, c Whole-mount of fuchsin-labeled embryo in ventral view. d, e Parasagittal and horizontal histological sections of embryo. f–h Magnified details of section shown in e. a Embryo has flattened and elongated and moves actively in the eggshell. A characteristic feature of stage 7 are the pigmented eyespots (eye) embedded in the brain (br). b, c Brain has condensed in medio-lateral axis (compare with corresponding views of stage 6 and stage 5 embryos shown in previous figures). The eyes are embedded in the dorsal brain cortex (cx in b). Main longitudinal cords (mln) can be seen leaving the brain posteriorly. Lumina of pharynx (ph) and gut (gtl) have become confluent (arrow in c). d, e Sections show epidermis (ep), brain with neuropile (np) and cortex (cx), pharynx (ph; arrow points at junction between lumina of gut and pharynx), gut epithelium (ge), and tail plate (tp). f Magnified view of body wall with epidermis (ep), gland cell (glc) and rhabdite (rh) filling external secretory duct of gland cell. g Ciliated pharyngeal epithelium. h Epidermis with cilia (ci), darkly staining terminal web (tw), basement membrane (bm), gland cell (glc) and body-wall muscle (sm, ge gut epithelium, gp gut primordium). Scale bars: 20 mm a–e; 10 mm f–h Pharynx and gut During stages 5 and 6, the pharynx primordium appears as a rosette-shaped structure that is closely attached to the posterior surface of the brain (Figs. 5b, c, 7b, c). Compared to other flatworms, the number of cells forming part of the pharynx primordium is not high, and a radial or- ganization of myoblasts that is so characteristic of the pharynx primordium of higher flatworms is absent. The pharynx primordium starts out as a cylinder three to four cell diameters high and six to eight cell diameters in perimeter. These cells form the epithelial lining of the pharynx. In the interior of the embryo, the pharyngeal lumen borders the gut primordium of the early embryo; 233 Fig. 11a–f Late phase of organogenesis (stage 8). a Photograph of living embryo, lateral view. b, c Whole-mount of fuchsin-labeled embryo in ventral view. d–f Cross sections of embryo labeled with acTub. a Embryo has elongated further and adopted shape of juvenile. b, c Brain has continued to condense in medio-lateral axis. Eyespots (eye) have come closer to each other (compared to stage 7) following brain (br) condensation. d–f Sections taken at level of anterior brain (d), pharynx (e) and gut (f). AcTub labels densely stacked cilia on epidermis (ep), pharynx (ph), gut epithelium (ge) and protonephridia (pn). Gland cells containing elongated rhabdites (rh) can be recognized. The terminal web (tw) forms a dark line along apical surface of epidermis (cx cortex, mln main longitudinal nerve cord, np neuropile, sm body-wall muscles, tp tail plate). Scale bars: 20 mm a, b, d–f; 10 mm c ventrally, the pharynx lumen initially seems to be closed off by hull cells or precursors of the epidermis (Fig. 5c, arrow). During stage 6, the pharynx elongates and forms an opening at the ventral surface (Fig. 7c, arrow). Pharynx epithelial cells resemble epidermal cells in their ultrastructural differentiation, including the apical ciliation, terminal web and junctional complex (Fig. 9e, g). In late embryos, gland cells with long secretory ducts can be seen to open into the pharynx lumen (Fig. 9g). A sparse network of circular muscle fibers, as well as neurons and axon fascicles, surround the pharynx epithelium (Fig. 9g). 234 235 Formation of the gut is difficult to follow, because cell boundaries are obscured by the high yolk content of gut precursors. From late stage 6 onward a narrow central lumen surrounded by cilia can be observed (Fig. 7e). Initially, the gut lumen is closed on all sides (Fig. 12k); during stage 7, the inner end of the pharynx connects to the gut, and the lumina of pharynx and gut become confluent (Fig. 10c, e, arrow). The gut epithelial cells that surround the lumen and carry cilia on their apical surface are few in number and reach a large size, compared to other cells of the late embryo. During intermediate stages of gut development (stages 4–6), cell borders are difficult to discern light microscopically. Whether the gut primordium represents a true dynamic syncytium will remain an interesting problem for future studies. Discussion In this paper, we have described the principal steps in morphogenesis that shape the embryonic development of the macrostomid flatworm, Macrostomum sp. We have employed a morphological staging system recently introduced for the rhabdocoel species Mesostoma lingua (Hartenstein and Ehlers 2000), and thereafter adapted to other flatworm taxa, including polyclads (YounossiHartenstein and Hartenstein 2000b) and acoels (Ramachandra et al. 2002). The stages facilitate the comparison of developmental events in different groups, and will add a useful tool to the description of gene expression patterns that form part of the molecular-genetic analysis of embryogenesis currently under way. Fig. 12a–l Organogenesis in Macrostomum sp.: development of the nervous system and protonephridia. a–l Confocal sections of wholemounts of embryos at stage 5 (a–c), stage 6 (d–f) and stage 8 (g–h) labeled with tyrTub antibody, which labels microtubular skeleton. Sections show brain primordium of one side; sections of upper row (a, d, g) were taken at dorsal level, middle row (b, e, h) at intermediate level, and lower row (c, f, I) at ventral level of developing neuropile. Labeling shows outline of neuronal cell bodies and axons. At stage 5 (a–c), several neuropile founder clusters can be distinguished: dorsal medial longitudinal cluster (mld), ventral medial longitudinal cluster (mlv), dorsal medial commissural cluster (mcd), ventral commissural cluster (mcv), and lateral commissural cluster (lc). Axon bundles formed by these clusters are later joined by more fibers, resulting in the growth and compaction of the neuropile. Axons forming the main longitudinal cord (mln) that extends into the trunk can be distinguished from stage 6 onward (not shown) and are prominent at late stages (g). TyrTub antibody also labels cilia of epidermis (ep), pharynx (ph) and protonephridia (pn). j, k Wholemount of stage 5 embryo labeled with acTub antibody labeling cilia of epidermis (j), pharynx (k; ph), gut lumen (gtl) and protonephridia (pn). l Whole-mount of stage 7 embryo labeled with acTub. Protonephridia (pn) are formed by tubules arranged in a row that extends on either side of the embryo dorsal of the main longitudinal nerve cord. Scale bars: 10 mm a–l; 20 mm j–l New aspects of the quartet-spiral development in platyhelminthes and the evolution of ectolecithal eggs of the neoophora Macrostomid development begins with a spiral cleavage of the entolecithal egg, a feature it shares with all other archoophoran “turbellarians”, including polyclads and acoels. Subsequently, during later cleavage (stage 2) the developmental path of Macrostomum sp. starts to deviate from that of typical spiralian embryos (as represented by the polyclads), in that some blastomeres extend around the embryonic primordium (a process called “inverse epiboly” by Thomas 1986) and give rise to a layer of yolk-rich hull cells that form an external yolk mantle (see Tyler 1981, for the M. hystricinum species group defined by Rieger 1977; Gehlen and Lochs 1990). In a Macrostomum species, such a yolk mantle originating from hull cells was first reported by Seilern-Aspang (1957 for M. appendiculatum) who described the formation of these cells from early vegetal blastomeres. For a similar species, Papi (1953) remarks that the fate of blastomeres could not be followed in vivo beyond the eight-cell stage. In other Macrostomum species hull cells have not been mentioned, but may have simply been overlooked (see Reisinger 1923, for M. viride). Blastomeres could be followed during a typical quartet-spiral cleavage up to the 64-cell stage by Bogomolow (1949, 1960) for M. viride and M. rossicum). Ax and Borkott (1968a, b, for M. romanicum) refer to a hull membrane that obscures the embryo after the 16-cell stage. Based on stylet morphology, all species of the genus Macrostomum used in these previous embryological studies appear to be related to the M. hystricinum species group (see Luther 1960, Fig. 17). Macrostomum sp., on the other hand, is a member of the M. tuba species group (Ladurner et al. 2004). Hull cells therefore appear to be a common feature within the genus Macrostomum. As mentioned by Ax (1961), the lack of hull cells in Macrostomum species described by Bogomolow (1949, 1960) warrants further investigation. Gastrulation in Macrostomum sp. also deviates significantly from the typical spiralian mode that prevails in other archoophorans. Previous studies had not provided any details on gastrulation because, as already stated by Seilern-Aspang (1957) for M. appendiculatum, the mode and type of gastrulation in living embryos cannot be observed due to the presence of the yolk mantle. Our data suggest a morphogenetic process that leaves out typical gastrulation movements and, like the formation of the external yolk mantle, bears resemblance to early developmental stages in neoophorans (Bresslau 1904; Hartenstein and Ehlers 2000). Thus, following the establishment of the yolk mantle, cells in the interior of the embryo sort out into a yolk-rich gut primordium, formed by large cells located more centrally, and a somatic primordium that consists of smaller cells located anteriorly. These cells gradually expand posteriorly and form plate-like mesenchymal cell masses on either side of the gut primordium. Only during late stages of embryogenesis, long after onset 236 Fig. 13a–d Muscle pattern of juvenile. Whole-mounts were labeled with phalloidin (red, labels myofilaments) and Sytox (green, labels nuclei); ventral view, anterior up . a, b Z-projection of five consecutive confocal sections (1-mm interval) showing pattern of epidermal nuclei (ep) and somatic muscle fibers (sm) of ventral body wall. b Magnified view illustrating one-to-one relationship between columns of epidermal nuclei and longitudinal muscle fibers. c Z-projection of 12 consecutive confocal sections (1-mm interval) showing deep systems of muscle fibers, consisting of brain-related muscles (brm), pharyngeal suspensors (psm), and internal pharyngeal muscles (phm). d Schematic diagram of juvenile, lateral view, clarifying location of muscle systems. (cx Cortex, gt gut, np neuropile, ph pharynx, vm visceral muscle; scale bar 20 mm) of cell differentiation, does the somatic primordium expand dorsally and ventrally to enclose the gut primordium. The difference between this peculiar mode of body wall formation and the corresponding process in polyclads and other spiralians is evident. Thus, in polyclads, hull cells are absent. The definitive epidermis primordium is formed at an early stage during gastrulation by animal micromeres, which by epibolic movements stretch over the large, yolk-rich cells at the vegetal pole (Surface 1908; Boyer et al. 1996, 1998; Younossi-Hartenstein and Hartenstein 2000b). The latter include the descendants of the fourth quartet micromere that form the mesoderm and the gut, and the macromeres providing yolk that will come to be located in the lumen of the gut, or (in case of polyclad flatworms) stay rather small and degenerate at an early stage (Surface 1908). The origin of hull cells from blastomeres may have evolutionary significance for the development of ectolecithal eggs, an issue that clearly needs further investigation. Ax (1961) drew attention to the different ontogenetic origin of hull cells in certain archoophora and neoophora. In the latter, hull cells have been described as descendants of yolk cells (vitellocytes; Bresslau 1904; Thomas 1986; Hartenstein and Ehlers 2000). Giesa (1966) also describes, for the proseriate Monocelis, a primary cover of the ectolecithal embryo formed by specialized yolk cells (“Vitellocytenepithel”). Later, a secondary set of hull cells that arise from blastomeres displace the vitellocytes. Such blastomere-derived hull cells have also been described for other proseriates (Minona, Bothrioplana, Otomesostoma) as well as for the lecithoepitheliate Xenoprorhynchus by Reisinger et al. (1974a, b). The formation of an external yolk mantle by hull cells and the absence of a characteristic gastrulation are two criteria which would place macrostomids between the more “basal” archoophorans (e.g., polyclads) and neoophorans. This is in contrast to other criteria, such as the structure of the pharynx, which, in macrostomids, is of the pharynx simplex type, whereas poIyclads show the more advanced pharynx plicatus type. More cytological studies on the hull cells are needed. In any event, the new data on the embryonic development of Macrostomum presented here open new avenues to investigate the origin of the heterocellular gonad with ectolecithal eggs and highly derived embryonic development from the basic quartetspiral cleavage of primitive archoophorans. The inverse epiboly seen in Macrostomum sp. (of the M. tuba species group) and certain other macrostomid species (of the M. hystricinum species group) poses an interesting phenomenon also from a developmental perspective. Spiralian cleavage is commonly seen as a developmental mechanism that generates fixed lineages. 237 Injecting early blastomeres with lineage tracers has revealed a high degree of invariance in regard to what organs and cell types of the hatching larva are derived from which blastomere (Costello and Henley 1976; Verdonk and van den Biggelaar 1983; Henry and Martindale 1998; Boyer et al. 1996, 1998; Henry et al. 2000). Experimental manipulations and molecular studies have also revealed intrinsic cell fate determinants that are expressed at an early stage in a distinct blastomere and are then distributed by means of an invariant pattern of cleavage divisions to the different cells whose fate they control (Bagu and Boyer 1990). As pointed out above, in Macrostomum sp., cleavage initially follows the conventional pattern, but then departs from this pattern when certain blastomeres, most likely vegetal cells, stretch out around the remaining blastomeres to form the outer yolk mantle. It is currently not clear exactly which cells contribute to the yolk mantle. Inverse epiboly begins at a stage when only two to three micromere quartets are formed. Seilern-Aspang (1957) suggested that either all four macromeres, or part of the macromeres plus part of the third quartet micromeres, give rise to the outer yolk mantle. In either way, the remaining blastomeres that become surrounded by the external yolk need to generate all of the cell types, including the mesoderm and the gut, with a reduced number of macromeres or no macromeres at all. Once surrounded by the yolk mantle, cell divisions in these inner blastomeres (the embryonic primordium) are difficult to observe. So far, we have no observations that any sort of spiral pattern is resumed; mitotic spindles appear to be random, and cell sizes initially do not suggest a distinction between macromeres and micromeres. Dye injection-based lineage studies are required to follow later cleavage and to reconstruct lineage relationships. It will also be interesting to establish how the expression of specific determinants in Macrostomum sp. has adapted to the novel types of lineages. For example, are endo- and mesodermal fate determinants, which in conventional spiralians appear in the macromeres and in specific fourth quartet micromeres, not expressed in early vegetal blastomeres, but rather come on at a later stage in distinct locations within the embryonic primordium? We expect that molecular-genetic studies will shed light on this and related questions that are important in reconstructing the evolution of developmental mechanisms. Organogenesis from organ primordia The developmental steps that lead from the early embryonic primordium of Macrostomum sp. to the body of the hatching juvenile correspond closely in most important aspects to what has been described for other flatworm taxa. Thus, following the establishment of the undifferentiated embryonic primordium, individual organ primordia appear. The first organ primordia invariably are those of the brain and the pharynx; in Macrostomum sp., as in the dalyellid (rhabdocoel) Gieysztoria superba and the temnocephalid (rhabdocoel) Craspedella pedum, the pharynx primordium appears directly posterior of the brain (Younossi-Hartenstein and Hartenstein 2000a, 2001); in the typhloplanoid (rhabdocoel) Mesostoma lingua the pharynx primordium is formed at mid-levels along the antero-posterior axis (Bresslau 1904; Hartenstein and Ehlers 2000), and in triclads (e.g., Schmidtea polychroa; Bennazzi and Gremigni 1982) it defines the posterior pole. The position of the pharynx primordium at early stages thus closely corresponds to the position where the pharynx ends up later. Aside from its location, the later small size and low complexity of the Macrostomum pharynx is also reflected in its early appearance. This supports the notion that the pharynx simplex coronatus, as defined by Doe (1981) for all Macrostomorpha, may be homologous to the other pharyngeal differentiations seen in higher rhabditophoran flatworms (see Ehlers 1985; Rieger et al. 1991, for summaries). Precursors of the massive arrays of radial muscle fibers characteristic of the pharynx bulbosus, the most advanced pharynx type, are visible from the very beginning; by contrast, in Macrostomum sp., pharynx muscle precursors in the pharynx simplex form only a thin, inconspicuous layer around the pharynx epithelium. The primordium of the body wall musculature in Macrostomum sp. first becomes apparent during stage 5 as an irregular, two to three cell diameters thick cell layer underneath the epidermis. A series of block-shaped myoblasts have been reported for M. hystricinum marinum (Reiter et al. 1996). These authors observed a close association between myoblasts and neuroblasts during differentiation, perhaps reflecting the intricate functional relationship of muscle and nerve cells in the body-wall musculature. Myoblasts subsequently flatten, elongate and send out muscle processes. Nothing can be said currently about the earlier origin of muscle precursors. In larval polyclads there is a different origin of longitudinal and circular muscles, the longitudinal fibers deriving from the vegetal 4d blastomere called ento-mesoderm, the circular fibers from cells deriving from the 2b micromere, called ecto-mesoderm or ecto-mesenchyme (Boyer et al. 1996). In Acoela all muscle cells are apparently derived from the ento-mesoderm (Henry et al. 2000). Dye injection-based lineage tracing experiments will be required to verify whether a similar distinction also exists for Macrostomum sp. Furthermore, we have recently cloned the homolog of the muscle specification gene Mef 2 (Morris et al., in preparation) which, in both vertebrates and Drosophila, is expressed in mesoderm, followed by muscle cells. Macrostomum sp. Mef 2 is expressed specifically in the adult musculature, and it is hoped that in situ hybridization to early embryos will provide us with a more detailed picture of the origin of muscles in this species. Specific molecular markers will also be needed to make progress in understanding the origin of the central nervous system in Macrostomum sp. or any other flatworm. Neuronal precursors can only be distinguished from myoblasts at the time when cell differentiation sets in, and neurons send out axons (see Reiter et al. 1996; Rieger 1998). The tyrTub marker allows one to study late aspects of neural differentiation (similar to acTub in other 238 species; Younossi-Hartenstein et al. 2000; YounossiHartenstein and Hartenstein 2001), but it does not help to follow crucial steps in early neurogenesis, because it labels all cells at this early stage. Given the close spatial relationship between differentiating muscle and nerve cells (see above), it is highly likely that precursors of these cells are intermingled at an earlier stage. This would be in stark contrast to “higher” animals, including arthropods and vertebrates, where these tissues originate apart from each other in different germ layers. With the present study on the embryogenesis of Macrostomum sp. we could not resolve the intriguing issue of the origin of the unique post-embryonic stem cell system (neoblast system) from embryonic stem cell precursors (see Ladurner et al. 2000; Peter et al. 2001, 2004). As summarized in Peter et al. (2001), circumstantial evidence even suggests that platyhelminths seem not to have a separate germ line. It is the general assumption in the literature that the possibly totiponent post-embryonic stem cell system (the neoblast system) originates from certain embryonic stem cells (see literature in Peter et al. 2001). Our investigation on Macrostomum sp., similar to many others on organ differentiation in platyhelminths, does support the notion that the organ primordia are clusters of embryonic stem cells that, like post-embryonic stem cells, are the only cells capable of mitotic proliferation. In analogy to post-embryonic stem cells (neoblasts), embryonic stem cells might either come from a single, totipotent stem cell pool in each organ primordium or each organ primordium may have specific subtypes of embryonic stem cells that differentiate only into specific tissue types. 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